Hindawi Publishing CorporationInternational Journal of Cell BiologyVolume 2013 Article ID 458727 10 pageshttpdxdoiorg1011552013458727
Review ArticleAlternative Splicing Programs in Prostate Cancer
Claudio Sette12
1 Department of Biomedicine and Prevention University of Rome ldquoTor Vergatardquo 00133 Rome Italy2 Laboratory of Neuroembryology Fondazione Santa Lucia IRCCS 00143 Rome Italy
Correspondence should be addressed to Claudio Sette claudiosetteuniroma2it
Received 31 May 2013 Accepted 11 July 2013
Academic Editor Michael Ladomery
Copyright copy 2013 Claudio SetteThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Prostate cancer (PCa) remains one of the most frequent causes of death for cancer in the male population Although the initialantiandrogenic therapies are efficacious PCa often evolves into a hormone-resistant incurable diseaseThe genetic and phenotypicheterogeneity of this type of cancer renders its diagnosis and cure particularly challenging Mounting evidence indicates thatalternative splicing the process that allows production of multiple mRNA variants from each gene contributes to the heterogeneityof the disease Key genes for the biology of normal and neoplastic prostate cells such as those encoding for the androgen receptorand cyclin D1 are alternatively spliced to yield protein isoforms with different or even opposing functions This review illustratessome examples of genes whose alternative splicing regulation is relevant to PCa biology and discusses the possibility to exploitalternative splicing regulation as a novel tool for prognosis diagnosis and therapeutic approaches to PCa
1 Introduction
Cancer cells are characterized by uncontrolled growth andability to migrate from the primary lesion and to establishmetastases in distant tissues Standard therapies involve sur-gical removal of the tumor mass radiation and chemother-apy which exploit the increased growth rate of cancer cellswith respect to surrounding cells More targeted approacheshave also been developed in the last decades by directlyinhibiting the function of the oncoproteins responsible forthe neoplastic transformation Nevertheless although manyhuman cancers initially respond to therapies and in somecases patients are cured most of them are characterized bydisease relapse that often occurs in more aggressive andincurable forms In this regard a clear example of aggressiverelapsing tumor is represented by prostate carcinoma (PCa)which remains one of the main causes of death for cancer inthe male population [1 2] Understanding the mechanismsthat lead to the acquisition of resistance to therapies in PCapatients might offer new molecular markers for earlier andmore accurate diagnoses Furthermore identification of thekey players involved in the transition to therapy-refractorystages may shed light on new targets for pharmaceuticalintervention and open the path for the development of noveland more efficacious therapies
PCa cells rely on androgens and on the androgen receptor(AR) for proliferation [1] Under normal conditions the ARis localized in the cytoplasm upon binding to androgens thereceptor dimerizes translocates to the nucleus and trans-activates genes containing androgen-responsive elements intheir promoter regions Clinical treatments are currentlybased on androgen ablation therapies obtained by chemicalcastration with drugs that block secretion of the hormoneor by directly targeting the AR with androgen antagonists[1 2] However after initial remission many patients developa hormone-resistant or castration-resistant form of PCa(CRPCa) for which no cure is available [1 2] Notablyin most cases CRPCa cells still require the AR but theycan bypass activation by androgens or be stimulated by thelow androgen levels present during the therapy or by theantagonists used for the therapy [3] Several mechanisms forthe development of androgen insensitivity of the AR havebeen documented [1] Among these recent evidence pointsto alternative splicing (AS) of AR as a key resource utilizedby PCa cells to evade the normal route of activation of thispathway [4]
AS is emerging as a key step in the regulation of crucialcellular and developmental pathways in higher eukaryotes[5] With regard to PCa it has been proposed that theldquosplicing signaturerdquo represents a more accurate parameter
2 International Journal of Cell Biology
to stratify patients than the ldquotranscriptome signaturerdquo whichis typically analysed by conventional microarray analyses [6]Thus understanding the regulation of splicing in normal andpathological prostate cells may help identify novel markersand targets for future therapeutic approaches to this neoplas-tic disease
2 Alternative Splicing and Cancer
The recent advent of high-throughput RNA sequencing hasunveiled new layers of regulation of gene expression andhighlighted the extreme complexity and versatility of thegenome The majority of human genes encode multiple tran-scripts through the use of alternative promoters AS andalternative polyadenylation [5 7 8] AS is a combinatorialmechanism that expands the coding potential of the genomeby allowing the production of protein isoforms with differentor even antagonistic functions from a single gene [5 7 8]Splicing is orchestrated by a ribonucleoprotein complexcalled ldquospliceosomerdquo which recognizes exon-intron junc-tions excises introns and ligates exons The lack of stringentconsensus sequences at exon-intron junctions in highereukaryotes allows flexibility in recognition by the spliceo-some Numerous RNA binding proteins (RBPs) interact withcomponents of the spliceosome and reinforce or weaken rec-ognition of exon-intron junctions The interplay betweenthese splicing factors determines the choice of variable exonsby the spliceosome and causes heterogeneity in pre-mRNAprocessing [5 7] As a consequence changes in the expressionlevels or in the activity of splicing factors can selectivelyinfluence AS of many genes [5 7] Although the flexibility ofAS regulation has represented an evolutionary advantage forhigher eukaryotes it also represents a risk factor In particu-lar mounting evidence illustrates how defective regulation ofAS correlates with onset and progression of human cancers[7 8]
Herein the literature describing the impact of AS inthe onset and progression of PCa will be reviewed High-throughput analyses of specimens from PCa patients havehighlighted more than 200 genes whose AS is differentiallyregulated in the neoplastic tissue [6] indicating that thismechanism can substantially contribute to the heterogeneityof gene expression in cancer cells Nevertheless the physi-ological consequences of the majority of these aberrant ASevents are still unknown and will require direct investigationThis review will focus on the regulation of genes and splicingregulators whose relevance for PCa has been firmly docu-mented
3 The Androgen Receptor
Several reports have documented the expression of alter-natively spliced AR variants lacking the C-terminal ligand-binding domain of the canonical receptor (reviewed in [4])Many of these AR splice variants are constitutively nuclearand active even in the absence of androgens (Figure 1) thusindicating their potential role in the acquisition of the CRPCaphenotype [4] Expression of most of these variants arises
from the inclusion of cryptic exons located in intron 2 and 3of theAR geneThe heterogeneity of the AR variants reportedin various studies is also due to the frequent amplificationof the exon 2-exon 3 genomic region of AR [4 9] In allreported variants however the splicing of these cryptic exonsinvariably introduces premature stop codons and terminationsites thereby yielding shorter AR proteins of 75ndash80 kDawhich lack the androgen-binding domain [4] In some casesthese truncated AR variants can function independentlyof the full-length AR and their selective knockdown wasshown to block the androgen-independent growth of CRPCacells while maintaining responsiveness to the hormone [10]Importantly expression of an AR variant containing a crypticexon located in intron 3 (CE3) in clinical PCa specimenspositively correlated with poor prognosis after surgery [11]This variant was also expressed at higher levels in CRPCawith respect to PCa patients [12] Furthermore constitutivelyactive truncated AR splice variants were recently shown toconfer resistance also to the next generation of AR inhibitorsthus limiting their therapeutic efficacy for many patients [13]However since the expression of these shorter AR variantswas also observed in normal prostate tissues it is unlikelythat they drive the initial steps of neoplastic transformationopening the possibility that AS of the AR gene plays also aphysiological role in the gland [4]
The mechanisms that lead to increased expression ofaberrant AR splice variants in PCa are still largely unknownOne possible cause of defective splicing is the alteration ofthe genomic AR locus which often occurs in CRPCa Forinstance disruption of the AR splicing pattern in the 22Rv1PCa cell line was linked to duplication of the genomicregion containing exon 3 and some of the cryptic exons [9]Alternatively aberrant expression of specific splicing factorsin PCa cells may also contribute to unbalanced splicing andaberrant recognition of cryptic exons in the AR gene Thusgiven the strong relevance of these constitutively active ARvariants for CRPCa progression further studies elucidatingthe regulation of their expression are strongly encouraged
4 KLF6
Kruppel-like factor 6 (KLF6) is a zinc finger transcriptionfactor that is mutated in a subset of human PCas [14 15]KLF6 is known to regulate cell proliferation by inducingthe expression of the cell cycle inhibitor p21 (WAF1CIP1)Notably this effect of KLF6 does not require p53 suggestingthat KLF6 is a tumor suppressor gene that functions as a p53-alternative brake for cell cycle progression in normal cells[14] One of the mutations found in PCa patients consistsof a single nucleotide change that creates a binding site forthe splicing factor SRSF5 (SRp40) and enhances splicing ofthree alternative mRNA variants encoding for truncated KLFproteins named KLF6-SV1 SV2 and SV3 [16] These splicevariants are upregulated in tumor versus normal prostatictissue A single G gt A mutation in intron 1 was shown torecapitulate the altered splicing pattern of KLF6 when itwas introduced in a minigene and it was found to correlatewith worse prognosis in patients [16] The KLF-SV1 variant
Cellular transformationEnhancement of AR-dependent transcription
1 32
Proapoptotic
1 32
AR gene
BCL-X gene
CCND1gene
BCL-XS
BCL-XL
5998400 5998400 3998400
Antiapoptotic chemotherapyresistance
Figure 1 Representative examples of genes whose alternative splicing affects prostate cancer cell biology The left side of the figure illustratesthe genomic structure of the alternatively spliced regions of the AR CCND1 and BCL-X genes Solid and dashed lines show the alternativesplicing events reported in the literature On the right side the alternative variants produced by splicing are shown The specific features ofthe protein isoforms produced by alternative splicing are summarized under the scheme of each variant
was characterized further and shown to function as adominant-negative protein which antagonizes the functionof full length KLF6 leading to decreased p21 expressionand enhanced cell growth [16] Increased expression ofthis splice variant in PCa patients predicted poorer out-come after surgery and was associated with development ofhormone-refractory metastatic PCa [17] Furthermore while
knockdown of the full length KLF6 promoted tumor forma-tion in nudemice selective silencing of the KLF6-SV1 variantinhibited it [18] Conversely PCa cells overexpressing KLF6-SV1 are more prone to develop metastases in various organsof the mouse models used in the study [17] Thus a mutationaffecting KLF6 AS represents a critical mechanism for theinactivation of a tumor suppressor gene in PCa suggesting
4 International Journal of Cell Biology
that interfering with this splicing event in PCa cells mightrestore the growth-inhibitory activity of this transcriptionfactor
5 Cyclin D1
CCND1 is a protooncogene that encodes for cyclin D1 whichassociates with the cyclin-dependent kinase 4 (CDK4) todrive progression through the G1 phase of the cell cycleImportantlyCCND1 expression is oftenderegulated in cancercells [19 20] This gene encodes for two alternative tran-scripts the common cyclin D1a isoform containing all fiveexons and cyclin D1b which derives from retention of intron4 and premature termination of the transcript (Figure 1) [1920] Unlike cyclin D1a cyclin D1b alone can promote cellulartransformation [21] and its expression has been associatedwith PCa progression and poor prognosis [22] Interestinglyrecent evidence indicated that cyclin D1b promotes AR-dependent transcription of genes involved in PCa metastaticpotential such as the transcription factor SLUG [23] CyclinD1a was instead reported to repress the transcriptionalactivity of AR (Figure 1) [19 20] Thus it is conceivable thata change in the ratio between the cyclin D1 variants willpotently enhance hormone-dependent growth of PCa cells
Given the relevance for PCa cell biology understandingthe regulation of CCND1 splicing is of crucial importanceIt was observed that a polymorphism (G870A) at theexon 4-intron 4 boundary predisposes cells to cyclin D1bsplicing [19 20] The splicing factor SRSF1 was shown tobind the exon4intron4 junction in the nascent CCND1 pre-mRNA thereby promoting intron 4 retention and cyclin D1bexpression [24] SRSF1 was hypothesized to favour intron 4retention by altering exon 4 definition and limiting assemblyof the spliceosome at the exon-intron junction [24] Anothersplicing factor promoting cyclin D1b expression in PCa cellsis SAM68 [25] In this case the binding site was identifiedwithin intron 4 in proximity of the termination site utilizedfor the cyclin D1b mRNA The binding of SAM68 to thisregion of the pre-mRNA was shown to compete with thatof the U1 snRNP [25] Since deposition of U1 snRNP nearcryptic polyadenylation sites located in introns is known toprevent premature termination of transcripts in a genome-wide fashion [26] it is possible that up-regulation of SAM68unmasks the cyclin D1b termination site by interfering withU1 snRNP binding in intron 4 Notably both SRSF1 andSAM68 display oncogenic features in several cell types andtissues [7 27] and their expression positively correlates withthat of cyclin D1b in clinical specimens of PCa patients [2425] Thus it is possible that interfering with the activity ofthese splicing factors will exert positive effects in therapeutictreatments of PCa through modulation of CCND1 splicingand expression
6 BCL-X
The BCL-X (BCL2L1) gene contains 3 exons and encodes fortwo splice variants [28] Two alternative 51015840 splice sites arepresent in exon 2 of the gene selection of the canonical
one at the end of the exon yields the long BCL-XL variantwhereas selection of the distal one located 220 bp upstreamin the exon produces the short BCL-XS variant Notablythese two splice variants have opposite effects in the cell withBCL-XL being prosurvival whereas BCL-XS is proapoptotic(Figure 1) [28] Thus regulation of BCL-X AS can finelymodulate cell viability illustrating the biological importanceof this splicing event In most cancer cells including PCacells the anti-apoptotic BCL-XL variant is overexpressed andconfers resistance to chemotherapeutic treatments [29 30]It is predictable that a full understanding of the mechanismsof regulation of BCL-X splicing will help develop tools toswitch it toward the proapoptotic BCL-XS variant therebyoffering a therapeutic opportunity to sensitize cancer cellsto treatments In line with this notion treatment of PCacells with an antisense oligonucleotide (ASO) masking theBCL-XL splice site effectively switched BCL-X splicing andinduced apoptosis [29] Interestingly the proapoptotic effectof the ASO was more pronounced in cancer cells whichdisplay high levels of expression of BCL-XL and it alsoenhanced their response to chemotherapeutic treatments[30] Thus ASOs targeting BCL-X splicing may have theadvantage of being selective for cancer cells with respect tonormal cells which is a positive feature for an antineoplasticdrug Unfortunately the delivery of ASOs to cancer cells isstill not efficient thus limiting their application in the cliniceven though development of vehicles favoring their deliverysuch as lipid nanoparticles [31] may aid in this direction
Although regulation of BCL-X splicing is highly relevantto PCa cell biology not much is known on the mechanism(s)of its regulation in prostate cells A possible regulator is theprotein phosphatase 1 (PP1) whose activity is required for theregulatory effect of ceramide on BCL-X splicing [32] IndeedPP1 activity was required also for induction of BCL-XSsplicing by emetine a protein synthesis inhibitor and otherproapoptotic drugs in PCa cells [33 34] Nevertheless themechanism by which PP1 modulates splicing of BCL-X is stillunknown PP1 is known to regulate splicing by modulatingthe activity of splicing factors either by direct binding tothem and regulation of their phosphorylation status [35] orindirectly by regulating kinases involved in their post-translational modifications [36]Thus activation of pathwaysimpinging on PP1may affect BCL-X splicing and cell viabilitythrough the regulation of the activity of specific splicingfactors in PCa cells
Several splicing factors have been shown to modulateBCL-X splicing Studies performed in a variety of cell modelsindicated that the heterogeneous nuclear ribonucleoprotein(hnRNP) H and F [37] and the splicing regulators SAM68[38] RBM25 [39] and RBM11 [40] promote splicing of theproapoptotic BCL-XS variant By contrast hnRNPK [41] theserine-arginine (SR) rich proteins SRSF1 [38 42] and SRSF9[43] and the splicing factor SAP155 [44] enhance splicing ofthe anti-apoptotic BCL-XLWhich of these factors contributeto the regulation of BCL-X splicing in PCa cells is stilllargely unknown SRSF1 [24] and SAM68 [45] were shownto be upregulated in PCa and might represent strong candi-dates for the regulation of this splicing event Intriguinglythese splicing factors normally modulate BCL-X splicing in
International Journal of Cell Biology 5
opposite directions [38] while the up-regulation of SRSF1 isin line with the high levels of BCL-XL in PCa cells SAM68should favour the proapoptotic short variant However thesplicing activity of SAM68 is finely tuned by phosphoryla-tion [46] and it was shown that tyrosine phosphorylationby the Src-related kinase FYN switched SAM68-dependentsplicing of BCL-X toward the anti-apoptotic variant [38 47]Since tyrosine phosphorylation of SAM68 is increased inspecimens of PCa patients [48] it is likely that this RBPcan also contribute to the upregulation of BCL-XL in PCacells In line with this hypothesis BCL-XL expression wasdecreased and sensitivity to genotoxic agents was increasedafter knockdown of SAM68 in the androgen-sensitive LNCaPcell line [45]
Thus based on the observations reported previouslyit is predictable that exogenous modulation of BCL-X ASthrough administration of ASOs or by interfering with theactivity of the splicing factors that promote the anti-apoptoticBCL-XL variant will enhance the efficacy of chemotherapy inadvanced PCa as suggested by preclinical studies in PCa celllines [30 31 45]
7 TMPRSS2ERG
ERG is a member of the ETS transcription factor family thatis expressed at very low levels in benign prostate epithelialcells However PCa patients often carry a fusion of theandrogen-responsiveTMPRSS2 genewith ERG which causesaberrantly high expression levels of the transcription factorin the neoplastic cells A detailed sequencing analysis of theTMPRSS2ERG transcripts isolated fromPCa tissues revealedthat fusion-derived transcripts underwent profound AS reg-ulation which yielded mRNA variants encoding both fulllength ERG proteins and isoforms lacking the ETS domainNotably an increase in the abundance of transcripts encodingfull length ERG correlated with less favorable outcome inpatients [49] These results support a possible functional rolefor this transcription factor in PCa pathology and suggest thatmodulation of AS events promoting less pathogenic variantsmay produce beneficial effects
This hypothesis is also supported by another study thattested the effects exerted by the expression of TMPRSS2ERGalternatively spliced transcripts in an immortalized prostatecell line [50] It was found that these TMPRSS2ERG splicevariants had different oncogenic activities in terms of pro-moting proliferation invasion and motility Notably coex-pression of different variants produced stronger effects thaneither variant alone suggesting that the presence of severalTMPRSS2ERG isoforms as it normally occurs in PCa cellsmight confer a more malignant phenotype [50] A furthercontribution of AS to the heterogeneity of TMPRSS2ERGexpression is provided by the extensive variability of the 51015840untranslated region (UTR) in the splice variants observedin patients [51] Indeed AS of the 51015840 UTR affects the onco-genic potential of the encoded proteins by regulating theirtranslation and activity Thus although a functional linkbetween TMPRSS2ERG expression and PCa pathology hasnot been firmly established yet this fusion gene appears
to be another suitable target for an AS-directed therapeuticapproach that would spare normal cells not expressing thechimeric proteins
8 Splicing Programs in Prostate Cancer
Cancer cells express a number of splice variants that conferthem higher resistance to chemotherapeutic drugs and sur-vival advantages When it was investigated in detail the spe-cific signature of splice variants expressed by cancer cells hasbeen recognized as a powerful diagnostic and prognostic tool[7 8] Evenmore importantly the existence of cancer-specificsplicing variants of key genes such as the AR or CCND1in PCa might offer a therapeutic opportunity for targetingproteins that are not expressed in healthy cells For instancedeveloping tools that specifically modulate the expressionof transcript variants preferentially or uniquely produced bycancer cells might slow down tumor growth andor promotecell death during therapy while sparing the healthy tissuesThus understanding AS regulation at the genome-wide levelin PCa cells may not only lead to the identification of noveldiagnostic or prognostic biomarkers but it could also helpfind tools for novel therapeutic approaches to this neoplasticdisease
A few studies have directly investigated the genome-wideregulation of AS in PCa cell lines and primary tumor tissuesUsing a splicing-sensitive microarray comprising a selectedsubset of genes and splice variants it was shown that splicingsignatures could efficiently segregate PCa cells lines from can-cer cell lines derived from other organs or tissues [6] Amongthe alternatively spliced genes the majority also showedvariation in expression levels [6] suggesting that regulationof splicing and transcriptionwere coupled as also observed incells exposed to DNA damage [52] Using the same splicing-sensitive platform it was also possible to identify splicingsignatures that were specific for normal or neoplastic prostatetissues obtained from biopsies [6] Although this approachwas limited to the genes and the splice variants selected for theplatform it provided a first indication that specific changes insplicing occur during prostate tumorigenesis and suggestedthat splicing variants can represent accurate biomarkers forPCaNevertheless how andwhen these changes occur aswellas to what extent they contribute to the acquisition of thetransformed phenotype are still open questions Given thetight association between transcription and splicing a spe-cific splicing program could result from the different activityof transcription factors splicing factors or both Mountingevidence indicates that all these events contribute to someextent to the acquisition of specific splicing signatures in PCa
The most relevant transcription factor involved in PCais the AR Several observations suggest that in addition toregulating the expression levels of target genes AR can alsoinfluence the transcript variants encoded by them Usingcomprehensive splicing-sensitive arrays it was demonstratedthat stimulation of LNCaP cells with androgens caused qual-itative changes in expression of splice variants [53] Many ofthe events altered by treatment with androgens were due tousage of alternative promoters within the transcription unit
6 International Journal of Cell Biology
of the target gene Some of these alternative transcripts werepredicted to influence the function of proteins with relevanceto PCa such as the mTOR regulator TSC2 Following andro-genic stimulation AR was recruited to a cryptic promoterupstream of exon 33 in the TSC2 gene thereby leading toexpression of a truncated transcript lacking the 51015840 exons of thegeneThis alternative TSC2 variant would encode a truncatedprotein lacking the domain required for the interaction withTSC1 which is needed to exert negative regulation of mTORThus androgens may lead to activation of mTOR by relievingthe repressive function of the TSC1TSC2 complex throughAR-dependent induction of a defective variant It is worthyof notice that activation of the mTOR pathway has beenlinked to both tumorigenesis and resistance to therapy in PCa[54] Thus AR might contribute to prostate tumorigenesisalso by causing mTOR activation through expression of thisalternative mRNA variant of TSC2
9 Splicing Regulators Contributing to AlteredGene Expression in Prostate Cancer
In addition to affecting recruitment of AR to alternativepromoters androgens also affected a number of AS events inseveral genes [53] Although the mechanism(s) involved inthese events and their potential relevance to PCa biology wasnot investigated it might involve the ability of AR to interactwith cofactors that modulate the transcriptional elongationrate andor the recognition of splicing enhancers or silencersin the pre-mRNA (Figure 2) For instance AR interacts withthe cofactor of BRCA1 (COBRA1) and this interaction wasshown to influence splicing of the nascent transcripts pro-duced from an androgen-dependent promoter [55] A similarregulation of AR activity was also documented for the DEADboxRNAhelicase p68 (DDX5) in the LNCaP cell line AR andDDX5 interact and are recruited to the promoter region of theandrogen-responsive prostate-specific antigen (PSA) geneThis interactionwas functionally relevant asDDX5 enhancedAR-dependent PSA expression In addition by using an AR-dependent minigene reporter it was shown that DDX5 andAR cooperated in repressing the splicing of variable exons inthe CD44 gene [56] DDX5 is involved in several steps of co-and posttranscriptional RNA processing including splicing[57] and some genes appear to be particularly sensitive to theintracellular levels of DDX5 [57 58] Hence since this RNAhelicase is upregulated in PCa [56] it will be interesting todetermine to what extent it contributes to RNA processing ofAR target genes in PCa cells
AR is also known to interact with several splicing fac-tors suggesting a direct link between androgen-regulatedtranscription initiation and pre-mRNA splicing in PCa cells(Figure 2) The PTB-associated splicing factor (PSF) and itscofactor p54nrb participate to androgen-dependent proteincomplexes containing the AR PSF and p54nrb inhibit thetranscriptional activity of AR by interfering with its bindingto androgen response element and by recruiting a his-tone deacetylase to AR-responsive promoters [59] Althoughdirect investigation of the effect of these splicing factors onAR-dependent splicing events was not addressed it is likely
that AS is also affected by this interaction Another splicingfactor that may participate to AR-dependent splicing regula-tion is SAM68 [60] which is frequently upregulated in PCa[25 45] SAM68 interacts with AR and is recruited to the PSApromoter [60] like DDX5 [55] Interestingly however theinteraction between SAM68 and AR exerted different effectson transcription and splicing as the two proteins cooperatedin transcriptional activation of AR-target genes but opposedeach other in splicing of the CD44 variable exons from areporter minigene [60] Unfortunately the direct effects ofall these RBPs on AR-dependent splicing of endogenoustranscripts have not been addressed yet Nevertheless it islikely that depending on the specific complex formedAR candifferentially influence splicing of its target genes in PCa cells
An additional layer of regulation of the aberrant splicingprogram in PCa might rely on the up-regulation of specificsplicing factors Beside the already mentioned SAM68 [2545] one likely candidate is SRSF1 a splicing factor thatis upregulated in many human cancers and was shown tobehave as an oncogene in mice and humans [61] In cancercells of other tissues SRSF1modulates the expression of splicevariants of the BIN1 and BIM genes that lack proapoptoticfunctions [61 62] Moreover SRSF1 promotes splicing ofMNK2b [61] a splice variant of the eIF4E kinase MNK2 thatwas shown to confer chemoresistance in pancreatic adenocar-cinoma cells [63] Importantly MNK-dependent phospho-rylation of eIF4E strongly contributes to PCa tumorigenesisboth in vitro and in vivo [64 65] and a tight balance betweenthe MNKeIF4E and the mTOR pathways is required tomaintain efficient protein synthesis in PCa cells therebyenhancing their proliferation rate [64] Thus it will be inter-esting to determine whether SRSF1 contributes to fine-tuningthe activation of these pathways in PCa cells through theregulation ofMNK2 AS
Other splicing factors may also contribute to the alteredsplicing programof PCa cells Indeed the activity of several ofthese RBPs ismodulated by signal transduction pathways thatare frequently turned on in cancer such as the PI3KAKT andthe RASERK pathways (see also [66]) For instance it wasshown that activation of AKT downstream of the epidermalgrowth factor (EGF) receptor modulated the activity ofthe SR protein-specific kinases and phosphorylation of SRproteins thereby affecting a large spectrum of AS events[67] Similarly the RASERK pathway modulates a numberof splicing factors involved in cancer such as SAM68 [68]and the alternative splicing factor 45 (SPF45) [69] whichin turn affect expression of splice variants that regulate cellmotility proliferation and survival Thus it is likely that theexamples reported above represent only a small picture of theoverall contribution of AS and splicing factors to the wideheterogeneity in gene expression observed in PCa cells andpatients
10 Conclusions and Perspectives
AS is widely recognized as a powerful tool that eukaryoticcells employ to expand the coding potential and the plasticityof their genomes The flexibility in the recognition of exons
International Journal of Cell Biology 7
DDX5
SAM68COBRA1
AR
AR splicing-responsive mRNAs
RNAPIIAR transcriptional-responsive gene
AR coactivators
PSF p54nrb
AR corepressor
ARRNAPII
AR transcriptional-responsive gene
Figure 2 Regulation of cotranscriptional splicing by proteins interacting with the androgen receptor Coregulators of the androgen receptor(AR) can affect splicing of target genes by direct interaction with AR and modulation of its activity COBRA1 SAM68 and DDX5 appear topromote the transcriptional activity of AR but differentially act on splicing of variable exons (red box in the left side of the figure) PSF andits interacting protein p54 (right side of the figure) repress the transcriptional activity of AR but their effect on splicing is currently unknown(see text for more details)
and introns within the transcription unit of the majority ofhuman genes offers the possibility to compose many mRNAvariants from each gene Subtle changes in the cellular envi-ronment or in external cues conveyed from the surroundingenvironment may result in global changes in the tran-scriptome which in part rely on the regulation of AS Aninteresting observation is that apparently homogenous cellpopulations actually display large differences in gene expres-sion This was recently exemplified by studies that appliedglobal RNA sequencing techniques to the analysis of singlecell transcriptomes After treatment of bone-marrow-deriveddendritic cells (BMDCs) with an inflammatory cue it wasfound that hundreds of key immune genes were differentiallyexpressed by single cells The heterogeneity in the responsewas particularly remarkable with regard to the splicing pat-terns expressed by these cells [70] suggesting that fine-tuningof AS regulation strongly contributes to the heterogeneity ofa cell population This aspect might be particularly relevantin the context of PCa which is a neoplastic disease charac-terized by extreme heterogeneity and unpredicted responseof patients to the therapy [1 2] The improvements in cellisolation techniques coupled to the higher sensitivity of thenext-generation sequencing techniques may soon allow ahighly detailed description of the transcriptome of patientswhich might result in more personalized treatments
The studies illustrated previously suggest that the upregu-lation of selected splicing regulators in PCa such as SAM68SRSF1 or DDX5 directly contributes to the phenotype byaltering the splicing profile of key genes Thus these RBPsmight represent potential therapeutic targets for interventionAlthough blocking the activity of a given splicing factor isnot necessarily an easy task some examples in this direction
have been provided For instance SAM68 can bind to RNAonly as a dimer By exploiting this requirement it was shownthat an RNA binding-defective SAM68 mutant exerted dom-inant negative effects on SAM68-mediated SMN2 splicing byassociating with the endogenous protein and preventing itsbinding to the pre-mRNA [71]This experiment suggests thatsmall molecules interfering with SAM68 function might dis-play therapeutic potential As homodimerization is a prereq-uisite for RNA binding one possibility is to target the SAM68dimerization domain which was restricted to a small regionwithin its Gld1-Sam68-Grp33 (GSG) homology domain [72]The potential value of targeting specific components of thesplicing machinery in cancer cells is also suggested by theantioncogenic properties of natural compounds such asspliceostatin A (SSA) in a variety of cancer cell modelsSSA targets the splicing factor 3B subunit 1 (SF3B1) of thespliceosome thus affecting a large number of splicing eventsconcomitantly [73] Perhaps more specific drugs targetingsplicing factors involved in subsets of oncogenic splicingevents in cancer cells as those described above might repre-sent more specific therapeutic approaches in the next future
Although the extreme flexibility of AS regulation is proneto errors that may concur to neoplastic transformation [7 8]it can also be exploited therapeutically Indeed examples ofAS modulation in selected genes by administering splicing-correcting ASOs to cells have been reported In some casesthis approach has also been challenged with a therapeuticapplication One of the most remarkable examples is repre-sented by the recovery of the phenotype observed in mousemodels of Spinal Muscular Atrophy (SMA) This neurode-generative disease is caused by inactivation of the SMN1 geneand skipping of exon 7 in the highly homologous SMN2 gene
8 International Journal of Cell Biology
[74] It was recently demonstrated that systemic injectionof a chemically modified ASO restored SMN2 splicing invitro and in vivo and profoundly ameliorated the viabilityand phenotypic features of mice affected by a severe form ofSMA [75] Although cancer is caused by multiple alterationsthus limiting the application of gene-specific ASOs it isconceivable that these tools could be used in combinationwith standard therapies to improve the clinical response ofpatients For instance an ASO that switched BCL-X splicingtoward the proapoptotic variant was effective in sensitizingcancer cells to drug-induced apoptosis and to reduce growthof tumors in nude mice [30 31] A similar effect was obtainedby switching expression of the 120572 to the 120573 variant of the signaltransducer and activator of transcription 3 (STAT3) genewhich modulates multiple oncogenic pathways [76] In thiscase administration of a modified ASO targeted to a splicingenhancer induced expression of the endogenous STAT3120573 andan anti-oncogenic response in vitro and in vivo [76] Thesestudies suggest that modulation of AS with synthetic drugsis possible and has entered a therapeutic perspective ASOsare particularly appealing in terms of high specificity andreduced side effects as theymay exploit their ability to annealwith specific sequences in the genomewithout affecting otherfeatures or target genes of the splicing factors involved in theoncogenicAS eventThus it is likely that thesemethods couldbe applied soon to the development of novel therapies aimedat fighting human cancers in which expression of specificoncogenic splice variants has been firmly confirmed
Conflict of Interests
The author declares no conflict of interests
Acknowledgments
The author wishes to thank Dr Chiara Naro for help with thepreparation of the figures and critical reading of the paperThe work in the laboratory of C Sette was supported byAssociation for International Cancer Research (AICR Grantno 12-0150) the Associazione Italiana Ricerca sul Cancro(AIRC) and Istituto Superiore della Sanita (ISS Grant Con-venzione 11US6A)
References
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[2] M Kohli and D J Tindall ldquoNew developments in the medicalmanagement of prostate cancerrdquo Mayo Clinic Proceedings vol85 no 1 pp 77ndash86 2010
[3] K E Knudsen and H I Scher ldquoStarving the addiction newopportunities for durable suppression of AR signaling in pros-tate cancerrdquo Clinical Cancer Research vol 15 no 15 pp 4792ndash4798 2009
[4] S M Dehm and D J Tindall ldquoAlternatively spliced androgenreceptor variantsrdquo Endocrine-Related Cancer vol 18 no 5 ppR183ndashR196 2011
[5] A Kalsotra and T A Cooper ldquoFunctional consequences ofdevelopmentally regulated alternative splicingrdquoNature ReviewsGenetics vol 12 no 10 pp 715ndash729 2011
