avb6 Integrin Promotes Castrate-ResistantProstate Cancer through JNK1-MediatedActivation of Androgen ReceptorHuimin Lu1,2, Tao Wang3, Jing Li1, Carmine Fedele1,2, Qin Liu1,4, Jianzhong Zhang1,Zhong Jiang5, Dhanpat Jain6, Renato V. Iozzo7, Shelia M. Violette8, Paul H.Weinreb8,Roger J. Davis9, Daniel Gioeli10, Thomas J. FitzGerald3, Dario C. Altieri1,11, andLucia R. Languino1,2
Abstract
Androgen receptor signaling fuels prostate cancer and is amajortherapeutic target. However, mechanisms of resistance to thera-peutic androgen ablation are not well understood. Here, using aprostate cancer mouse model, Ptenpc�/�, carrying a prostateepithelial-specific Pten deletion, we show that the avb6 integrinis required for tumor growth in vivo of castrated as well as ofnoncastrated mice. We describe a novel signaling pathway thatcouples the avb6 integrin cell surface receptor to androgenreceptor via activation of JNK1 and causes increased nuclearlocalization and activity of androgen receptor. This downstreamkinase activation by avb6 is specific for JNK1, with no involve-ment of p38 or ERK kinase. In addition, differential phosphor-ylation of Akt is not observed under these conditions, nor is cellmorphology affected by avb6 expression. This pathway, which is
specific for avb6, because it is not regulated by a different av-containing integrin, avb3, promotes upregulation of survivin,which in turn supports anchorage-independent growth of avb6-expressing cells. Consistently, both avb6 and survivin are signif-icantly increased in prostatic adenocarcinoma, but are notdetected in normal prostatic epithelium. Neither XIAP nor Bcl-2 is affected byavb6 expression. In conclusion,we show thatavb6expression is required for prostate cancer progression, includingcastrate-resistant prostate cancer; mechanistically, by promotingactivation of JNK1, the avb6 integrin causes androgen receptor–increased activity in the absence of androgen and consequentupregulation of survivin. These preclinical results pave theway forfurther clinical development of avb6 antagonists for prostatecancer therapy. Cancer Res; 76(17); 5163–74. �2016 AACR.
IntroductionDespite considerable progress in the successful management of
early-stage disease, prostate cancer remains a life-threatening
disease in its advanced stage (1–3). Most, if not all, of aberrantcellular signaling in prostate cancer can be traced back toaberrant androgen receptor (AR) signaling, which dictates dis-ease onset and progression. AR belongs to the nuclear receptorgene superfamily and, in response to binding to its ligand, i.e.,androgen, mediates transcription of various genes importantfor disease maintenance (4). The gain of resistance to thera-peutic androgen ablation in its advanced phases, castrate-resis-tant prostate cancer (CRPC; refs. 5, 6), does not indicatedispensability of AR activity, but rather the acquisition ofancillary mechanism(s) that fuel AR signaling in the absenceof androgen (7), such as AR locus amplification, AR variants ormutants that display transcriptional activity without ligandbinding, or inappropriate AR activation by ligand-independentand non–androgen-mediated pathways (4, 8).
Changes in the cell matrix interaction during prostate tumor-igenesis are involved in disease behaviors and resistance totherapy (9, 10). In this context, prostate cancer cells often over-express members of the integrin family of adhesion receptors.Integrins not only mediate cell-to-cell communication, but alsoplay a pivotal role in transducing environmental cues to down-stream signaling molecules of cell proliferation, survival, andinvasiveness, including MAPK, focal adhesion kinase (FAK), src,Akt, and JNK (11, 12). In particular, theavb6 integrin is associatedwith neoplastic and metastatic phenotypes in various cancers(13, 14). It functions as a receptor for extracellular matrix (ECM)proteins, including latency associated peptide transforminggrowth factor b (LAP-TGFb), fibronectin (FN), tenascin, and
1Prostate Cancer Discovery and Development Program, Thomas Jef-ferson University, Philadelphia, Pennsylvania. 2Department of CancerBiology, Sidney Kimmel Cancer Center, Thomas Jefferson University,Philadelphia, Pennsylvania. 3Department of Radiation Oncology, Uni-versity of Massachusetts Medical School, Worcester, Massachusetts.4Molecular and Cellular Oncogenesis Program, The Wistar Institute,Philadelphia, Pennsylvania. 5Department of Pathology, University ofMassachusetts Medical School, Worcester, Massachusetts. 6Depart-ment of Pathology, Yale University School of Medicine, New Haven,Connecticut. 7Department of Pathology, Anatomy and Cell Biology,Thomas JeffersonUniversity, Philadelphia, Pennsylvania. 8Biogen Inc.,Cambridge, Massachusetts. 9Program in Molecular Medicine andHowardHughesMedical Institute,UniversityofMassachusettsMedicalSchool, Worcester, Massachusetts. 10Department of Microbiology,Immunology, and Cancer Biology, University of Virginia, Charlottes-ville,Virginia. 11Tumor Microenvironment and Metastasis Program,TheWistar Institute, Philadelphia, Pennsylvania.