[6] C Zhang H-R Li J-B Fan et al ldquoProfiling alternativelysplicedmRNA isoforms for prostate cancer classificationrdquo BMCBioinformatics vol 7 article 202 2006
[7] C J David and J L Manley ldquoAlternative pre-mRNA splicingregulation in cancer pathways and programs unhingedrdquo Genesand Development vol 24 no 21 pp 2343ndash2364 2010
[8] S Pal R Gupta and R V Davuluri ldquoAlternative transcriptionand alternative splicing in cancerrdquo Pharmacology amp Therapeu-tics vol 136 no 3 pp 283ndash294 2012
[9] Y Li M Alsagabi D Fan G S Bova A H Tewfik and S MDehm ldquoIntragenic rearrangement and altered RNA splicing ofthe androgen receptor in a cell-based model of prostate cancerprogressionrdquo Cancer Research vol 71 no 6 pp 2108ndash2117 2011
[10] S M Dehm L J Schmidt H V Heemers R L Vessella and DJ Tindall ldquoSplicing of a novel androgen receptor exon generatesa constitutively active androgen receptor that mediates prostatecancer therapy resistancerdquo Cancer Research vol 68 no 13 pp5469ndash5477 2008
[11] R Hu T A Dunn SWei et al ldquoLigand-independent androgenreceptor variants derived from splicing of cryptic exons signifyhormone-refractory prostate cancerrdquo Cancer Research vol 69no 1 pp 16ndash22 2009
[12] Z Guo X Yang F Sun et al ldquoA novel androgen receptor splicevariant is up-regulated during prostate cancer progressionand promotes androgen depletion-resistant growthrdquo CancerResearch vol 69 no 6 pp 2305ndash2313 2009
[13] Y Li S C Chan L J Brand T H Hwang K A Silverstein andS M Dehm ldquoAndrogen receptor splice variants mediate enza-lutamide resistance in castration-resistant prostate cancer celllinesrdquo Cancer Research vol 73 no 2 pp 483ndash489 2013
[14] G Narla K E Heath H L Reeves et al ldquoKLF6 a candidatetumor suppressor genemutated in prostate cancerrdquo Science vol294 no 5551 pp 2563ndash2566 2001
[15] X Liu A Gomez-Pinillos C Loder et al ldquoKLF6 loss of func-tion in human prostate cancer progression is implicated inresistance to androgen deprivationrdquo The American Journal ofPathology vol 181 no 3 pp 1007ndash1016 2012
[16] G Narla A DiFeo H L Reeves et al ldquoA germline DNApolymorphism enhances alternative splicing of the KLF6 tumorsuppressor gene and is associatedwith increased prostate cancerriskrdquo Cancer Research vol 65 no 4 pp 1213ndash1222 2005
[17] G Narla A DiFeo Y Fernandez et al ldquoKLF6-SV1 overexpres-sion accelerates human and mouse prostate cancer progressionand metastasisrdquo Journal of Clinical Investigation vol 118 no 8pp 2711ndash2721 2008
[18] G Narla A Difeo S Yao et al ldquoTargeted inhibition of theKLF6 splice variant KLF6 SV1 suppresses prostate cancer cellgrowth and spreadrdquo Cancer Research vol 65 no 13 pp 5761ndash5768 2005
[19] K E Knudsen ldquoThe cyclin D1b splice variant an old oncogenelearns new tricksrdquo Cell Division vol 1 article 15 2006
[20] K E Knudsen J A Diehl C A Haiman and E S KnudsenldquoCyclin D1 polymorphism aberrant splicing and cancer riskrdquoOncogene vol 25 no 11 pp 1620ndash1628 2006
[21] F Lu A B Gladden and J A Diehl ldquoAn alternatively splicedcyclin D1 isoform cyclin D1b is a nuclear oncogenerdquo CancerResearch vol 63 no 21 pp 7056ndash7061 2003
International Journal of Cell Biology 9
[22] C E S Comstock M A Augello R Pe Benito et al ldquoCyclinD1 splice variants polymorphism risk and isoform-specificregulation in prostate cancerrdquo Clinical Cancer Research vol 15no 17 pp 5338ndash5349 2009
[23] M A Augello C J Burd R Birbe et al ldquoConvergence of onco-genic and hormone receptor pathways promotes metastaticphenotypesrdquo The Journal of Clinical Investigation vol 123 no1 pp 493ndash508 2013
[24] N A Olshavsky C E S Comstock M J Schiewer et al ldquoIden-tification of ASFSF2 as a critical allele-specific effector of thecyclin D1b oncogenerdquoCancer Research vol 70 no 10 pp 3975ndash3984 2010
[25] M P Paronetto M Cappellari R Busa et al ldquoAlternative splic-ing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68rdquoCancer Research vol 70 no 1 pp 229ndash239 2010
[26] D Kaida M G Berg I Younis et al ldquoU1 snRNP protects pre-mRNAs frompremature cleavage and polyadenylationrdquoNaturevol 468 no 7324 pp 664ndash668 2010
[27] P Bielli R Busa M P Paronetto and C Sette ldquoThe RNA-binding protein Sam68 is a multifunctional player in humancancerrdquo Endocrine-Related Cancer vol 18 no 4 pp R91ndashR1022011
[28] L H Boise M Gonzalez-Garcia C E Postema et al ldquobcl-xA bcl-2-related gene that functions as a dominant regulator ofapoptotic cell deathrdquo Cell vol 74 no 4 pp 597ndash608 1993
[29] D R Mercatante C D Bortner J A Cidlowski and R KoleldquoModification of alternative splicing of Bcl-x Pre-mRNA inprostate and breast cancer cells analysis of apoptosis and celldeathrdquo Journal of Biological Chemistry vol 276 no 19 pp 16411ndash16417 2001
[30] D R Mercatante J L Mohler and R Kole ldquoCellular responseto an antisense-mediated shift of Bcl-x pre-mRNA splicing andantineoplastic agentsrdquo Journal of Biological Chemistry vol 277no 51 pp 49374ndash49382 2002
[31] J A Bauman S-D Li A Yang L Huang and R Kole ldquoAnti-tumor activity of splice-switching oligonucleotidesrdquo NucleicAcids Research vol 38 no 22 pp 8348ndash8356 2010
[32] C E Chalfant K Rathman R L Pinkerman et al ldquoDe novoceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells Dependence on proteinphosphatase-1rdquo Journal of Biological Chemistry vol 277 no 15pp 12587ndash12595 2002
[33] K Boon-Unge Q Yu T Zou A Zhou P Govitrapong andJ Zhou ldquoEmetine regulates the alternative splicing of Bcl-xthrough a protein phosphatase 1-dependent mechanismrdquoChemistry and Biology vol 14 no 12 pp 1386ndash1392 2007
[34] M H Kim ldquoProtein phosphatase 1 activation and alternativesplicing of Bcl-X and Mcl-1 by EGCG + ibuprofenrdquo Journal ofCellular Biochemistry vol 104 no 4 pp 1491ndash1499 2008
[35] T Novoyatleva B Heinrich Y Tang et al ldquoProtein phosphatase1 binds to the RNA recognition motif of several splicing fac-tors and regulates alternative pre-mRNA processingrdquo HumanMolecular Genetics vol 17 no 1 pp 52ndash70 2008
[36] N Ghosh N Patel K Jiang et al ldquoCeramide-activated pro-tein phosphatase involvement in insulin resistance via Aktserinearginine-rich protein 40 and ribonucleic acid splicing inL6 skeletal muscle cellsrdquo Endocrinology vol 148 no 3 pp 1359ndash1366 2007
[37] D Garneau T Revil J-F Fisette and B Chabot ldquoHetero-geneous nuclear ribonucleoprotein FH proteins modulate
the alternative splicing of the apoptotic mediator Bcl-xrdquo Journalof Biological Chemistry vol 280 no 24 pp 22641ndash22650 2005
[38] M P Paronetto T Achsel A Massiello C E Chalfant andC Sette ldquoThe RNA-binding protein Sam68 modulates thealternative splicing of Bcl-xrdquo Journal of Cell Biology vol 176 no7 pp 929ndash939 2007
[39] A Zhou A C Ou A Cho E J Benz Jr and S-C HuangldquoNovel splicing factor RBM25 modulates Bcl-x Pre-mRNA 51015840splice site selectionrdquoMolecular and Cellular Biology vol 28 no19 pp 5924ndash5936 2008
[40] S Pedrotti R Busa C Compagnucci and C Sette ldquoThe RNArecognition motif protein RBM11 is a novel tissue-specificsplicing regulatorrdquo Nucleic Acids Research vol 40 no 3 pp1021ndash1032 2012
[41] T Revil J Pelletier J Toutant A Cloutier and B ChabotldquoHeterogeneous nuclear ribonucleoprotein K represses theproduction of pro-apoptotic Bcl-xS splice isoformrdquo Journal ofBiological Chemistry vol 284 no 32 pp 21458ndash21467 2009
[42] M J Moore Q Wang C J Kennedy and P A Silver ldquoAn alter-native splicing network links cell-cycle control to apoptosisrdquoCell vol 142 no 4 pp 625ndash636 2010
[43] P Cloutier J Toutant L Shkreta S Goekjian T Revil and BChabot ldquoAntagonistic effects of the SRp30c protein and cryptic51015840 splice sites on the alternative splicing of the apoptoticregulator Bcl-xrdquo Journal of Biological Chemistry vol 283 no 31pp 21315ndash21324 2008
[44] A Massiello J R Roesser and C E Chalfant ldquoSAP155 Bindsto ceramide-responsive RNA cis-element 1 and regulates thealternative 51015840 splice site selection of Bcl-x pre-mRNArdquo TheFASEB Journal vol 20 no 10 pp 1680ndash1682 2006
[45] R BusaM P Paronetto D Farini et al ldquoTheRNA-binding pro-tein Sam68 contributes to proliferation and survival of humanprostate cancer cellsrdquo Oncogene vol 26 no 30 pp 4372ndash43822007
[46] C Sette ldquoPost-translational regulation of star proteins andeffects on their biological functionsrdquo Advances in ExperimentalMedicine and Biology vol 693 pp 54ndash66 2010
[47] C Brignatz M P Paronetto S Opi et al ldquoAlternative splicingmodulates autoinhibition and SH3 accessibility in the Src kinaseFynrdquo Molecular and Cellular Biology vol 29 no 24 pp 6438ndash6448 2009
[48] M P Paronetto D Farini I Sammarco et al ldquoExpression of atruncated form of the c-Kit tyrosine kinase receptor and activa-tion of Src kinase in human prostatic cancerrdquo American Journalof Pathology vol 164 no 4 pp 1243ndash1251 2004
[49] Y Hu A Dobi T Sreenath et al ldquoDelineation of TMPRSS2-ERG splice variants in prostate cancerrdquo Clinical CancerResearch vol 14 no 15 pp 4719ndash4725 2008
[50] J Wang Y Cai W Yu C Ren D M Spencer and M Itt-mann ldquoPleiotropic biological activities of alternatively splicedTMPRSS2ERG fusion gene transcriptsrdquo Cancer Research vol68 no 20 pp 8516ndash8524 2008
[51] F Zammarchi G Boutsalis and L Cartegni ldquo51015840 UTR controlof native ERG and of Tmprss2 ERG variants activity in prostatecancerrdquo PLoS One vol 8 no 3 Article ID e49721 2013
[52] M J Munoz M S P Santangelo M P Paronetto et al ldquoDNAdamage regulates alternative splicing through inhibition ofRNApolymerase II elongationrdquoCell vol 137 no 4 pp 708ndash7202009
[53] P Rajan C Dalgliesh P J Carling et al ldquoIdentification of novelandrogen-regulated pathways and mrna isoforms through
10 International Journal of Cell Biology
genome-wide exon-specific profiling of the LNCaP transcrip-tomerdquo PLoS One vol 6 no 12 Article ID e29088 2011
[54] T M Morgan T D Koreckij and E Corey ldquoTargeted therapyfor advanced prostate cancer inhibition of the PI3KAktmTORpathwayrdquoCurrent CancerDrug Targets vol 9 no 2 pp 237ndash2492009
[55] J Sun A L Blair S E Aiyar and R Li ldquoCofactor of BRCA1modulates androgen-dependent transcription and alternativesplicingrdquo Journal of Steroid Biochemistry andMolecular Biologyvol 107 no 3ndash5 pp 131ndash139 2007
[56] E L Clark A Coulson C Dalgliesh et al ldquoThe RNA helicasep68 is a novel androgen receptor coactivator involved in splicingand is overexpressed in prostate cancerrdquo Cancer Research vol68 no 19 pp 7938ndash7946 2008
[57] E Zonta D Bittencourt S Samaan S Germann M Dutertreand D Auboeuf ldquoThe RNA helicase DDX5p68 is a key factorpromoting c-fos expression at different levels from transcriptionto mRNA exportrdquoNucleic Acids Research vol 41 no 1 pp 554ndash564 2013
[58] A Honig D Auboeuf M M Parker B W OrsquoMalley andS M Berget ldquoRegulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72rdquo Molecular and Cel-lular Biology vol 22 no 16 pp 5698ndash5707 2002
[59] X Dong J Sweet J R G Challis T Brown and S J Lye ldquoTran-scriptional activity of androgen receptor is modulated by twoRNA splicing factors PSF and p54nrbrdquoMolecular and CellularBiology vol 27 no 13 pp 4863ndash4875 2007
[60] P Rajan L Gaughan C Dalgliesh et al ldquoThe RNA-binding andadaptor protein Sam68 modulates signal-dependent splicingand transcriptional activity of the androgen receptorrdquo Journalof Pathology vol 215 no 1 pp 67ndash77 2008
[61] R Karni E De Stanchina S W Lowe R Sinha D Mu and AR Krainer ldquoThe gene encoding the splicing factor SF2ASF is aproto-oncogenerdquo Nature Structural and Molecular Biology vol14 no 3 pp 185ndash193 2007
[62] OAnczukowA Z RosenbergMAkerman et al ldquoThe splicingfactor SRSF1 regulates apoptosis and proliferation to promotemammary epithelial cell transformationrdquoNature Structural andMolecular Biology vol 19 no 2 pp 220ndash228 2012
[63] L Adesso S Calabretta F Barbagallo et al ldquoGemcitabine trig-gers a pro-survival response in pancreatic cancer cells throughactivation of the MNK2eIF4E pathwayrdquo Oncogene vol 32 pp2848ndash2857 2013
[64] A Bianchini M Loiarro P Bielli et al ldquoPhosphorylation ofeIF4E by MNKs supports protein synthesis cell cycle progres-sion and proliferation in prostate cancer cellsrdquo Carcinogenesisvol 29 no 12 pp 2279ndash2288 2008
[65] L Furic L RongO Larsson et al ldquoEIF4Ephosphorylation pro-motes tumorigenesis and is associated with prostate cancer pro-gressionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 32 pp 14134ndash14139 2010
[66] C Naro and C Sette ldquoPhosphorylation-mediated regulation ofalternative splicing in cancerrdquo International Journal of Cell Bio-logy In press
[67] Z Zhou J QiuW Liu et al ldquoTheAkt-SRPK-SR axis constitutesa major pathway in transducing EGF signaling to regulatealternative splicing in the nucleusrdquo Molecular Cell vol 47 no3 pp 422ndash433 2012
[68] NMatter P Herrlich andH Konig ldquoSignal-dependent regula-tion of splicing via phosphorylation of Sam68rdquoNature vol 420no 6916 pp 691ndash695 2002
[69] A M Al-Ayoubi H Zheng Y Liu T Bai and S T EblenldquoMitogen-activated protein kinase phosphorylation of splicingfactor 45 (SPF45) regulates SPF45 alternative splicing siteutilization proliferation and cell adhesionrdquoMolecular and Cel-lular Biology vol 32 no 14 pp 2880ndash2893 2012
[70] A K Shalek R Satija X Adiconis et al ldquoSingle-cell transcrip-tomics reveals bimodality in expression and splicing in immunecellsrdquo Nature 2013
[71] S Pedrotti P BielliM P Paronetto et al ldquoThe splicing regulatorSam68 binds to a novel exonic splicing silencer and functions inSMN2 alternative splicing in spinal muscular atrophyrdquo EMBOJournal vol 29 no 7 pp 1235ndash1247 2010
[72] N H Meyer K Tripsianes M Vincendeau et al ldquoStructuralbasis for homodimerization of the Src-associated during mito-sis 68-kDa protein (Sam68)Qua1 domainrdquo Journal of BiologicalChemistry vol 285 no 37 pp 28893ndash28901 2010
[73] S Bonnal L Vigevani and J Valcarcel ldquoThe spliceosome asa target of novel antitumour drugsrdquo Nature Reviews Drug Dis-covery vol 11 no 11 pp 847ndash859 2012
[74] S Pedrotti and C Sette ldquoSpinal muscular atrophy a new playerjoins the battle for SMN2 exon 7 splicingrdquo Cell Cycle vol 9 no19 pp 3874ndash3879 2010
[75] Y Hua K Sahashi F Rigo et al ldquoPeripheral SMN restoration isessential for long-term rescue of a severe spinal muscular atro-phy mouse modelrdquo Nature vol 478 no 7367 pp 123ndash126 2011
[76] F Zammarchi E De Stanchina E Bournazou et al ldquoAntitu-morigenic potential of STAT3 alternative splicing modulationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 43 pp 17779ndash17784 2011
to stratify patients than the ldquotranscriptome signaturerdquo whichis typically analysed by conventional microarray analyses [6]Thus understanding the regulation of splicing in normal andpathological prostate cells may help identify novel markersand targets for future therapeutic approaches to this neoplas-tic disease
2 Alternative Splicing and Cancer
The recent advent of high-throughput RNA sequencing hasunveiled new layers of regulation of gene expression andhighlighted the extreme complexity and versatility of thegenome The majority of human genes encode multiple tran-scripts through the use of alternative promoters AS andalternative polyadenylation [5 7 8] AS is a combinatorialmechanism that expands the coding potential of the genomeby allowing the production of protein isoforms with differentor even antagonistic functions from a single gene [5 7 8]Splicing is orchestrated by a ribonucleoprotein complexcalled ldquospliceosomerdquo which recognizes exon-intron junc-tions excises introns and ligates exons The lack of stringentconsensus sequences at exon-intron junctions in highereukaryotes allows flexibility in recognition by the spliceo-some Numerous RNA binding proteins (RBPs) interact withcomponents of the spliceosome and reinforce or weaken rec-ognition of exon-intron junctions The interplay betweenthese splicing factors determines the choice of variable exonsby the spliceosome and causes heterogeneity in pre-mRNAprocessing [5 7] As a consequence changes in the expressionlevels or in the activity of splicing factors can selectivelyinfluence AS of many genes [5 7] Although the flexibility ofAS regulation has represented an evolutionary advantage forhigher eukaryotes it also represents a risk factor In particu-lar mounting evidence illustrates how defective regulation ofAS correlates with onset and progression of human cancers[7 8]
Herein the literature describing the impact of AS inthe onset and progression of PCa will be reviewed High-throughput analyses of specimens from PCa patients havehighlighted more than 200 genes whose AS is differentiallyregulated in the neoplastic tissue [6] indicating that thismechanism can substantially contribute to the heterogeneityof gene expression in cancer cells Nevertheless the physi-ological consequences of the majority of these aberrant ASevents are still unknown and will require direct investigationThis review will focus on the regulation of genes and splicingregulators whose relevance for PCa has been firmly docu-mented
3 The Androgen Receptor
Several reports have documented the expression of alter-natively spliced AR variants lacking the C-terminal ligand-binding domain of the canonical receptor (reviewed in [4])Many of these AR splice variants are constitutively nuclearand active even in the absence of androgens (Figure 1) thusindicating their potential role in the acquisition of the CRPCaphenotype [4] Expression of most of these variants arises
from the inclusion of cryptic exons located in intron 2 and 3of theAR geneThe heterogeneity of the AR variants reportedin various studies is also due to the frequent amplificationof the exon 2-exon 3 genomic region of AR [4 9] In allreported variants however the splicing of these cryptic exonsinvariably introduces premature stop codons and terminationsites thereby yielding shorter AR proteins of 75ndash80 kDawhich lack the androgen-binding domain [4] In some casesthese truncated AR variants can function independentlyof the full-length AR and their selective knockdown wasshown to block the androgen-independent growth of CRPCacells while maintaining responsiveness to the hormone [10]Importantly expression of an AR variant containing a crypticexon located in intron 3 (CE3) in clinical PCa specimenspositively correlated with poor prognosis after surgery [11]This variant was also expressed at higher levels in CRPCawith respect to PCa patients [12] Furthermore constitutivelyactive truncated AR splice variants were recently shown toconfer resistance also to the next generation of AR inhibitorsthus limiting their therapeutic efficacy for many patients [13]However since the expression of these shorter AR variantswas also observed in normal prostate tissues it is unlikelythat they drive the initial steps of neoplastic transformationopening the possibility that AS of the AR gene plays also aphysiological role in the gland [4]
The mechanisms that lead to increased expression ofaberrant AR splice variants in PCa are still largely unknownOne possible cause of defective splicing is the alteration ofthe genomic AR locus which often occurs in CRPCa Forinstance disruption of the AR splicing pattern in the 22Rv1PCa cell line was linked to duplication of the genomicregion containing exon 3 and some of the cryptic exons [9]Alternatively aberrant expression of specific splicing factorsin PCa cells may also contribute to unbalanced splicing andaberrant recognition of cryptic exons in the AR gene Thusgiven the strong relevance of these constitutively active ARvariants for CRPCa progression further studies elucidatingthe regulation of their expression are strongly encouraged
4 KLF6
Kruppel-like factor 6 (KLF6) is a zinc finger transcriptionfactor that is mutated in a subset of human PCas [14 15]KLF6 is known to regulate cell proliferation by inducingthe expression of the cell cycle inhibitor p21 (WAF1CIP1)Notably this effect of KLF6 does not require p53 suggestingthat KLF6 is a tumor suppressor gene that functions as a p53-alternative brake for cell cycle progression in normal cells[14] One of the mutations found in PCa patients consistsof a single nucleotide change that creates a binding site forthe splicing factor SRSF5 (SRp40) and enhances splicing ofthree alternative mRNA variants encoding for truncated KLFproteins named KLF6-SV1 SV2 and SV3 [16] These splicevariants are upregulated in tumor versus normal prostatictissue A single G gt A mutation in intron 1 was shown torecapitulate the altered splicing pattern of KLF6 when itwas introduced in a minigene and it was found to correlatewith worse prognosis in patients [16] The KLF-SV1 variant
Cellular transformationEnhancement of AR-dependent transcription
1 32
Proapoptotic
1 32
AR gene
BCL-X gene
CCND1gene
BCL-XS
BCL-XL
5998400 5998400 3998400
Antiapoptotic chemotherapyresistance
Figure 1 Representative examples of genes whose alternative splicing affects prostate cancer cell biology The left side of the figure illustratesthe genomic structure of the alternatively spliced regions of the AR CCND1 and BCL-X genes Solid and dashed lines show the alternativesplicing events reported in the literature On the right side the alternative variants produced by splicing are shown The specific features ofthe protein isoforms produced by alternative splicing are summarized under the scheme of each variant
was characterized further and shown to function as adominant-negative protein which antagonizes the functionof full length KLF6 leading to decreased p21 expressionand enhanced cell growth [16] Increased expression ofthis splice variant in PCa patients predicted poorer out-come after surgery and was associated with development ofhormone-refractory metastatic PCa [17] Furthermore while
knockdown of the full length KLF6 promoted tumor forma-tion in nudemice selective silencing of the KLF6-SV1 variantinhibited it [18] Conversely PCa cells overexpressing KLF6-SV1 are more prone to develop metastases in various organsof the mouse models used in the study [17] Thus a mutationaffecting KLF6 AS represents a critical mechanism for theinactivation of a tumor suppressor gene in PCa suggesting
4 International Journal of Cell Biology
that interfering with this splicing event in PCa cells mightrestore the growth-inhibitory activity of this transcriptionfactor
5 Cyclin D1
CCND1 is a protooncogene that encodes for cyclin D1 whichassociates with the cyclin-dependent kinase 4 (CDK4) todrive progression through the G1 phase of the cell cycleImportantlyCCND1 expression is oftenderegulated in cancercells [19 20] This gene encodes for two alternative tran-scripts the common cyclin D1a isoform containing all fiveexons and cyclin D1b which derives from retention of intron4 and premature termination of the transcript (Figure 1) [1920] Unlike cyclin D1a cyclin D1b alone can promote cellulartransformation [21] and its expression has been associatedwith PCa progression and poor prognosis [22] Interestinglyrecent evidence indicated that cyclin D1b promotes AR-dependent transcription of genes involved in PCa metastaticpotential such as the transcription factor SLUG [23] CyclinD1a was instead reported to repress the transcriptionalactivity of AR (Figure 1) [19 20] Thus it is conceivable thata change in the ratio between the cyclin D1 variants willpotently enhance hormone-dependent growth of PCa cells
Given the relevance for PCa cell biology understandingthe regulation of CCND1 splicing is of crucial importanceIt was observed that a polymorphism (G870A) at theexon 4-intron 4 boundary predisposes cells to cyclin D1bsplicing [19 20] The splicing factor SRSF1 was shown tobind the exon4intron4 junction in the nascent CCND1 pre-mRNA thereby promoting intron 4 retention and cyclin D1bexpression [24] SRSF1 was hypothesized to favour intron 4retention by altering exon 4 definition and limiting assemblyof the spliceosome at the exon-intron junction [24] Anothersplicing factor promoting cyclin D1b expression in PCa cellsis SAM68 [25] In this case the binding site was identifiedwithin intron 4 in proximity of the termination site utilizedfor the cyclin D1b mRNA The binding of SAM68 to thisregion of the pre-mRNA was shown to compete with thatof the U1 snRNP [25] Since deposition of U1 snRNP nearcryptic polyadenylation sites located in introns is known toprevent premature termination of transcripts in a genome-wide fashion [26] it is possible that up-regulation of SAM68unmasks the cyclin D1b termination site by interfering withU1 snRNP binding in intron 4 Notably both SRSF1 andSAM68 display oncogenic features in several cell types andtissues [7 27] and their expression positively correlates withthat of cyclin D1b in clinical specimens of PCa patients [2425] Thus it is possible that interfering with the activity ofthese splicing factors will exert positive effects in therapeutictreatments of PCa through modulation of CCND1 splicingand expression
6 BCL-X
The BCL-X (BCL2L1) gene contains 3 exons and encodes fortwo splice variants [28] Two alternative 51015840 splice sites arepresent in exon 2 of the gene selection of the canonical
one at the end of the exon yields the long BCL-XL variantwhereas selection of the distal one located 220 bp upstreamin the exon produces the short BCL-XS variant Notablythese two splice variants have opposite effects in the cell withBCL-XL being prosurvival whereas BCL-XS is proapoptotic(Figure 1) [28] Thus regulation of BCL-X AS can finelymodulate cell viability illustrating the biological importanceof this splicing event In most cancer cells including PCacells the anti-apoptotic BCL-XL variant is overexpressed andconfers resistance to chemotherapeutic treatments [29 30]It is predictable that a full understanding of the mechanismsof regulation of BCL-X splicing will help develop tools toswitch it toward the proapoptotic BCL-XS variant therebyoffering a therapeutic opportunity to sensitize cancer cellsto treatments In line with this notion treatment of PCacells with an antisense oligonucleotide (ASO) masking theBCL-XL splice site effectively switched BCL-X splicing andinduced apoptosis [29] Interestingly the proapoptotic effectof the ASO was more pronounced in cancer cells whichdisplay high levels of expression of BCL-XL and it alsoenhanced their response to chemotherapeutic treatments[30] Thus ASOs targeting BCL-X splicing may have theadvantage of being selective for cancer cells with respect tonormal cells which is a positive feature for an antineoplasticdrug Unfortunately the delivery of ASOs to cancer cells isstill not efficient thus limiting their application in the cliniceven though development of vehicles favoring their deliverysuch as lipid nanoparticles [31] may aid in this direction
Although regulation of BCL-X splicing is highly relevantto PCa cell biology not much is known on the mechanism(s)of its regulation in prostate cells A possible regulator is theprotein phosphatase 1 (PP1) whose activity is required for theregulatory effect of ceramide on BCL-X splicing [32] IndeedPP1 activity was required also for induction of BCL-XSsplicing by emetine a protein synthesis inhibitor and otherproapoptotic drugs in PCa cells [33 34] Nevertheless themechanism by which PP1 modulates splicing of BCL-X is stillunknown PP1 is known to regulate splicing by modulatingthe activity of splicing factors either by direct binding tothem and regulation of their phosphorylation status [35] orindirectly by regulating kinases involved in their post-translational modifications [36]Thus activation of pathwaysimpinging on PP1may affect BCL-X splicing and cell viabilitythrough the regulation of the activity of specific splicingfactors in PCa cells
Several splicing factors have been shown to modulateBCL-X splicing Studies performed in a variety of cell modelsindicated that the heterogeneous nuclear ribonucleoprotein(hnRNP) H and F [37] and the splicing regulators SAM68[38] RBM25 [39] and RBM11 [40] promote splicing of theproapoptotic BCL-XS variant By contrast hnRNPK [41] theserine-arginine (SR) rich proteins SRSF1 [38 42] and SRSF9[43] and the splicing factor SAP155 [44] enhance splicing ofthe anti-apoptotic BCL-XLWhich of these factors contributeto the regulation of BCL-X splicing in PCa cells is stilllargely unknown SRSF1 [24] and SAM68 [45] were shownto be upregulated in PCa and might represent strong candi-dates for the regulation of this splicing event Intriguinglythese splicing factors normally modulate BCL-X splicing in
International Journal of Cell Biology 5
opposite directions [38] while the up-regulation of SRSF1 isin line with the high levels of BCL-XL in PCa cells SAM68should favour the proapoptotic short variant However thesplicing activity of SAM68 is finely tuned by phosphoryla-tion [46] and it was shown that tyrosine phosphorylationby the Src-related kinase FYN switched SAM68-dependentsplicing of BCL-X toward the anti-apoptotic variant [38 47]Since tyrosine phosphorylation of SAM68 is increased inspecimens of PCa patients [48] it is likely that this RBPcan also contribute to the upregulation of BCL-XL in PCacells In line with this hypothesis BCL-XL expression wasdecreased and sensitivity to genotoxic agents was increasedafter knockdown of SAM68 in the androgen-sensitive LNCaPcell line [45]
Thus based on the observations reported previouslyit is predictable that exogenous modulation of BCL-X ASthrough administration of ASOs or by interfering with theactivity of the splicing factors that promote the anti-apoptoticBCL-XL variant will enhance the efficacy of chemotherapy inadvanced PCa as suggested by preclinical studies in PCa celllines [30 31 45]
7 TMPRSS2ERG
ERG is a member of the ETS transcription factor family thatis expressed at very low levels in benign prostate epithelialcells However PCa patients often carry a fusion of theandrogen-responsiveTMPRSS2 genewith ERG which causesaberrantly high expression levels of the transcription factorin the neoplastic cells A detailed sequencing analysis of theTMPRSS2ERG transcripts isolated fromPCa tissues revealedthat fusion-derived transcripts underwent profound AS reg-ulation which yielded mRNA variants encoding both fulllength ERG proteins and isoforms lacking the ETS domainNotably an increase in the abundance of transcripts encodingfull length ERG correlated with less favorable outcome inpatients [49] These results support a possible functional rolefor this transcription factor in PCa pathology and suggest thatmodulation of AS events promoting less pathogenic variantsmay produce beneficial effects
This hypothesis is also supported by another study thattested the effects exerted by the expression of TMPRSS2ERGalternatively spliced transcripts in an immortalized prostatecell line [50] It was found that these TMPRSS2ERG splicevariants had different oncogenic activities in terms of pro-moting proliferation invasion and motility Notably coex-pression of different variants produced stronger effects thaneither variant alone suggesting that the presence of severalTMPRSS2ERG isoforms as it normally occurs in PCa cellsmight confer a more malignant phenotype [50] A furthercontribution of AS to the heterogeneity of TMPRSS2ERGexpression is provided by the extensive variability of the 51015840untranslated region (UTR) in the splice variants observedin patients [51] Indeed AS of the 51015840 UTR affects the onco-genic potential of the encoded proteins by regulating theirtranslation and activity Thus although a functional linkbetween TMPRSS2ERG expression and PCa pathology hasnot been firmly established yet this fusion gene appears
to be another suitable target for an AS-directed therapeuticapproach that would spare normal cells not expressing thechimeric proteins
8 Splicing Programs in Prostate Cancer
Cancer cells express a number of splice variants that conferthem higher resistance to chemotherapeutic drugs and sur-vival advantages When it was investigated in detail the spe-cific signature of splice variants expressed by cancer cells hasbeen recognized as a powerful diagnostic and prognostic tool[7 8] Evenmore importantly the existence of cancer-specificsplicing variants of key genes such as the AR or CCND1in PCa might offer a therapeutic opportunity for targetingproteins that are not expressed in healthy cells For instancedeveloping tools that specifically modulate the expressionof transcript variants preferentially or uniquely produced bycancer cells might slow down tumor growth andor promotecell death during therapy while sparing the healthy tissuesThus understanding AS regulation at the genome-wide levelin PCa cells may not only lead to the identification of noveldiagnostic or prognostic biomarkers but it could also helpfind tools for novel therapeutic approaches to this neoplasticdisease
A few studies have directly investigated the genome-wideregulation of AS in PCa cell lines and primary tumor tissuesUsing a splicing-sensitive microarray comprising a selectedsubset of genes and splice variants it was shown that splicingsignatures could efficiently segregate PCa cells lines from can-cer cell lines derived from other organs or tissues [6] Amongthe alternatively spliced genes the majority also showedvariation in expression levels [6] suggesting that regulationof splicing and transcriptionwere coupled as also observed incells exposed to DNA damage [52] Using the same splicing-sensitive platform it was also possible to identify splicingsignatures that were specific for normal or neoplastic prostatetissues obtained from biopsies [6] Although this approachwas limited to the genes and the splice variants selected for theplatform it provided a first indication that specific changes insplicing occur during prostate tumorigenesis and suggestedthat splicing variants can represent accurate biomarkers forPCaNevertheless how andwhen these changes occur aswellas to what extent they contribute to the acquisition of thetransformed phenotype are still open questions Given thetight association between transcription and splicing a spe-cific splicing program could result from the different activityof transcription factors splicing factors or both Mountingevidence indicates that all these events contribute to someextent to the acquisition of specific splicing signatures in PCa
The most relevant transcription factor involved in PCais the AR Several observations suggest that in addition toregulating the expression levels of target genes AR can alsoinfluence the transcript variants encoded by them Usingcomprehensive splicing-sensitive arrays it was demonstratedthat stimulation of LNCaP cells with androgens caused qual-itative changes in expression of splice variants [53] Many ofthe events altered by treatment with androgens were due tousage of alternative promoters within the transcription unit
6 International Journal of Cell Biology
of the target gene Some of these alternative transcripts werepredicted to influence the function of proteins with relevanceto PCa such as the mTOR regulator TSC2 Following andro-genic stimulation AR was recruited to a cryptic promoterupstream of exon 33 in the TSC2 gene thereby leading toexpression of a truncated transcript lacking the 51015840 exons of thegeneThis alternative TSC2 variant would encode a truncatedprotein lacking the domain required for the interaction withTSC1 which is needed to exert negative regulation of mTORThus androgens may lead to activation of mTOR by relievingthe repressive function of the TSC1TSC2 complex throughAR-dependent induction of a defective variant It is worthyof notice that activation of the mTOR pathway has beenlinked to both tumorigenesis and resistance to therapy in PCa[54] Thus AR might contribute to prostate tumorigenesisalso by causing mTOR activation through expression of thisalternative mRNA variant of TSC2
9 Splicing Regulators Contributing to AlteredGene Expression in Prostate Cancer
In addition to affecting recruitment of AR to alternativepromoters androgens also affected a number of AS events inseveral genes [53] Although the mechanism(s) involved inthese events and their potential relevance to PCa biology wasnot investigated it might involve the ability of AR to interactwith cofactors that modulate the transcriptional elongationrate andor the recognition of splicing enhancers or silencersin the pre-mRNA (Figure 2) For instance AR interacts withthe cofactor of BRCA1 (COBRA1) and this interaction wasshown to influence splicing of the nascent transcripts pro-duced from an androgen-dependent promoter [55] A similarregulation of AR activity was also documented for the DEADboxRNAhelicase p68 (DDX5) in the LNCaP cell line AR andDDX5 interact and are recruited to the promoter region of theandrogen-responsive prostate-specific antigen (PSA) geneThis interactionwas functionally relevant asDDX5 enhancedAR-dependent PSA expression In addition by using an AR-dependent minigene reporter it was shown that DDX5 andAR cooperated in repressing the splicing of variable exons inthe CD44 gene [56] DDX5 is involved in several steps of co-and posttranscriptional RNA processing including splicing[57] and some genes appear to be particularly sensitive to theintracellular levels of DDX5 [57 58] Hence since this RNAhelicase is upregulated in PCa [56] it will be interesting todetermine to what extent it contributes to RNA processing ofAR target genes in PCa cells
AR is also known to interact with several splicing fac-tors suggesting a direct link between androgen-regulatedtranscription initiation and pre-mRNA splicing in PCa cells(Figure 2) The PTB-associated splicing factor (PSF) and itscofactor p54nrb participate to androgen-dependent proteincomplexes containing the AR PSF and p54nrb inhibit thetranscriptional activity of AR by interfering with its bindingto androgen response element and by recruiting a his-tone deacetylase to AR-responsive promoters [59] Althoughdirect investigation of the effect of these splicing factors onAR-dependent splicing events was not addressed it is likely
that AS is also affected by this interaction Another splicingfactor that may participate to AR-dependent splicing regula-tion is SAM68 [60] which is frequently upregulated in PCa[25 45] SAM68 interacts with AR and is recruited to the PSApromoter [60] like DDX5 [55] Interestingly however theinteraction between SAM68 and AR exerted different effectson transcription and splicing as the two proteins cooperatedin transcriptional activation of AR-target genes but opposedeach other in splicing of the CD44 variable exons from areporter minigene [60] Unfortunately the direct effects ofall these RBPs on AR-dependent splicing of endogenoustranscripts have not been addressed yet Nevertheless it islikely that depending on the specific complex formedAR candifferentially influence splicing of its target genes in PCa cells
An additional layer of regulation of the aberrant splicingprogram in PCa might rely on the up-regulation of specificsplicing factors Beside the already mentioned SAM68 [2545] one likely candidate is SRSF1 a splicing factor thatis upregulated in many human cancers and was shown tobehave as an oncogene in mice and humans [61] In cancercells of other tissues SRSF1modulates the expression of splicevariants of the BIN1 and BIM genes that lack proapoptoticfunctions [61 62] Moreover SRSF1 promotes splicing ofMNK2b [61] a splice variant of the eIF4E kinase MNK2 thatwas shown to confer chemoresistance in pancreatic adenocar-cinoma cells [63] Importantly MNK-dependent phospho-rylation of eIF4E strongly contributes to PCa tumorigenesisboth in vitro and in vivo [64 65] and a tight balance betweenthe MNKeIF4E and the mTOR pathways is required tomaintain efficient protein synthesis in PCa cells therebyenhancing their proliferation rate [64] Thus it will be inter-esting to determine whether SRSF1 contributes to fine-tuningthe activation of these pathways in PCa cells through theregulation ofMNK2 AS
Other splicing factors may also contribute to the alteredsplicing programof PCa cells Indeed the activity of several ofthese RBPs ismodulated by signal transduction pathways thatare frequently turned on in cancer such as the PI3KAKT andthe RASERK pathways (see also [66]) For instance it wasshown that activation of AKT downstream of the epidermalgrowth factor (EGF) receptor modulated the activity ofthe SR protein-specific kinases and phosphorylation of SRproteins thereby affecting a large spectrum of AS events[67] Similarly the RASERK pathway modulates a numberof splicing factors involved in cancer such as SAM68 [68]and the alternative splicing factor 45 (SPF45) [69] whichin turn affect expression of splice variants that regulate cellmotility proliferation and survival Thus it is likely that theexamples reported above represent only a small picture of theoverall contribution of AS and splicing factors to the wideheterogeneity in gene expression observed in PCa cells andpatients
10 Conclusions and Perspectives
AS is widely recognized as a powerful tool that eukaryoticcells employ to expand the coding potential and the plasticityof their genomes The flexibility in the recognition of exons
International Journal of Cell Biology 7
DDX5
SAM68COBRA1
AR
AR splicing-responsive mRNAs
RNAPIIAR transcriptional-responsive gene
AR coactivators
PSF p54nrb
AR corepressor
ARRNAPII
AR transcriptional-responsive gene
Figure 2 Regulation of cotranscriptional splicing by proteins interacting with the androgen receptor Coregulators of the androgen receptor(AR) can affect splicing of target genes by direct interaction with AR and modulation of its activity COBRA1 SAM68 and DDX5 appear topromote the transcriptional activity of AR but differentially act on splicing of variable exons (red box in the left side of the figure) PSF andits interacting protein p54 (right side of the figure) repress the transcriptional activity of AR but their effect on splicing is currently unknown(see text for more details)
and introns within the transcription unit of the majority ofhuman genes offers the possibility to compose many mRNAvariants from each gene Subtle changes in the cellular envi-ronment or in external cues conveyed from the surroundingenvironment may result in global changes in the tran-scriptome which in part rely on the regulation of AS Aninteresting observation is that apparently homogenous cellpopulations actually display large differences in gene expres-sion This was recently exemplified by studies that appliedglobal RNA sequencing techniques to the analysis of singlecell transcriptomes After treatment of bone-marrow-deriveddendritic cells (BMDCs) with an inflammatory cue it wasfound that hundreds of key immune genes were differentiallyexpressed by single cells The heterogeneity in the responsewas particularly remarkable with regard to the splicing pat-terns expressed by these cells [70] suggesting that fine-tuningof AS regulation strongly contributes to the heterogeneity ofa cell population This aspect might be particularly relevantin the context of PCa which is a neoplastic disease charac-terized by extreme heterogeneity and unpredicted responseof patients to the therapy [1 2] The improvements in cellisolation techniques coupled to the higher sensitivity of thenext-generation sequencing techniques may soon allow ahighly detailed description of the transcriptome of patientswhich might result in more personalized treatments
The studies illustrated previously suggest that the upregu-lation of selected splicing regulators in PCa such as SAM68SRSF1 or DDX5 directly contributes to the phenotype byaltering the splicing profile of key genes Thus these RBPsmight represent potential therapeutic targets for interventionAlthough blocking the activity of a given splicing factor isnot necessarily an easy task some examples in this direction
have been provided For instance SAM68 can bind to RNAonly as a dimer By exploiting this requirement it was shownthat an RNA binding-defective SAM68 mutant exerted dom-inant negative effects on SAM68-mediated SMN2 splicing byassociating with the endogenous protein and preventing itsbinding to the pre-mRNA [71]This experiment suggests thatsmall molecules interfering with SAM68 function might dis-play therapeutic potential As homodimerization is a prereq-uisite for RNA binding one possibility is to target the SAM68dimerization domain which was restricted to a small regionwithin its Gld1-Sam68-Grp33 (GSG) homology domain [72]The potential value of targeting specific components of thesplicing machinery in cancer cells is also suggested by theantioncogenic properties of natural compounds such asspliceostatin A (SSA) in a variety of cancer cell modelsSSA targets the splicing factor 3B subunit 1 (SF3B1) of thespliceosome thus affecting a large number of splicing eventsconcomitantly [73] Perhaps more specific drugs targetingsplicing factors involved in subsets of oncogenic splicingevents in cancer cells as those described above might repre-sent more specific therapeutic approaches in the next future
Although the extreme flexibility of AS regulation is proneto errors that may concur to neoplastic transformation [7 8]it can also be exploited therapeutically Indeed examples ofAS modulation in selected genes by administering splicing-correcting ASOs to cells have been reported In some casesthis approach has also been challenged with a therapeuticapplication One of the most remarkable examples is repre-sented by the recovery of the phenotype observed in mousemodels of Spinal Muscular Atrophy (SMA) This neurode-generative disease is caused by inactivation of the SMN1 geneand skipping of exon 7 in the highly homologous SMN2 gene
8 International Journal of Cell Biology
[74] It was recently demonstrated that systemic injectionof a chemically modified ASO restored SMN2 splicing invitro and in vivo and profoundly ameliorated the viabilityand phenotypic features of mice affected by a severe form ofSMA [75] Although cancer is caused by multiple alterationsthus limiting the application of gene-specific ASOs it isconceivable that these tools could be used in combinationwith standard therapies to improve the clinical response ofpatients For instance an ASO that switched BCL-X splicingtoward the proapoptotic variant was effective in sensitizingcancer cells to drug-induced apoptosis and to reduce growthof tumors in nude mice [30 31] A similar effect was obtainedby switching expression of the 120572 to the 120573 variant of the signaltransducer and activator of transcription 3 (STAT3) genewhich modulates multiple oncogenic pathways [76] In thiscase administration of a modified ASO targeted to a splicingenhancer induced expression of the endogenous STAT3120573 andan anti-oncogenic response in vitro and in vivo [76] Thesestudies suggest that modulation of AS with synthetic drugsis possible and has entered a therapeutic perspective ASOsare particularly appealing in terms of high specificity andreduced side effects as theymay exploit their ability to annealwith specific sequences in the genomewithout affecting otherfeatures or target genes of the splicing factors involved in theoncogenicAS eventThus it is likely that thesemethods couldbe applied soon to the development of novel therapies aimedat fighting human cancers in which expression of specificoncogenic splice variants has been firmly confirmed
Conflict of Interests
The author declares no conflict of interests
Acknowledgments
The author wishes to thank Dr Chiara Naro for help with thepreparation of the figures and critical reading of the paperThe work in the laboratory of C Sette was supported byAssociation for International Cancer Research (AICR Grantno 12-0150) the Associazione Italiana Ricerca sul Cancro(AIRC) and Istituto Superiore della Sanita (ISS Grant Con-venzione 11US6A)
References
[1] B J Feldman and D Feldman ldquoThe development of androgen-independent prostate cancerrdquoNature Reviews Cancer vol 1 no1 pp 34ndash45 2001
[2] M Kohli and D J Tindall ldquoNew developments in the medicalmanagement of prostate cancerrdquo Mayo Clinic Proceedings vol85 no 1 pp 77ndash86 2010
[3] K E Knudsen and H I Scher ldquoStarving the addiction newopportunities for durable suppression of AR signaling in pros-tate cancerrdquo Clinical Cancer Research vol 15 no 15 pp 4792ndash4798 2009
[4] S M Dehm and D J Tindall ldquoAlternatively spliced androgenreceptor variantsrdquo Endocrine-Related Cancer vol 18 no 5 ppR183ndashR196 2011
[5] A Kalsotra and T A Cooper ldquoFunctional consequences ofdevelopmentally regulated alternative splicingrdquoNature ReviewsGenetics vol 12 no 10 pp 715ndash729 2011
[6] C Zhang H-R Li J-B Fan et al ldquoProfiling alternativelysplicedmRNA isoforms for prostate cancer classificationrdquo BMCBioinformatics vol 7 article 202 2006
[7] C J David and J L Manley ldquoAlternative pre-mRNA splicingregulation in cancer pathways and programs unhingedrdquo Genesand Development vol 24 no 21 pp 2343ndash2364 2010
[8] S Pal R Gupta and R V Davuluri ldquoAlternative transcriptionand alternative splicing in cancerrdquo Pharmacology amp Therapeu-tics vol 136 no 3 pp 283ndash294 2012
[9] Y Li M Alsagabi D Fan G S Bova A H Tewfik and S MDehm ldquoIntragenic rearrangement and altered RNA splicing ofthe androgen receptor in a cell-based model of prostate cancerprogressionrdquo Cancer Research vol 71 no 6 pp 2108ndash2117 2011
[10] S M Dehm L J Schmidt H V Heemers R L Vessella and DJ Tindall ldquoSplicing of a novel androgen receptor exon generatesa constitutively active androgen receptor that mediates prostatecancer therapy resistancerdquo Cancer Research vol 68 no 13 pp5469ndash5477 2008
[11] R Hu T A Dunn SWei et al ldquoLigand-independent androgenreceptor variants derived from splicing of cryptic exons signifyhormone-refractory prostate cancerrdquo Cancer Research vol 69no 1 pp 16ndash22 2009
[12] Z Guo X Yang F Sun et al ldquoA novel androgen receptor splicevariant is up-regulated during prostate cancer progressionand promotes androgen depletion-resistant growthrdquo CancerResearch vol 69 no 6 pp 2305ndash2313 2009
[13] Y Li S C Chan L J Brand T H Hwang K A Silverstein andS M Dehm ldquoAndrogen receptor splice variants mediate enza-lutamide resistance in castration-resistant prostate cancer celllinesrdquo Cancer Research vol 73 no 2 pp 483ndash489 2013
[14] G Narla K E Heath H L Reeves et al ldquoKLF6 a candidatetumor suppressor genemutated in prostate cancerrdquo Science vol294 no 5551 pp 2563ndash2566 2001
[15] X Liu A Gomez-Pinillos C Loder et al ldquoKLF6 loss of func-tion in human prostate cancer progression is implicated inresistance to androgen deprivationrdquo The American Journal ofPathology vol 181 no 3 pp 1007ndash1016 2012
[16] G Narla A DiFeo H L Reeves et al ldquoA germline DNApolymorphism enhances alternative splicing of the KLF6 tumorsuppressor gene and is associatedwith increased prostate cancerriskrdquo Cancer Research vol 65 no 4 pp 1213ndash1222 2005
[17] G Narla A DiFeo Y Fernandez et al ldquoKLF6-SV1 overexpres-sion accelerates human and mouse prostate cancer progressionand metastasisrdquo Journal of Clinical Investigation vol 118 no 8pp 2711ndash2721 2008
[18] G Narla A Difeo S Yao et al ldquoTargeted inhibition of theKLF6 splice variant KLF6 SV1 suppresses prostate cancer cellgrowth and spreadrdquo Cancer Research vol 65 no 13 pp 5761ndash5768 2005
[19] K E Knudsen ldquoThe cyclin D1b splice variant an old oncogenelearns new tricksrdquo Cell Division vol 1 article 15 2006
[20] K E Knudsen J A Diehl C A Haiman and E S KnudsenldquoCyclin D1 polymorphism aberrant splicing and cancer riskrdquoOncogene vol 25 no 11 pp 1620ndash1628 2006
[21] F Lu A B Gladden and J A Diehl ldquoAn alternatively splicedcyclin D1 isoform cyclin D1b is a nuclear oncogenerdquo CancerResearch vol 63 no 21 pp 7056ndash7061 2003
International Journal of Cell Biology 9
[22] C E S Comstock M A Augello R Pe Benito et al ldquoCyclinD1 splice variants polymorphism risk and isoform-specificregulation in prostate cancerrdquo Clinical Cancer Research vol 15no 17 pp 5338ndash5349 2009
[23] M A Augello C J Burd R Birbe et al ldquoConvergence of onco-genic and hormone receptor pathways promotes metastaticphenotypesrdquo The Journal of Clinical Investigation vol 123 no1 pp 493ndash508 2013
[24] N A Olshavsky C E S Comstock M J Schiewer et al ldquoIden-tification of ASFSF2 as a critical allele-specific effector of thecyclin D1b oncogenerdquoCancer Research vol 70 no 10 pp 3975ndash3984 2010
[25] M P Paronetto M Cappellari R Busa et al ldquoAlternative splic-ing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68rdquoCancer Research vol 70 no 1 pp 229ndash239 2010
[26] D Kaida M G Berg I Younis et al ldquoU1 snRNP protects pre-mRNAs frompremature cleavage and polyadenylationrdquoNaturevol 468 no 7324 pp 664ndash668 2010
[27] P Bielli R Busa M P Paronetto and C Sette ldquoThe RNA-binding protein Sam68 is a multifunctional player in humancancerrdquo Endocrine-Related Cancer vol 18 no 4 pp R91ndashR1022011
[28] L H Boise M Gonzalez-Garcia C E Postema et al ldquobcl-xA bcl-2-related gene that functions as a dominant regulator ofapoptotic cell deathrdquo Cell vol 74 no 4 pp 597ndash608 1993
[29] D R Mercatante C D Bortner J A Cidlowski and R KoleldquoModification of alternative splicing of Bcl-x Pre-mRNA inprostate and breast cancer cells analysis of apoptosis and celldeathrdquo Journal of Biological Chemistry vol 276 no 19 pp 16411ndash16417 2001
[30] D R Mercatante J L Mohler and R Kole ldquoCellular responseto an antisense-mediated shift of Bcl-x pre-mRNA splicing andantineoplastic agentsrdquo Journal of Biological Chemistry vol 277no 51 pp 49374ndash49382 2002
[31] J A Bauman S-D Li A Yang L Huang and R Kole ldquoAnti-tumor activity of splice-switching oligonucleotidesrdquo NucleicAcids Research vol 38 no 22 pp 8348ndash8356 2010
[32] C E Chalfant K Rathman R L Pinkerman et al ldquoDe novoceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells Dependence on proteinphosphatase-1rdquo Journal of Biological Chemistry vol 277 no 15pp 12587ndash12595 2002
[33] K Boon-Unge Q Yu T Zou A Zhou P Govitrapong andJ Zhou ldquoEmetine regulates the alternative splicing of Bcl-xthrough a protein phosphatase 1-dependent mechanismrdquoChemistry and Biology vol 14 no 12 pp 1386ndash1392 2007
[34] M H Kim ldquoProtein phosphatase 1 activation and alternativesplicing of Bcl-X and Mcl-1 by EGCG + ibuprofenrdquo Journal ofCellular Biochemistry vol 104 no 4 pp 1491ndash1499 2008
[35] T Novoyatleva B Heinrich Y Tang et al ldquoProtein phosphatase1 binds to the RNA recognition motif of several splicing fac-tors and regulates alternative pre-mRNA processingrdquo HumanMolecular Genetics vol 17 no 1 pp 52ndash70 2008
[36] N Ghosh N Patel K Jiang et al ldquoCeramide-activated pro-tein phosphatase involvement in insulin resistance via Aktserinearginine-rich protein 40 and ribonucleic acid splicing inL6 skeletal muscle cellsrdquo Endocrinology vol 148 no 3 pp 1359ndash1366 2007
[37] D Garneau T Revil J-F Fisette and B Chabot ldquoHetero-geneous nuclear ribonucleoprotein FH proteins modulate
the alternative splicing of the apoptotic mediator Bcl-xrdquo Journalof Biological Chemistry vol 280 no 24 pp 22641ndash22650 2005
[38] M P Paronetto T Achsel A Massiello C E Chalfant andC Sette ldquoThe RNA-binding protein Sam68 modulates thealternative splicing of Bcl-xrdquo Journal of Cell Biology vol 176 no7 pp 929ndash939 2007
[39] A Zhou A C Ou A Cho E J Benz Jr and S-C HuangldquoNovel splicing factor RBM25 modulates Bcl-x Pre-mRNA 51015840splice site selectionrdquoMolecular and Cellular Biology vol 28 no19 pp 5924ndash5936 2008
[40] S Pedrotti R Busa C Compagnucci and C Sette ldquoThe RNArecognition motif protein RBM11 is a novel tissue-specificsplicing regulatorrdquo Nucleic Acids Research vol 40 no 3 pp1021ndash1032 2012
[41] T Revil J Pelletier J Toutant A Cloutier and B ChabotldquoHeterogeneous nuclear ribonucleoprotein K represses theproduction of pro-apoptotic Bcl-xS splice isoformrdquo Journal ofBiological Chemistry vol 284 no 32 pp 21458ndash21467 2009
[42] M J Moore Q Wang C J Kennedy and P A Silver ldquoAn alter-native splicing network links cell-cycle control to apoptosisrdquoCell vol 142 no 4 pp 625ndash636 2010
[43] P Cloutier J Toutant L Shkreta S Goekjian T Revil and BChabot ldquoAntagonistic effects of the SRp30c protein and cryptic51015840 splice sites on the alternative splicing of the apoptoticregulator Bcl-xrdquo Journal of Biological Chemistry vol 283 no 31pp 21315ndash21324 2008
[44] A Massiello J R Roesser and C E Chalfant ldquoSAP155 Bindsto ceramide-responsive RNA cis-element 1 and regulates thealternative 51015840 splice site selection of Bcl-x pre-mRNArdquo TheFASEB Journal vol 20 no 10 pp 1680ndash1682 2006
[45] R BusaM P Paronetto D Farini et al ldquoTheRNA-binding pro-tein Sam68 contributes to proliferation and survival of humanprostate cancer cellsrdquo Oncogene vol 26 no 30 pp 4372ndash43822007
[46] C Sette ldquoPost-translational regulation of star proteins andeffects on their biological functionsrdquo Advances in ExperimentalMedicine and Biology vol 693 pp 54ndash66 2010
[47] C Brignatz M P Paronetto S Opi et al ldquoAlternative splicingmodulates autoinhibition and SH3 accessibility in the Src kinaseFynrdquo Molecular and Cellular Biology vol 29 no 24 pp 6438ndash6448 2009
[48] M P Paronetto D Farini I Sammarco et al ldquoExpression of atruncated form of the c-Kit tyrosine kinase receptor and activa-tion of Src kinase in human prostatic cancerrdquo American Journalof Pathology vol 164 no 4 pp 1243ndash1251 2004
[49] Y Hu A Dobi T Sreenath et al ldquoDelineation of TMPRSS2-ERG splice variants in prostate cancerrdquo Clinical CancerResearch vol 14 no 15 pp 4719ndash4725 2008
[50] J Wang Y Cai W Yu C Ren D M Spencer and M Itt-mann ldquoPleiotropic biological activities of alternatively splicedTMPRSS2ERG fusion gene transcriptsrdquo Cancer Research vol68 no 20 pp 8516ndash8524 2008
[51] F Zammarchi G Boutsalis and L Cartegni ldquo51015840 UTR controlof native ERG and of Tmprss2 ERG variants activity in prostatecancerrdquo PLoS One vol 8 no 3 Article ID e49721 2013
[52] M J Munoz M S P Santangelo M P Paronetto et al ldquoDNAdamage regulates alternative splicing through inhibition ofRNApolymerase II elongationrdquoCell vol 137 no 4 pp 708ndash7202009
[53] P Rajan C Dalgliesh P J Carling et al ldquoIdentification of novelandrogen-regulated pathways and mrna isoforms through
10 International Journal of Cell Biology
genome-wide exon-specific profiling of the LNCaP transcrip-tomerdquo PLoS One vol 6 no 12 Article ID e29088 2011
[54] T M Morgan T D Koreckij and E Corey ldquoTargeted therapyfor advanced prostate cancer inhibition of the PI3KAktmTORpathwayrdquoCurrent CancerDrug Targets vol 9 no 2 pp 237ndash2492009
[55] J Sun A L Blair S E Aiyar and R Li ldquoCofactor of BRCA1modulates androgen-dependent transcription and alternativesplicingrdquo Journal of Steroid Biochemistry andMolecular Biologyvol 107 no 3ndash5 pp 131ndash139 2007
[56] E L Clark A Coulson C Dalgliesh et al ldquoThe RNA helicasep68 is a novel androgen receptor coactivator involved in splicingand is overexpressed in prostate cancerrdquo Cancer Research vol68 no 19 pp 7938ndash7946 2008
[57] E Zonta D Bittencourt S Samaan S Germann M Dutertreand D Auboeuf ldquoThe RNA helicase DDX5p68 is a key factorpromoting c-fos expression at different levels from transcriptionto mRNA exportrdquoNucleic Acids Research vol 41 no 1 pp 554ndash564 2013
[58] A Honig D Auboeuf M M Parker B W OrsquoMalley andS M Berget ldquoRegulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72rdquo Molecular and Cel-lular Biology vol 22 no 16 pp 5698ndash5707 2002
[59] X Dong J Sweet J R G Challis T Brown and S J Lye ldquoTran-scriptional activity of androgen receptor is modulated by twoRNA splicing factors PSF and p54nrbrdquoMolecular and CellularBiology vol 27 no 13 pp 4863ndash4875 2007
[60] P Rajan L Gaughan C Dalgliesh et al ldquoThe RNA-binding andadaptor protein Sam68 modulates signal-dependent splicingand transcriptional activity of the androgen receptorrdquo Journalof Pathology vol 215 no 1 pp 67ndash77 2008
[61] R Karni E De Stanchina S W Lowe R Sinha D Mu and AR Krainer ldquoThe gene encoding the splicing factor SF2ASF is aproto-oncogenerdquo Nature Structural and Molecular Biology vol14 no 3 pp 185ndash193 2007
[62] OAnczukowA Z RosenbergMAkerman et al ldquoThe splicingfactor SRSF1 regulates apoptosis and proliferation to promotemammary epithelial cell transformationrdquoNature Structural andMolecular Biology vol 19 no 2 pp 220ndash228 2012
[63] L Adesso S Calabretta F Barbagallo et al ldquoGemcitabine trig-gers a pro-survival response in pancreatic cancer cells throughactivation of the MNK2eIF4E pathwayrdquo Oncogene vol 32 pp2848ndash2857 2013
[64] A Bianchini M Loiarro P Bielli et al ldquoPhosphorylation ofeIF4E by MNKs supports protein synthesis cell cycle progres-sion and proliferation in prostate cancer cellsrdquo Carcinogenesisvol 29 no 12 pp 2279ndash2288 2008
[65] L Furic L RongO Larsson et al ldquoEIF4Ephosphorylation pro-motes tumorigenesis and is associated with prostate cancer pro-gressionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 32 pp 14134ndash14139 2010
[66] C Naro and C Sette ldquoPhosphorylation-mediated regulation ofalternative splicing in cancerrdquo International Journal of Cell Bio-logy In press
[67] Z Zhou J QiuW Liu et al ldquoTheAkt-SRPK-SR axis constitutesa major pathway in transducing EGF signaling to regulatealternative splicing in the nucleusrdquo Molecular Cell vol 47 no3 pp 422ndash433 2012
[68] NMatter P Herrlich andH Konig ldquoSignal-dependent regula-tion of splicing via phosphorylation of Sam68rdquoNature vol 420no 6916 pp 691ndash695 2002
[69] A M Al-Ayoubi H Zheng Y Liu T Bai and S T EblenldquoMitogen-activated protein kinase phosphorylation of splicingfactor 45 (SPF45) regulates SPF45 alternative splicing siteutilization proliferation and cell adhesionrdquoMolecular and Cel-lular Biology vol 32 no 14 pp 2880ndash2893 2012
[70] A K Shalek R Satija X Adiconis et al ldquoSingle-cell transcrip-tomics reveals bimodality in expression and splicing in immunecellsrdquo Nature 2013
[71] S Pedrotti P BielliM P Paronetto et al ldquoThe splicing regulatorSam68 binds to a novel exonic splicing silencer and functions inSMN2 alternative splicing in spinal muscular atrophyrdquo EMBOJournal vol 29 no 7 pp 1235ndash1247 2010
[72] N H Meyer K Tripsianes M Vincendeau et al ldquoStructuralbasis for homodimerization of the Src-associated during mito-sis 68-kDa protein (Sam68)Qua1 domainrdquo Journal of BiologicalChemistry vol 285 no 37 pp 28893ndash28901 2010
[73] S Bonnal L Vigevani and J Valcarcel ldquoThe spliceosome asa target of novel antitumour drugsrdquo Nature Reviews Drug Dis-covery vol 11 no 11 pp 847ndash859 2012
[74] S Pedrotti and C Sette ldquoSpinal muscular atrophy a new playerjoins the battle for SMN2 exon 7 splicingrdquo Cell Cycle vol 9 no19 pp 3874ndash3879 2010
[75] Y Hua K Sahashi F Rigo et al ldquoPeripheral SMN restoration isessential for long-term rescue of a severe spinal muscular atro-phy mouse modelrdquo Nature vol 478 no 7367 pp 123ndash126 2011
[76] F Zammarchi E De Stanchina E Bournazou et al ldquoAntitu-morigenic potential of STAT3 alternative splicing modulationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 43 pp 17779ndash17784 2011
Cellular transformationEnhancement of AR-dependent transcription
1 32
Proapoptotic
1 32
AR gene
BCL-X gene
CCND1gene
BCL-XS
BCL-XL
5998400 5998400 3998400
Antiapoptotic chemotherapyresistance
Figure 1 Representative examples of genes whose alternative splicing affects prostate cancer cell biology The left side of the figure illustratesthe genomic structure of the alternatively spliced regions of the AR CCND1 and BCL-X genes Solid and dashed lines show the alternativesplicing events reported in the literature On the right side the alternative variants produced by splicing are shown The specific features ofthe protein isoforms produced by alternative splicing are summarized under the scheme of each variant
was characterized further and shown to function as adominant-negative protein which antagonizes the functionof full length KLF6 leading to decreased p21 expressionand enhanced cell growth [16] Increased expression ofthis splice variant in PCa patients predicted poorer out-come after surgery and was associated with development ofhormone-refractory metastatic PCa [17] Furthermore while
knockdown of the full length KLF6 promoted tumor forma-tion in nudemice selective silencing of the KLF6-SV1 variantinhibited it [18] Conversely PCa cells overexpressing KLF6-SV1 are more prone to develop metastases in various organsof the mouse models used in the study [17] Thus a mutationaffecting KLF6 AS represents a critical mechanism for theinactivation of a tumor suppressor gene in PCa suggesting
4 International Journal of Cell Biology
that interfering with this splicing event in PCa cells mightrestore the growth-inhibitory activity of this transcriptionfactor
5 Cyclin D1
CCND1 is a protooncogene that encodes for cyclin D1 whichassociates with the cyclin-dependent kinase 4 (CDK4) todrive progression through the G1 phase of the cell cycleImportantlyCCND1 expression is oftenderegulated in cancercells [19 20] This gene encodes for two alternative tran-scripts the common cyclin D1a isoform containing all fiveexons and cyclin D1b which derives from retention of intron4 and premature termination of the transcript (Figure 1) [1920] Unlike cyclin D1a cyclin D1b alone can promote cellulartransformation [21] and its expression has been associatedwith PCa progression and poor prognosis [22] Interestinglyrecent evidence indicated that cyclin D1b promotes AR-dependent transcription of genes involved in PCa metastaticpotential such as the transcription factor SLUG [23] CyclinD1a was instead reported to repress the transcriptionalactivity of AR (Figure 1) [19 20] Thus it is conceivable thata change in the ratio between the cyclin D1 variants willpotently enhance hormone-dependent growth of PCa cells
Given the relevance for PCa cell biology understandingthe regulation of CCND1 splicing is of crucial importanceIt was observed that a polymorphism (G870A) at theexon 4-intron 4 boundary predisposes cells to cyclin D1bsplicing [19 20] The splicing factor SRSF1 was shown tobind the exon4intron4 junction in the nascent CCND1 pre-mRNA thereby promoting intron 4 retention and cyclin D1bexpression [24] SRSF1 was hypothesized to favour intron 4retention by altering exon 4 definition and limiting assemblyof the spliceosome at the exon-intron junction [24] Anothersplicing factor promoting cyclin D1b expression in PCa cellsis SAM68 [25] In this case the binding site was identifiedwithin intron 4 in proximity of the termination site utilizedfor the cyclin D1b mRNA The binding of SAM68 to thisregion of the pre-mRNA was shown to compete with thatof the U1 snRNP [25] Since deposition of U1 snRNP nearcryptic polyadenylation sites located in introns is known toprevent premature termination of transcripts in a genome-wide fashion [26] it is possible that up-regulation of SAM68unmasks the cyclin D1b termination site by interfering withU1 snRNP binding in intron 4 Notably both SRSF1 andSAM68 display oncogenic features in several cell types andtissues [7 27] and their expression positively correlates withthat of cyclin D1b in clinical specimens of PCa patients [2425] Thus it is possible that interfering with the activity ofthese splicing factors will exert positive effects in therapeutictreatments of PCa through modulation of CCND1 splicingand expression
6 BCL-X
The BCL-X (BCL2L1) gene contains 3 exons and encodes fortwo splice variants [28] Two alternative 51015840 splice sites arepresent in exon 2 of the gene selection of the canonical
one at the end of the exon yields the long BCL-XL variantwhereas selection of the distal one located 220 bp upstreamin the exon produces the short BCL-XS variant Notablythese two splice variants have opposite effects in the cell withBCL-XL being prosurvival whereas BCL-XS is proapoptotic(Figure 1) [28] Thus regulation of BCL-X AS can finelymodulate cell viability illustrating the biological importanceof this splicing event In most cancer cells including PCacells the anti-apoptotic BCL-XL variant is overexpressed andconfers resistance to chemotherapeutic treatments [29 30]It is predictable that a full understanding of the mechanismsof regulation of BCL-X splicing will help develop tools toswitch it toward the proapoptotic BCL-XS variant therebyoffering a therapeutic opportunity to sensitize cancer cellsto treatments In line with this notion treatment of PCacells with an antisense oligonucleotide (ASO) masking theBCL-XL splice site effectively switched BCL-X splicing andinduced apoptosis [29] Interestingly the proapoptotic effectof the ASO was more pronounced in cancer cells whichdisplay high levels of expression of BCL-XL and it alsoenhanced their response to chemotherapeutic treatments[30] Thus ASOs targeting BCL-X splicing may have theadvantage of being selective for cancer cells with respect tonormal cells which is a positive feature for an antineoplasticdrug Unfortunately the delivery of ASOs to cancer cells isstill not efficient thus limiting their application in the cliniceven though development of vehicles favoring their deliverysuch as lipid nanoparticles [31] may aid in this direction
Although regulation of BCL-X splicing is highly relevantto PCa cell biology not much is known on the mechanism(s)of its regulation in prostate cells A possible regulator is theprotein phosphatase 1 (PP1) whose activity is required for theregulatory effect of ceramide on BCL-X splicing [32] IndeedPP1 activity was required also for induction of BCL-XSsplicing by emetine a protein synthesis inhibitor and otherproapoptotic drugs in PCa cells [33 34] Nevertheless themechanism by which PP1 modulates splicing of BCL-X is stillunknown PP1 is known to regulate splicing by modulatingthe activity of splicing factors either by direct binding tothem and regulation of their phosphorylation status [35] orindirectly by regulating kinases involved in their post-translational modifications [36]Thus activation of pathwaysimpinging on PP1may affect BCL-X splicing and cell viabilitythrough the regulation of the activity of specific splicingfactors in PCa cells
Several splicing factors have been shown to modulateBCL-X splicing Studies performed in a variety of cell modelsindicated that the heterogeneous nuclear ribonucleoprotein(hnRNP) H and F [37] and the splicing regulators SAM68[38] RBM25 [39] and RBM11 [40] promote splicing of theproapoptotic BCL-XS variant By contrast hnRNPK [41] theserine-arginine (SR) rich proteins SRSF1 [38 42] and SRSF9[43] and the splicing factor SAP155 [44] enhance splicing ofthe anti-apoptotic BCL-XLWhich of these factors contributeto the regulation of BCL-X splicing in PCa cells is stilllargely unknown SRSF1 [24] and SAM68 [45] were shownto be upregulated in PCa and might represent strong candi-dates for the regulation of this splicing event Intriguinglythese splicing factors normally modulate BCL-X splicing in
International Journal of Cell Biology 5
opposite directions [38] while the up-regulation of SRSF1 isin line with the high levels of BCL-XL in PCa cells SAM68should favour the proapoptotic short variant However thesplicing activity of SAM68 is finely tuned by phosphoryla-tion [46] and it was shown that tyrosine phosphorylationby the Src-related kinase FYN switched SAM68-dependentsplicing of BCL-X toward the anti-apoptotic variant [38 47]Since tyrosine phosphorylation of SAM68 is increased inspecimens of PCa patients [48] it is likely that this RBPcan also contribute to the upregulation of BCL-XL in PCacells In line with this hypothesis BCL-XL expression wasdecreased and sensitivity to genotoxic agents was increasedafter knockdown of SAM68 in the androgen-sensitive LNCaPcell line [45]
Thus based on the observations reported previouslyit is predictable that exogenous modulation of BCL-X ASthrough administration of ASOs or by interfering with theactivity of the splicing factors that promote the anti-apoptoticBCL-XL variant will enhance the efficacy of chemotherapy inadvanced PCa as suggested by preclinical studies in PCa celllines [30 31 45]
7 TMPRSS2ERG
ERG is a member of the ETS transcription factor family thatis expressed at very low levels in benign prostate epithelialcells However PCa patients often carry a fusion of theandrogen-responsiveTMPRSS2 genewith ERG which causesaberrantly high expression levels of the transcription factorin the neoplastic cells A detailed sequencing analysis of theTMPRSS2ERG transcripts isolated fromPCa tissues revealedthat fusion-derived transcripts underwent profound AS reg-ulation which yielded mRNA variants encoding both fulllength ERG proteins and isoforms lacking the ETS domainNotably an increase in the abundance of transcripts encodingfull length ERG correlated with less favorable outcome inpatients [49] These results support a possible functional rolefor this transcription factor in PCa pathology and suggest thatmodulation of AS events promoting less pathogenic variantsmay produce beneficial effects
This hypothesis is also supported by another study thattested the effects exerted by the expression of TMPRSS2ERGalternatively spliced transcripts in an immortalized prostatecell line [50] It was found that these TMPRSS2ERG splicevariants had different oncogenic activities in terms of pro-moting proliferation invasion and motility Notably coex-pression of different variants produced stronger effects thaneither variant alone suggesting that the presence of severalTMPRSS2ERG isoforms as it normally occurs in PCa cellsmight confer a more malignant phenotype [50] A furthercontribution of AS to the heterogeneity of TMPRSS2ERGexpression is provided by the extensive variability of the 51015840untranslated region (UTR) in the splice variants observedin patients [51] Indeed AS of the 51015840 UTR affects the onco-genic potential of the encoded proteins by regulating theirtranslation and activity Thus although a functional linkbetween TMPRSS2ERG expression and PCa pathology hasnot been firmly established yet this fusion gene appears
to be another suitable target for an AS-directed therapeuticapproach that would spare normal cells not expressing thechimeric proteins
8 Splicing Programs in Prostate Cancer
Cancer cells express a number of splice variants that conferthem higher resistance to chemotherapeutic drugs and sur-vival advantages When it was investigated in detail the spe-cific signature of splice variants expressed by cancer cells hasbeen recognized as a powerful diagnostic and prognostic tool[7 8] Evenmore importantly the existence of cancer-specificsplicing variants of key genes such as the AR or CCND1in PCa might offer a therapeutic opportunity for targetingproteins that are not expressed in healthy cells For instancedeveloping tools that specifically modulate the expressionof transcript variants preferentially or uniquely produced bycancer cells might slow down tumor growth andor promotecell death during therapy while sparing the healthy tissuesThus understanding AS regulation at the genome-wide levelin PCa cells may not only lead to the identification of noveldiagnostic or prognostic biomarkers but it could also helpfind tools for novel therapeutic approaches to this neoplasticdisease