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
H. Lu and T. Wang contributed equally to this article.
Corresponding Author: Lucia R. Languino, Thomas Jefferson University, 233South 10th Street, BLSB 506, Philadelphia, PA 19107. Phone: 215-503-3442; Fax:215-503-1307; E-mail: [email protected]
vitronectin (VN), and has been linked to activation of FAK andMAPK (15). However, how the precise arrangement of integrinsignaling contributes to resistance to androgen ablation is stilllargely unknown.
Another downstream protein associated with integrin activa-tion is survivin (16). Survivin is a member of the inhibitor ofapoptosis (IAP) family whose expression is associated with con-servation of cell division (17). Survivin is not usually expressed innormal prostate epithelium, but gradually increases duringtumorigenesis and shows the highest level in lymph node metas-tasis (18). It has been also reported that androgen induces survivinexpression and AR inhibitors prevent survivin expression (19).Intratumoral injection of an antisense reagent against survivinmRNA or an adenovirus expressing a dominant negative form ofsurvivin, T34A, inhibits tumor growth and enhances sensitivity toandrogen ablation (19, 20).
In this study, we investigated a potential cross-talk betweenintegrins, AR signaling and survivin in prostate cancermodels.Wefound thatavb6 integrin expression is required for prostate cancerprogression in vivo, including CRPC, and this pathway mediatesactivation of JNK1, nuclear accumulation and increased activity ofAR in the absence of androgen.
Materials and MethodsReagents and antibodies
Synthetic androgen R1881 was from PerkinElmer. Enzaluta-mide was from Selleck Chemicals. The following rabbit Abs(pAbs) were used: AR (N20), phospho-ERK1 (Santa Cruz Bio-technology); Smad3 (Invitrogen); p-src (Y416), Akt, p-Akt, p-JNKand p-p38 (Cell Signaling Technology); prostate-specific antigen(PSA; DAKOCytomation). A goat Ab against p38 was from SantaCruz Biotechnology. A rabbit monoclonal Ab against b6 integrin,B1, was from Dr. Dean Sheppard. The following mouse mono-clonal Abs (mAbs): AR (441; Santa Cruz Biotechnology); b1integrin (C-18) and JNK (BD Biosciences); c-src (Cell SignalingTechnology); b1 integrin (TS2/16) and b3 integrin (AP3) are fromATCC;b5 integrin (P1F6) fromLife Technologies, Inc.;av integrin(VNR147) and a5 integrin (P1D6) are from Life Technologies;CK8 (Boehringer Mannheim), CK18 (Sigma); b6 integrin Abs6.2A1, ch2A1 and 6.3G9 were previously described (21); b6integrin Ab 10D5 from Chemicon. Purified nonimmune mouseIgGs (mIgG) fromPierce, 1E6 (IgG1) fromBiogen and 1C10wereused as negative controls.
Cells and culture conditionsLNCaP, PC3, and RWPE cells were obtained from the ATCC,
and C4-2B was obtained from UroCor and cultured as previ-ously described (22, 23). LNCaP stable cell lines expressingfull-length b3, b6, or empty vector have been described previ-ously (24). PC3 and C4-2B cells stably transfected with humanb6-integrin cDNA in pBABE retroviral vector were used (23).Authentication of the cell lines was provided with their pur-chase. Cellular morphology and anchorage-independentgrowth assay were visualized using an Olympus IX71 invertedmicroscope with IPLab V3.55 (Scanalytics, Inc.).
Human tissue specimensAll tissues in this study were discarded, coded, and unidenti-
fiable specimens. Forty-seven human adenocarcinoma tissue spe-cimens from radical prostatectomies provided by: The Coopera-
tive Human Tissue Network, which is funded by the NationalCancer Institute (other investigatorsmay have received specimensfrom the same subjects); theDepartment of Pathology, Universityof Massachusetts Medical School and the Department of Pathol-ogy, Yale University and were processed according to Institution-approved protocols. Eight normal prostatic tissue sections wereobtained from human biopsies. Thirteen additional prostatespecimens were snap-frozen in liquid nitrogen for protein extrac-tion. Tissues were processed as previously described (25).