A few studies have directly investigated the genome-wideregulation of AS in PCa cell lines and primary tumor tissuesUsing a splicing-sensitive microarray comprising a selectedsubset of genes and splice variants it was shown that splicingsignatures could efficiently segregate PCa cells lines from can-cer cell lines derived from other organs or tissues [6] Amongthe alternatively spliced genes the majority also showedvariation in expression levels [6] suggesting that regulationof splicing and transcriptionwere coupled as also observed incells exposed to DNA damage [52] Using the same splicing-sensitive platform it was also possible to identify splicingsignatures that were specific for normal or neoplastic prostatetissues obtained from biopsies [6] Although this approachwas limited to the genes and the splice variants selected for theplatform it provided a first indication that specific changes insplicing occur during prostate tumorigenesis and suggestedthat splicing variants can represent accurate biomarkers forPCaNevertheless how andwhen these changes occur aswellas to what extent they contribute to the acquisition of thetransformed phenotype are still open questions Given thetight association between transcription and splicing a spe-cific splicing program could result from the different activityof transcription factors splicing factors or both Mountingevidence indicates that all these events contribute to someextent to the acquisition of specific splicing signatures in PCa
The most relevant transcription factor involved in PCais the AR Several observations suggest that in addition toregulating the expression levels of target genes AR can alsoinfluence the transcript variants encoded by them Usingcomprehensive splicing-sensitive arrays it was demonstratedthat stimulation of LNCaP cells with androgens caused qual-itative changes in expression of splice variants [53] Many ofthe events altered by treatment with androgens were due tousage of alternative promoters within the transcription unit
6 International Journal of Cell Biology
of the target gene Some of these alternative transcripts werepredicted to influence the function of proteins with relevanceto PCa such as the mTOR regulator TSC2 Following andro-genic stimulation AR was recruited to a cryptic promoterupstream of exon 33 in the TSC2 gene thereby leading toexpression of a truncated transcript lacking the 51015840 exons of thegeneThis alternative TSC2 variant would encode a truncatedprotein lacking the domain required for the interaction withTSC1 which is needed to exert negative regulation of mTORThus androgens may lead to activation of mTOR by relievingthe repressive function of the TSC1TSC2 complex throughAR-dependent induction of a defective variant It is worthyof notice that activation of the mTOR pathway has beenlinked to both tumorigenesis and resistance to therapy in PCa[54] Thus AR might contribute to prostate tumorigenesisalso by causing mTOR activation through expression of thisalternative mRNA variant of TSC2
9 Splicing Regulators Contributing to AlteredGene Expression in Prostate Cancer
In addition to affecting recruitment of AR to alternativepromoters androgens also affected a number of AS events inseveral genes [53] Although the mechanism(s) involved inthese events and their potential relevance to PCa biology wasnot investigated it might involve the ability of AR to interactwith cofactors that modulate the transcriptional elongationrate andor the recognition of splicing enhancers or silencersin the pre-mRNA (Figure 2) For instance AR interacts withthe cofactor of BRCA1 (COBRA1) and this interaction wasshown to influence splicing of the nascent transcripts pro-duced from an androgen-dependent promoter [55] A similarregulation of AR activity was also documented for the DEADboxRNAhelicase p68 (DDX5) in the LNCaP cell line AR andDDX5 interact and are recruited to the promoter region of theandrogen-responsive prostate-specific antigen (PSA) geneThis interactionwas functionally relevant asDDX5 enhancedAR-dependent PSA expression In addition by using an AR-dependent minigene reporter it was shown that DDX5 andAR cooperated in repressing the splicing of variable exons inthe CD44 gene [56] DDX5 is involved in several steps of co-and posttranscriptional RNA processing including splicing[57] and some genes appear to be particularly sensitive to theintracellular levels of DDX5 [57 58] Hence since this RNAhelicase is upregulated in PCa [56] it will be interesting todetermine to what extent it contributes to RNA processing ofAR target genes in PCa cells
AR is also known to interact with several splicing fac-tors suggesting a direct link between androgen-regulatedtranscription initiation and pre-mRNA splicing in PCa cells(Figure 2) The PTB-associated splicing factor (PSF) and itscofactor p54nrb participate to androgen-dependent proteincomplexes containing the AR PSF and p54nrb inhibit thetranscriptional activity of AR by interfering with its bindingto androgen response element and by recruiting a his-tone deacetylase to AR-responsive promoters [59] Althoughdirect investigation of the effect of these splicing factors onAR-dependent splicing events was not addressed it is likely
that AS is also affected by this interaction Another splicingfactor that may participate to AR-dependent splicing regula-tion is SAM68 [60] which is frequently upregulated in PCa[25 45] SAM68 interacts with AR and is recruited to the PSApromoter [60] like DDX5 [55] Interestingly however theinteraction between SAM68 and AR exerted different effectson transcription and splicing as the two proteins cooperatedin transcriptional activation of AR-target genes but opposedeach other in splicing of the CD44 variable exons from areporter minigene [60] Unfortunately the direct effects ofall these RBPs on AR-dependent splicing of endogenoustranscripts have not been addressed yet Nevertheless it islikely that depending on the specific complex formedAR candifferentially influence splicing of its target genes in PCa cells
An additional layer of regulation of the aberrant splicingprogram in PCa might rely on the up-regulation of specificsplicing factors Beside the already mentioned SAM68 [2545] one likely candidate is SRSF1 a splicing factor thatis upregulated in many human cancers and was shown tobehave as an oncogene in mice and humans [61] In cancercells of other tissues SRSF1modulates the expression of splicevariants of the BIN1 and BIM genes that lack proapoptoticfunctions [61 62] Moreover SRSF1 promotes splicing ofMNK2b [61] a splice variant of the eIF4E kinase MNK2 thatwas shown to confer chemoresistance in pancreatic adenocar-cinoma cells [63] Importantly MNK-dependent phospho-rylation of eIF4E strongly contributes to PCa tumorigenesisboth in vitro and in vivo [64 65] and a tight balance betweenthe MNKeIF4E and the mTOR pathways is required tomaintain efficient protein synthesis in PCa cells therebyenhancing their proliferation rate [64] Thus it will be inter-esting to determine whether SRSF1 contributes to fine-tuningthe activation of these pathways in PCa cells through theregulation ofMNK2 AS
Other splicing factors may also contribute to the alteredsplicing programof PCa cells Indeed the activity of several ofthese RBPs ismodulated by signal transduction pathways thatare frequently turned on in cancer such as the PI3KAKT andthe RASERK pathways (see also [66]) For instance it wasshown that activation of AKT downstream of the epidermalgrowth factor (EGF) receptor modulated the activity ofthe SR protein-specific kinases and phosphorylation of SRproteins thereby affecting a large spectrum of AS events[67] Similarly the RASERK pathway modulates a numberof splicing factors involved in cancer such as SAM68 [68]and the alternative splicing factor 45 (SPF45) [69] whichin turn affect expression of splice variants that regulate cellmotility proliferation and survival Thus it is likely that theexamples reported above represent only a small picture of theoverall contribution of AS and splicing factors to the wideheterogeneity in gene expression observed in PCa cells andpatients
10 Conclusions and Perspectives
AS is widely recognized as a powerful tool that eukaryoticcells employ to expand the coding potential and the plasticityof their genomes The flexibility in the recognition of exons
International Journal of Cell Biology 7
DDX5
SAM68COBRA1
AR
AR splicing-responsive mRNAs
RNAPIIAR transcriptional-responsive gene
AR coactivators
PSF p54nrb
AR corepressor
ARRNAPII
AR transcriptional-responsive gene
Figure 2 Regulation of cotranscriptional splicing by proteins interacting with the androgen receptor Coregulators of the androgen receptor(AR) can affect splicing of target genes by direct interaction with AR and modulation of its activity COBRA1 SAM68 and DDX5 appear topromote the transcriptional activity of AR but differentially act on splicing of variable exons (red box in the left side of the figure) PSF andits interacting protein p54 (right side of the figure) repress the transcriptional activity of AR but their effect on splicing is currently unknown(see text for more details)
and introns within the transcription unit of the majority ofhuman genes offers the possibility to compose many mRNAvariants from each gene Subtle changes in the cellular envi-ronment or in external cues conveyed from the surroundingenvironment may result in global changes in the tran-scriptome which in part rely on the regulation of AS Aninteresting observation is that apparently homogenous cellpopulations actually display large differences in gene expres-sion This was recently exemplified by studies that appliedglobal RNA sequencing techniques to the analysis of singlecell transcriptomes After treatment of bone-marrow-deriveddendritic cells (BMDCs) with an inflammatory cue it wasfound that hundreds of key immune genes were differentiallyexpressed by single cells The heterogeneity in the responsewas particularly remarkable with regard to the splicing pat-terns expressed by these cells [70] suggesting that fine-tuningof AS regulation strongly contributes to the heterogeneity ofa cell population This aspect might be particularly relevantin the context of PCa which is a neoplastic disease charac-terized by extreme heterogeneity and unpredicted responseof patients to the therapy [1 2] The improvements in cellisolation techniques coupled to the higher sensitivity of thenext-generation sequencing techniques may soon allow ahighly detailed description of the transcriptome of patientswhich might result in more personalized treatments
The studies illustrated previously suggest that the upregu-lation of selected splicing regulators in PCa such as SAM68SRSF1 or DDX5 directly contributes to the phenotype byaltering the splicing profile of key genes Thus these RBPsmight represent potential therapeutic targets for interventionAlthough blocking the activity of a given splicing factor isnot necessarily an easy task some examples in this direction
have been provided For instance SAM68 can bind to RNAonly as a dimer By exploiting this requirement it was shownthat an RNA binding-defective SAM68 mutant exerted dom-inant negative effects on SAM68-mediated SMN2 splicing byassociating with the endogenous protein and preventing itsbinding to the pre-mRNA [71]This experiment suggests thatsmall molecules interfering with SAM68 function might dis-play therapeutic potential As homodimerization is a prereq-uisite for RNA binding one possibility is to target the SAM68dimerization domain which was restricted to a small regionwithin its Gld1-Sam68-Grp33 (GSG) homology domain [72]The potential value of targeting specific components of thesplicing machinery in cancer cells is also suggested by theantioncogenic properties of natural compounds such asspliceostatin A (SSA) in a variety of cancer cell modelsSSA targets the splicing factor 3B subunit 1 (SF3B1) of thespliceosome thus affecting a large number of splicing eventsconcomitantly [73] Perhaps more specific drugs targetingsplicing factors involved in subsets of oncogenic splicingevents in cancer cells as those described above might repre-sent more specific therapeutic approaches in the next future
Although the extreme flexibility of AS regulation is proneto errors that may concur to neoplastic transformation [7 8]it can also be exploited therapeutically Indeed examples ofAS modulation in selected genes by administering splicing-correcting ASOs to cells have been reported In some casesthis approach has also been challenged with a therapeuticapplication One of the most remarkable examples is repre-sented by the recovery of the phenotype observed in mousemodels of Spinal Muscular Atrophy (SMA) This neurode-generative disease is caused by inactivation of the SMN1 geneand skipping of exon 7 in the highly homologous SMN2 gene
8 International Journal of Cell Biology
[74] It was recently demonstrated that systemic injectionof a chemically modified ASO restored SMN2 splicing invitro and in vivo and profoundly ameliorated the viabilityand phenotypic features of mice affected by a severe form ofSMA [75] Although cancer is caused by multiple alterationsthus limiting the application of gene-specific ASOs it isconceivable that these tools could be used in combinationwith standard therapies to improve the clinical response ofpatients For instance an ASO that switched BCL-X splicingtoward the proapoptotic variant was effective in sensitizingcancer cells to drug-induced apoptosis and to reduce growthof tumors in nude mice [30 31] A similar effect was obtainedby switching expression of the 120572 to the 120573 variant of the signaltransducer and activator of transcription 3 (STAT3) genewhich modulates multiple oncogenic pathways [76] In thiscase administration of a modified ASO targeted to a splicingenhancer induced expression of the endogenous STAT3120573 andan anti-oncogenic response in vitro and in vivo [76] Thesestudies suggest that modulation of AS with synthetic drugsis possible and has entered a therapeutic perspective ASOsare particularly appealing in terms of high specificity andreduced side effects as theymay exploit their ability to annealwith specific sequences in the genomewithout affecting otherfeatures or target genes of the splicing factors involved in theoncogenicAS eventThus it is likely that thesemethods couldbe applied soon to the development of novel therapies aimedat fighting human cancers in which expression of specificoncogenic splice variants has been firmly confirmed
Conflict of Interests
The author declares no conflict of interests
Acknowledgments
The author wishes to thank Dr Chiara Naro for help with thepreparation of the figures and critical reading of the paperThe work in the laboratory of C Sette was supported byAssociation for International Cancer Research (AICR Grantno 12-0150) the Associazione Italiana Ricerca sul Cancro(AIRC) and Istituto Superiore della Sanita (ISS Grant Con-venzione 11US6A)
References
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[2] M Kohli and D J Tindall ldquoNew developments in the medicalmanagement of prostate cancerrdquo Mayo Clinic Proceedings vol85 no 1 pp 77ndash86 2010
[3] K E Knudsen and H I Scher ldquoStarving the addiction newopportunities for durable suppression of AR signaling in pros-tate cancerrdquo Clinical Cancer Research vol 15 no 15 pp 4792ndash4798 2009
[4] S M Dehm and D J Tindall ldquoAlternatively spliced androgenreceptor variantsrdquo Endocrine-Related Cancer vol 18 no 5 ppR183ndashR196 2011
[5] A Kalsotra and T A Cooper ldquoFunctional consequences ofdevelopmentally regulated alternative splicingrdquoNature ReviewsGenetics vol 12 no 10 pp 715ndash729 2011
[6] C Zhang H-R Li J-B Fan et al ldquoProfiling alternativelysplicedmRNA isoforms for prostate cancer classificationrdquo BMCBioinformatics vol 7 article 202 2006
[7] C J David and J L Manley ldquoAlternative pre-mRNA splicingregulation in cancer pathways and programs unhingedrdquo Genesand Development vol 24 no 21 pp 2343ndash2364 2010
[8] S Pal R Gupta and R V Davuluri ldquoAlternative transcriptionand alternative splicing in cancerrdquo Pharmacology amp Therapeu-tics vol 136 no 3 pp 283ndash294 2012
[9] Y Li M Alsagabi D Fan G S Bova A H Tewfik and S MDehm ldquoIntragenic rearrangement and altered RNA splicing ofthe androgen receptor in a cell-based model of prostate cancerprogressionrdquo Cancer Research vol 71 no 6 pp 2108ndash2117 2011
[10] S M Dehm L J Schmidt H V Heemers R L Vessella and DJ Tindall ldquoSplicing of a novel androgen receptor exon generatesa constitutively active androgen receptor that mediates prostatecancer therapy resistancerdquo Cancer Research vol 68 no 13 pp5469ndash5477 2008
[11] R Hu T A Dunn SWei et al ldquoLigand-independent androgenreceptor variants derived from splicing of cryptic exons signifyhormone-refractory prostate cancerrdquo Cancer Research vol 69no 1 pp 16ndash22 2009
[12] Z Guo X Yang F Sun et al ldquoA novel androgen receptor splicevariant is up-regulated during prostate cancer progressionand promotes androgen depletion-resistant growthrdquo CancerResearch vol 69 no 6 pp 2305ndash2313 2009
[13] Y Li S C Chan L J Brand T H Hwang K A Silverstein andS M Dehm ldquoAndrogen receptor splice variants mediate enza-lutamide resistance in castration-resistant prostate cancer celllinesrdquo Cancer Research vol 73 no 2 pp 483ndash489 2013
[14] G Narla K E Heath H L Reeves et al ldquoKLF6 a candidatetumor suppressor genemutated in prostate cancerrdquo Science vol294 no 5551 pp 2563ndash2566 2001
[15] X Liu A Gomez-Pinillos C Loder et al ldquoKLF6 loss of func-tion in human prostate cancer progression is implicated inresistance to androgen deprivationrdquo The American Journal ofPathology vol 181 no 3 pp 1007ndash1016 2012
[16] G Narla A DiFeo H L Reeves et al ldquoA germline DNApolymorphism enhances alternative splicing of the KLF6 tumorsuppressor gene and is associatedwith increased prostate cancerriskrdquo Cancer Research vol 65 no 4 pp 1213ndash1222 2005
[17] G Narla A DiFeo Y Fernandez et al ldquoKLF6-SV1 overexpres-sion accelerates human and mouse prostate cancer progressionand metastasisrdquo Journal of Clinical Investigation vol 118 no 8pp 2711ndash2721 2008
[18] G Narla A Difeo S Yao et al ldquoTargeted inhibition of theKLF6 splice variant KLF6 SV1 suppresses prostate cancer cellgrowth and spreadrdquo Cancer Research vol 65 no 13 pp 5761ndash5768 2005
[19] K E Knudsen ldquoThe cyclin D1b splice variant an old oncogenelearns new tricksrdquo Cell Division vol 1 article 15 2006
[20] K E Knudsen J A Diehl C A Haiman and E S KnudsenldquoCyclin D1 polymorphism aberrant splicing and cancer riskrdquoOncogene vol 25 no 11 pp 1620ndash1628 2006
[21] F Lu A B Gladden and J A Diehl ldquoAn alternatively splicedcyclin D1 isoform cyclin D1b is a nuclear oncogenerdquo CancerResearch vol 63 no 21 pp 7056ndash7061 2003
International Journal of Cell Biology 9
[22] C E S Comstock M A Augello R Pe Benito et al ldquoCyclinD1 splice variants polymorphism risk and isoform-specificregulation in prostate cancerrdquo Clinical Cancer Research vol 15no 17 pp 5338ndash5349 2009
[23] M A Augello C J Burd R Birbe et al ldquoConvergence of onco-genic and hormone receptor pathways promotes metastaticphenotypesrdquo The Journal of Clinical Investigation vol 123 no1 pp 493ndash508 2013
[24] N A Olshavsky C E S Comstock M J Schiewer et al ldquoIden-tification of ASFSF2 as a critical allele-specific effector of thecyclin D1b oncogenerdquoCancer Research vol 70 no 10 pp 3975ndash3984 2010
[25] M P Paronetto M Cappellari R Busa et al ldquoAlternative splic-ing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68rdquoCancer Research vol 70 no 1 pp 229ndash239 2010
[26] D Kaida M G Berg I Younis et al ldquoU1 snRNP protects pre-mRNAs frompremature cleavage and polyadenylationrdquoNaturevol 468 no 7324 pp 664ndash668 2010
[27] P Bielli R Busa M P Paronetto and C Sette ldquoThe RNA-binding protein Sam68 is a multifunctional player in humancancerrdquo Endocrine-Related Cancer vol 18 no 4 pp R91ndashR1022011
[28] L H Boise M Gonzalez-Garcia C E Postema et al ldquobcl-xA bcl-2-related gene that functions as a dominant regulator ofapoptotic cell deathrdquo Cell vol 74 no 4 pp 597ndash608 1993
[29] D R Mercatante C D Bortner J A Cidlowski and R KoleldquoModification of alternative splicing of Bcl-x Pre-mRNA inprostate and breast cancer cells analysis of apoptosis and celldeathrdquo Journal of Biological Chemistry vol 276 no 19 pp 16411ndash16417 2001
[30] D R Mercatante J L Mohler and R Kole ldquoCellular responseto an antisense-mediated shift of Bcl-x pre-mRNA splicing andantineoplastic agentsrdquo Journal of Biological Chemistry vol 277no 51 pp 49374ndash49382 2002
[31] J A Bauman S-D Li A Yang L Huang and R Kole ldquoAnti-tumor activity of splice-switching oligonucleotidesrdquo NucleicAcids Research vol 38 no 22 pp 8348ndash8356 2010
[32] C E Chalfant K Rathman R L Pinkerman et al ldquoDe novoceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells Dependence on proteinphosphatase-1rdquo Journal of Biological Chemistry vol 277 no 15pp 12587ndash12595 2002
[33] K Boon-Unge Q Yu T Zou A Zhou P Govitrapong andJ Zhou ldquoEmetine regulates the alternative splicing of Bcl-xthrough a protein phosphatase 1-dependent mechanismrdquoChemistry and Biology vol 14 no 12 pp 1386ndash1392 2007
[34] M H Kim ldquoProtein phosphatase 1 activation and alternativesplicing of Bcl-X and Mcl-1 by EGCG + ibuprofenrdquo Journal ofCellular Biochemistry vol 104 no 4 pp 1491ndash1499 2008
[35] T Novoyatleva B Heinrich Y Tang et al ldquoProtein phosphatase1 binds to the RNA recognition motif of several splicing fac-tors and regulates alternative pre-mRNA processingrdquo HumanMolecular Genetics vol 17 no 1 pp 52ndash70 2008
[36] N Ghosh N Patel K Jiang et al ldquoCeramide-activated pro-tein phosphatase involvement in insulin resistance via Aktserinearginine-rich protein 40 and ribonucleic acid splicing inL6 skeletal muscle cellsrdquo Endocrinology vol 148 no 3 pp 1359ndash1366 2007
[37] D Garneau T Revil J-F Fisette and B Chabot ldquoHetero-geneous nuclear ribonucleoprotein FH proteins modulate
the alternative splicing of the apoptotic mediator Bcl-xrdquo Journalof Biological Chemistry vol 280 no 24 pp 22641ndash22650 2005
[38] M P Paronetto T Achsel A Massiello C E Chalfant andC Sette ldquoThe RNA-binding protein Sam68 modulates thealternative splicing of Bcl-xrdquo Journal of Cell Biology vol 176 no7 pp 929ndash939 2007
[39] A Zhou A C Ou A Cho E J Benz Jr and S-C HuangldquoNovel splicing factor RBM25 modulates Bcl-x Pre-mRNA 51015840splice site selectionrdquoMolecular and Cellular Biology vol 28 no19 pp 5924ndash5936 2008
[40] S Pedrotti R Busa C Compagnucci and C Sette ldquoThe RNArecognition motif protein RBM11 is a novel tissue-specificsplicing regulatorrdquo Nucleic Acids Research vol 40 no 3 pp1021ndash1032 2012
[41] T Revil J Pelletier J Toutant A Cloutier and B ChabotldquoHeterogeneous nuclear ribonucleoprotein K represses theproduction of pro-apoptotic Bcl-xS splice isoformrdquo Journal ofBiological Chemistry vol 284 no 32 pp 21458ndash21467 2009
[42] M J Moore Q Wang C J Kennedy and P A Silver ldquoAn alter-native splicing network links cell-cycle control to apoptosisrdquoCell vol 142 no 4 pp 625ndash636 2010
[43] P Cloutier J Toutant L Shkreta S Goekjian T Revil and BChabot ldquoAntagonistic effects of the SRp30c protein and cryptic51015840 splice sites on the alternative splicing of the apoptoticregulator Bcl-xrdquo Journal of Biological Chemistry vol 283 no 31pp 21315ndash21324 2008
[44] A Massiello J R Roesser and C E Chalfant ldquoSAP155 Bindsto ceramide-responsive RNA cis-element 1 and regulates thealternative 51015840 splice site selection of Bcl-x pre-mRNArdquo TheFASEB Journal vol 20 no 10 pp 1680ndash1682 2006
[45] R BusaM P Paronetto D Farini et al ldquoTheRNA-binding pro-tein Sam68 contributes to proliferation and survival of humanprostate cancer cellsrdquo Oncogene vol 26 no 30 pp 4372ndash43822007
[46] C Sette ldquoPost-translational regulation of star proteins andeffects on their biological functionsrdquo Advances in ExperimentalMedicine and Biology vol 693 pp 54ndash66 2010
[47] C Brignatz M P Paronetto S Opi et al ldquoAlternative splicingmodulates autoinhibition and SH3 accessibility in the Src kinaseFynrdquo Molecular and Cellular Biology vol 29 no 24 pp 6438ndash6448 2009
[48] M P Paronetto D Farini I Sammarco et al ldquoExpression of atruncated form of the c-Kit tyrosine kinase receptor and activa-tion of Src kinase in human prostatic cancerrdquo American Journalof Pathology vol 164 no 4 pp 1243ndash1251 2004
[49] Y Hu A Dobi T Sreenath et al ldquoDelineation of TMPRSS2-ERG splice variants in prostate cancerrdquo Clinical CancerResearch vol 14 no 15 pp 4719ndash4725 2008
[50] J Wang Y Cai W Yu C Ren D M Spencer and M Itt-mann ldquoPleiotropic biological activities of alternatively splicedTMPRSS2ERG fusion gene transcriptsrdquo Cancer Research vol68 no 20 pp 8516ndash8524 2008
[51] F Zammarchi G Boutsalis and L Cartegni ldquo51015840 UTR controlof native ERG and of Tmprss2 ERG variants activity in prostatecancerrdquo PLoS One vol 8 no 3 Article ID e49721 2013
[52] M J Munoz M S P Santangelo M P Paronetto et al ldquoDNAdamage regulates alternative splicing through inhibition ofRNApolymerase II elongationrdquoCell vol 137 no 4 pp 708ndash7202009
[53] P Rajan C Dalgliesh P J Carling et al ldquoIdentification of novelandrogen-regulated pathways and mrna isoforms through
10 International Journal of Cell Biology
genome-wide exon-specific profiling of the LNCaP transcrip-tomerdquo PLoS One vol 6 no 12 Article ID e29088 2011
[54] T M Morgan T D Koreckij and E Corey ldquoTargeted therapyfor advanced prostate cancer inhibition of the PI3KAktmTORpathwayrdquoCurrent CancerDrug Targets vol 9 no 2 pp 237ndash2492009
[55] J Sun A L Blair S E Aiyar and R Li ldquoCofactor of BRCA1modulates androgen-dependent transcription and alternativesplicingrdquo Journal of Steroid Biochemistry andMolecular Biologyvol 107 no 3ndash5 pp 131ndash139 2007
[56] E L Clark A Coulson C Dalgliesh et al ldquoThe RNA helicasep68 is a novel androgen receptor coactivator involved in splicingand is overexpressed in prostate cancerrdquo Cancer Research vol68 no 19 pp 7938ndash7946 2008
[57] E Zonta D Bittencourt S Samaan S Germann M Dutertreand D Auboeuf ldquoThe RNA helicase DDX5p68 is a key factorpromoting c-fos expression at different levels from transcriptionto mRNA exportrdquoNucleic Acids Research vol 41 no 1 pp 554ndash564 2013
[58] A Honig D Auboeuf M M Parker B W OrsquoMalley andS M Berget ldquoRegulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72rdquo Molecular and Cel-lular Biology vol 22 no 16 pp 5698ndash5707 2002
[59] X Dong J Sweet J R G Challis T Brown and S J Lye ldquoTran-scriptional activity of androgen receptor is modulated by twoRNA splicing factors PSF and p54nrbrdquoMolecular and CellularBiology vol 27 no 13 pp 4863ndash4875 2007
[60] P Rajan L Gaughan C Dalgliesh et al ldquoThe RNA-binding andadaptor protein Sam68 modulates signal-dependent splicingand transcriptional activity of the androgen receptorrdquo Journalof Pathology vol 215 no 1 pp 67ndash77 2008
[61] R Karni E De Stanchina S W Lowe R Sinha D Mu and AR Krainer ldquoThe gene encoding the splicing factor SF2ASF is aproto-oncogenerdquo Nature Structural and Molecular Biology vol14 no 3 pp 185ndash193 2007
[62] OAnczukowA Z RosenbergMAkerman et al ldquoThe splicingfactor SRSF1 regulates apoptosis and proliferation to promotemammary epithelial cell transformationrdquoNature Structural andMolecular Biology vol 19 no 2 pp 220ndash228 2012
[63] L Adesso S Calabretta F Barbagallo et al ldquoGemcitabine trig-gers a pro-survival response in pancreatic cancer cells throughactivation of the MNK2eIF4E pathwayrdquo Oncogene vol 32 pp2848ndash2857 2013
[64] A Bianchini M Loiarro P Bielli et al ldquoPhosphorylation ofeIF4E by MNKs supports protein synthesis cell cycle progres-sion and proliferation in prostate cancer cellsrdquo Carcinogenesisvol 29 no 12 pp 2279ndash2288 2008
[65] L Furic L RongO Larsson et al ldquoEIF4Ephosphorylation pro-motes tumorigenesis and is associated with prostate cancer pro-gressionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 32 pp 14134ndash14139 2010
[66] C Naro and C Sette ldquoPhosphorylation-mediated regulation ofalternative splicing in cancerrdquo International Journal of Cell Bio-logy In press
[67] Z Zhou J QiuW Liu et al ldquoTheAkt-SRPK-SR axis constitutesa major pathway in transducing EGF signaling to regulatealternative splicing in the nucleusrdquo Molecular Cell vol 47 no3 pp 422ndash433 2012
[68] NMatter P Herrlich andH Konig ldquoSignal-dependent regula-tion of splicing via phosphorylation of Sam68rdquoNature vol 420no 6916 pp 691ndash695 2002
[69] A M Al-Ayoubi H Zheng Y Liu T Bai and S T EblenldquoMitogen-activated protein kinase phosphorylation of splicingfactor 45 (SPF45) regulates SPF45 alternative splicing siteutilization proliferation and cell adhesionrdquoMolecular and Cel-lular Biology vol 32 no 14 pp 2880ndash2893 2012
[70] A K Shalek R Satija X Adiconis et al ldquoSingle-cell transcrip-tomics reveals bimodality in expression and splicing in immunecellsrdquo Nature 2013
[71] S Pedrotti P BielliM P Paronetto et al ldquoThe splicing regulatorSam68 binds to a novel exonic splicing silencer and functions inSMN2 alternative splicing in spinal muscular atrophyrdquo EMBOJournal vol 29 no 7 pp 1235ndash1247 2010
[72] N H Meyer K Tripsianes M Vincendeau et al ldquoStructuralbasis for homodimerization of the Src-associated during mito-sis 68-kDa protein (Sam68)Qua1 domainrdquo Journal of BiologicalChemistry vol 285 no 37 pp 28893ndash28901 2010
[73] S Bonnal L Vigevani and J Valcarcel ldquoThe spliceosome asa target of novel antitumour drugsrdquo Nature Reviews Drug Dis-covery vol 11 no 11 pp 847ndash859 2012
[74] S Pedrotti and C Sette ldquoSpinal muscular atrophy a new playerjoins the battle for SMN2 exon 7 splicingrdquo Cell Cycle vol 9 no19 pp 3874ndash3879 2010
[75] Y Hua K Sahashi F Rigo et al ldquoPeripheral SMN restoration isessential for long-term rescue of a severe spinal muscular atro-phy mouse modelrdquo Nature vol 478 no 7367 pp 123ndash126 2011
[76] F Zammarchi E De Stanchina E Bournazou et al ldquoAntitu-morigenic potential of STAT3 alternative splicing modulationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 43 pp 17779ndash17784 2011
that interfering with this splicing event in PCa cells mightrestore the growth-inhibitory activity of this transcriptionfactor
5 Cyclin D1
CCND1 is a protooncogene that encodes for cyclin D1 whichassociates with the cyclin-dependent kinase 4 (CDK4) todrive progression through the G1 phase of the cell cycleImportantlyCCND1 expression is oftenderegulated in cancercells [19 20] This gene encodes for two alternative tran-scripts the common cyclin D1a isoform containing all fiveexons and cyclin D1b which derives from retention of intron4 and premature termination of the transcript (Figure 1) [1920] Unlike cyclin D1a cyclin D1b alone can promote cellulartransformation [21] and its expression has been associatedwith PCa progression and poor prognosis [22] Interestinglyrecent evidence indicated that cyclin D1b promotes AR-dependent transcription of genes involved in PCa metastaticpotential such as the transcription factor SLUG [23] CyclinD1a was instead reported to repress the transcriptionalactivity of AR (Figure 1) [19 20] Thus it is conceivable thata change in the ratio between the cyclin D1 variants willpotently enhance hormone-dependent growth of PCa cells
Given the relevance for PCa cell biology understandingthe regulation of CCND1 splicing is of crucial importanceIt was observed that a polymorphism (G870A) at theexon 4-intron 4 boundary predisposes cells to cyclin D1bsplicing [19 20] The splicing factor SRSF1 was shown tobind the exon4intron4 junction in the nascent CCND1 pre-mRNA thereby promoting intron 4 retention and cyclin D1bexpression [24] SRSF1 was hypothesized to favour intron 4retention by altering exon 4 definition and limiting assemblyof the spliceosome at the exon-intron junction [24] Anothersplicing factor promoting cyclin D1b expression in PCa cellsis SAM68 [25] In this case the binding site was identifiedwithin intron 4 in proximity of the termination site utilizedfor the cyclin D1b mRNA The binding of SAM68 to thisregion of the pre-mRNA was shown to compete with thatof the U1 snRNP [25] Since deposition of U1 snRNP nearcryptic polyadenylation sites located in introns is known toprevent premature termination of transcripts in a genome-wide fashion [26] it is possible that up-regulation of SAM68unmasks the cyclin D1b termination site by interfering withU1 snRNP binding in intron 4 Notably both SRSF1 andSAM68 display oncogenic features in several cell types andtissues [7 27] and their expression positively correlates withthat of cyclin D1b in clinical specimens of PCa patients [2425] Thus it is possible that interfering with the activity ofthese splicing factors will exert positive effects in therapeutictreatments of PCa through modulation of CCND1 splicingand expression
6 BCL-X
The BCL-X (BCL2L1) gene contains 3 exons and encodes fortwo splice variants [28] Two alternative 51015840 splice sites arepresent in exon 2 of the gene selection of the canonical
one at the end of the exon yields the long BCL-XL variantwhereas selection of the distal one located 220 bp upstreamin the exon produces the short BCL-XS variant Notablythese two splice variants have opposite effects in the cell withBCL-XL being prosurvival whereas BCL-XS is proapoptotic(Figure 1) [28] Thus regulation of BCL-X AS can finelymodulate cell viability illustrating the biological importanceof this splicing event In most cancer cells including PCacells the anti-apoptotic BCL-XL variant is overexpressed andconfers resistance to chemotherapeutic treatments [29 30]It is predictable that a full understanding of the mechanismsof regulation of BCL-X splicing will help develop tools toswitch it toward the proapoptotic BCL-XS variant therebyoffering a therapeutic opportunity to sensitize cancer cellsto treatments In line with this notion treatment of PCacells with an antisense oligonucleotide (ASO) masking theBCL-XL splice site effectively switched BCL-X splicing andinduced apoptosis [29] Interestingly the proapoptotic effectof the ASO was more pronounced in cancer cells whichdisplay high levels of expression of BCL-XL and it alsoenhanced their response to chemotherapeutic treatments[30] Thus ASOs targeting BCL-X splicing may have theadvantage of being selective for cancer cells with respect tonormal cells which is a positive feature for an antineoplasticdrug Unfortunately the delivery of ASOs to cancer cells isstill not efficient thus limiting their application in the cliniceven though development of vehicles favoring their deliverysuch as lipid nanoparticles [31] may aid in this direction
Although regulation of BCL-X splicing is highly relevantto PCa cell biology not much is known on the mechanism(s)of its regulation in prostate cells A possible regulator is theprotein phosphatase 1 (PP1) whose activity is required for theregulatory effect of ceramide on BCL-X splicing [32] IndeedPP1 activity was required also for induction of BCL-XSsplicing by emetine a protein synthesis inhibitor and otherproapoptotic drugs in PCa cells [33 34] Nevertheless themechanism by which PP1 modulates splicing of BCL-X is stillunknown PP1 is known to regulate splicing by modulatingthe activity of splicing factors either by direct binding tothem and regulation of their phosphorylation status [35] orindirectly by regulating kinases involved in their post-translational modifications [36]Thus activation of pathwaysimpinging on PP1may affect BCL-X splicing and cell viabilitythrough the regulation of the activity of specific splicingfactors in PCa cells
Several splicing factors have been shown to modulateBCL-X splicing Studies performed in a variety of cell modelsindicated that the heterogeneous nuclear ribonucleoprotein(hnRNP) H and F [37] and the splicing regulators SAM68[38] RBM25 [39] and RBM11 [40] promote splicing of theproapoptotic BCL-XS variant By contrast hnRNPK [41] theserine-arginine (SR) rich proteins SRSF1 [38 42] and SRSF9[43] and the splicing factor SAP155 [44] enhance splicing ofthe anti-apoptotic BCL-XLWhich of these factors contributeto the regulation of BCL-X splicing in PCa cells is stilllargely unknown SRSF1 [24] and SAM68 [45] were shownto be upregulated in PCa and might represent strong candi-dates for the regulation of this splicing event Intriguinglythese splicing factors normally modulate BCL-X splicing in
International Journal of Cell Biology 5
opposite directions [38] while the up-regulation of SRSF1 isin line with the high levels of BCL-XL in PCa cells SAM68should favour the proapoptotic short variant However thesplicing activity of SAM68 is finely tuned by phosphoryla-tion [46] and it was shown that tyrosine phosphorylationby the Src-related kinase FYN switched SAM68-dependentsplicing of BCL-X toward the anti-apoptotic variant [38 47]Since tyrosine phosphorylation of SAM68 is increased inspecimens of PCa patients [48] it is likely that this RBPcan also contribute to the upregulation of BCL-XL in PCacells In line with this hypothesis BCL-XL expression wasdecreased and sensitivity to genotoxic agents was increasedafter knockdown of SAM68 in the androgen-sensitive LNCaPcell line [45]
Thus based on the observations reported previouslyit is predictable that exogenous modulation of BCL-X ASthrough administration of ASOs or by interfering with theactivity of the splicing factors that promote the anti-apoptoticBCL-XL variant will enhance the efficacy of chemotherapy inadvanced PCa as suggested by preclinical studies in PCa celllines [30 31 45]
7 TMPRSS2ERG
ERG is a member of the ETS transcription factor family thatis expressed at very low levels in benign prostate epithelialcells However PCa patients often carry a fusion of theandrogen-responsiveTMPRSS2 genewith ERG which causesaberrantly high expression levels of the transcription factorin the neoplastic cells A detailed sequencing analysis of theTMPRSS2ERG transcripts isolated fromPCa tissues revealedthat fusion-derived transcripts underwent profound AS reg-ulation which yielded mRNA variants encoding both fulllength ERG proteins and isoforms lacking the ETS domainNotably an increase in the abundance of transcripts encodingfull length ERG correlated with less favorable outcome inpatients [49] These results support a possible functional rolefor this transcription factor in PCa pathology and suggest thatmodulation of AS events promoting less pathogenic variantsmay produce beneficial effects
This hypothesis is also supported by another study thattested the effects exerted by the expression of TMPRSS2ERGalternatively spliced transcripts in an immortalized prostatecell line [50] It was found that these TMPRSS2ERG splicevariants had different oncogenic activities in terms of pro-moting proliferation invasion and motility Notably coex-pression of different variants produced stronger effects thaneither variant alone suggesting that the presence of severalTMPRSS2ERG isoforms as it normally occurs in PCa cellsmight confer a more malignant phenotype [50] A furthercontribution of AS to the heterogeneity of TMPRSS2ERGexpression is provided by the extensive variability of the 51015840untranslated region (UTR) in the splice variants observedin patients [51] Indeed AS of the 51015840 UTR affects the onco-genic potential of the encoded proteins by regulating theirtranslation and activity Thus although a functional linkbetween TMPRSS2ERG expression and PCa pathology hasnot been firmly established yet this fusion gene appears
to be another suitable target for an AS-directed therapeuticapproach that would spare normal cells not expressing thechimeric proteins
8 Splicing Programs in Prostate Cancer
Cancer cells express a number of splice variants that conferthem higher resistance to chemotherapeutic drugs and sur-vival advantages When it was investigated in detail the spe-cific signature of splice variants expressed by cancer cells hasbeen recognized as a powerful diagnostic and prognostic tool[7 8] Evenmore importantly the existence of cancer-specificsplicing variants of key genes such as the AR or CCND1in PCa might offer a therapeutic opportunity for targetingproteins that are not expressed in healthy cells For instancedeveloping tools that specifically modulate the expressionof transcript variants preferentially or uniquely produced bycancer cells might slow down tumor growth andor promotecell death during therapy while sparing the healthy tissuesThus understanding AS regulation at the genome-wide levelin PCa cells may not only lead to the identification of noveldiagnostic or prognostic biomarkers but it could also helpfind tools for novel therapeutic approaches to this neoplasticdisease
A few studies have directly investigated the genome-wideregulation of AS in PCa cell lines and primary tumor tissuesUsing a splicing-sensitive microarray comprising a selectedsubset of genes and splice variants it was shown that splicingsignatures could efficiently segregate PCa cells lines from can-cer cell lines derived from other organs or tissues [6] Amongthe alternatively spliced genes the majority also showedvariation in expression levels [6] suggesting that regulationof splicing and transcriptionwere coupled as also observed incells exposed to DNA damage [52] Using the same splicing-sensitive platform it was also possible to identify splicingsignatures that were specific for normal or neoplastic prostatetissues obtained from biopsies [6] Although this approachwas limited to the genes and the splice variants selected for theplatform it provided a first indication that specific changes insplicing occur during prostate tumorigenesis and suggestedthat splicing variants can represent accurate biomarkers forPCaNevertheless how andwhen these changes occur aswellas to what extent they contribute to the acquisition of thetransformed phenotype are still open questions Given thetight association between transcription and splicing a spe-cific splicing program could result from the different activityof transcription factors splicing factors or both Mountingevidence indicates that all these events contribute to someextent to the acquisition of specific splicing signatures in PCa
The most relevant transcription factor involved in PCais the AR Several observations suggest that in addition toregulating the expression levels of target genes AR can alsoinfluence the transcript variants encoded by them Usingcomprehensive splicing-sensitive arrays it was demonstratedthat stimulation of LNCaP cells with androgens caused qual-itative changes in expression of splice variants [53] Many ofthe events altered by treatment with androgens were due tousage of alternative promoters within the transcription unit
6 International Journal of Cell Biology
of the target gene Some of these alternative transcripts werepredicted to influence the function of proteins with relevanceto PCa such as the mTOR regulator TSC2 Following andro-genic stimulation AR was recruited to a cryptic promoterupstream of exon 33 in the TSC2 gene thereby leading toexpression of a truncated transcript lacking the 51015840 exons of thegeneThis alternative TSC2 variant would encode a truncatedprotein lacking the domain required for the interaction withTSC1 which is needed to exert negative regulation of mTORThus androgens may lead to activation of mTOR by relievingthe repressive function of the TSC1TSC2 complex throughAR-dependent induction of a defective variant It is worthyof notice that activation of the mTOR pathway has beenlinked to both tumorigenesis and resistance to therapy in PCa[54] Thus AR might contribute to prostate tumorigenesisalso by causing mTOR activation through expression of thisalternative mRNA variant of TSC2
9 Splicing Regulators Contributing to AlteredGene Expression in Prostate Cancer
In addition to affecting recruitment of AR to alternativepromoters androgens also affected a number of AS events inseveral genes [53] Although the mechanism(s) involved inthese events and their potential relevance to PCa biology wasnot investigated it might involve the ability of AR to interactwith cofactors that modulate the transcriptional elongationrate andor the recognition of splicing enhancers or silencersin the pre-mRNA (Figure 2) For instance AR interacts withthe cofactor of BRCA1 (COBRA1) and this interaction wasshown to influence splicing of the nascent transcripts pro-duced from an androgen-dependent promoter [55] A similarregulation of AR activity was also documented for the DEADboxRNAhelicase p68 (DDX5) in the LNCaP cell line AR andDDX5 interact and are recruited to the promoter region of theandrogen-responsive prostate-specific antigen (PSA) geneThis interactionwas functionally relevant asDDX5 enhancedAR-dependent PSA expression In addition by using an AR-dependent minigene reporter it was shown that DDX5 andAR cooperated in repressing the splicing of variable exons inthe CD44 gene [56] DDX5 is involved in several steps of co-and posttranscriptional RNA processing including splicing[57] and some genes appear to be particularly sensitive to theintracellular levels of DDX5 [57 58] Hence since this RNAhelicase is upregulated in PCa [56] it will be interesting todetermine to what extent it contributes to RNA processing ofAR target genes in PCa cells
AR is also known to interact with several splicing fac-tors suggesting a direct link between androgen-regulatedtranscription initiation and pre-mRNA splicing in PCa cells(Figure 2) The PTB-associated splicing factor (PSF) and itscofactor p54nrb participate to androgen-dependent proteincomplexes containing the AR PSF and p54nrb inhibit thetranscriptional activity of AR by interfering with its bindingto androgen response element and by recruiting a his-tone deacetylase to AR-responsive promoters [59] Althoughdirect investigation of the effect of these splicing factors onAR-dependent splicing events was not addressed it is likely
that AS is also affected by this interaction Another splicingfactor that may participate to AR-dependent splicing regula-tion is SAM68 [60] which is frequently upregulated in PCa[25 45] SAM68 interacts with AR and is recruited to the PSApromoter [60] like DDX5 [55] Interestingly however theinteraction between SAM68 and AR exerted different effectson transcription and splicing as the two proteins cooperatedin transcriptional activation of AR-target genes but opposedeach other in splicing of the CD44 variable exons from areporter minigene [60] Unfortunately the direct effects ofall these RBPs on AR-dependent splicing of endogenoustranscripts have not been addressed yet Nevertheless it islikely that depending on the specific complex formedAR candifferentially influence splicing of its target genes in PCa cells
An additional layer of regulation of the aberrant splicingprogram in PCa might rely on the up-regulation of specificsplicing factors Beside the already mentioned SAM68 [2545] one likely candidate is SRSF1 a splicing factor thatis upregulated in many human cancers and was shown tobehave as an oncogene in mice and humans [61] In cancercells of other tissues SRSF1modulates the expression of splicevariants of the BIN1 and BIM genes that lack proapoptoticfunctions [61 62] Moreover SRSF1 promotes splicing ofMNK2b [61] a splice variant of the eIF4E kinase MNK2 thatwas shown to confer chemoresistance in pancreatic adenocar-cinoma cells [63] Importantly MNK-dependent phospho-rylation of eIF4E strongly contributes to PCa tumorigenesisboth in vitro and in vivo [64 65] and a tight balance betweenthe MNKeIF4E and the mTOR pathways is required tomaintain efficient protein synthesis in PCa cells therebyenhancing their proliferation rate [64] Thus it will be inter-esting to determine whether SRSF1 contributes to fine-tuningthe activation of these pathways in PCa cells through theregulation ofMNK2 AS
Other splicing factors may also contribute to the alteredsplicing programof PCa cells Indeed the activity of several ofthese RBPs ismodulated by signal transduction pathways thatare frequently turned on in cancer such as the PI3KAKT andthe RASERK pathways (see also [66]) For instance it wasshown that activation of AKT downstream of the epidermalgrowth factor (EGF) receptor modulated the activity ofthe SR protein-specific kinases and phosphorylation of SRproteins thereby affecting a large spectrum of AS events[67] Similarly the RASERK pathway modulates a numberof splicing factors involved in cancer such as SAM68 [68]and the alternative splicing factor 45 (SPF45) [69] whichin turn affect expression of splice variants that regulate cellmotility proliferation and survival Thus it is likely that theexamples reported above represent only a small picture of theoverall contribution of AS and splicing factors to the wideheterogeneity in gene expression observed in PCa cells andpatients
10 Conclusions and Perspectives
AS is widely recognized as a powerful tool that eukaryoticcells employ to expand the coding potential and the plasticityof their genomes The flexibility in the recognition of exons
International Journal of Cell Biology 7
DDX5
SAM68COBRA1
AR
AR splicing-responsive mRNAs
RNAPIIAR transcriptional-responsive gene
AR coactivators
PSF p54nrb
AR corepressor
ARRNAPII
AR transcriptional-responsive gene
Figure 2 Regulation of cotranscriptional splicing by proteins interacting with the androgen receptor Coregulators of the androgen receptor(AR) can affect splicing of target genes by direct interaction with AR and modulation of its activity COBRA1 SAM68 and DDX5 appear topromote the transcriptional activity of AR but differentially act on splicing of variable exons (red box in the left side of the figure) PSF andits interacting protein p54 (right side of the figure) repress the transcriptional activity of AR but their effect on splicing is currently unknown(see text for more details)
and introns within the transcription unit of the majority ofhuman genes offers the possibility to compose many mRNAvariants from each gene Subtle changes in the cellular envi-ronment or in external cues conveyed from the surroundingenvironment may result in global changes in the tran-scriptome which in part rely on the regulation of AS Aninteresting observation is that apparently homogenous cellpopulations actually display large differences in gene expres-sion This was recently exemplified by studies that appliedglobal RNA sequencing techniques to the analysis of singlecell transcriptomes After treatment of bone-marrow-deriveddendritic cells (BMDCs) with an inflammatory cue it wasfound that hundreds of key immune genes were differentiallyexpressed by single cells The heterogeneity in the responsewas particularly remarkable with regard to the splicing pat-terns expressed by these cells [70] suggesting that fine-tuningof AS regulation strongly contributes to the heterogeneity ofa cell population This aspect might be particularly relevantin the context of PCa which is a neoplastic disease charac-terized by extreme heterogeneity and unpredicted responseof patients to the therapy [1 2] The improvements in cellisolation techniques coupled to the higher sensitivity of thenext-generation sequencing techniques may soon allow ahighly detailed description of the transcriptome of patientswhich might result in more personalized treatments
The studies illustrated previously suggest that the upregu-lation of selected splicing regulators in PCa such as SAM68SRSF1 or DDX5 directly contributes to the phenotype byaltering the splicing profile of key genes Thus these RBPsmight represent potential therapeutic targets for interventionAlthough blocking the activity of a given splicing factor isnot necessarily an easy task some examples in this direction
have been provided For instance SAM68 can bind to RNAonly as a dimer By exploiting this requirement it was shownthat an RNA binding-defective SAM68 mutant exerted dom-inant negative effects on SAM68-mediated SMN2 splicing byassociating with the endogenous protein and preventing itsbinding to the pre-mRNA [71]This experiment suggests thatsmall molecules interfering with SAM68 function might dis-play therapeutic potential As homodimerization is a prereq-uisite for RNA binding one possibility is to target the SAM68dimerization domain which was restricted to a small regionwithin its Gld1-Sam68-Grp33 (GSG) homology domain [72]The potential value of targeting specific components of thesplicing machinery in cancer cells is also suggested by theantioncogenic properties of natural compounds such asspliceostatin A (SSA) in a variety of cancer cell modelsSSA targets the splicing factor 3B subunit 1 (SF3B1) of thespliceosome thus affecting a large number of splicing eventsconcomitantly [73] Perhaps more specific drugs targetingsplicing factors involved in subsets of oncogenic splicingevents in cancer cells as those described above might repre-sent more specific therapeutic approaches in the next future
Although the extreme flexibility of AS regulation is proneto errors that may concur to neoplastic transformation [7 8]it can also be exploited therapeutically Indeed examples ofAS modulation in selected genes by administering splicing-correcting ASOs to cells have been reported In some casesthis approach has also been challenged with a therapeuticapplication One of the most remarkable examples is repre-sented by the recovery of the phenotype observed in mousemodels of Spinal Muscular Atrophy (SMA) This neurode-generative disease is caused by inactivation of the SMN1 geneand skipping of exon 7 in the highly homologous SMN2 gene
8 International Journal of Cell Biology
[74] It was recently demonstrated that systemic injectionof a chemically modified ASO restored SMN2 splicing invitro and in vivo and profoundly ameliorated the viabilityand phenotypic features of mice affected by a severe form ofSMA [75] Although cancer is caused by multiple alterationsthus limiting the application of gene-specific ASOs it isconceivable that these tools could be used in combinationwith standard therapies to improve the clinical response ofpatients For instance an ASO that switched BCL-X splicingtoward the proapoptotic variant was effective in sensitizingcancer cells to drug-induced apoptosis and to reduce growthof tumors in nude mice [30 31] A similar effect was obtainedby switching expression of the 120572 to the 120573 variant of the signaltransducer and activator of transcription 3 (STAT3) genewhich modulates multiple oncogenic pathways [76] In thiscase administration of a modified ASO targeted to a splicingenhancer induced expression of the endogenous STAT3120573 andan anti-oncogenic response in vitro and in vivo [76] Thesestudies suggest that modulation of AS with synthetic drugsis possible and has entered a therapeutic perspective ASOsare particularly appealing in terms of high specificity andreduced side effects as theymay exploit their ability to annealwith specific sequences in the genomewithout affecting otherfeatures or target genes of the splicing factors involved in theoncogenicAS eventThus it is likely that thesemethods couldbe applied soon to the development of novel therapies aimedat fighting human cancers in which expression of specificoncogenic splice variants has been firmly confirmed
Conflict of Interests
The author declares no conflict of interests
Acknowledgments
The author wishes to thank Dr Chiara Naro for help with thepreparation of the figures and critical reading of the paperThe work in the laboratory of C Sette was supported byAssociation for International Cancer Research (AICR Grantno 12-0150) the Associazione Italiana Ricerca sul Cancro(AIRC) and Istituto Superiore della Sanita (ISS Grant Con-venzione 11US6A)
References
[1] B J Feldman and D Feldman ldquoThe development of androgen-independent prostate cancerrdquoNature Reviews Cancer vol 1 no1 pp 34ndash45 2001
[2] M Kohli and D J Tindall ldquoNew developments in the medicalmanagement of prostate cancerrdquo Mayo Clinic Proceedings vol85 no 1 pp 77ndash86 2010
[3] K E Knudsen and H I Scher ldquoStarving the addiction newopportunities for durable suppression of AR signaling in pros-tate cancerrdquo Clinical Cancer Research vol 15 no 15 pp 4792ndash4798 2009
[4] S M Dehm and D J Tindall ldquoAlternatively spliced androgenreceptor variantsrdquo Endocrine-Related Cancer vol 18 no 5 ppR183ndashR196 2011
[5] A Kalsotra and T A Cooper ldquoFunctional consequences ofdevelopmentally regulated alternative splicingrdquoNature ReviewsGenetics vol 12 no 10 pp 715ndash729 2011
[6] C Zhang H-R Li J-B Fan et al ldquoProfiling alternativelysplicedmRNA isoforms for prostate cancer classificationrdquo BMCBioinformatics vol 7 article 202 2006
[7] C J David and J L Manley ldquoAlternative pre-mRNA splicingregulation in cancer pathways and programs unhingedrdquo Genesand Development vol 24 no 21 pp 2343ndash2364 2010
[8] S Pal R Gupta and R V Davuluri ldquoAlternative transcriptionand alternative splicing in cancerrdquo Pharmacology amp Therapeu-tics vol 136 no 3 pp 283ndash294 2012
[9] Y Li M Alsagabi D Fan G S Bova A H Tewfik and S MDehm ldquoIntragenic rearrangement and altered RNA splicing ofthe androgen receptor in a cell-based model of prostate cancerprogressionrdquo Cancer Research vol 71 no 6 pp 2108ndash2117 2011
[10] S M Dehm L J Schmidt H V Heemers R L Vessella and DJ Tindall ldquoSplicing of a novel androgen receptor exon generatesa constitutively active androgen receptor that mediates prostatecancer therapy resistancerdquo Cancer Research vol 68 no 13 pp5469ndash5477 2008
[11] R Hu T A Dunn SWei et al ldquoLigand-independent androgenreceptor variants derived from splicing of cryptic exons signifyhormone-refractory prostate cancerrdquo Cancer Research vol 69no 1 pp 16ndash22 2009
[12] Z Guo X Yang F Sun et al ldquoA novel androgen receptor splicevariant is up-regulated during prostate cancer progressionand promotes androgen depletion-resistant growthrdquo CancerResearch vol 69 no 6 pp 2305ndash2313 2009
[13] Y Li S C Chan L J Brand T H Hwang K A Silverstein andS M Dehm ldquoAndrogen receptor splice variants mediate enza-lutamide resistance in castration-resistant prostate cancer celllinesrdquo Cancer Research vol 73 no 2 pp 483ndash489 2013
[14] G Narla K E Heath H L Reeves et al ldquoKLF6 a candidatetumor suppressor genemutated in prostate cancerrdquo Science vol294 no 5551 pp 2563ndash2566 2001
[15] X Liu A Gomez-Pinillos C Loder et al ldquoKLF6 loss of func-tion in human prostate cancer progression is implicated inresistance to androgen deprivationrdquo The American Journal ofPathology vol 181 no 3 pp 1007ndash1016 2012
[16] G Narla A DiFeo H L Reeves et al ldquoA germline DNApolymorphism enhances alternative splicing of the KLF6 tumorsuppressor gene and is associatedwith increased prostate cancerriskrdquo Cancer Research vol 65 no 4 pp 1213ndash1222 2005
[17] G Narla A DiFeo Y Fernandez et al ldquoKLF6-SV1 overexpres-sion accelerates human and mouse prostate cancer progressionand metastasisrdquo Journal of Clinical Investigation vol 118 no 8pp 2711ndash2721 2008
[18] G Narla A Difeo S Yao et al ldquoTargeted inhibition of theKLF6 splice variant KLF6 SV1 suppresses prostate cancer cellgrowth and spreadrdquo Cancer Research vol 65 no 13 pp 5761ndash5768 2005
[19] K E Knudsen ldquoThe cyclin D1b splice variant an old oncogenelearns new tricksrdquo Cell Division vol 1 article 15 2006
[20] K E Knudsen J A Diehl C A Haiman and E S KnudsenldquoCyclin D1 polymorphism aberrant splicing and cancer riskrdquoOncogene vol 25 no 11 pp 1620ndash1628 2006
[21] F Lu A B Gladden and J A Diehl ldquoAn alternatively splicedcyclin D1 isoform cyclin D1b is a nuclear oncogenerdquo CancerResearch vol 63 no 21 pp 7056ndash7061 2003
International Journal of Cell Biology 9
[22] C E S Comstock M A Augello R Pe Benito et al ldquoCyclinD1 splice variants polymorphism risk and isoform-specificregulation in prostate cancerrdquo Clinical Cancer Research vol 15no 17 pp 5338ndash5349 2009
[23] M A Augello C J Burd R Birbe et al ldquoConvergence of onco-genic and hormone receptor pathways promotes metastaticphenotypesrdquo The Journal of Clinical Investigation vol 123 no1 pp 493ndash508 2013
[24] N A Olshavsky C E S Comstock M J Schiewer et al ldquoIden-tification of ASFSF2 as a critical allele-specific effector of thecyclin D1b oncogenerdquoCancer Research vol 70 no 10 pp 3975ndash3984 2010
[25] M P Paronetto M Cappellari R Busa et al ldquoAlternative splic-ing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68rdquoCancer Research vol 70 no 1 pp 229ndash239 2010
[26] D Kaida M G Berg I Younis et al ldquoU1 snRNP protects pre-mRNAs frompremature cleavage and polyadenylationrdquoNaturevol 468 no 7324 pp 664ndash668 2010
[27] P Bielli R Busa M P Paronetto and C Sette ldquoThe RNA-binding protein Sam68 is a multifunctional player in humancancerrdquo Endocrine-Related Cancer vol 18 no 4 pp R91ndashR1022011
[28] L H Boise M Gonzalez-Garcia C E Postema et al ldquobcl-xA bcl-2-related gene that functions as a dominant regulator ofapoptotic cell deathrdquo Cell vol 74 no 4 pp 597ndash608 1993
[29] D R Mercatante C D Bortner J A Cidlowski and R KoleldquoModification of alternative splicing of Bcl-x Pre-mRNA inprostate and breast cancer cells analysis of apoptosis and celldeathrdquo Journal of Biological Chemistry vol 276 no 19 pp 16411ndash16417 2001
[30] D R Mercatante J L Mohler and R Kole ldquoCellular responseto an antisense-mediated shift of Bcl-x pre-mRNA splicing andantineoplastic agentsrdquo Journal of Biological Chemistry vol 277no 51 pp 49374ndash49382 2002
[31] J A Bauman S-D Li A Yang L Huang and R Kole ldquoAnti-tumor activity of splice-switching oligonucleotidesrdquo NucleicAcids Research vol 38 no 22 pp 8348ndash8356 2010
[32] C E Chalfant K Rathman R L Pinkerman et al ldquoDe novoceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells Dependence on proteinphosphatase-1rdquo Journal of Biological Chemistry vol 277 no 15pp 12587ndash12595 2002
[33] K Boon-Unge Q Yu T Zou A Zhou P Govitrapong andJ Zhou ldquoEmetine regulates the alternative splicing of Bcl-xthrough a protein phosphatase 1-dependent mechanismrdquoChemistry and Biology vol 14 no 12 pp 1386ndash1392 2007
[34] M H Kim ldquoProtein phosphatase 1 activation and alternativesplicing of Bcl-X and Mcl-1 by EGCG + ibuprofenrdquo Journal ofCellular Biochemistry vol 104 no 4 pp 1491ndash1499 2008
[35] T Novoyatleva B Heinrich Y Tang et al ldquoProtein phosphatase1 binds to the RNA recognition motif of several splicing fac-tors and regulates alternative pre-mRNA processingrdquo HumanMolecular Genetics vol 17 no 1 pp 52ndash70 2008
[36] N Ghosh N Patel K Jiang et al ldquoCeramide-activated pro-tein phosphatase involvement in insulin resistance via Aktserinearginine-rich protein 40 and ribonucleic acid splicing inL6 skeletal muscle cellsrdquo Endocrinology vol 148 no 3 pp 1359ndash1366 2007
[37] D Garneau T Revil J-F Fisette and B Chabot ldquoHetero-geneous nuclear ribonucleoprotein FH proteins modulate
the alternative splicing of the apoptotic mediator Bcl-xrdquo Journalof Biological Chemistry vol 280 no 24 pp 22641ndash22650 2005
[38] M P Paronetto T Achsel A Massiello C E Chalfant andC Sette ldquoThe RNA-binding protein Sam68 modulates thealternative splicing of Bcl-xrdquo Journal of Cell Biology vol 176 no7 pp 929ndash939 2007
[39] A Zhou A C Ou A Cho E J Benz Jr and S-C HuangldquoNovel splicing factor RBM25 modulates Bcl-x Pre-mRNA 51015840splice site selectionrdquoMolecular and Cellular Biology vol 28 no19 pp 5924ndash5936 2008
[40] S Pedrotti R Busa C Compagnucci and C Sette ldquoThe RNArecognition motif protein RBM11 is a novel tissue-specificsplicing regulatorrdquo Nucleic Acids Research vol 40 no 3 pp1021ndash1032 2012
[41] T Revil J Pelletier J Toutant A Cloutier and B ChabotldquoHeterogeneous nuclear ribonucleoprotein K represses theproduction of pro-apoptotic Bcl-xS splice isoformrdquo Journal ofBiological Chemistry vol 284 no 32 pp 21458ndash21467 2009
[42] M J Moore Q Wang C J Kennedy and P A Silver ldquoAn alter-native splicing network links cell-cycle control to apoptosisrdquoCell vol 142 no 4 pp 625ndash636 2010
[43] P Cloutier J Toutant L Shkreta S Goekjian T Revil and BChabot ldquoAntagonistic effects of the SRp30c protein and cryptic51015840 splice sites on the alternative splicing of the apoptoticregulator Bcl-xrdquo Journal of Biological Chemistry vol 283 no 31pp 21315ndash21324 2008
[44] A Massiello J R Roesser and C E Chalfant ldquoSAP155 Bindsto ceramide-responsive RNA cis-element 1 and regulates thealternative 51015840 splice site selection of Bcl-x pre-mRNArdquo TheFASEB Journal vol 20 no 10 pp 1680ndash1682 2006
[45] R BusaM P Paronetto D Farini et al ldquoTheRNA-binding pro-tein Sam68 contributes to proliferation and survival of humanprostate cancer cellsrdquo Oncogene vol 26 no 30 pp 4372ndash43822007
[46] C Sette ldquoPost-translational regulation of star proteins andeffects on their biological functionsrdquo Advances in ExperimentalMedicine and Biology vol 693 pp 54ndash66 2010
[47] C Brignatz M P Paronetto S Opi et al ldquoAlternative splicingmodulates autoinhibition and SH3 accessibility in the Src kinaseFynrdquo Molecular and Cellular Biology vol 29 no 24 pp 6438ndash6448 2009
[48] M P Paronetto D Farini I Sammarco et al ldquoExpression of atruncated form of the c-Kit tyrosine kinase receptor and activa-tion of Src kinase in human prostatic cancerrdquo American Journalof Pathology vol 164 no 4 pp 1243ndash1251 2004
[49] Y Hu A Dobi T Sreenath et al ldquoDelineation of TMPRSS2-ERG splice variants in prostate cancerrdquo Clinical CancerResearch vol 14 no 15 pp 4719ndash4725 2008
[50] J Wang Y Cai W Yu C Ren D M Spencer and M Itt-mann ldquoPleiotropic biological activities of alternatively splicedTMPRSS2ERG fusion gene transcriptsrdquo Cancer Research vol68 no 20 pp 8516ndash8524 2008
[51] F Zammarchi G Boutsalis and L Cartegni ldquo51015840 UTR controlof native ERG and of Tmprss2 ERG variants activity in prostatecancerrdquo PLoS One vol 8 no 3 Article ID e49721 2013
[52] M J Munoz M S P Santangelo M P Paronetto et al ldquoDNAdamage regulates alternative splicing through inhibition ofRNApolymerase II elongationrdquoCell vol 137 no 4 pp 708ndash7202009
[53] P Rajan C Dalgliesh P J Carling et al ldquoIdentification of novelandrogen-regulated pathways and mrna isoforms through
10 International Journal of Cell Biology
genome-wide exon-specific profiling of the LNCaP transcrip-tomerdquo PLoS One vol 6 no 12 Article ID e29088 2011
[54] T M Morgan T D Koreckij and E Corey ldquoTargeted therapyfor advanced prostate cancer inhibition of the PI3KAktmTORpathwayrdquoCurrent CancerDrug Targets vol 9 no 2 pp 237ndash2492009
[55] J Sun A L Blair S E Aiyar and R Li ldquoCofactor of BRCA1modulates androgen-dependent transcription and alternativesplicingrdquo Journal of Steroid Biochemistry andMolecular Biologyvol 107 no 3ndash5 pp 131ndash139 2007
[56] E L Clark A Coulson C Dalgliesh et al ldquoThe RNA helicasep68 is a novel androgen receptor coactivator involved in splicingand is overexpressed in prostate cancerrdquo Cancer Research vol68 no 19 pp 7938ndash7946 2008
[57] E Zonta D Bittencourt S Samaan S Germann M Dutertreand D Auboeuf ldquoThe RNA helicase DDX5p68 is a key factorpromoting c-fos expression at different levels from transcriptionto mRNA exportrdquoNucleic Acids Research vol 41 no 1 pp 554ndash564 2013
[58] A Honig D Auboeuf M M Parker B W OrsquoMalley andS M Berget ldquoRegulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72rdquo Molecular and Cel-lular Biology vol 22 no 16 pp 5698ndash5707 2002
[59] X Dong J Sweet J R G Challis T Brown and S J Lye ldquoTran-scriptional activity of androgen receptor is modulated by twoRNA splicing factors PSF and p54nrbrdquoMolecular and CellularBiology vol 27 no 13 pp 4863ndash4875 2007
[60] P Rajan L Gaughan C Dalgliesh et al ldquoThe RNA-binding andadaptor protein Sam68 modulates signal-dependent splicingand transcriptional activity of the androgen receptorrdquo Journalof Pathology vol 215 no 1 pp 67ndash77 2008
[61] R Karni E De Stanchina S W Lowe R Sinha D Mu and AR Krainer ldquoThe gene encoding the splicing factor SF2ASF is aproto-oncogenerdquo Nature Structural and Molecular Biology vol14 no 3 pp 185ndash193 2007
[62] OAnczukowA Z RosenbergMAkerman et al ldquoThe splicingfactor SRSF1 regulates apoptosis and proliferation to promotemammary epithelial cell transformationrdquoNature Structural andMolecular Biology vol 19 no 2 pp 220ndash228 2012
[63] L Adesso S Calabretta F Barbagallo et al ldquoGemcitabine trig-gers a pro-survival response in pancreatic cancer cells throughactivation of the MNK2eIF4E pathwayrdquo Oncogene vol 32 pp2848ndash2857 2013
[64] A Bianchini M Loiarro P Bielli et al ldquoPhosphorylation ofeIF4E by MNKs supports protein synthesis cell cycle progres-sion and proliferation in prostate cancer cellsrdquo Carcinogenesisvol 29 no 12 pp 2279ndash2288 2008
[65] L Furic L RongO Larsson et al ldquoEIF4Ephosphorylation pro-motes tumorigenesis and is associated with prostate cancer pro-gressionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 32 pp 14134ndash14139 2010
[66] C Naro and C Sette ldquoPhosphorylation-mediated regulation ofalternative splicing in cancerrdquo International Journal of Cell Bio-logy In press
[67] Z Zhou J QiuW Liu et al ldquoTheAkt-SRPK-SR axis constitutesa major pathway in transducing EGF signaling to regulatealternative splicing in the nucleusrdquo Molecular Cell vol 47 no3 pp 422ndash433 2012
[68] NMatter P Herrlich andH Konig ldquoSignal-dependent regula-tion of splicing via phosphorylation of Sam68rdquoNature vol 420no 6916 pp 691ndash695 2002
[69] A M Al-Ayoubi H Zheng Y Liu T Bai and S T EblenldquoMitogen-activated protein kinase phosphorylation of splicingfactor 45 (SPF45) regulates SPF45 alternative splicing siteutilization proliferation and cell adhesionrdquoMolecular and Cel-lular Biology vol 32 no 14 pp 2880ndash2893 2012
[70] A K Shalek R Satija X Adiconis et al ldquoSingle-cell transcrip-tomics reveals bimodality in expression and splicing in immunecellsrdquo Nature 2013
[71] S Pedrotti P BielliM P Paronetto et al ldquoThe splicing regulatorSam68 binds to a novel exonic splicing silencer and functions inSMN2 alternative splicing in spinal muscular atrophyrdquo EMBOJournal vol 29 no 7 pp 1235ndash1247 2010
[72] N H Meyer K Tripsianes M Vincendeau et al ldquoStructuralbasis for homodimerization of the Src-associated during mito-sis 68-kDa protein (Sam68)Qua1 domainrdquo Journal of BiologicalChemistry vol 285 no 37 pp 28893ndash28901 2010
[73] S Bonnal L Vigevani and J Valcarcel ldquoThe spliceosome asa target of novel antitumour drugsrdquo Nature Reviews Drug Dis-covery vol 11 no 11 pp 847ndash859 2012
[74] S Pedrotti and C Sette ldquoSpinal muscular atrophy a new playerjoins the battle for SMN2 exon 7 splicingrdquo Cell Cycle vol 9 no19 pp 3874ndash3879 2010
[75] Y Hua K Sahashi F Rigo et al ldquoPeripheral SMN restoration isessential for long-term rescue of a severe spinal muscular atro-phy mouse modelrdquo Nature vol 478 no 7367 pp 123ndash126 2011
[76] F Zammarchi E De Stanchina E Bournazou et al ldquoAntitu-morigenic potential of STAT3 alternative splicing modulationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 43 pp 17779ndash17784 2011
opposite directions [38] while the up-regulation of SRSF1 isin line with the high levels of BCL-XL in PCa cells SAM68should favour the proapoptotic short variant However thesplicing activity of SAM68 is finely tuned by phosphoryla-tion [46] and it was shown that tyrosine phosphorylationby the Src-related kinase FYN switched SAM68-dependentsplicing of BCL-X toward the anti-apoptotic variant [38 47]Since tyrosine phosphorylation of SAM68 is increased inspecimens of PCa patients [48] it is likely that this RBPcan also contribute to the upregulation of BCL-XL in PCacells In line with this hypothesis BCL-XL expression wasdecreased and sensitivity to genotoxic agents was increasedafter knockdown of SAM68 in the androgen-sensitive LNCaPcell line [45]
Thus based on the observations reported previouslyit is predictable that exogenous modulation of BCL-X ASthrough administration of ASOs or by interfering with theactivity of the splicing factors that promote the anti-apoptoticBCL-XL variant will enhance the efficacy of chemotherapy inadvanced PCa as suggested by preclinical studies in PCa celllines [30 31 45]
7 TMPRSS2ERG
ERG is a member of the ETS transcription factor family thatis expressed at very low levels in benign prostate epithelialcells However PCa patients often carry a fusion of theandrogen-responsiveTMPRSS2 genewith ERG which causesaberrantly high expression levels of the transcription factorin the neoplastic cells A detailed sequencing analysis of theTMPRSS2ERG transcripts isolated fromPCa tissues revealedthat fusion-derived transcripts underwent profound AS reg-ulation which yielded mRNA variants encoding both fulllength ERG proteins and isoforms lacking the ETS domainNotably an increase in the abundance of transcripts encodingfull length ERG correlated with less favorable outcome inpatients [49] These results support a possible functional rolefor this transcription factor in PCa pathology and suggest thatmodulation of AS events promoting less pathogenic variantsmay produce beneficial effects
This hypothesis is also supported by another study thattested the effects exerted by the expression of TMPRSS2ERGalternatively spliced transcripts in an immortalized prostatecell line [50] It was found that these TMPRSS2ERG splicevariants had different oncogenic activities in terms of pro-moting proliferation invasion and motility Notably coex-pression of different variants produced stronger effects thaneither variant alone suggesting that the presence of severalTMPRSS2ERG isoforms as it normally occurs in PCa cellsmight confer a more malignant phenotype [50] A furthercontribution of AS to the heterogeneity of TMPRSS2ERGexpression is provided by the extensive variability of the 51015840untranslated region (UTR) in the splice variants observedin patients [51] Indeed AS of the 51015840 UTR affects the onco-genic potential of the encoded proteins by regulating theirtranslation and activity Thus although a functional linkbetween TMPRSS2ERG expression and PCa pathology hasnot been firmly established yet this fusion gene appears
to be another suitable target for an AS-directed therapeuticapproach that would spare normal cells not expressing thechimeric proteins
8 Splicing Programs in Prostate Cancer
Cancer cells express a number of splice variants that conferthem higher resistance to chemotherapeutic drugs and sur-vival advantages When it was investigated in detail the spe-cific signature of splice variants expressed by cancer cells hasbeen recognized as a powerful diagnostic and prognostic tool[7 8] Evenmore importantly the existence of cancer-specificsplicing variants of key genes such as the AR or CCND1in PCa might offer a therapeutic opportunity for targetingproteins that are not expressed in healthy cells For instancedeveloping tools that specifically modulate the expressionof transcript variants preferentially or uniquely produced bycancer cells might slow down tumor growth andor promotecell death during therapy while sparing the healthy tissuesThus understanding AS regulation at the genome-wide levelin PCa cells may not only lead to the identification of noveldiagnostic or prognostic biomarkers but it could also helpfind tools for novel therapeutic approaches to this neoplasticdisease
A few studies have directly investigated the genome-wideregulation of AS in PCa cell lines and primary tumor tissuesUsing a splicing-sensitive microarray comprising a selectedsubset of genes and splice variants it was shown that splicingsignatures could efficiently segregate PCa cells lines from can-cer cell lines derived from other organs or tissues [6] Amongthe alternatively spliced genes the majority also showedvariation in expression levels [6] suggesting that regulationof splicing and transcriptionwere coupled as also observed incells exposed to DNA damage [52] Using the same splicing-sensitive platform it was also possible to identify splicingsignatures that were specific for normal or neoplastic prostatetissues obtained from biopsies [6] Although this approachwas limited to the genes and the splice variants selected for theplatform it provided a first indication that specific changes insplicing occur during prostate tumorigenesis and suggestedthat splicing variants can represent accurate biomarkers forPCaNevertheless how andwhen these changes occur aswellas to what extent they contribute to the acquisition of thetransformed phenotype are still open questions Given thetight association between transcription and splicing a spe-cific splicing program could result from the different activityof transcription factors splicing factors or both Mountingevidence indicates that all these events contribute to someextent to the acquisition of specific splicing signatures in PCa