Constructs and cell transfectionAdenoviruses, containing wild-type survivin (WT), dominant
negative survivin (T34A) or empty vector (pAd) have beendescribed previously (26). avb6-LNCaP cells were transientlytransfected with adenoviral constructs for 17 hours. All threeconstructs contain the GFP gene for determination of transfectionefficiency. C4-2B cells were stably transfected human b6 and b3integrin cDNA generated as described before (24). Clones wereselected and maintained in culture medium containing 5% FBSand 100 mg/mL G418. Retroviral constructs, containing wild-typeJNK1 or an NH2-terminal JNK-binding domain (JBD), and thetransient transfections have been described previously (27).avb6-C4-2B, avb3-C4-2B or mock-C4-2B cells were transientlytransfected with WT-JNK- or JBD-viral construct to determine theeffect of JNK on AR activity. WT-JNK (pBABE-puro-JNK1) orJBD containing retrovirus (babe-puro-JBD) were wrapped withVSVG and GAGPol using phoenix cells (ATCC, CRL-3213). Thetransient transfection was performed with 8 mg/mL of poly-brene for 48 hours, then cells were starved in 2% charcoal-stripped (CS) serum-containing medium for 24 hours followedby stimulation with 1 nmol/L R1881 or ethanol for 24 hours.
siRNA transfectionCells were transfected with b6 integrin siRNA duplexes (IDT
Inc.). Two b6 siRNA duplexes, D1 and D2, were described before(22). siRNA duplexes were transfected using oligofectamine at afinal concentration of 20 nmol/L. Forty-eight hours after trans-fection, cells were harvested and analyzed by FACS to confirmdownregulation of b6 integrin and to evaluate anchorage-inde-pendent cell growth. For downregulation of AR, siRNA smartpooland nonsilencing siRNA (Dharmacon)were used according to themanufacturer's instruction. The protocol used for downregulationof survivin by siRNA inavb6-LNCaP cells was based on publishedmethods (28).
Luciferase assay and cell adhesion assayLuciferase activity was measured as described previously (29).
Cell adhesion protocols used here have been described previously(30). For JNK activation assay, LNCaP, C4-2B or PC3 cells stablytransfected with pBabe-b6-integrin or pBabe-empty vector werestarved overnight in serum-free RPMI culture medium. Cells wereseeded on culture plates coated with FN (3 mg/mL), LAP-TGFb1(0.5 mg/mL) or BSA. Cells (1� 106) were plated on each substratefor 1 hour in serum-free RPMI culture medium before cell lysis.
Cell proliferation assayCells were seeded in a 96-well plate for 24 hours, starved with
medium containing 2% CS serum for 24 hours. Cells werestimulated with either 1 nmol/L R1881 or incubated with thesame volume of ethanol and then cultured for 72 hours beforeperforming Sulforhodamine B (SRB) staining.
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IHCIHC staining using formalin-fixed paraffin-embedded speci-
menswas performed as described elsewhere (31). For detection ofsurvivin, IHC was performed according to published protocols(32).
ImmunofluorescencePrimary epithelial cells isolated as described (30), were probed
using Abs specific to CK8 or CK18, markers of prostate epithelialcells by indirect IF staining. AR nuclear localization was analyzedusing IF staining. Cells were seeded on poly-L-Lysine (1 mg/mL)coated coverslips for 2 days. After starvation for 24 hours in 2%CS serum contained medium, cells were grown in the presenceof ethanol or R1881 (1 nmol/L) for 2 hours. Cells were probedwith an AR-specific Ab N20 (1:500) and 50 cells/condition werecounted to determine the localization of AR in nucleus.
FACSFACS analysis was performed to determine surface expression
of integrins using Abs specific to b6 (10D5) or b3 (AP3); non-immune mIgG, 12CA5, or IC10 were used as negative controls.
Generation of Ptenpc�/� mice and castrationProstate epithelial-specific Pten conditional knockout mice
(Ptenpc�/�) mice were generated as previously reported (33).Ptenpc�/� mice were surgically castrated using published method(33).
Statistical analysisUnless otherwise indicated, data in the figures are presented as
mean � SEM, and significant differences between experimentalgroups were determined using the two-tailed Student t test. TheFisher's exact two-tailed test was used to compare the percentageof b6-positive cells. Mantel-extension of Mantel–Haenszel Statis-tic stratified by subject was used to test the correlation between b6expression and the types of lesion. TheWilcoxon–Mann–Whitneytest was used to analyze the difference in tumor growth between6.3G9 and control group in vivo. To examine the association
between b6 and survivin expression in human specimens, theSpearman correlation coefficientwas calculated and tested. A two-sided P value of less than 0.05 was considered statistically signif-icant. SAS statistical software 9.1.2 (SAS Institute, Inc.) was usedfor statistical analysis.