The most relevant transcription factor involved in PCais the AR Several observations suggest that in addition toregulating the expression levels of target genes AR can alsoinfluence the transcript variants encoded by them Usingcomprehensive splicing-sensitive arrays it was demonstratedthat stimulation of LNCaP cells with androgens caused qual-itative changes in expression of splice variants [53] Many ofthe events altered by treatment with androgens were due tousage of alternative promoters within the transcription unit
6 International Journal of Cell Biology
of the target gene Some of these alternative transcripts werepredicted to influence the function of proteins with relevanceto PCa such as the mTOR regulator TSC2 Following andro-genic stimulation AR was recruited to a cryptic promoterupstream of exon 33 in the TSC2 gene thereby leading toexpression of a truncated transcript lacking the 51015840 exons of thegeneThis alternative TSC2 variant would encode a truncatedprotein lacking the domain required for the interaction withTSC1 which is needed to exert negative regulation of mTORThus androgens may lead to activation of mTOR by relievingthe repressive function of the TSC1TSC2 complex throughAR-dependent induction of a defective variant It is worthyof notice that activation of the mTOR pathway has beenlinked to both tumorigenesis and resistance to therapy in PCa[54] Thus AR might contribute to prostate tumorigenesisalso by causing mTOR activation through expression of thisalternative mRNA variant of TSC2
9 Splicing Regulators Contributing to AlteredGene Expression in Prostate Cancer
In addition to affecting recruitment of AR to alternativepromoters androgens also affected a number of AS events inseveral genes [53] Although the mechanism(s) involved inthese events and their potential relevance to PCa biology wasnot investigated it might involve the ability of AR to interactwith cofactors that modulate the transcriptional elongationrate andor the recognition of splicing enhancers or silencersin the pre-mRNA (Figure 2) For instance AR interacts withthe cofactor of BRCA1 (COBRA1) and this interaction wasshown to influence splicing of the nascent transcripts pro-duced from an androgen-dependent promoter [55] A similarregulation of AR activity was also documented for the DEADboxRNAhelicase p68 (DDX5) in the LNCaP cell line AR andDDX5 interact and are recruited to the promoter region of theandrogen-responsive prostate-specific antigen (PSA) geneThis interactionwas functionally relevant asDDX5 enhancedAR-dependent PSA expression In addition by using an AR-dependent minigene reporter it was shown that DDX5 andAR cooperated in repressing the splicing of variable exons inthe CD44 gene [56] DDX5 is involved in several steps of co-and posttranscriptional RNA processing including splicing[57] and some genes appear to be particularly sensitive to theintracellular levels of DDX5 [57 58] Hence since this RNAhelicase is upregulated in PCa [56] it will be interesting todetermine to what extent it contributes to RNA processing ofAR target genes in PCa cells
AR is also known to interact with several splicing fac-tors suggesting a direct link between androgen-regulatedtranscription initiation and pre-mRNA splicing in PCa cells(Figure 2) The PTB-associated splicing factor (PSF) and itscofactor p54nrb participate to androgen-dependent proteincomplexes containing the AR PSF and p54nrb inhibit thetranscriptional activity of AR by interfering with its bindingto androgen response element and by recruiting a his-tone deacetylase to AR-responsive promoters [59] Althoughdirect investigation of the effect of these splicing factors onAR-dependent splicing events was not addressed it is likely
that AS is also affected by this interaction Another splicingfactor that may participate to AR-dependent splicing regula-tion is SAM68 [60] which is frequently upregulated in PCa[25 45] SAM68 interacts with AR and is recruited to the PSApromoter [60] like DDX5 [55] Interestingly however theinteraction between SAM68 and AR exerted different effectson transcription and splicing as the two proteins cooperatedin transcriptional activation of AR-target genes but opposedeach other in splicing of the CD44 variable exons from areporter minigene [60] Unfortunately the direct effects ofall these RBPs on AR-dependent splicing of endogenoustranscripts have not been addressed yet Nevertheless it islikely that depending on the specific complex formedAR candifferentially influence splicing of its target genes in PCa cells
An additional layer of regulation of the aberrant splicingprogram in PCa might rely on the up-regulation of specificsplicing factors Beside the already mentioned SAM68 [2545] one likely candidate is SRSF1 a splicing factor thatis upregulated in many human cancers and was shown tobehave as an oncogene in mice and humans [61] In cancercells of other tissues SRSF1modulates the expression of splicevariants of the BIN1 and BIM genes that lack proapoptoticfunctions [61 62] Moreover SRSF1 promotes splicing ofMNK2b [61] a splice variant of the eIF4E kinase MNK2 thatwas shown to confer chemoresistance in pancreatic adenocar-cinoma cells [63] Importantly MNK-dependent phospho-rylation of eIF4E strongly contributes to PCa tumorigenesisboth in vitro and in vivo [64 65] and a tight balance betweenthe MNKeIF4E and the mTOR pathways is required tomaintain efficient protein synthesis in PCa cells therebyenhancing their proliferation rate [64] Thus it will be inter-esting to determine whether SRSF1 contributes to fine-tuningthe activation of these pathways in PCa cells through theregulation ofMNK2 AS
Other splicing factors may also contribute to the alteredsplicing programof PCa cells Indeed the activity of several ofthese RBPs ismodulated by signal transduction pathways thatare frequently turned on in cancer such as the PI3KAKT andthe RASERK pathways (see also [66]) For instance it wasshown that activation of AKT downstream of the epidermalgrowth factor (EGF) receptor modulated the activity ofthe SR protein-specific kinases and phosphorylation of SRproteins thereby affecting a large spectrum of AS events[67] Similarly the RASERK pathway modulates a numberof splicing factors involved in cancer such as SAM68 [68]and the alternative splicing factor 45 (SPF45) [69] whichin turn affect expression of splice variants that regulate cellmotility proliferation and survival Thus it is likely that theexamples reported above represent only a small picture of theoverall contribution of AS and splicing factors to the wideheterogeneity in gene expression observed in PCa cells andpatients
10 Conclusions and Perspectives
AS is widely recognized as a powerful tool that eukaryoticcells employ to expand the coding potential and the plasticityof their genomes The flexibility in the recognition of exons
International Journal of Cell Biology 7
DDX5
SAM68COBRA1
AR
AR splicing-responsive mRNAs
RNAPIIAR transcriptional-responsive gene
AR coactivators
PSF p54nrb
AR corepressor
ARRNAPII
AR transcriptional-responsive gene
Figure 2 Regulation of cotranscriptional splicing by proteins interacting with the androgen receptor Coregulators of the androgen receptor(AR) can affect splicing of target genes by direct interaction with AR and modulation of its activity COBRA1 SAM68 and DDX5 appear topromote the transcriptional activity of AR but differentially act on splicing of variable exons (red box in the left side of the figure) PSF andits interacting protein p54 (right side of the figure) repress the transcriptional activity of AR but their effect on splicing is currently unknown(see text for more details)
and introns within the transcription unit of the majority ofhuman genes offers the possibility to compose many mRNAvariants from each gene Subtle changes in the cellular envi-ronment or in external cues conveyed from the surroundingenvironment may result in global changes in the tran-scriptome which in part rely on the regulation of AS Aninteresting observation is that apparently homogenous cellpopulations actually display large differences in gene expres-sion This was recently exemplified by studies that appliedglobal RNA sequencing techniques to the analysis of singlecell transcriptomes After treatment of bone-marrow-deriveddendritic cells (BMDCs) with an inflammatory cue it wasfound that hundreds of key immune genes were differentiallyexpressed by single cells The heterogeneity in the responsewas particularly remarkable with regard to the splicing pat-terns expressed by these cells [70] suggesting that fine-tuningof AS regulation strongly contributes to the heterogeneity ofa cell population This aspect might be particularly relevantin the context of PCa which is a neoplastic disease charac-terized by extreme heterogeneity and unpredicted responseof patients to the therapy [1 2] The improvements in cellisolation techniques coupled to the higher sensitivity of thenext-generation sequencing techniques may soon allow ahighly detailed description of the transcriptome of patientswhich might result in more personalized treatments
The studies illustrated previously suggest that the upregu-lation of selected splicing regulators in PCa such as SAM68SRSF1 or DDX5 directly contributes to the phenotype byaltering the splicing profile of key genes Thus these RBPsmight represent potential therapeutic targets for interventionAlthough blocking the activity of a given splicing factor isnot necessarily an easy task some examples in this direction
have been provided For instance SAM68 can bind to RNAonly as a dimer By exploiting this requirement it was shownthat an RNA binding-defective SAM68 mutant exerted dom-inant negative effects on SAM68-mediated SMN2 splicing byassociating with the endogenous protein and preventing itsbinding to the pre-mRNA [71]This experiment suggests thatsmall molecules interfering with SAM68 function might dis-play therapeutic potential As homodimerization is a prereq-uisite for RNA binding one possibility is to target the SAM68dimerization domain which was restricted to a small regionwithin its Gld1-Sam68-Grp33 (GSG) homology domain [72]The potential value of targeting specific components of thesplicing machinery in cancer cells is also suggested by theantioncogenic properties of natural compounds such asspliceostatin A (SSA) in a variety of cancer cell modelsSSA targets the splicing factor 3B subunit 1 (SF3B1) of thespliceosome thus affecting a large number of splicing eventsconcomitantly [73] Perhaps more specific drugs targetingsplicing factors involved in subsets of oncogenic splicingevents in cancer cells as those described above might repre-sent more specific therapeutic approaches in the next future
Although the extreme flexibility of AS regulation is proneto errors that may concur to neoplastic transformation [7 8]it can also be exploited therapeutically Indeed examples ofAS modulation in selected genes by administering splicing-correcting ASOs to cells have been reported In some casesthis approach has also been challenged with a therapeuticapplication One of the most remarkable examples is repre-sented by the recovery of the phenotype observed in mousemodels of Spinal Muscular Atrophy (SMA) This neurode-generative disease is caused by inactivation of the SMN1 geneand skipping of exon 7 in the highly homologous SMN2 gene
8 International Journal of Cell Biology
[74] It was recently demonstrated that systemic injectionof a chemically modified ASO restored SMN2 splicing invitro and in vivo and profoundly ameliorated the viabilityand phenotypic features of mice affected by a severe form ofSMA [75] Although cancer is caused by multiple alterationsthus limiting the application of gene-specific ASOs it isconceivable that these tools could be used in combinationwith standard therapies to improve the clinical response ofpatients For instance an ASO that switched BCL-X splicingtoward the proapoptotic variant was effective in sensitizingcancer cells to drug-induced apoptosis and to reduce growthof tumors in nude mice [30 31] A similar effect was obtainedby switching expression of the 120572 to the 120573 variant of the signaltransducer and activator of transcription 3 (STAT3) genewhich modulates multiple oncogenic pathways [76] In thiscase administration of a modified ASO targeted to a splicingenhancer induced expression of the endogenous STAT3120573 andan anti-oncogenic response in vitro and in vivo [76] Thesestudies suggest that modulation of AS with synthetic drugsis possible and has entered a therapeutic perspective ASOsare particularly appealing in terms of high specificity andreduced side effects as theymay exploit their ability to annealwith specific sequences in the genomewithout affecting otherfeatures or target genes of the splicing factors involved in theoncogenicAS eventThus it is likely that thesemethods couldbe applied soon to the development of novel therapies aimedat fighting human cancers in which expression of specificoncogenic splice variants has been firmly confirmed
Conflict of Interests
The author declares no conflict of interests
Acknowledgments
The author wishes to thank Dr Chiara Naro for help with thepreparation of the figures and critical reading of the paperThe work in the laboratory of C Sette was supported byAssociation for International Cancer Research (AICR Grantno 12-0150) the Associazione Italiana Ricerca sul Cancro(AIRC) and Istituto Superiore della Sanita (ISS Grant Con-venzione 11US6A)
References
[1] B J Feldman and D Feldman ldquoThe development of androgen-independent prostate cancerrdquoNature Reviews Cancer vol 1 no1 pp 34ndash45 2001
[2] M Kohli and D J Tindall ldquoNew developments in the medicalmanagement of prostate cancerrdquo Mayo Clinic Proceedings vol85 no 1 pp 77ndash86 2010
[3] K E Knudsen and H I Scher ldquoStarving the addiction newopportunities for durable suppression of AR signaling in pros-tate cancerrdquo Clinical Cancer Research vol 15 no 15 pp 4792ndash4798 2009
[4] S M Dehm and D J Tindall ldquoAlternatively spliced androgenreceptor variantsrdquo Endocrine-Related Cancer vol 18 no 5 ppR183ndashR196 2011
[5] A Kalsotra and T A Cooper ldquoFunctional consequences ofdevelopmentally regulated alternative splicingrdquoNature ReviewsGenetics vol 12 no 10 pp 715ndash729 2011
[6] C Zhang H-R Li J-B Fan et al ldquoProfiling alternativelysplicedmRNA isoforms for prostate cancer classificationrdquo BMCBioinformatics vol 7 article 202 2006
[7] C J David and J L Manley ldquoAlternative pre-mRNA splicingregulation in cancer pathways and programs unhingedrdquo Genesand Development vol 24 no 21 pp 2343ndash2364 2010
[8] S Pal R Gupta and R V Davuluri ldquoAlternative transcriptionand alternative splicing in cancerrdquo Pharmacology amp Therapeu-tics vol 136 no 3 pp 283ndash294 2012
[9] Y Li M Alsagabi D Fan G S Bova A H Tewfik and S MDehm ldquoIntragenic rearrangement and altered RNA splicing ofthe androgen receptor in a cell-based model of prostate cancerprogressionrdquo Cancer Research vol 71 no 6 pp 2108ndash2117 2011
[10] S M Dehm L J Schmidt H V Heemers R L Vessella and DJ Tindall ldquoSplicing of a novel androgen receptor exon generatesa constitutively active androgen receptor that mediates prostatecancer therapy resistancerdquo Cancer Research vol 68 no 13 pp5469ndash5477 2008
[11] R Hu T A Dunn SWei et al ldquoLigand-independent androgenreceptor variants derived from splicing of cryptic exons signifyhormone-refractory prostate cancerrdquo Cancer Research vol 69no 1 pp 16ndash22 2009
[12] Z Guo X Yang F Sun et al ldquoA novel androgen receptor splicevariant is up-regulated during prostate cancer progressionand promotes androgen depletion-resistant growthrdquo CancerResearch vol 69 no 6 pp 2305ndash2313 2009
[13] Y Li S C Chan L J Brand T H Hwang K A Silverstein andS M Dehm ldquoAndrogen receptor splice variants mediate enza-lutamide resistance in castration-resistant prostate cancer celllinesrdquo Cancer Research vol 73 no 2 pp 483ndash489 2013
[14] G Narla K E Heath H L Reeves et al ldquoKLF6 a candidatetumor suppressor genemutated in prostate cancerrdquo Science vol294 no 5551 pp 2563ndash2566 2001
[15] X Liu A Gomez-Pinillos C Loder et al ldquoKLF6 loss of func-tion in human prostate cancer progression is implicated inresistance to androgen deprivationrdquo The American Journal ofPathology vol 181 no 3 pp 1007ndash1016 2012
[16] G Narla A DiFeo H L Reeves et al ldquoA germline DNApolymorphism enhances alternative splicing of the KLF6 tumorsuppressor gene and is associatedwith increased prostate cancerriskrdquo Cancer Research vol 65 no 4 pp 1213ndash1222 2005
[17] G Narla A DiFeo Y Fernandez et al ldquoKLF6-SV1 overexpres-sion accelerates human and mouse prostate cancer progressionand metastasisrdquo Journal of Clinical Investigation vol 118 no 8pp 2711ndash2721 2008
[18] G Narla A Difeo S Yao et al ldquoTargeted inhibition of theKLF6 splice variant KLF6 SV1 suppresses prostate cancer cellgrowth and spreadrdquo Cancer Research vol 65 no 13 pp 5761ndash5768 2005
[19] K E Knudsen ldquoThe cyclin D1b splice variant an old oncogenelearns new tricksrdquo Cell Division vol 1 article 15 2006
[20] K E Knudsen J A Diehl C A Haiman and E S KnudsenldquoCyclin D1 polymorphism aberrant splicing and cancer riskrdquoOncogene vol 25 no 11 pp 1620ndash1628 2006
[21] F Lu A B Gladden and J A Diehl ldquoAn alternatively splicedcyclin D1 isoform cyclin D1b is a nuclear oncogenerdquo CancerResearch vol 63 no 21 pp 7056ndash7061 2003
International Journal of Cell Biology 9
[22] C E S Comstock M A Augello R Pe Benito et al ldquoCyclinD1 splice variants polymorphism risk and isoform-specificregulation in prostate cancerrdquo Clinical Cancer Research vol 15no 17 pp 5338ndash5349 2009
[23] M A Augello C J Burd R Birbe et al ldquoConvergence of onco-genic and hormone receptor pathways promotes metastaticphenotypesrdquo The Journal of Clinical Investigation vol 123 no1 pp 493ndash508 2013
[24] N A Olshavsky C E S Comstock M J Schiewer et al ldquoIden-tification of ASFSF2 as a critical allele-specific effector of thecyclin D1b oncogenerdquoCancer Research vol 70 no 10 pp 3975ndash3984 2010
[25] M P Paronetto M Cappellari R Busa et al ldquoAlternative splic-ing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68rdquoCancer Research vol 70 no 1 pp 229ndash239 2010
[26] D Kaida M G Berg I Younis et al ldquoU1 snRNP protects pre-mRNAs frompremature cleavage and polyadenylationrdquoNaturevol 468 no 7324 pp 664ndash668 2010
[27] P Bielli R Busa M P Paronetto and C Sette ldquoThe RNA-binding protein Sam68 is a multifunctional player in humancancerrdquo Endocrine-Related Cancer vol 18 no 4 pp R91ndashR1022011
[28] L H Boise M Gonzalez-Garcia C E Postema et al ldquobcl-xA bcl-2-related gene that functions as a dominant regulator ofapoptotic cell deathrdquo Cell vol 74 no 4 pp 597ndash608 1993
[29] D R Mercatante C D Bortner J A Cidlowski and R KoleldquoModification of alternative splicing of Bcl-x Pre-mRNA inprostate and breast cancer cells analysis of apoptosis and celldeathrdquo Journal of Biological Chemistry vol 276 no 19 pp 16411ndash16417 2001
[30] D R Mercatante J L Mohler and R Kole ldquoCellular responseto an antisense-mediated shift of Bcl-x pre-mRNA splicing andantineoplastic agentsrdquo Journal of Biological Chemistry vol 277no 51 pp 49374ndash49382 2002
[31] J A Bauman S-D Li A Yang L Huang and R Kole ldquoAnti-tumor activity of splice-switching oligonucleotidesrdquo NucleicAcids Research vol 38 no 22 pp 8348ndash8356 2010
[32] C E Chalfant K Rathman R L Pinkerman et al ldquoDe novoceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells Dependence on proteinphosphatase-1rdquo Journal of Biological Chemistry vol 277 no 15pp 12587ndash12595 2002
[33] K Boon-Unge Q Yu T Zou A Zhou P Govitrapong andJ Zhou ldquoEmetine regulates the alternative splicing of Bcl-xthrough a protein phosphatase 1-dependent mechanismrdquoChemistry and Biology vol 14 no 12 pp 1386ndash1392 2007
[34] M H Kim ldquoProtein phosphatase 1 activation and alternativesplicing of Bcl-X and Mcl-1 by EGCG + ibuprofenrdquo Journal ofCellular Biochemistry vol 104 no 4 pp 1491ndash1499 2008
[35] T Novoyatleva B Heinrich Y Tang et al ldquoProtein phosphatase1 binds to the RNA recognition motif of several splicing fac-tors and regulates alternative pre-mRNA processingrdquo HumanMolecular Genetics vol 17 no 1 pp 52ndash70 2008
[36] N Ghosh N Patel K Jiang et al ldquoCeramide-activated pro-tein phosphatase involvement in insulin resistance via Aktserinearginine-rich protein 40 and ribonucleic acid splicing inL6 skeletal muscle cellsrdquo Endocrinology vol 148 no 3 pp 1359ndash1366 2007
[37] D Garneau T Revil J-F Fisette and B Chabot ldquoHetero-geneous nuclear ribonucleoprotein FH proteins modulate
the alternative splicing of the apoptotic mediator Bcl-xrdquo Journalof Biological Chemistry vol 280 no 24 pp 22641ndash22650 2005
[38] M P Paronetto T Achsel A Massiello C E Chalfant andC Sette ldquoThe RNA-binding protein Sam68 modulates thealternative splicing of Bcl-xrdquo Journal of Cell Biology vol 176 no7 pp 929ndash939 2007
[39] A Zhou A C Ou A Cho E J Benz Jr and S-C HuangldquoNovel splicing factor RBM25 modulates Bcl-x Pre-mRNA 51015840splice site selectionrdquoMolecular and Cellular Biology vol 28 no19 pp 5924ndash5936 2008
[40] S Pedrotti R Busa C Compagnucci and C Sette ldquoThe RNArecognition motif protein RBM11 is a novel tissue-specificsplicing regulatorrdquo Nucleic Acids Research vol 40 no 3 pp1021ndash1032 2012
[41] T Revil J Pelletier J Toutant A Cloutier and B ChabotldquoHeterogeneous nuclear ribonucleoprotein K represses theproduction of pro-apoptotic Bcl-xS splice isoformrdquo Journal ofBiological Chemistry vol 284 no 32 pp 21458ndash21467 2009
[42] M J Moore Q Wang C J Kennedy and P A Silver ldquoAn alter-native splicing network links cell-cycle control to apoptosisrdquoCell vol 142 no 4 pp 625ndash636 2010
[43] P Cloutier J Toutant L Shkreta S Goekjian T Revil and BChabot ldquoAntagonistic effects of the SRp30c protein and cryptic51015840 splice sites on the alternative splicing of the apoptoticregulator Bcl-xrdquo Journal of Biological Chemistry vol 283 no 31pp 21315ndash21324 2008
[44] A Massiello J R Roesser and C E Chalfant ldquoSAP155 Bindsto ceramide-responsive RNA cis-element 1 and regulates thealternative 51015840 splice site selection of Bcl-x pre-mRNArdquo TheFASEB Journal vol 20 no 10 pp 1680ndash1682 2006
[45] R BusaM P Paronetto D Farini et al ldquoTheRNA-binding pro-tein Sam68 contributes to proliferation and survival of humanprostate cancer cellsrdquo Oncogene vol 26 no 30 pp 4372ndash43822007
[46] C Sette ldquoPost-translational regulation of star proteins andeffects on their biological functionsrdquo Advances in ExperimentalMedicine and Biology vol 693 pp 54ndash66 2010
[47] C Brignatz M P Paronetto S Opi et al ldquoAlternative splicingmodulates autoinhibition and SH3 accessibility in the Src kinaseFynrdquo Molecular and Cellular Biology vol 29 no 24 pp 6438ndash6448 2009
[48] M P Paronetto D Farini I Sammarco et al ldquoExpression of atruncated form of the c-Kit tyrosine kinase receptor and activa-tion of Src kinase in human prostatic cancerrdquo American Journalof Pathology vol 164 no 4 pp 1243ndash1251 2004
[49] Y Hu A Dobi T Sreenath et al ldquoDelineation of TMPRSS2-ERG splice variants in prostate cancerrdquo Clinical CancerResearch vol 14 no 15 pp 4719ndash4725 2008
[50] J Wang Y Cai W Yu C Ren D M Spencer and M Itt-mann ldquoPleiotropic biological activities of alternatively splicedTMPRSS2ERG fusion gene transcriptsrdquo Cancer Research vol68 no 20 pp 8516ndash8524 2008
[51] F Zammarchi G Boutsalis and L Cartegni ldquo51015840 UTR controlof native ERG and of Tmprss2 ERG variants activity in prostatecancerrdquo PLoS One vol 8 no 3 Article ID e49721 2013
[52] M J Munoz M S P Santangelo M P Paronetto et al ldquoDNAdamage regulates alternative splicing through inhibition ofRNApolymerase II elongationrdquoCell vol 137 no 4 pp 708ndash7202009
[53] P Rajan C Dalgliesh P J Carling et al ldquoIdentification of novelandrogen-regulated pathways and mrna isoforms through
10 International Journal of Cell Biology
genome-wide exon-specific profiling of the LNCaP transcrip-tomerdquo PLoS One vol 6 no 12 Article ID e29088 2011
[54] T M Morgan T D Koreckij and E Corey ldquoTargeted therapyfor advanced prostate cancer inhibition of the PI3KAktmTORpathwayrdquoCurrent CancerDrug Targets vol 9 no 2 pp 237ndash2492009
[55] J Sun A L Blair S E Aiyar and R Li ldquoCofactor of BRCA1modulates androgen-dependent transcription and alternativesplicingrdquo Journal of Steroid Biochemistry andMolecular Biologyvol 107 no 3ndash5 pp 131ndash139 2007
[56] E L Clark A Coulson C Dalgliesh et al ldquoThe RNA helicasep68 is a novel androgen receptor coactivator involved in splicingand is overexpressed in prostate cancerrdquo Cancer Research vol68 no 19 pp 7938ndash7946 2008
[57] E Zonta D Bittencourt S Samaan S Germann M Dutertreand D Auboeuf ldquoThe RNA helicase DDX5p68 is a key factorpromoting c-fos expression at different levels from transcriptionto mRNA exportrdquoNucleic Acids Research vol 41 no 1 pp 554ndash564 2013
[58] A Honig D Auboeuf M M Parker B W OrsquoMalley andS M Berget ldquoRegulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72rdquo Molecular and Cel-lular Biology vol 22 no 16 pp 5698ndash5707 2002
[59] X Dong J Sweet J R G Challis T Brown and S J Lye ldquoTran-scriptional activity of androgen receptor is modulated by twoRNA splicing factors PSF and p54nrbrdquoMolecular and CellularBiology vol 27 no 13 pp 4863ndash4875 2007
[60] P Rajan L Gaughan C Dalgliesh et al ldquoThe RNA-binding andadaptor protein Sam68 modulates signal-dependent splicingand transcriptional activity of the androgen receptorrdquo Journalof Pathology vol 215 no 1 pp 67ndash77 2008
[61] R Karni E De Stanchina S W Lowe R Sinha D Mu and AR Krainer ldquoThe gene encoding the splicing factor SF2ASF is aproto-oncogenerdquo Nature Structural and Molecular Biology vol14 no 3 pp 185ndash193 2007
[62] OAnczukowA Z RosenbergMAkerman et al ldquoThe splicingfactor SRSF1 regulates apoptosis and proliferation to promotemammary epithelial cell transformationrdquoNature Structural andMolecular Biology vol 19 no 2 pp 220ndash228 2012
[63] L Adesso S Calabretta F Barbagallo et al ldquoGemcitabine trig-gers a pro-survival response in pancreatic cancer cells throughactivation of the MNK2eIF4E pathwayrdquo Oncogene vol 32 pp2848ndash2857 2013
[64] A Bianchini M Loiarro P Bielli et al ldquoPhosphorylation ofeIF4E by MNKs supports protein synthesis cell cycle progres-sion and proliferation in prostate cancer cellsrdquo Carcinogenesisvol 29 no 12 pp 2279ndash2288 2008
[65] L Furic L RongO Larsson et al ldquoEIF4Ephosphorylation pro-motes tumorigenesis and is associated with prostate cancer pro-gressionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 32 pp 14134ndash14139 2010
[66] C Naro and C Sette ldquoPhosphorylation-mediated regulation ofalternative splicing in cancerrdquo International Journal of Cell Bio-logy In press
[67] Z Zhou J QiuW Liu et al ldquoTheAkt-SRPK-SR axis constitutesa major pathway in transducing EGF signaling to regulatealternative splicing in the nucleusrdquo Molecular Cell vol 47 no3 pp 422ndash433 2012
[68] NMatter P Herrlich andH Konig ldquoSignal-dependent regula-tion of splicing via phosphorylation of Sam68rdquoNature vol 420no 6916 pp 691ndash695 2002
[69] A M Al-Ayoubi H Zheng Y Liu T Bai and S T EblenldquoMitogen-activated protein kinase phosphorylation of splicingfactor 45 (SPF45) regulates SPF45 alternative splicing siteutilization proliferation and cell adhesionrdquoMolecular and Cel-lular Biology vol 32 no 14 pp 2880ndash2893 2012
[70] A K Shalek R Satija X Adiconis et al ldquoSingle-cell transcrip-tomics reveals bimodality in expression and splicing in immunecellsrdquo Nature 2013
[71] S Pedrotti P BielliM P Paronetto et al ldquoThe splicing regulatorSam68 binds to a novel exonic splicing silencer and functions inSMN2 alternative splicing in spinal muscular atrophyrdquo EMBOJournal vol 29 no 7 pp 1235ndash1247 2010
[72] N H Meyer K Tripsianes M Vincendeau et al ldquoStructuralbasis for homodimerization of the Src-associated during mito-sis 68-kDa protein (Sam68)Qua1 domainrdquo Journal of BiologicalChemistry vol 285 no 37 pp 28893ndash28901 2010
[73] S Bonnal L Vigevani and J Valcarcel ldquoThe spliceosome asa target of novel antitumour drugsrdquo Nature Reviews Drug Dis-covery vol 11 no 11 pp 847ndash859 2012
[74] S Pedrotti and C Sette ldquoSpinal muscular atrophy a new playerjoins the battle for SMN2 exon 7 splicingrdquo Cell Cycle vol 9 no19 pp 3874ndash3879 2010
[75] Y Hua K Sahashi F Rigo et al ldquoPeripheral SMN restoration isessential for long-term rescue of a severe spinal muscular atro-phy mouse modelrdquo Nature vol 478 no 7367 pp 123ndash126 2011
[76] F Zammarchi E De Stanchina E Bournazou et al ldquoAntitu-morigenic potential of STAT3 alternative splicing modulationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 43 pp 17779ndash17784 2011
of the target gene Some of these alternative transcripts werepredicted to influence the function of proteins with relevanceto PCa such as the mTOR regulator TSC2 Following andro-genic stimulation AR was recruited to a cryptic promoterupstream of exon 33 in the TSC2 gene thereby leading toexpression of a truncated transcript lacking the 51015840 exons of thegeneThis alternative TSC2 variant would encode a truncatedprotein lacking the domain required for the interaction withTSC1 which is needed to exert negative regulation of mTORThus androgens may lead to activation of mTOR by relievingthe repressive function of the TSC1TSC2 complex throughAR-dependent induction of a defective variant It is worthyof notice that activation of the mTOR pathway has beenlinked to both tumorigenesis and resistance to therapy in PCa[54] Thus AR might contribute to prostate tumorigenesisalso by causing mTOR activation through expression of thisalternative mRNA variant of TSC2
9 Splicing Regulators Contributing to AlteredGene Expression in Prostate Cancer
In addition to affecting recruitment of AR to alternativepromoters androgens also affected a number of AS events inseveral genes [53] Although the mechanism(s) involved inthese events and their potential relevance to PCa biology wasnot investigated it might involve the ability of AR to interactwith cofactors that modulate the transcriptional elongationrate andor the recognition of splicing enhancers or silencersin the pre-mRNA (Figure 2) For instance AR interacts withthe cofactor of BRCA1 (COBRA1) and this interaction wasshown to influence splicing of the nascent transcripts pro-duced from an androgen-dependent promoter [55] A similarregulation of AR activity was also documented for the DEADboxRNAhelicase p68 (DDX5) in the LNCaP cell line AR andDDX5 interact and are recruited to the promoter region of theandrogen-responsive prostate-specific antigen (PSA) geneThis interactionwas functionally relevant asDDX5 enhancedAR-dependent PSA expression In addition by using an AR-dependent minigene reporter it was shown that DDX5 andAR cooperated in repressing the splicing of variable exons inthe CD44 gene [56] DDX5 is involved in several steps of co-and posttranscriptional RNA processing including splicing[57] and some genes appear to be particularly sensitive to theintracellular levels of DDX5 [57 58] Hence since this RNAhelicase is upregulated in PCa [56] it will be interesting todetermine to what extent it contributes to RNA processing ofAR target genes in PCa cells
AR is also known to interact with several splicing fac-tors suggesting a direct link between androgen-regulatedtranscription initiation and pre-mRNA splicing in PCa cells(Figure 2) The PTB-associated splicing factor (PSF) and itscofactor p54nrb participate to androgen-dependent proteincomplexes containing the AR PSF and p54nrb inhibit thetranscriptional activity of AR by interfering with its bindingto androgen response element and by recruiting a his-tone deacetylase to AR-responsive promoters [59] Althoughdirect investigation of the effect of these splicing factors onAR-dependent splicing events was not addressed it is likely
that AS is also affected by this interaction Another splicingfactor that may participate to AR-dependent splicing regula-tion is SAM68 [60] which is frequently upregulated in PCa[25 45] SAM68 interacts with AR and is recruited to the PSApromoter [60] like DDX5 [55] Interestingly however theinteraction between SAM68 and AR exerted different effectson transcription and splicing as the two proteins cooperatedin transcriptional activation of AR-target genes but opposedeach other in splicing of the CD44 variable exons from areporter minigene [60] Unfortunately the direct effects ofall these RBPs on AR-dependent splicing of endogenoustranscripts have not been addressed yet Nevertheless it islikely that depending on the specific complex formedAR candifferentially influence splicing of its target genes in PCa cells
An additional layer of regulation of the aberrant splicingprogram in PCa might rely on the up-regulation of specificsplicing factors Beside the already mentioned SAM68 [2545] one likely candidate is SRSF1 a splicing factor thatis upregulated in many human cancers and was shown tobehave as an oncogene in mice and humans [61] In cancercells of other tissues SRSF1modulates the expression of splicevariants of the BIN1 and BIM genes that lack proapoptoticfunctions [61 62] Moreover SRSF1 promotes splicing ofMNK2b [61] a splice variant of the eIF4E kinase MNK2 thatwas shown to confer chemoresistance in pancreatic adenocar-cinoma cells [63] Importantly MNK-dependent phospho-rylation of eIF4E strongly contributes to PCa tumorigenesisboth in vitro and in vivo [64 65] and a tight balance betweenthe MNKeIF4E and the mTOR pathways is required tomaintain efficient protein synthesis in PCa cells therebyenhancing their proliferation rate [64] Thus it will be inter-esting to determine whether SRSF1 contributes to fine-tuningthe activation of these pathways in PCa cells through theregulation ofMNK2 AS
Other splicing factors may also contribute to the alteredsplicing programof PCa cells Indeed the activity of several ofthese RBPs ismodulated by signal transduction pathways thatare frequently turned on in cancer such as the PI3KAKT andthe RASERK pathways (see also [66]) For instance it wasshown that activation of AKT downstream of the epidermalgrowth factor (EGF) receptor modulated the activity ofthe SR protein-specific kinases and phosphorylation of SRproteins thereby affecting a large spectrum of AS events[67] Similarly the RASERK pathway modulates a numberof splicing factors involved in cancer such as SAM68 [68]and the alternative splicing factor 45 (SPF45) [69] whichin turn affect expression of splice variants that regulate cellmotility proliferation and survival Thus it is likely that theexamples reported above represent only a small picture of theoverall contribution of AS and splicing factors to the wideheterogeneity in gene expression observed in PCa cells andpatients
10 Conclusions and Perspectives
AS is widely recognized as a powerful tool that eukaryoticcells employ to expand the coding potential and the plasticityof their genomes The flexibility in the recognition of exons
International Journal of Cell Biology 7
DDX5
SAM68COBRA1
AR
AR splicing-responsive mRNAs
RNAPIIAR transcriptional-responsive gene
AR coactivators
PSF p54nrb
AR corepressor
ARRNAPII
AR transcriptional-responsive gene
Figure 2 Regulation of cotranscriptional splicing by proteins interacting with the androgen receptor Coregulators of the androgen receptor(AR) can affect splicing of target genes by direct interaction with AR and modulation of its activity COBRA1 SAM68 and DDX5 appear topromote the transcriptional activity of AR but differentially act on splicing of variable exons (red box in the left side of the figure) PSF andits interacting protein p54 (right side of the figure) repress the transcriptional activity of AR but their effect on splicing is currently unknown(see text for more details)
and introns within the transcription unit of the majority ofhuman genes offers the possibility to compose many mRNAvariants from each gene Subtle changes in the cellular envi-ronment or in external cues conveyed from the surroundingenvironment may result in global changes in the tran-scriptome which in part rely on the regulation of AS Aninteresting observation is that apparently homogenous cellpopulations actually display large differences in gene expres-sion This was recently exemplified by studies that appliedglobal RNA sequencing techniques to the analysis of singlecell transcriptomes After treatment of bone-marrow-deriveddendritic cells (BMDCs) with an inflammatory cue it wasfound that hundreds of key immune genes were differentiallyexpressed by single cells The heterogeneity in the responsewas particularly remarkable with regard to the splicing pat-terns expressed by these cells [70] suggesting that fine-tuningof AS regulation strongly contributes to the heterogeneity ofa cell population This aspect might be particularly relevantin the context of PCa which is a neoplastic disease charac-terized by extreme heterogeneity and unpredicted responseof patients to the therapy [1 2] The improvements in cellisolation techniques coupled to the higher sensitivity of thenext-generation sequencing techniques may soon allow ahighly detailed description of the transcriptome of patientswhich might result in more personalized treatments
The studies illustrated previously suggest that the upregu-lation of selected splicing regulators in PCa such as SAM68SRSF1 or DDX5 directly contributes to the phenotype byaltering the splicing profile of key genes Thus these RBPsmight represent potential therapeutic targets for interventionAlthough blocking the activity of a given splicing factor isnot necessarily an easy task some examples in this direction
have been provided For instance SAM68 can bind to RNAonly as a dimer By exploiting this requirement it was shownthat an RNA binding-defective SAM68 mutant exerted dom-inant negative effects on SAM68-mediated SMN2 splicing byassociating with the endogenous protein and preventing itsbinding to the pre-mRNA [71]This experiment suggests thatsmall molecules interfering with SAM68 function might dis-play therapeutic potential As homodimerization is a prereq-uisite for RNA binding one possibility is to target the SAM68dimerization domain which was restricted to a small regionwithin its Gld1-Sam68-Grp33 (GSG) homology domain [72]The potential value of targeting specific components of thesplicing machinery in cancer cells is also suggested by theantioncogenic properties of natural compounds such asspliceostatin A (SSA) in a variety of cancer cell modelsSSA targets the splicing factor 3B subunit 1 (SF3B1) of thespliceosome thus affecting a large number of splicing eventsconcomitantly [73] Perhaps more specific drugs targetingsplicing factors involved in subsets of oncogenic splicingevents in cancer cells as those described above might repre-sent more specific therapeutic approaches in the next future
Although the extreme flexibility of AS regulation is proneto errors that may concur to neoplastic transformation [7 8]it can also be exploited therapeutically Indeed examples ofAS modulation in selected genes by administering splicing-correcting ASOs to cells have been reported In some casesthis approach has also been challenged with a therapeuticapplication One of the most remarkable examples is repre-sented by the recovery of the phenotype observed in mousemodels of Spinal Muscular Atrophy (SMA) This neurode-generative disease is caused by inactivation of the SMN1 geneand skipping of exon 7 in the highly homologous SMN2 gene
8 International Journal of Cell Biology
[74] It was recently demonstrated that systemic injectionof a chemically modified ASO restored SMN2 splicing invitro and in vivo and profoundly ameliorated the viabilityand phenotypic features of mice affected by a severe form ofSMA [75] Although cancer is caused by multiple alterationsthus limiting the application of gene-specific ASOs it isconceivable that these tools could be used in combinationwith standard therapies to improve the clinical response ofpatients For instance an ASO that switched BCL-X splicingtoward the proapoptotic variant was effective in sensitizingcancer cells to drug-induced apoptosis and to reduce growthof tumors in nude mice [30 31] A similar effect was obtainedby switching expression of the 120572 to the 120573 variant of the signaltransducer and activator of transcription 3 (STAT3) genewhich modulates multiple oncogenic pathways [76] In thiscase administration of a modified ASO targeted to a splicingenhancer induced expression of the endogenous STAT3120573 andan anti-oncogenic response in vitro and in vivo [76] Thesestudies suggest that modulation of AS with synthetic drugsis possible and has entered a therapeutic perspective ASOsare particularly appealing in terms of high specificity andreduced side effects as theymay exploit their ability to annealwith specific sequences in the genomewithout affecting otherfeatures or target genes of the splicing factors involved in theoncogenicAS eventThus it is likely that thesemethods couldbe applied soon to the development of novel therapies aimedat fighting human cancers in which expression of specificoncogenic splice variants has been firmly confirmed
Conflict of Interests
The author declares no conflict of interests
Acknowledgments
The author wishes to thank Dr Chiara Naro for help with thepreparation of the figures and critical reading of the paperThe work in the laboratory of C Sette was supported byAssociation for International Cancer Research (AICR Grantno 12-0150) the Associazione Italiana Ricerca sul Cancro(AIRC) and Istituto Superiore della Sanita (ISS Grant Con-venzione 11US6A)
References
[1] B J Feldman and D Feldman ldquoThe development of androgen-independent prostate cancerrdquoNature Reviews Cancer vol 1 no1 pp 34ndash45 2001
[2] M Kohli and D J Tindall ldquoNew developments in the medicalmanagement of prostate cancerrdquo Mayo Clinic Proceedings vol85 no 1 pp 77ndash86 2010
[3] K E Knudsen and H I Scher ldquoStarving the addiction newopportunities for durable suppression of AR signaling in pros-tate cancerrdquo Clinical Cancer Research vol 15 no 15 pp 4792ndash4798 2009
[4] S M Dehm and D J Tindall ldquoAlternatively spliced androgenreceptor variantsrdquo Endocrine-Related Cancer vol 18 no 5 ppR183ndashR196 2011
[5] A Kalsotra and T A Cooper ldquoFunctional consequences ofdevelopmentally regulated alternative splicingrdquoNature ReviewsGenetics vol 12 no 10 pp 715ndash729 2011
[6] C Zhang H-R Li J-B Fan et al ldquoProfiling alternativelysplicedmRNA isoforms for prostate cancer classificationrdquo BMCBioinformatics vol 7 article 202 2006
[7] C J David and J L Manley ldquoAlternative pre-mRNA splicingregulation in cancer pathways and programs unhingedrdquo Genesand Development vol 24 no 21 pp 2343ndash2364 2010
[8] S Pal R Gupta and R V Davuluri ldquoAlternative transcriptionand alternative splicing in cancerrdquo Pharmacology amp Therapeu-tics vol 136 no 3 pp 283ndash294 2012
[9] Y Li M Alsagabi D Fan G S Bova A H Tewfik and S MDehm ldquoIntragenic rearrangement and altered RNA splicing ofthe androgen receptor in a cell-based model of prostate cancerprogressionrdquo Cancer Research vol 71 no 6 pp 2108ndash2117 2011
[10] S M Dehm L J Schmidt H V Heemers R L Vessella and DJ Tindall ldquoSplicing of a novel androgen receptor exon generatesa constitutively active androgen receptor that mediates prostatecancer therapy resistancerdquo Cancer Research vol 68 no 13 pp5469ndash5477 2008
[11] R Hu T A Dunn SWei et al ldquoLigand-independent androgenreceptor variants derived from splicing of cryptic exons signifyhormone-refractory prostate cancerrdquo Cancer Research vol 69no 1 pp 16ndash22 2009
[12] Z Guo X Yang F Sun et al ldquoA novel androgen receptor splicevariant is up-regulated during prostate cancer progressionand promotes androgen depletion-resistant growthrdquo CancerResearch vol 69 no 6 pp 2305ndash2313 2009
[13] Y Li S C Chan L J Brand T H Hwang K A Silverstein andS M Dehm ldquoAndrogen receptor splice variants mediate enza-lutamide resistance in castration-resistant prostate cancer celllinesrdquo Cancer Research vol 73 no 2 pp 483ndash489 2013
[14] G Narla K E Heath H L Reeves et al ldquoKLF6 a candidatetumor suppressor genemutated in prostate cancerrdquo Science vol294 no 5551 pp 2563ndash2566 2001
[15] X Liu A Gomez-Pinillos C Loder et al ldquoKLF6 loss of func-tion in human prostate cancer progression is implicated inresistance to androgen deprivationrdquo The American Journal ofPathology vol 181 no 3 pp 1007ndash1016 2012
[16] G Narla A DiFeo H L Reeves et al ldquoA germline DNApolymorphism enhances alternative splicing of the KLF6 tumorsuppressor gene and is associatedwith increased prostate cancerriskrdquo Cancer Research vol 65 no 4 pp 1213ndash1222 2005
[17] G Narla A DiFeo Y Fernandez et al ldquoKLF6-SV1 overexpres-sion accelerates human and mouse prostate cancer progressionand metastasisrdquo Journal of Clinical Investigation vol 118 no 8pp 2711ndash2721 2008
[18] G Narla A Difeo S Yao et al ldquoTargeted inhibition of theKLF6 splice variant KLF6 SV1 suppresses prostate cancer cellgrowth and spreadrdquo Cancer Research vol 65 no 13 pp 5761ndash5768 2005
[19] K E Knudsen ldquoThe cyclin D1b splice variant an old oncogenelearns new tricksrdquo Cell Division vol 1 article 15 2006
[20] K E Knudsen J A Diehl C A Haiman and E S KnudsenldquoCyclin D1 polymorphism aberrant splicing and cancer riskrdquoOncogene vol 25 no 11 pp 1620ndash1628 2006
[21] F Lu A B Gladden and J A Diehl ldquoAn alternatively splicedcyclin D1 isoform cyclin D1b is a nuclear oncogenerdquo CancerResearch vol 63 no 21 pp 7056ndash7061 2003
International Journal of Cell Biology 9
[22] C E S Comstock M A Augello R Pe Benito et al ldquoCyclinD1 splice variants polymorphism risk and isoform-specificregulation in prostate cancerrdquo Clinical Cancer Research vol 15no 17 pp 5338ndash5349 2009
[23] M A Augello C J Burd R Birbe et al ldquoConvergence of onco-genic and hormone receptor pathways promotes metastaticphenotypesrdquo The Journal of Clinical Investigation vol 123 no1 pp 493ndash508 2013
[24] N A Olshavsky C E S Comstock M J Schiewer et al ldquoIden-tification of ASFSF2 as a critical allele-specific effector of thecyclin D1b oncogenerdquoCancer Research vol 70 no 10 pp 3975ndash3984 2010
[25] M P Paronetto M Cappellari R Busa et al ldquoAlternative splic-ing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68rdquoCancer Research vol 70 no 1 pp 229ndash239 2010
[26] D Kaida M G Berg I Younis et al ldquoU1 snRNP protects pre-mRNAs frompremature cleavage and polyadenylationrdquoNaturevol 468 no 7324 pp 664ndash668 2010
[27] P Bielli R Busa M P Paronetto and C Sette ldquoThe RNA-binding protein Sam68 is a multifunctional player in humancancerrdquo Endocrine-Related Cancer vol 18 no 4 pp R91ndashR1022011
[28] L H Boise M Gonzalez-Garcia C E Postema et al ldquobcl-xA bcl-2-related gene that functions as a dominant regulator ofapoptotic cell deathrdquo Cell vol 74 no 4 pp 597ndash608 1993
[29] D R Mercatante C D Bortner J A Cidlowski and R KoleldquoModification of alternative splicing of Bcl-x Pre-mRNA inprostate and breast cancer cells analysis of apoptosis and celldeathrdquo Journal of Biological Chemistry vol 276 no 19 pp 16411ndash16417 2001
[30] D R Mercatante J L Mohler and R Kole ldquoCellular responseto an antisense-mediated shift of Bcl-x pre-mRNA splicing andantineoplastic agentsrdquo Journal of Biological Chemistry vol 277no 51 pp 49374ndash49382 2002
[31] J A Bauman S-D Li A Yang L Huang and R Kole ldquoAnti-tumor activity of splice-switching oligonucleotidesrdquo NucleicAcids Research vol 38 no 22 pp 8348ndash8356 2010
[32] C E Chalfant K Rathman R L Pinkerman et al ldquoDe novoceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells Dependence on proteinphosphatase-1rdquo Journal of Biological Chemistry vol 277 no 15pp 12587ndash12595 2002
[33] K Boon-Unge Q Yu T Zou A Zhou P Govitrapong andJ Zhou ldquoEmetine regulates the alternative splicing of Bcl-xthrough a protein phosphatase 1-dependent mechanismrdquoChemistry and Biology vol 14 no 12 pp 1386ndash1392 2007
[34] M H Kim ldquoProtein phosphatase 1 activation and alternativesplicing of Bcl-X and Mcl-1 by EGCG + ibuprofenrdquo Journal ofCellular Biochemistry vol 104 no 4 pp 1491ndash1499 2008
[35] T Novoyatleva B Heinrich Y Tang et al ldquoProtein phosphatase1 binds to the RNA recognition motif of several splicing fac-tors and regulates alternative pre-mRNA processingrdquo HumanMolecular Genetics vol 17 no 1 pp 52ndash70 2008
[36] N Ghosh N Patel K Jiang et al ldquoCeramide-activated pro-tein phosphatase involvement in insulin resistance via Aktserinearginine-rich protein 40 and ribonucleic acid splicing inL6 skeletal muscle cellsrdquo Endocrinology vol 148 no 3 pp 1359ndash1366 2007
[37] D Garneau T Revil J-F Fisette and B Chabot ldquoHetero-geneous nuclear ribonucleoprotein FH proteins modulate
the alternative splicing of the apoptotic mediator Bcl-xrdquo Journalof Biological Chemistry vol 280 no 24 pp 22641ndash22650 2005
[38] M P Paronetto T Achsel A Massiello C E Chalfant andC Sette ldquoThe RNA-binding protein Sam68 modulates thealternative splicing of Bcl-xrdquo Journal of Cell Biology vol 176 no7 pp 929ndash939 2007
[39] A Zhou A C Ou A Cho E J Benz Jr and S-C HuangldquoNovel splicing factor RBM25 modulates Bcl-x Pre-mRNA 51015840splice site selectionrdquoMolecular and Cellular Biology vol 28 no19 pp 5924ndash5936 2008
[40] S Pedrotti R Busa C Compagnucci and C Sette ldquoThe RNArecognition motif protein RBM11 is a novel tissue-specificsplicing regulatorrdquo Nucleic Acids Research vol 40 no 3 pp1021ndash1032 2012
[41] T Revil J Pelletier J Toutant A Cloutier and B ChabotldquoHeterogeneous nuclear ribonucleoprotein K represses theproduction of pro-apoptotic Bcl-xS splice isoformrdquo Journal ofBiological Chemistry vol 284 no 32 pp 21458ndash21467 2009
[42] M J Moore Q Wang C J Kennedy and P A Silver ldquoAn alter-native splicing network links cell-cycle control to apoptosisrdquoCell vol 142 no 4 pp 625ndash636 2010
[43] P Cloutier J Toutant L Shkreta S Goekjian T Revil and BChabot ldquoAntagonistic effects of the SRp30c protein and cryptic51015840 splice sites on the alternative splicing of the apoptoticregulator Bcl-xrdquo Journal of Biological Chemistry vol 283 no 31pp 21315ndash21324 2008
[44] A Massiello J R Roesser and C E Chalfant ldquoSAP155 Bindsto ceramide-responsive RNA cis-element 1 and regulates thealternative 51015840 splice site selection of Bcl-x pre-mRNArdquo TheFASEB Journal vol 20 no 10 pp 1680ndash1682 2006
[45] R BusaM P Paronetto D Farini et al ldquoTheRNA-binding pro-tein Sam68 contributes to proliferation and survival of humanprostate cancer cellsrdquo Oncogene vol 26 no 30 pp 4372ndash43822007
[46] C Sette ldquoPost-translational regulation of star proteins andeffects on their biological functionsrdquo Advances in ExperimentalMedicine and Biology vol 693 pp 54ndash66 2010
[47] C Brignatz M P Paronetto S Opi et al ldquoAlternative splicingmodulates autoinhibition and SH3 accessibility in the Src kinaseFynrdquo Molecular and Cellular Biology vol 29 no 24 pp 6438ndash6448 2009
[48] M P Paronetto D Farini I Sammarco et al ldquoExpression of atruncated form of the c-Kit tyrosine kinase receptor and activa-tion of Src kinase in human prostatic cancerrdquo American Journalof Pathology vol 164 no 4 pp 1243ndash1251 2004
[49] Y Hu A Dobi T Sreenath et al ldquoDelineation of TMPRSS2-ERG splice variants in prostate cancerrdquo Clinical CancerResearch vol 14 no 15 pp 4719ndash4725 2008
[50] J Wang Y Cai W Yu C Ren D M Spencer and M Itt-mann ldquoPleiotropic biological activities of alternatively splicedTMPRSS2ERG fusion gene transcriptsrdquo Cancer Research vol68 no 20 pp 8516ndash8524 2008
[51] F Zammarchi G Boutsalis and L Cartegni ldquo51015840 UTR controlof native ERG and of Tmprss2 ERG variants activity in prostatecancerrdquo PLoS One vol 8 no 3 Article ID e49721 2013
[52] M J Munoz M S P Santangelo M P Paronetto et al ldquoDNAdamage regulates alternative splicing through inhibition ofRNApolymerase II elongationrdquoCell vol 137 no 4 pp 708ndash7202009
[53] P Rajan C Dalgliesh P J Carling et al ldquoIdentification of novelandrogen-regulated pathways and mrna isoforms through
10 International Journal of Cell Biology
genome-wide exon-specific profiling of the LNCaP transcrip-tomerdquo PLoS One vol 6 no 12 Article ID e29088 2011
[54] T M Morgan T D Koreckij and E Corey ldquoTargeted therapyfor advanced prostate cancer inhibition of the PI3KAktmTORpathwayrdquoCurrent CancerDrug Targets vol 9 no 2 pp 237ndash2492009
[55] J Sun A L Blair S E Aiyar and R Li ldquoCofactor of BRCA1modulates androgen-dependent transcription and alternativesplicingrdquo Journal of Steroid Biochemistry andMolecular Biologyvol 107 no 3ndash5 pp 131ndash139 2007
[56] E L Clark A Coulson C Dalgliesh et al ldquoThe RNA helicasep68 is a novel androgen receptor coactivator involved in splicingand is overexpressed in prostate cancerrdquo Cancer Research vol68 no 19 pp 7938ndash7946 2008
[57] E Zonta D Bittencourt S Samaan S Germann M Dutertreand D Auboeuf ldquoThe RNA helicase DDX5p68 is a key factorpromoting c-fos expression at different levels from transcriptionto mRNA exportrdquoNucleic Acids Research vol 41 no 1 pp 554ndash564 2013
[58] A Honig D Auboeuf M M Parker B W OrsquoMalley andS M Berget ldquoRegulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72rdquo Molecular and Cel-lular Biology vol 22 no 16 pp 5698ndash5707 2002
[59] X Dong J Sweet J R G Challis T Brown and S J Lye ldquoTran-scriptional activity of androgen receptor is modulated by twoRNA splicing factors PSF and p54nrbrdquoMolecular and CellularBiology vol 27 no 13 pp 4863ndash4875 2007
[60] P Rajan L Gaughan C Dalgliesh et al ldquoThe RNA-binding andadaptor protein Sam68 modulates signal-dependent splicingand transcriptional activity of the androgen receptorrdquo Journalof Pathology vol 215 no 1 pp 67ndash77 2008
[61] R Karni E De Stanchina S W Lowe R Sinha D Mu and AR Krainer ldquoThe gene encoding the splicing factor SF2ASF is aproto-oncogenerdquo Nature Structural and Molecular Biology vol14 no 3 pp 185ndash193 2007
[62] OAnczukowA Z RosenbergMAkerman et al ldquoThe splicingfactor SRSF1 regulates apoptosis and proliferation to promotemammary epithelial cell transformationrdquoNature Structural andMolecular Biology vol 19 no 2 pp 220ndash228 2012
[63] L Adesso S Calabretta F Barbagallo et al ldquoGemcitabine trig-gers a pro-survival response in pancreatic cancer cells throughactivation of the MNK2eIF4E pathwayrdquo Oncogene vol 32 pp2848ndash2857 2013
[64] A Bianchini M Loiarro P Bielli et al ldquoPhosphorylation ofeIF4E by MNKs supports protein synthesis cell cycle progres-sion and proliferation in prostate cancer cellsrdquo Carcinogenesisvol 29 no 12 pp 2279ndash2288 2008
[65] L Furic L RongO Larsson et al ldquoEIF4Ephosphorylation pro-motes tumorigenesis and is associated with prostate cancer pro-gressionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 32 pp 14134ndash14139 2010
[66] C Naro and C Sette ldquoPhosphorylation-mediated regulation ofalternative splicing in cancerrdquo International Journal of Cell Bio-logy In press
[67] Z Zhou J QiuW Liu et al ldquoTheAkt-SRPK-SR axis constitutesa major pathway in transducing EGF signaling to regulatealternative splicing in the nucleusrdquo Molecular Cell vol 47 no3 pp 422ndash433 2012
[68] NMatter P Herrlich andH Konig ldquoSignal-dependent regula-tion of splicing via phosphorylation of Sam68rdquoNature vol 420no 6916 pp 691ndash695 2002
[69] A M Al-Ayoubi H Zheng Y Liu T Bai and S T EblenldquoMitogen-activated protein kinase phosphorylation of splicingfactor 45 (SPF45) regulates SPF45 alternative splicing siteutilization proliferation and cell adhesionrdquoMolecular and Cel-lular Biology vol 32 no 14 pp 2880ndash2893 2012
[70] A K Shalek R Satija X Adiconis et al ldquoSingle-cell transcrip-tomics reveals bimodality in expression and splicing in immunecellsrdquo Nature 2013
[71] S Pedrotti P BielliM P Paronetto et al ldquoThe splicing regulatorSam68 binds to a novel exonic splicing silencer and functions inSMN2 alternative splicing in spinal muscular atrophyrdquo EMBOJournal vol 29 no 7 pp 1235ndash1247 2010
[72] N H Meyer K Tripsianes M Vincendeau et al ldquoStructuralbasis for homodimerization of the Src-associated during mito-sis 68-kDa protein (Sam68)Qua1 domainrdquo Journal of BiologicalChemistry vol 285 no 37 pp 28893ndash28901 2010
[73] S Bonnal L Vigevani and J Valcarcel ldquoThe spliceosome asa target of novel antitumour drugsrdquo Nature Reviews Drug Dis-covery vol 11 no 11 pp 847ndash859 2012
[74] S Pedrotti and C Sette ldquoSpinal muscular atrophy a new playerjoins the battle for SMN2 exon 7 splicingrdquo Cell Cycle vol 9 no19 pp 3874ndash3879 2010
[75] Y Hua K Sahashi F Rigo et al ldquoPeripheral SMN restoration isessential for long-term rescue of a severe spinal muscular atro-phy mouse modelrdquo Nature vol 478 no 7367 pp 123ndash126 2011
[76] F Zammarchi E De Stanchina E Bournazou et al ldquoAntitu-morigenic potential of STAT3 alternative splicing modulationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 43 pp 17779ndash17784 2011
Figure 2 Regulation of cotranscriptional splicing by proteins interacting with the androgen receptor Coregulators of the androgen receptor(AR) can affect splicing of target genes by direct interaction with AR and modulation of its activity COBRA1 SAM68 and DDX5 appear topromote the transcriptional activity of AR but differentially act on splicing of variable exons (red box in the left side of the figure) PSF andits interacting protein p54 (right side of the figure) repress the transcriptional activity of AR but their effect on splicing is currently unknown(see text for more details)
and introns within the transcription unit of the majority ofhuman genes offers the possibility to compose many mRNAvariants from each gene Subtle changes in the cellular envi-ronment or in external cues conveyed from the surroundingenvironment may result in global changes in the tran-scriptome which in part rely on the regulation of AS Aninteresting observation is that apparently homogenous cellpopulations actually display large differences in gene expres-sion This was recently exemplified by studies that appliedglobal RNA sequencing techniques to the analysis of singlecell transcriptomes After treatment of bone-marrow-deriveddendritic cells (BMDCs) with an inflammatory cue it wasfound that hundreds of key immune genes were differentiallyexpressed by single cells The heterogeneity in the responsewas particularly remarkable with regard to the splicing pat-terns expressed by these cells [70] suggesting that fine-tuningof AS regulation strongly contributes to the heterogeneity ofa cell population This aspect might be particularly relevantin the context of PCa which is a neoplastic disease charac-terized by extreme heterogeneity and unpredicted responseof patients to the therapy [1 2] The improvements in cellisolation techniques coupled to the higher sensitivity of thenext-generation sequencing techniques may soon allow ahighly detailed description of the transcriptome of patientswhich might result in more personalized treatments
The studies illustrated previously suggest that the upregu-lation of selected splicing regulators in PCa such as SAM68SRSF1 or DDX5 directly contributes to the phenotype byaltering the splicing profile of key genes Thus these RBPsmight represent potential therapeutic targets for interventionAlthough blocking the activity of a given splicing factor isnot necessarily an easy task some examples in this direction
have been provided For instance SAM68 can bind to RNAonly as a dimer By exploiting this requirement it was shownthat an RNA binding-defective SAM68 mutant exerted dom-inant negative effects on SAM68-mediated SMN2 splicing byassociating with the endogenous protein and preventing itsbinding to the pre-mRNA [71]This experiment suggests thatsmall molecules interfering with SAM68 function might dis-play therapeutic potential As homodimerization is a prereq-uisite for RNA binding one possibility is to target the SAM68dimerization domain which was restricted to a small regionwithin its Gld1-Sam68-Grp33 (GSG) homology domain [72]The potential value of targeting specific components of thesplicing machinery in cancer cells is also suggested by theantioncogenic properties of natural compounds such asspliceostatin A (SSA) in a variety of cancer cell modelsSSA targets the splicing factor 3B subunit 1 (SF3B1) of thespliceosome thus affecting a large number of splicing eventsconcomitantly [73] Perhaps more specific drugs targetingsplicing factors involved in subsets of oncogenic splicingevents in cancer cells as those described above might repre-sent more specific therapeutic approaches in the next future
Although the extreme flexibility of AS regulation is proneto errors that may concur to neoplastic transformation [7 8]it can also be exploited therapeutically Indeed examples ofAS modulation in selected genes by administering splicing-correcting ASOs to cells have been reported In some casesthis approach has also been challenged with a therapeuticapplication One of the most remarkable examples is repre-sented by the recovery of the phenotype observed in mousemodels of Spinal Muscular Atrophy (SMA) This neurode-generative disease is caused by inactivation of the SMN1 geneand skipping of exon 7 in the highly homologous SMN2 gene
8 International Journal of Cell Biology
[74] It was recently demonstrated that systemic injectionof a chemically modified ASO restored SMN2 splicing invitro and in vivo and profoundly ameliorated the viabilityand phenotypic features of mice affected by a severe form ofSMA [75] Although cancer is caused by multiple alterationsthus limiting the application of gene-specific ASOs it isconceivable that these tools could be used in combinationwith standard therapies to improve the clinical response ofpatients For instance an ASO that switched BCL-X splicingtoward the proapoptotic variant was effective in sensitizingcancer cells to drug-induced apoptosis and to reduce growthof tumors in nude mice [30 31] A similar effect was obtainedby switching expression of the 120572 to the 120573 variant of the signaltransducer and activator of transcription 3 (STAT3) genewhich modulates multiple oncogenic pathways [76] In thiscase administration of a modified ASO targeted to a splicingenhancer induced expression of the endogenous STAT3120573 andan anti-oncogenic response in vitro and in vivo [76] Thesestudies suggest that modulation of AS with synthetic drugsis possible and has entered a therapeutic perspective ASOsare particularly appealing in terms of high specificity andreduced side effects as theymay exploit their ability to annealwith specific sequences in the genomewithout affecting otherfeatures or target genes of the splicing factors involved in theoncogenicAS eventThus it is likely that thesemethods couldbe applied soon to the development of novel therapies aimedat fighting human cancers in which expression of specificoncogenic splice variants has been firmly confirmed
Conflict of Interests
The author declares no conflict of interests
Acknowledgments
The author wishes to thank Dr Chiara Naro for help with thepreparation of the figures and critical reading of the paperThe work in the laboratory of C Sette was supported byAssociation for International Cancer Research (AICR Grantno 12-0150) the Associazione Italiana Ricerca sul Cancro(AIRC) and Istituto Superiore della Sanita (ISS Grant Con-venzione 11US6A)
References
[1] B J Feldman and D Feldman ldquoThe development of androgen-independent prostate cancerrdquoNature Reviews Cancer vol 1 no1 pp 34ndash45 2001
[2] M Kohli and D J Tindall ldquoNew developments in the medicalmanagement of prostate cancerrdquo Mayo Clinic Proceedings vol85 no 1 pp 77ndash86 2010
[3] K E Knudsen and H I Scher ldquoStarving the addiction newopportunities for durable suppression of AR signaling in pros-tate cancerrdquo Clinical Cancer Research vol 15 no 15 pp 4792ndash4798 2009
[4] S M Dehm and D J Tindall ldquoAlternatively spliced androgenreceptor variantsrdquo Endocrine-Related Cancer vol 18 no 5 ppR183ndashR196 2011
[5] A Kalsotra and T A Cooper ldquoFunctional consequences ofdevelopmentally regulated alternative splicingrdquoNature ReviewsGenetics vol 12 no 10 pp 715ndash729 2011
[6] C Zhang H-R Li J-B Fan et al ldquoProfiling alternativelysplicedmRNA isoforms for prostate cancer classificationrdquo BMCBioinformatics vol 7 article 202 2006
[7] C J David and J L Manley ldquoAlternative pre-mRNA splicingregulation in cancer pathways and programs unhingedrdquo Genesand Development vol 24 no 21 pp 2343ndash2364 2010
[8] S Pal R Gupta and R V Davuluri ldquoAlternative transcriptionand alternative splicing in cancerrdquo Pharmacology amp Therapeu-tics vol 136 no 3 pp 283ndash294 2012
[9] Y Li M Alsagabi D Fan G S Bova A H Tewfik and S MDehm ldquoIntragenic rearrangement and altered RNA splicing ofthe androgen receptor in a cell-based model of prostate cancerprogressionrdquo Cancer Research vol 71 no 6 pp 2108ndash2117 2011
[10] S M Dehm L J Schmidt H V Heemers R L Vessella and DJ Tindall ldquoSplicing of a novel androgen receptor exon generatesa constitutively active androgen receptor that mediates prostatecancer therapy resistancerdquo Cancer Research vol 68 no 13 pp5469ndash5477 2008
[11] R Hu T A Dunn SWei et al ldquoLigand-independent androgenreceptor variants derived from splicing of cryptic exons signifyhormone-refractory prostate cancerrdquo Cancer Research vol 69no 1 pp 16ndash22 2009
[12] Z Guo X Yang F Sun et al ldquoA novel androgen receptor splicevariant is up-regulated during prostate cancer progressionand promotes androgen depletion-resistant growthrdquo CancerResearch vol 69 no 6 pp 2305ndash2313 2009
[13] Y Li S C Chan L J Brand T H Hwang K A Silverstein andS M Dehm ldquoAndrogen receptor splice variants mediate enza-lutamide resistance in castration-resistant prostate cancer celllinesrdquo Cancer Research vol 73 no 2 pp 483ndash489 2013
[14] G Narla K E Heath H L Reeves et al ldquoKLF6 a candidatetumor suppressor genemutated in prostate cancerrdquo Science vol294 no 5551 pp 2563ndash2566 2001
[15] X Liu A Gomez-Pinillos C Loder et al ldquoKLF6 loss of func-tion in human prostate cancer progression is implicated inresistance to androgen deprivationrdquo The American Journal ofPathology vol 181 no 3 pp 1007ndash1016 2012
[16] G Narla A DiFeo H L Reeves et al ldquoA germline DNApolymorphism enhances alternative splicing of the KLF6 tumorsuppressor gene and is associatedwith increased prostate cancerriskrdquo Cancer Research vol 65 no 4 pp 1213ndash1222 2005
[17] G Narla A DiFeo Y Fernandez et al ldquoKLF6-SV1 overexpres-sion accelerates human and mouse prostate cancer progressionand metastasisrdquo Journal of Clinical Investigation vol 118 no 8pp 2711ndash2721 2008
[18] G Narla A Difeo S Yao et al ldquoTargeted inhibition of theKLF6 splice variant KLF6 SV1 suppresses prostate cancer cellgrowth and spreadrdquo Cancer Research vol 65 no 13 pp 5761ndash5768 2005
[19] K E Knudsen ldquoThe cyclin D1b splice variant an old oncogenelearns new tricksrdquo Cell Division vol 1 article 15 2006
[20] K E Knudsen J A Diehl C A Haiman and E S KnudsenldquoCyclin D1 polymorphism aberrant splicing and cancer riskrdquoOncogene vol 25 no 11 pp 1620ndash1628 2006
[21] F Lu A B Gladden and J A Diehl ldquoAn alternatively splicedcyclin D1 isoform cyclin D1b is a nuclear oncogenerdquo CancerResearch vol 63 no 21 pp 7056ndash7061 2003
International Journal of Cell Biology 9
[22] C E S Comstock M A Augello R Pe Benito et al ldquoCyclinD1 splice variants polymorphism risk and isoform-specificregulation in prostate cancerrdquo Clinical Cancer Research vol 15no 17 pp 5338ndash5349 2009
[23] M A Augello C J Burd R Birbe et al ldquoConvergence of onco-genic and hormone receptor pathways promotes metastaticphenotypesrdquo The Journal of Clinical Investigation vol 123 no1 pp 493ndash508 2013
[24] N A Olshavsky C E S Comstock M J Schiewer et al ldquoIden-tification of ASFSF2 as a critical allele-specific effector of thecyclin D1b oncogenerdquoCancer Research vol 70 no 10 pp 3975ndash3984 2010
[25] M P Paronetto M Cappellari R Busa et al ldquoAlternative splic-ing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68rdquoCancer Research vol 70 no 1 pp 229ndash239 2010
[26] D Kaida M G Berg I Younis et al ldquoU1 snRNP protects pre-mRNAs frompremature cleavage and polyadenylationrdquoNaturevol 468 no 7324 pp 664ndash668 2010
[27] P Bielli R Busa M P Paronetto and C Sette ldquoThe RNA-binding protein Sam68 is a multifunctional player in humancancerrdquo Endocrine-Related Cancer vol 18 no 4 pp R91ndashR1022011
[28] L H Boise M Gonzalez-Garcia C E Postema et al ldquobcl-xA bcl-2-related gene that functions as a dominant regulator ofapoptotic cell deathrdquo Cell vol 74 no 4 pp 597ndash608 1993
[29] D R Mercatante C D Bortner J A Cidlowski and R KoleldquoModification of alternative splicing of Bcl-x Pre-mRNA inprostate and breast cancer cells analysis of apoptosis and celldeathrdquo Journal of Biological Chemistry vol 276 no 19 pp 16411ndash16417 2001
[30] D R Mercatante J L Mohler and R Kole ldquoCellular responseto an antisense-mediated shift of Bcl-x pre-mRNA splicing andantineoplastic agentsrdquo Journal of Biological Chemistry vol 277no 51 pp 49374ndash49382 2002
[31] J A Bauman S-D Li A Yang L Huang and R Kole ldquoAnti-tumor activity of splice-switching oligonucleotidesrdquo NucleicAcids Research vol 38 no 22 pp 8348ndash8356 2010
[32] C E Chalfant K Rathman R L Pinkerman et al ldquoDe novoceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells Dependence on proteinphosphatase-1rdquo Journal of Biological Chemistry vol 277 no 15pp 12587ndash12595 2002
[33] K Boon-Unge Q Yu T Zou A Zhou P Govitrapong andJ Zhou ldquoEmetine regulates the alternative splicing of Bcl-xthrough a protein phosphatase 1-dependent mechanismrdquoChemistry and Biology vol 14 no 12 pp 1386ndash1392 2007
[34] M H Kim ldquoProtein phosphatase 1 activation and alternativesplicing of Bcl-X and Mcl-1 by EGCG + ibuprofenrdquo Journal ofCellular Biochemistry vol 104 no 4 pp 1491ndash1499 2008
[35] T Novoyatleva B Heinrich Y Tang et al ldquoProtein phosphatase1 binds to the RNA recognition motif of several splicing fac-tors and regulates alternative pre-mRNA processingrdquo HumanMolecular Genetics vol 17 no 1 pp 52ndash70 2008
[36] N Ghosh N Patel K Jiang et al ldquoCeramide-activated pro-tein phosphatase involvement in insulin resistance via Aktserinearginine-rich protein 40 and ribonucleic acid splicing inL6 skeletal muscle cellsrdquo Endocrinology vol 148 no 3 pp 1359ndash1366 2007
[37] D Garneau T Revil J-F Fisette and B Chabot ldquoHetero-geneous nuclear ribonucleoprotein FH proteins modulate
the alternative splicing of the apoptotic mediator Bcl-xrdquo Journalof Biological Chemistry vol 280 no 24 pp 22641ndash22650 2005
[38] M P Paronetto T Achsel A Massiello C E Chalfant andC Sette ldquoThe RNA-binding protein Sam68 modulates thealternative splicing of Bcl-xrdquo Journal of Cell Biology vol 176 no7 pp 929ndash939 2007
[39] A Zhou A C Ou A Cho E J Benz Jr and S-C HuangldquoNovel splicing factor RBM25 modulates Bcl-x Pre-mRNA 51015840splice site selectionrdquoMolecular and Cellular Biology vol 28 no19 pp 5924ndash5936 2008
[40] S Pedrotti R Busa C Compagnucci and C Sette ldquoThe RNArecognition motif protein RBM11 is a novel tissue-specificsplicing regulatorrdquo Nucleic Acids Research vol 40 no 3 pp1021ndash1032 2012
[41] T Revil J Pelletier J Toutant A Cloutier and B ChabotldquoHeterogeneous nuclear ribonucleoprotein K represses theproduction of pro-apoptotic Bcl-xS splice isoformrdquo Journal ofBiological Chemistry vol 284 no 32 pp 21458ndash21467 2009
[42] M J Moore Q Wang C J Kennedy and P A Silver ldquoAn alter-native splicing network links cell-cycle control to apoptosisrdquoCell vol 142 no 4 pp 625ndash636 2010
[43] P Cloutier J Toutant L Shkreta S Goekjian T Revil and BChabot ldquoAntagonistic effects of the SRp30c protein and cryptic51015840 splice sites on the alternative splicing of the apoptoticregulator Bcl-xrdquo Journal of Biological Chemistry vol 283 no 31pp 21315ndash21324 2008
[44] A Massiello J R Roesser and C E Chalfant ldquoSAP155 Bindsto ceramide-responsive RNA cis-element 1 and regulates thealternative 51015840 splice site selection of Bcl-x pre-mRNArdquo TheFASEB Journal vol 20 no 10 pp 1680ndash1682 2006
[45] R BusaM P Paronetto D Farini et al ldquoTheRNA-binding pro-tein Sam68 contributes to proliferation and survival of humanprostate cancer cellsrdquo Oncogene vol 26 no 30 pp 4372ndash43822007
[46] C Sette ldquoPost-translational regulation of star proteins andeffects on their biological functionsrdquo Advances in ExperimentalMedicine and Biology vol 693 pp 54ndash66 2010
[47] C Brignatz M P Paronetto S Opi et al ldquoAlternative splicingmodulates autoinhibition and SH3 accessibility in the Src kinaseFynrdquo Molecular and Cellular Biology vol 29 no 24 pp 6438ndash6448 2009
[48] M P Paronetto D Farini I Sammarco et al ldquoExpression of atruncated form of the c-Kit tyrosine kinase receptor and activa-tion of Src kinase in human prostatic cancerrdquo American Journalof Pathology vol 164 no 4 pp 1243ndash1251 2004
[49] Y Hu A Dobi T Sreenath et al ldquoDelineation of TMPRSS2-ERG splice variants in prostate cancerrdquo Clinical CancerResearch vol 14 no 15 pp 4719ndash4725 2008
[50] J Wang Y Cai W Yu C Ren D M Spencer and M Itt-mann ldquoPleiotropic biological activities of alternatively splicedTMPRSS2ERG fusion gene transcriptsrdquo Cancer Research vol68 no 20 pp 8516ndash8524 2008
[51] F Zammarchi G Boutsalis and L Cartegni ldquo51015840 UTR controlof native ERG and of Tmprss2 ERG variants activity in prostatecancerrdquo PLoS One vol 8 no 3 Article ID e49721 2013
[52] M J Munoz M S P Santangelo M P Paronetto et al ldquoDNAdamage regulates alternative splicing through inhibition ofRNApolymerase II elongationrdquoCell vol 137 no 4 pp 708ndash7202009
[53] P Rajan C Dalgliesh P J Carling et al ldquoIdentification of novelandrogen-regulated pathways and mrna isoforms through
10 International Journal of Cell Biology
genome-wide exon-specific profiling of the LNCaP transcrip-tomerdquo PLoS One vol 6 no 12 Article ID e29088 2011
[54] T M Morgan T D Koreckij and E Corey ldquoTargeted therapyfor advanced prostate cancer inhibition of the PI3KAktmTORpathwayrdquoCurrent CancerDrug Targets vol 9 no 2 pp 237ndash2492009
[55] J Sun A L Blair S E Aiyar and R Li ldquoCofactor of BRCA1modulates androgen-dependent transcription and alternativesplicingrdquo Journal of Steroid Biochemistry andMolecular Biologyvol 107 no 3ndash5 pp 131ndash139 2007
[56] E L Clark A Coulson C Dalgliesh et al ldquoThe RNA helicasep68 is a novel androgen receptor coactivator involved in splicingand is overexpressed in prostate cancerrdquo Cancer Research vol68 no 19 pp 7938ndash7946 2008
[57] E Zonta D Bittencourt S Samaan S Germann M Dutertreand D Auboeuf ldquoThe RNA helicase DDX5p68 is a key factorpromoting c-fos expression at different levels from transcriptionto mRNA exportrdquoNucleic Acids Research vol 41 no 1 pp 554ndash564 2013
[58] A Honig D Auboeuf M M Parker B W OrsquoMalley andS M Berget ldquoRegulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72rdquo Molecular and Cel-lular Biology vol 22 no 16 pp 5698ndash5707 2002
[59] X Dong J Sweet J R G Challis T Brown and S J Lye ldquoTran-scriptional activity of androgen receptor is modulated by twoRNA splicing factors PSF and p54nrbrdquoMolecular and CellularBiology vol 27 no 13 pp 4863ndash4875 2007
[60] P Rajan L Gaughan C Dalgliesh et al ldquoThe RNA-binding andadaptor protein Sam68 modulates signal-dependent splicingand transcriptional activity of the androgen receptorrdquo Journalof Pathology vol 215 no 1 pp 67ndash77 2008
[61] R Karni E De Stanchina S W Lowe R Sinha D Mu and AR Krainer ldquoThe gene encoding the splicing factor SF2ASF is aproto-oncogenerdquo Nature Structural and Molecular Biology vol14 no 3 pp 185ndash193 2007
[62] OAnczukowA Z RosenbergMAkerman et al ldquoThe splicingfactor SRSF1 regulates apoptosis and proliferation to promotemammary epithelial cell transformationrdquoNature Structural andMolecular Biology vol 19 no 2 pp 220ndash228 2012
[63] L Adesso S Calabretta F Barbagallo et al ldquoGemcitabine trig-gers a pro-survival response in pancreatic cancer cells throughactivation of the MNK2eIF4E pathwayrdquo Oncogene vol 32 pp2848ndash2857 2013
[64] A Bianchini M Loiarro P Bielli et al ldquoPhosphorylation ofeIF4E by MNKs supports protein synthesis cell cycle progres-sion and proliferation in prostate cancer cellsrdquo Carcinogenesisvol 29 no 12 pp 2279ndash2288 2008
[65] L Furic L RongO Larsson et al ldquoEIF4Ephosphorylation pro-motes tumorigenesis and is associated with prostate cancer pro-gressionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 32 pp 14134ndash14139 2010
[66] C Naro and C Sette ldquoPhosphorylation-mediated regulation ofalternative splicing in cancerrdquo International Journal of Cell Bio-logy In press
[67] Z Zhou J QiuW Liu et al ldquoTheAkt-SRPK-SR axis constitutesa major pathway in transducing EGF signaling to regulatealternative splicing in the nucleusrdquo Molecular Cell vol 47 no3 pp 422ndash433 2012
[68] NMatter P Herrlich andH Konig ldquoSignal-dependent regula-tion of splicing via phosphorylation of Sam68rdquoNature vol 420no 6916 pp 691ndash695 2002
[69] A M Al-Ayoubi H Zheng Y Liu T Bai and S T EblenldquoMitogen-activated protein kinase phosphorylation of splicingfactor 45 (SPF45) regulates SPF45 alternative splicing siteutilization proliferation and cell adhesionrdquoMolecular and Cel-lular Biology vol 32 no 14 pp 2880ndash2893 2012
[70] A K Shalek R Satija X Adiconis et al ldquoSingle-cell transcrip-tomics reveals bimodality in expression and splicing in immunecellsrdquo Nature 2013
[71] S Pedrotti P BielliM P Paronetto et al ldquoThe splicing regulatorSam68 binds to a novel exonic splicing silencer and functions inSMN2 alternative splicing in spinal muscular atrophyrdquo EMBOJournal vol 29 no 7 pp 1235ndash1247 2010
[72] N H Meyer K Tripsianes M Vincendeau et al ldquoStructuralbasis for homodimerization of the Src-associated during mito-sis 68-kDa protein (Sam68)Qua1 domainrdquo Journal of BiologicalChemistry vol 285 no 37 pp 28893ndash28901 2010
[73] S Bonnal L Vigevani and J Valcarcel ldquoThe spliceosome asa target of novel antitumour drugsrdquo Nature Reviews Drug Dis-covery vol 11 no 11 pp 847ndash859 2012
[74] S Pedrotti and C Sette ldquoSpinal muscular atrophy a new playerjoins the battle for SMN2 exon 7 splicingrdquo Cell Cycle vol 9 no19 pp 3874ndash3879 2010
[75] Y Hua K Sahashi F Rigo et al ldquoPeripheral SMN restoration isessential for long-term rescue of a severe spinal muscular atro-phy mouse modelrdquo Nature vol 478 no 7367 pp 123ndash126 2011
[76] F Zammarchi E De Stanchina E Bournazou et al ldquoAntitu-morigenic potential of STAT3 alternative splicing modulationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 43 pp 17779ndash17784 2011
[74] It was recently demonstrated that systemic injectionof a chemically modified ASO restored SMN2 splicing invitro and in vivo and profoundly ameliorated the viabilityand phenotypic features of mice affected by a severe form ofSMA [75] Although cancer is caused by multiple alterationsthus limiting the application of gene-specific ASOs it isconceivable that these tools could be used in combinationwith standard therapies to improve the clinical response ofpatients For instance an ASO that switched BCL-X splicingtoward the proapoptotic variant was effective in sensitizingcancer cells to drug-induced apoptosis and to reduce growthof tumors in nude mice [30 31] A similar effect was obtainedby switching expression of the 120572 to the 120573 variant of the signaltransducer and activator of transcription 3 (STAT3) genewhich modulates multiple oncogenic pathways [76] In thiscase administration of a modified ASO targeted to a splicingenhancer induced expression of the endogenous STAT3120573 andan anti-oncogenic response in vitro and in vivo [76] Thesestudies suggest that modulation of AS with synthetic drugsis possible and has entered a therapeutic perspective ASOsare particularly appealing in terms of high specificity andreduced side effects as theymay exploit their ability to annealwith specific sequences in the genomewithout affecting otherfeatures or target genes of the splicing factors involved in theoncogenicAS eventThus it is likely that thesemethods couldbe applied soon to the development of novel therapies aimedat fighting human cancers in which expression of specificoncogenic splice variants has been firmly confirmed
Conflict of Interests
The author declares no conflict of interests
Acknowledgments
The author wishes to thank Dr Chiara Naro for help with thepreparation of the figures and critical reading of the paperThe work in the laboratory of C Sette was supported byAssociation for International Cancer Research (AICR Grantno 12-0150) the Associazione Italiana Ricerca sul Cancro(AIRC) and Istituto Superiore della Sanita (ISS Grant Con-venzione 11US6A)
References
[1] B J Feldman and D Feldman ldquoThe development of androgen-independent prostate cancerrdquoNature Reviews Cancer vol 1 no1 pp 34ndash45 2001
[2] M Kohli and D J Tindall ldquoNew developments in the medicalmanagement of prostate cancerrdquo Mayo Clinic Proceedings vol85 no 1 pp 77ndash86 2010
[3] K E Knudsen and H I Scher ldquoStarving the addiction newopportunities for durable suppression of AR signaling in pros-tate cancerrdquo Clinical Cancer Research vol 15 no 15 pp 4792ndash4798 2009
[4] S M Dehm and D J Tindall ldquoAlternatively spliced androgenreceptor variantsrdquo Endocrine-Related Cancer vol 18 no 5 ppR183ndashR196 2011
[5] A Kalsotra and T A Cooper ldquoFunctional consequences ofdevelopmentally regulated alternative splicingrdquoNature ReviewsGenetics vol 12 no 10 pp 715ndash729 2011
[6] C Zhang H-R Li J-B Fan et al ldquoProfiling alternativelysplicedmRNA isoforms for prostate cancer classificationrdquo BMCBioinformatics vol 7 article 202 2006
[7] C J David and J L Manley ldquoAlternative pre-mRNA splicingregulation in cancer pathways and programs unhingedrdquo Genesand Development vol 24 no 21 pp 2343ndash2364 2010
[8] S Pal R Gupta and R V Davuluri ldquoAlternative transcriptionand alternative splicing in cancerrdquo Pharmacology amp Therapeu-tics vol 136 no 3 pp 283ndash294 2012
[9] Y Li M Alsagabi D Fan G S Bova A H Tewfik and S MDehm ldquoIntragenic rearrangement and altered RNA splicing ofthe androgen receptor in a cell-based model of prostate cancerprogressionrdquo Cancer Research vol 71 no 6 pp 2108ndash2117 2011
[10] S M Dehm L J Schmidt H V Heemers R L Vessella and DJ Tindall ldquoSplicing of a novel androgen receptor exon generatesa constitutively active androgen receptor that mediates prostatecancer therapy resistancerdquo Cancer Research vol 68 no 13 pp5469ndash5477 2008
[11] R Hu T A Dunn SWei et al ldquoLigand-independent androgenreceptor variants derived from splicing of cryptic exons signifyhormone-refractory prostate cancerrdquo Cancer Research vol 69no 1 pp 16ndash22 2009
[12] Z Guo X Yang F Sun et al ldquoA novel androgen receptor splicevariant is up-regulated during prostate cancer progressionand promotes androgen depletion-resistant growthrdquo CancerResearch vol 69 no 6 pp 2305ndash2313 2009
[13] Y Li S C Chan L J Brand T H Hwang K A Silverstein andS M Dehm ldquoAndrogen receptor splice variants mediate enza-lutamide resistance in castration-resistant prostate cancer celllinesrdquo Cancer Research vol 73 no 2 pp 483ndash489 2013
[14] G Narla K E Heath H L Reeves et al ldquoKLF6 a candidatetumor suppressor genemutated in prostate cancerrdquo Science vol294 no 5551 pp 2563ndash2566 2001
[15] X Liu A Gomez-Pinillos C Loder et al ldquoKLF6 loss of func-tion in human prostate cancer progression is implicated inresistance to androgen deprivationrdquo The American Journal ofPathology vol 181 no 3 pp 1007ndash1016 2012
[16] G Narla A DiFeo H L Reeves et al ldquoA germline DNApolymorphism enhances alternative splicing of the KLF6 tumorsuppressor gene and is associatedwith increased prostate cancerriskrdquo Cancer Research vol 65 no 4 pp 1213ndash1222 2005
[17] G Narla A DiFeo Y Fernandez et al ldquoKLF6-SV1 overexpres-sion accelerates human and mouse prostate cancer progressionand metastasisrdquo Journal of Clinical Investigation vol 118 no 8pp 2711ndash2721 2008
[18] G Narla A Difeo S Yao et al ldquoTargeted inhibition of theKLF6 splice variant KLF6 SV1 suppresses prostate cancer cellgrowth and spreadrdquo Cancer Research vol 65 no 13 pp 5761ndash5768 2005
[19] K E Knudsen ldquoThe cyclin D1b splice variant an old oncogenelearns new tricksrdquo Cell Division vol 1 article 15 2006
[20] K E Knudsen J A Diehl C A Haiman and E S KnudsenldquoCyclin D1 polymorphism aberrant splicing and cancer riskrdquoOncogene vol 25 no 11 pp 1620ndash1628 2006
[21] F Lu A B Gladden and J A Diehl ldquoAn alternatively splicedcyclin D1 isoform cyclin D1b is a nuclear oncogenerdquo CancerResearch vol 63 no 21 pp 7056ndash7061 2003
International Journal of Cell Biology 9
[22] C E S Comstock M A Augello R Pe Benito et al ldquoCyclinD1 splice variants polymorphism risk and isoform-specificregulation in prostate cancerrdquo Clinical Cancer Research vol 15no 17 pp 5338ndash5349 2009
[23] M A Augello C J Burd R Birbe et al ldquoConvergence of onco-genic and hormone receptor pathways promotes metastaticphenotypesrdquo The Journal of Clinical Investigation vol 123 no1 pp 493ndash508 2013
[24] N A Olshavsky C E S Comstock M J Schiewer et al ldquoIden-tification of ASFSF2 as a critical allele-specific effector of thecyclin D1b oncogenerdquoCancer Research vol 70 no 10 pp 3975ndash3984 2010
[25] M P Paronetto M Cappellari R Busa et al ldquoAlternative splic-ing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68rdquoCancer Research vol 70 no 1 pp 229ndash239 2010
[26] D Kaida M G Berg I Younis et al ldquoU1 snRNP protects pre-mRNAs frompremature cleavage and polyadenylationrdquoNaturevol 468 no 7324 pp 664ndash668 2010
[27] P Bielli R Busa M P Paronetto and C Sette ldquoThe RNA-binding protein Sam68 is a multifunctional player in humancancerrdquo Endocrine-Related Cancer vol 18 no 4 pp R91ndashR1022011
[28] L H Boise M Gonzalez-Garcia C E Postema et al ldquobcl-xA bcl-2-related gene that functions as a dominant regulator ofapoptotic cell deathrdquo Cell vol 74 no 4 pp 597ndash608 1993
[29] D R Mercatante C D Bortner J A Cidlowski and R KoleldquoModification of alternative splicing of Bcl-x Pre-mRNA inprostate and breast cancer cells analysis of apoptosis and celldeathrdquo Journal of Biological Chemistry vol 276 no 19 pp 16411ndash16417 2001
[30] D R Mercatante J L Mohler and R Kole ldquoCellular responseto an antisense-mediated shift of Bcl-x pre-mRNA splicing andantineoplastic agentsrdquo Journal of Biological Chemistry vol 277no 51 pp 49374ndash49382 2002
[31] J A Bauman S-D Li A Yang L Huang and R Kole ldquoAnti-tumor activity of splice-switching oligonucleotidesrdquo NucleicAcids Research vol 38 no 22 pp 8348ndash8356 2010
[32] C E Chalfant K Rathman R L Pinkerman et al ldquoDe novoceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells Dependence on proteinphosphatase-1rdquo Journal of Biological Chemistry vol 277 no 15pp 12587ndash12595 2002
[33] K Boon-Unge Q Yu T Zou A Zhou P Govitrapong andJ Zhou ldquoEmetine regulates the alternative splicing of Bcl-xthrough a protein phosphatase 1-dependent mechanismrdquoChemistry and Biology vol 14 no 12 pp 1386ndash1392 2007
[34] M H Kim ldquoProtein phosphatase 1 activation and alternativesplicing of Bcl-X and Mcl-1 by EGCG + ibuprofenrdquo Journal ofCellular Biochemistry vol 104 no 4 pp 1491ndash1499 2008
[35] T Novoyatleva B Heinrich Y Tang et al ldquoProtein phosphatase1 binds to the RNA recognition motif of several splicing fac-tors and regulates alternative pre-mRNA processingrdquo HumanMolecular Genetics vol 17 no 1 pp 52ndash70 2008
[36] N Ghosh N Patel K Jiang et al ldquoCeramide-activated pro-tein phosphatase involvement in insulin resistance via Aktserinearginine-rich protein 40 and ribonucleic acid splicing inL6 skeletal muscle cellsrdquo Endocrinology vol 148 no 3 pp 1359ndash1366 2007
[37] D Garneau T Revil J-F Fisette and B Chabot ldquoHetero-geneous nuclear ribonucleoprotein FH proteins modulate
the alternative splicing of the apoptotic mediator Bcl-xrdquo Journalof Biological Chemistry vol 280 no 24 pp 22641ndash22650 2005
[38] M P Paronetto T Achsel A Massiello C E Chalfant andC Sette ldquoThe RNA-binding protein Sam68 modulates thealternative splicing of Bcl-xrdquo Journal of Cell Biology vol 176 no7 pp 929ndash939 2007
[39] A Zhou A C Ou A Cho E J Benz Jr and S-C HuangldquoNovel splicing factor RBM25 modulates Bcl-x Pre-mRNA 51015840splice site selectionrdquoMolecular and Cellular Biology vol 28 no19 pp 5924ndash5936 2008
[40] S Pedrotti R Busa C Compagnucci and C Sette ldquoThe RNArecognition motif protein RBM11 is a novel tissue-specificsplicing regulatorrdquo Nucleic Acids Research vol 40 no 3 pp1021ndash1032 2012
[41] T Revil J Pelletier J Toutant A Cloutier and B ChabotldquoHeterogeneous nuclear ribonucleoprotein K represses theproduction of pro-apoptotic Bcl-xS splice isoformrdquo Journal ofBiological Chemistry vol 284 no 32 pp 21458ndash21467 2009
[42] M J Moore Q Wang C J Kennedy and P A Silver ldquoAn alter-native splicing network links cell-cycle control to apoptosisrdquoCell vol 142 no 4 pp 625ndash636 2010
[43] P Cloutier J Toutant L Shkreta S Goekjian T Revil and BChabot ldquoAntagonistic effects of the SRp30c protein and cryptic51015840 splice sites on the alternative splicing of the apoptoticregulator Bcl-xrdquo Journal of Biological Chemistry vol 283 no 31pp 21315ndash21324 2008
[44] A Massiello J R Roesser and C E Chalfant ldquoSAP155 Bindsto ceramide-responsive RNA cis-element 1 and regulates thealternative 51015840 splice site selection of Bcl-x pre-mRNArdquo TheFASEB Journal vol 20 no 10 pp 1680ndash1682 2006
[45] R BusaM P Paronetto D Farini et al ldquoTheRNA-binding pro-tein Sam68 contributes to proliferation and survival of humanprostate cancer cellsrdquo Oncogene vol 26 no 30 pp 4372ndash43822007
[46] C Sette ldquoPost-translational regulation of star proteins andeffects on their biological functionsrdquo Advances in ExperimentalMedicine and Biology vol 693 pp 54ndash66 2010
[47] C Brignatz M P Paronetto S Opi et al ldquoAlternative splicingmodulates autoinhibition and SH3 accessibility in the Src kinaseFynrdquo Molecular and Cellular Biology vol 29 no 24 pp 6438ndash6448 2009
[48] M P Paronetto D Farini I Sammarco et al ldquoExpression of atruncated form of the c-Kit tyrosine kinase receptor and activa-tion of Src kinase in human prostatic cancerrdquo American Journalof Pathology vol 164 no 4 pp 1243ndash1251 2004
[49] Y Hu A Dobi T Sreenath et al ldquoDelineation of TMPRSS2-ERG splice variants in prostate cancerrdquo Clinical CancerResearch vol 14 no 15 pp 4719ndash4725 2008
[50] J Wang Y Cai W Yu C Ren D M Spencer and M Itt-mann ldquoPleiotropic biological activities of alternatively splicedTMPRSS2ERG fusion gene transcriptsrdquo Cancer Research vol68 no 20 pp 8516ndash8524 2008
[51] F Zammarchi G Boutsalis and L Cartegni ldquo51015840 UTR controlof native ERG and of Tmprss2 ERG variants activity in prostatecancerrdquo PLoS One vol 8 no 3 Article ID e49721 2013
[52] M J Munoz M S P Santangelo M P Paronetto et al ldquoDNAdamage regulates alternative splicing through inhibition ofRNApolymerase II elongationrdquoCell vol 137 no 4 pp 708ndash7202009
[53] P Rajan C Dalgliesh P J Carling et al ldquoIdentification of novelandrogen-regulated pathways and mrna isoforms through
10 International Journal of Cell Biology
genome-wide exon-specific profiling of the LNCaP transcrip-tomerdquo PLoS One vol 6 no 12 Article ID e29088 2011
[54] T M Morgan T D Koreckij and E Corey ldquoTargeted therapyfor advanced prostate cancer inhibition of the PI3KAktmTORpathwayrdquoCurrent CancerDrug Targets vol 9 no 2 pp 237ndash2492009
[55] J Sun A L Blair S E Aiyar and R Li ldquoCofactor of BRCA1modulates androgen-dependent transcription and alternativesplicingrdquo Journal of Steroid Biochemistry andMolecular Biologyvol 107 no 3ndash5 pp 131ndash139 2007
[56] E L Clark A Coulson C Dalgliesh et al ldquoThe RNA helicasep68 is a novel androgen receptor coactivator involved in splicingand is overexpressed in prostate cancerrdquo Cancer Research vol68 no 19 pp 7938ndash7946 2008
[57] E Zonta D Bittencourt S Samaan S Germann M Dutertreand D Auboeuf ldquoThe RNA helicase DDX5p68 is a key factorpromoting c-fos expression at different levels from transcriptionto mRNA exportrdquoNucleic Acids Research vol 41 no 1 pp 554ndash564 2013
[58] A Honig D Auboeuf M M Parker B W OrsquoMalley andS M Berget ldquoRegulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72rdquo Molecular and Cel-lular Biology vol 22 no 16 pp 5698ndash5707 2002
[59] X Dong J Sweet J R G Challis T Brown and S J Lye ldquoTran-scriptional activity of androgen receptor is modulated by twoRNA splicing factors PSF and p54nrbrdquoMolecular and CellularBiology vol 27 no 13 pp 4863ndash4875 2007
[60] P Rajan L Gaughan C Dalgliesh et al ldquoThe RNA-binding andadaptor protein Sam68 modulates signal-dependent splicingand transcriptional activity of the androgen receptorrdquo Journalof Pathology vol 215 no 1 pp 67ndash77 2008
[61] R Karni E De Stanchina S W Lowe R Sinha D Mu and AR Krainer ldquoThe gene encoding the splicing factor SF2ASF is aproto-oncogenerdquo Nature Structural and Molecular Biology vol14 no 3 pp 185ndash193 2007
[62] OAnczukowA Z RosenbergMAkerman et al ldquoThe splicingfactor SRSF1 regulates apoptosis and proliferation to promotemammary epithelial cell transformationrdquoNature Structural andMolecular Biology vol 19 no 2 pp 220ndash228 2012
[63] L Adesso S Calabretta F Barbagallo et al ldquoGemcitabine trig-gers a pro-survival response in pancreatic cancer cells throughactivation of the MNK2eIF4E pathwayrdquo Oncogene vol 32 pp2848ndash2857 2013
[64] A Bianchini M Loiarro P Bielli et al ldquoPhosphorylation ofeIF4E by MNKs supports protein synthesis cell cycle progres-sion and proliferation in prostate cancer cellsrdquo Carcinogenesisvol 29 no 12 pp 2279ndash2288 2008
[65] L Furic L RongO Larsson et al ldquoEIF4Ephosphorylation pro-motes tumorigenesis and is associated with prostate cancer pro-gressionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 32 pp 14134ndash14139 2010
[66] C Naro and C Sette ldquoPhosphorylation-mediated regulation ofalternative splicing in cancerrdquo International Journal of Cell Bio-logy In press
[67] Z Zhou J QiuW Liu et al ldquoTheAkt-SRPK-SR axis constitutesa major pathway in transducing EGF signaling to regulatealternative splicing in the nucleusrdquo Molecular Cell vol 47 no3 pp 422ndash433 2012
[68] NMatter P Herrlich andH Konig ldquoSignal-dependent regula-tion of splicing via phosphorylation of Sam68rdquoNature vol 420no 6916 pp 691ndash695 2002
[69] A M Al-Ayoubi H Zheng Y Liu T Bai and S T EblenldquoMitogen-activated protein kinase phosphorylation of splicingfactor 45 (SPF45) regulates SPF45 alternative splicing siteutilization proliferation and cell adhesionrdquoMolecular and Cel-lular Biology vol 32 no 14 pp 2880ndash2893 2012
[70] A K Shalek R Satija X Adiconis et al ldquoSingle-cell transcrip-tomics reveals bimodality in expression and splicing in immunecellsrdquo Nature 2013
[71] S Pedrotti P BielliM P Paronetto et al ldquoThe splicing regulatorSam68 binds to a novel exonic splicing silencer and functions inSMN2 alternative splicing in spinal muscular atrophyrdquo EMBOJournal vol 29 no 7 pp 1235ndash1247 2010
[72] N H Meyer K Tripsianes M Vincendeau et al ldquoStructuralbasis for homodimerization of the Src-associated during mito-sis 68-kDa protein (Sam68)Qua1 domainrdquo Journal of BiologicalChemistry vol 285 no 37 pp 28893ndash28901 2010
[73] S Bonnal L Vigevani and J Valcarcel ldquoThe spliceosome asa target of novel antitumour drugsrdquo Nature Reviews Drug Dis-covery vol 11 no 11 pp 847ndash859 2012
[74] S Pedrotti and C Sette ldquoSpinal muscular atrophy a new playerjoins the battle for SMN2 exon 7 splicingrdquo Cell Cycle vol 9 no19 pp 3874ndash3879 2010
[75] Y Hua K Sahashi F Rigo et al ldquoPeripheral SMN restoration isessential for long-term rescue of a severe spinal muscular atro-phy mouse modelrdquo Nature vol 478 no 7367 pp 123ndash126 2011
[76] F Zammarchi E De Stanchina E Bournazou et al ldquoAntitu-morigenic potential of STAT3 alternative splicing modulationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 43 pp 17779ndash17784 2011
[22] C E S Comstock M A Augello R Pe Benito et al ldquoCyclinD1 splice variants polymorphism risk and isoform-specificregulation in prostate cancerrdquo Clinical Cancer Research vol 15no 17 pp 5338ndash5349 2009
[23] M A Augello C J Burd R Birbe et al ldquoConvergence of onco-genic and hormone receptor pathways promotes metastaticphenotypesrdquo The Journal of Clinical Investigation vol 123 no1 pp 493ndash508 2013
[24] N A Olshavsky C E S Comstock M J Schiewer et al ldquoIden-tification of ASFSF2 as a critical allele-specific effector of thecyclin D1b oncogenerdquoCancer Research vol 70 no 10 pp 3975ndash3984 2010
[25] M P Paronetto M Cappellari R Busa et al ldquoAlternative splic-ing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68rdquoCancer Research vol 70 no 1 pp 229ndash239 2010
[26] D Kaida M G Berg I Younis et al ldquoU1 snRNP protects pre-mRNAs frompremature cleavage and polyadenylationrdquoNaturevol 468 no 7324 pp 664ndash668 2010
[27] P Bielli R Busa M P Paronetto and C Sette ldquoThe RNA-binding protein Sam68 is a multifunctional player in humancancerrdquo Endocrine-Related Cancer vol 18 no 4 pp R91ndashR1022011
[28] L H Boise M Gonzalez-Garcia C E Postema et al ldquobcl-xA bcl-2-related gene that functions as a dominant regulator ofapoptotic cell deathrdquo Cell vol 74 no 4 pp 597ndash608 1993
[29] D R Mercatante C D Bortner J A Cidlowski and R KoleldquoModification of alternative splicing of Bcl-x Pre-mRNA inprostate and breast cancer cells analysis of apoptosis and celldeathrdquo Journal of Biological Chemistry vol 276 no 19 pp 16411ndash16417 2001
[30] D R Mercatante J L Mohler and R Kole ldquoCellular responseto an antisense-mediated shift of Bcl-x pre-mRNA splicing andantineoplastic agentsrdquo Journal of Biological Chemistry vol 277no 51 pp 49374ndash49382 2002
[31] J A Bauman S-D Li A Yang L Huang and R Kole ldquoAnti-tumor activity of splice-switching oligonucleotidesrdquo NucleicAcids Research vol 38 no 22 pp 8348ndash8356 2010
[32] C E Chalfant K Rathman R L Pinkerman et al ldquoDe novoceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells Dependence on proteinphosphatase-1rdquo Journal of Biological Chemistry vol 277 no 15pp 12587ndash12595 2002
[33] K Boon-Unge Q Yu T Zou A Zhou P Govitrapong andJ Zhou ldquoEmetine regulates the alternative splicing of Bcl-xthrough a protein phosphatase 1-dependent mechanismrdquoChemistry and Biology vol 14 no 12 pp 1386ndash1392 2007
[34] M H Kim ldquoProtein phosphatase 1 activation and alternativesplicing of Bcl-X and Mcl-1 by EGCG + ibuprofenrdquo Journal ofCellular Biochemistry vol 104 no 4 pp 1491ndash1499 2008
[35] T Novoyatleva B Heinrich Y Tang et al ldquoProtein phosphatase1 binds to the RNA recognition motif of several splicing fac-tors and regulates alternative pre-mRNA processingrdquo HumanMolecular Genetics vol 17 no 1 pp 52ndash70 2008
[36] N Ghosh N Patel K Jiang et al ldquoCeramide-activated pro-tein phosphatase involvement in insulin resistance via Aktserinearginine-rich protein 40 and ribonucleic acid splicing inL6 skeletal muscle cellsrdquo Endocrinology vol 148 no 3 pp 1359ndash1366 2007
[37] D Garneau T Revil J-F Fisette and B Chabot ldquoHetero-geneous nuclear ribonucleoprotein FH proteins modulate
the alternative splicing of the apoptotic mediator Bcl-xrdquo Journalof Biological Chemistry vol 280 no 24 pp 22641ndash22650 2005
[38] M P Paronetto T Achsel A Massiello C E Chalfant andC Sette ldquoThe RNA-binding protein Sam68 modulates thealternative splicing of Bcl-xrdquo Journal of Cell Biology vol 176 no7 pp 929ndash939 2007
[39] A Zhou A C Ou A Cho E J Benz Jr and S-C HuangldquoNovel splicing factor RBM25 modulates Bcl-x Pre-mRNA 51015840splice site selectionrdquoMolecular and Cellular Biology vol 28 no19 pp 5924ndash5936 2008
[40] S Pedrotti R Busa C Compagnucci and C Sette ldquoThe RNArecognition motif protein RBM11 is a novel tissue-specificsplicing regulatorrdquo Nucleic Acids Research vol 40 no 3 pp1021ndash1032 2012
[41] T Revil J Pelletier J Toutant A Cloutier and B ChabotldquoHeterogeneous nuclear ribonucleoprotein K represses theproduction of pro-apoptotic Bcl-xS splice isoformrdquo Journal ofBiological Chemistry vol 284 no 32 pp 21458ndash21467 2009
[42] M J Moore Q Wang C J Kennedy and P A Silver ldquoAn alter-native splicing network links cell-cycle control to apoptosisrdquoCell vol 142 no 4 pp 625ndash636 2010
[43] P Cloutier J Toutant L Shkreta S Goekjian T Revil and BChabot ldquoAntagonistic effects of the SRp30c protein and cryptic51015840 splice sites on the alternative splicing of the apoptoticregulator Bcl-xrdquo Journal of Biological Chemistry vol 283 no 31pp 21315ndash21324 2008
[44] A Massiello J R Roesser and C E Chalfant ldquoSAP155 Bindsto ceramide-responsive RNA cis-element 1 and regulates thealternative 51015840 splice site selection of Bcl-x pre-mRNArdquo TheFASEB Journal vol 20 no 10 pp 1680ndash1682 2006
[45] R BusaM P Paronetto D Farini et al ldquoTheRNA-binding pro-tein Sam68 contributes to proliferation and survival of humanprostate cancer cellsrdquo Oncogene vol 26 no 30 pp 4372ndash43822007
[46] C Sette ldquoPost-translational regulation of star proteins andeffects on their biological functionsrdquo Advances in ExperimentalMedicine and Biology vol 693 pp 54ndash66 2010
[47] C Brignatz M P Paronetto S Opi et al ldquoAlternative splicingmodulates autoinhibition and SH3 accessibility in the Src kinaseFynrdquo Molecular and Cellular Biology vol 29 no 24 pp 6438ndash6448 2009
[48] M P Paronetto D Farini I Sammarco et al ldquoExpression of atruncated form of the c-Kit tyrosine kinase receptor and activa-tion of Src kinase in human prostatic cancerrdquo American Journalof Pathology vol 164 no 4 pp 1243ndash1251 2004
[49] Y Hu A Dobi T Sreenath et al ldquoDelineation of TMPRSS2-ERG splice variants in prostate cancerrdquo Clinical CancerResearch vol 14 no 15 pp 4719ndash4725 2008
[50] J Wang Y Cai W Yu C Ren D M Spencer and M Itt-mann ldquoPleiotropic biological activities of alternatively splicedTMPRSS2ERG fusion gene transcriptsrdquo Cancer Research vol68 no 20 pp 8516ndash8524 2008
[51] F Zammarchi G Boutsalis and L Cartegni ldquo51015840 UTR controlof native ERG and of Tmprss2 ERG variants activity in prostatecancerrdquo PLoS One vol 8 no 3 Article ID e49721 2013
[52] M J Munoz M S P Santangelo M P Paronetto et al ldquoDNAdamage regulates alternative splicing through inhibition ofRNApolymerase II elongationrdquoCell vol 137 no 4 pp 708ndash7202009
[53] P Rajan C Dalgliesh P J Carling et al ldquoIdentification of novelandrogen-regulated pathways and mrna isoforms through
10 International Journal of Cell Biology
genome-wide exon-specific profiling of the LNCaP transcrip-tomerdquo PLoS One vol 6 no 12 Article ID e29088 2011
[54] T M Morgan T D Koreckij and E Corey ldquoTargeted therapyfor advanced prostate cancer inhibition of the PI3KAktmTORpathwayrdquoCurrent CancerDrug Targets vol 9 no 2 pp 237ndash2492009
[55] J Sun A L Blair S E Aiyar and R Li ldquoCofactor of BRCA1modulates androgen-dependent transcription and alternativesplicingrdquo Journal of Steroid Biochemistry andMolecular Biologyvol 107 no 3ndash5 pp 131ndash139 2007
[56] E L Clark A Coulson C Dalgliesh et al ldquoThe RNA helicasep68 is a novel androgen receptor coactivator involved in splicingand is overexpressed in prostate cancerrdquo Cancer Research vol68 no 19 pp 7938ndash7946 2008
[57] E Zonta D Bittencourt S Samaan S Germann M Dutertreand D Auboeuf ldquoThe RNA helicase DDX5p68 is a key factorpromoting c-fos expression at different levels from transcriptionto mRNA exportrdquoNucleic Acids Research vol 41 no 1 pp 554ndash564 2013
[58] A Honig D Auboeuf M M Parker B W OrsquoMalley andS M Berget ldquoRegulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72rdquo Molecular and Cel-lular Biology vol 22 no 16 pp 5698ndash5707 2002
[59] X Dong J Sweet J R G Challis T Brown and S J Lye ldquoTran-scriptional activity of androgen receptor is modulated by twoRNA splicing factors PSF and p54nrbrdquoMolecular and CellularBiology vol 27 no 13 pp 4863ndash4875 2007
[60] P Rajan L Gaughan C Dalgliesh et al ldquoThe RNA-binding andadaptor protein Sam68 modulates signal-dependent splicingand transcriptional activity of the androgen receptorrdquo Journalof Pathology vol 215 no 1 pp 67ndash77 2008
[61] R Karni E De Stanchina S W Lowe R Sinha D Mu and AR Krainer ldquoThe gene encoding the splicing factor SF2ASF is aproto-oncogenerdquo Nature Structural and Molecular Biology vol14 no 3 pp 185ndash193 2007
[62] OAnczukowA Z RosenbergMAkerman et al ldquoThe splicingfactor SRSF1 regulates apoptosis and proliferation to promotemammary epithelial cell transformationrdquoNature Structural andMolecular Biology vol 19 no 2 pp 220ndash228 2012
[63] L Adesso S Calabretta F Barbagallo et al ldquoGemcitabine trig-gers a pro-survival response in pancreatic cancer cells throughactivation of the MNK2eIF4E pathwayrdquo Oncogene vol 32 pp2848ndash2857 2013
[64] A Bianchini M Loiarro P Bielli et al ldquoPhosphorylation ofeIF4E by MNKs supports protein synthesis cell cycle progres-sion and proliferation in prostate cancer cellsrdquo Carcinogenesisvol 29 no 12 pp 2279ndash2288 2008
[65] L Furic L RongO Larsson et al ldquoEIF4Ephosphorylation pro-motes tumorigenesis and is associated with prostate cancer pro-gressionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 32 pp 14134ndash14139 2010
[66] C Naro and C Sette ldquoPhosphorylation-mediated regulation ofalternative splicing in cancerrdquo International Journal of Cell Bio-logy In press
[67] Z Zhou J QiuW Liu et al ldquoTheAkt-SRPK-SR axis constitutesa major pathway in transducing EGF signaling to regulatealternative splicing in the nucleusrdquo Molecular Cell vol 47 no3 pp 422ndash433 2012
[68] NMatter P Herrlich andH Konig ldquoSignal-dependent regula-tion of splicing via phosphorylation of Sam68rdquoNature vol 420no 6916 pp 691ndash695 2002
[69] A M Al-Ayoubi H Zheng Y Liu T Bai and S T EblenldquoMitogen-activated protein kinase phosphorylation of splicingfactor 45 (SPF45) regulates SPF45 alternative splicing siteutilization proliferation and cell adhesionrdquoMolecular and Cel-lular Biology vol 32 no 14 pp 2880ndash2893 2012
[70] A K Shalek R Satija X Adiconis et al ldquoSingle-cell transcrip-tomics reveals bimodality in expression and splicing in immunecellsrdquo Nature 2013
[71] S Pedrotti P BielliM P Paronetto et al ldquoThe splicing regulatorSam68 binds to a novel exonic splicing silencer and functions inSMN2 alternative splicing in spinal muscular atrophyrdquo EMBOJournal vol 29 no 7 pp 1235ndash1247 2010
[72] N H Meyer K Tripsianes M Vincendeau et al ldquoStructuralbasis for homodimerization of the Src-associated during mito-sis 68-kDa protein (Sam68)Qua1 domainrdquo Journal of BiologicalChemistry vol 285 no 37 pp 28893ndash28901 2010
[73] S Bonnal L Vigevani and J Valcarcel ldquoThe spliceosome asa target of novel antitumour drugsrdquo Nature Reviews Drug Dis-covery vol 11 no 11 pp 847ndash859 2012
[74] S Pedrotti and C Sette ldquoSpinal muscular atrophy a new playerjoins the battle for SMN2 exon 7 splicingrdquo Cell Cycle vol 9 no19 pp 3874ndash3879 2010
[75] Y Hua K Sahashi F Rigo et al ldquoPeripheral SMN restoration isessential for long-term rescue of a severe spinal muscular atro-phy mouse modelrdquo Nature vol 478 no 7367 pp 123ndash126 2011
[76] F Zammarchi E De Stanchina E Bournazou et al ldquoAntitu-morigenic potential of STAT3 alternative splicing modulationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 43 pp 17779ndash17784 2011
genome-wide exon-specific profiling of the LNCaP transcrip-tomerdquo PLoS One vol 6 no 12 Article ID e29088 2011
[54] T M Morgan T D Koreckij and E Corey ldquoTargeted therapyfor advanced prostate cancer inhibition of the PI3KAktmTORpathwayrdquoCurrent CancerDrug Targets vol 9 no 2 pp 237ndash2492009
[55] J Sun A L Blair S E Aiyar and R Li ldquoCofactor of BRCA1modulates androgen-dependent transcription and alternativesplicingrdquo Journal of Steroid Biochemistry andMolecular Biologyvol 107 no 3ndash5 pp 131ndash139 2007
[56] E L Clark A Coulson C Dalgliesh et al ldquoThe RNA helicasep68 is a novel androgen receptor coactivator involved in splicingand is overexpressed in prostate cancerrdquo Cancer Research vol68 no 19 pp 7938ndash7946 2008
[57] E Zonta D Bittencourt S Samaan S Germann M Dutertreand D Auboeuf ldquoThe RNA helicase DDX5p68 is a key factorpromoting c-fos expression at different levels from transcriptionto mRNA exportrdquoNucleic Acids Research vol 41 no 1 pp 554ndash564 2013
[58] A Honig D Auboeuf M M Parker B W OrsquoMalley andS M Berget ldquoRegulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72rdquo Molecular and Cel-lular Biology vol 22 no 16 pp 5698ndash5707 2002
[59] X Dong J Sweet J R G Challis T Brown and S J Lye ldquoTran-scriptional activity of androgen receptor is modulated by twoRNA splicing factors PSF and p54nrbrdquoMolecular and CellularBiology vol 27 no 13 pp 4863ndash4875 2007
[60] P Rajan L Gaughan C Dalgliesh et al ldquoThe RNA-binding andadaptor protein Sam68 modulates signal-dependent splicingand transcriptional activity of the androgen receptorrdquo Journalof Pathology vol 215 no 1 pp 67ndash77 2008
[61] R Karni E De Stanchina S W Lowe R Sinha D Mu and AR Krainer ldquoThe gene encoding the splicing factor SF2ASF is aproto-oncogenerdquo Nature Structural and Molecular Biology vol14 no 3 pp 185ndash193 2007
[62] OAnczukowA Z RosenbergMAkerman et al ldquoThe splicingfactor SRSF1 regulates apoptosis and proliferation to promotemammary epithelial cell transformationrdquoNature Structural andMolecular Biology vol 19 no 2 pp 220ndash228 2012
[63] L Adesso S Calabretta F Barbagallo et al ldquoGemcitabine trig-gers a pro-survival response in pancreatic cancer cells throughactivation of the MNK2eIF4E pathwayrdquo Oncogene vol 32 pp2848ndash2857 2013
[64] A Bianchini M Loiarro P Bielli et al ldquoPhosphorylation ofeIF4E by MNKs supports protein synthesis cell cycle progres-sion and proliferation in prostate cancer cellsrdquo Carcinogenesisvol 29 no 12 pp 2279ndash2288 2008
[65] L Furic L RongO Larsson et al ldquoEIF4Ephosphorylation pro-motes tumorigenesis and is associated with prostate cancer pro-gressionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 32 pp 14134ndash14139 2010
[66] C Naro and C Sette ldquoPhosphorylation-mediated regulation ofalternative splicing in cancerrdquo International Journal of Cell Bio-logy In press
[67] Z Zhou J QiuW Liu et al ldquoTheAkt-SRPK-SR axis constitutesa major pathway in transducing EGF signaling to regulatealternative splicing in the nucleusrdquo Molecular Cell vol 47 no3 pp 422ndash433 2012
[68] NMatter P Herrlich andH Konig ldquoSignal-dependent regula-tion of splicing via phosphorylation of Sam68rdquoNature vol 420no 6916 pp 691ndash695 2002
[69] A M Al-Ayoubi H Zheng Y Liu T Bai and S T EblenldquoMitogen-activated protein kinase phosphorylation of splicingfactor 45 (SPF45) regulates SPF45 alternative splicing siteutilization proliferation and cell adhesionrdquoMolecular and Cel-lular Biology vol 32 no 14 pp 2880ndash2893 2012
[70] A K Shalek R Satija X Adiconis et al ldquoSingle-cell transcrip-tomics reveals bimodality in expression and splicing in immunecellsrdquo Nature 2013
[71] S Pedrotti P BielliM P Paronetto et al ldquoThe splicing regulatorSam68 binds to a novel exonic splicing silencer and functions inSMN2 alternative splicing in spinal muscular atrophyrdquo EMBOJournal vol 29 no 7 pp 1235ndash1247 2010
[72] N H Meyer K Tripsianes M Vincendeau et al ldquoStructuralbasis for homodimerization of the Src-associated during mito-sis 68-kDa protein (Sam68)Qua1 domainrdquo Journal of BiologicalChemistry vol 285 no 37 pp 28893ndash28901 2010
[73] S Bonnal L Vigevani and J Valcarcel ldquoThe spliceosome asa target of novel antitumour drugsrdquo Nature Reviews Drug Dis-covery vol 11 no 11 pp 847ndash859 2012
[74] S Pedrotti and C Sette ldquoSpinal muscular atrophy a new playerjoins the battle for SMN2 exon 7 splicingrdquo Cell Cycle vol 9 no19 pp 3874ndash3879 2010
[75] Y Hua K Sahashi F Rigo et al ldquoPeripheral SMN restoration isessential for long-term rescue of a severe spinal muscular atro-phy mouse modelrdquo Nature vol 478 no 7367 pp 123ndash126 2011
[76] F Zammarchi E De Stanchina E Bournazou et al ldquoAntitu-morigenic potential of STAT3 alternative splicing modulationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 43 pp 17779ndash17784 2011
![Paraquat Modulates Alternative Pre-mRNA Splicing by ...muehlemann.dcb.unibe.ch/publications/Vivarelli.pdf · alternative pre-mRNA splicing (AS) [2]. Pre-mRNA splicing is a crucial](https://static.documents.pub/doc/80x56/606212ed67e7345b4269ee34/paraquat-modulates-alternative-pre-mrna-splicing-by-alternative-pre-mrna-splicing.jpg)