Resultsavb6 integrin promotes CRPC growth in vivo
To determine the effect of avb6 expression in vivo, we used aCRPCmodel that uses surgically castrated Ptenpc�/� as delineatedin Fig. 1A. avb6 expression pattern in castrate-resistant tumors(n¼5)was comparedby IHCwithnoncastrated tumors (n¼5) inPtenpc�/� mice. We show here that avb6 is expressed in mouseCRPC; specifically, we demonstrate that the expression of avb6 ishighly heterogeneous (18% of the tumor cells) in tumors fromnoncastrated mice while homogenous (90% of tumor cells) intumors from castratedmice, indicating a significant enrichment ofthis integrin upon castration. Accordingly, JNK nuclear localiza-tion is increased inavb6positive cells innoncastrated tumors (3%of tumor cells), and becomes dominant in castrated tumors (75%of tumor cells; Supplementary Fig. S1).
Upon intraperitoneal administration for 5 weeks of 6.3G9, anon ligand-mimetic blocking mAb that does not get internalizedupon binding (21), to castrated Ptenpc�/� mice, tumor progres-sion is inhibited and the prostate glands do not show evidence ofinvasive adenocarcinoma; in contrast, upon intraperitonealadministration of a negative control mAb, 1E6, the prostateglands show invasive adenocarcinoma (Fig. 1B). The results showa significant decrease of tumor weight in castrated Ptenpc�/�micetreated with 6.3G9 (25.3 � 1.8 mg) as compared with the grouptreated with 1E6 (39.8 � 1.5 mg; Fig. 1C). We also carried outxenograft tumor growth experiments in castrated athymic nudemice (Supplementary Fig. S2). For this purpose, we used ARþ
LNCaP cells that stably express human b6 (avb6-LNCaP), humanb3 (avb3-LNCaP) or empty vector (mock-LNCaP). These cellsexhibit no difference in the expression of other endogenousintegrin subunits (Supplementary Fig. S2A). Upon subcutaneousinjection, avb6-LNCaP xenograft tumors continue to grow
Figure 1.
Inhibitory effect of mAb 6.3G9 on tumor growth incastrated Ptenpc�/� mice. A, scheme of surgicalcastration and Ab treatment of Ptenpc�/� mice. B and C,mAb 6.3G9 against avb6 or a negative control mAb1E6 were injected in castrated Ptenpc�/� miceintraperitoneally both at 10 mg/kg weekly for 5 weeks,then mice (n ¼ 11) were sacrificed and tumor weightswere quantified. B, hematoxylin and eosin analysis oftumor specimens from 6.3G9-treated mice (top) or 1E6-treated mice (bottom). Arrow, top right, epithelial cells.Arrowhead, cancer cells sloughed in the lumen. Asterisk,microinvasion. Originalmagnification, left column,�200;right column, �400. C, individual tumor weight isplotted; lines are mean � SEM. P ¼ 0.004 (Wilcoxonrank-sum two-sided test).
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significantly (Supplementary Fig. S2B), suggesting that avb6expression is sufficient to confer a resistant phenotype to prostatecancer cells, in vivo. In contrast, avb3-LNCaP transfectants do notsignificantly grow as subcutaneous tumors in castrated mice(Supplementary Fig. S2B).
We in parallel investigated whether avb6 is required for pros-tate cancer growth in noncastrated mice. We used Ptenpc�/� miceto independently validate a requirement of avb6 for tumorgrowth in vivo. Intraperitoneal administration of the 6.3G9 Abfor 5 weeks to Ptenpc�/� mice results in acute disruption ofepithelial layers of prostate adenocarcinoma, with appearance ofnecrotic cells filling the glandular lumen (Fig. 2A) and in asignificant decrease in tumor weight as compared with micetreated with the 1E6 mAb (P ¼ 0.0042; Fig. 2B). In contrast,prostate specimens collected from Ptenpc�/� mice treated with1E6 show an intact structure of transformed prostatic glands (Fig.2A). In addition, histologically normal glands of Ptenpc�/� micereceiving 6.3G9mAb donot show a disruption of epithelial layers(Fig. 2A). Subcutaneous injection of avb6-LNCaP cells in theflanks of immunocompromised SCID mice gives rise to expo-nentially growing tumors with a statistically significant difference(P < 0.01) compared withavb3-LNCaP tumors starting at 54 days
after injection (Fig. 2C). In two additional independent experi-ments, avb3-LNCaP cells or mock-LNCaP cells (SupplementaryFig. S3A) produce tumors with statistically slower growth kinetics(P < 0.01) as compared with avb6-LNCaP tumors. Accordingly,expression of avb3- or avb6-integrins on the cell surface wasconfirmed by FACS (Supplementary Fig. S3B). Tumors generatedby the various transfectants retain the expression of the respectiveb6 or b3 integrins as detected by immunoblotting, whereastumors generated by mock transfectants remain negative (Sup-plementary Fig. S3C). Together, these data demonstrate thatavb6is required for accelerated prostate cancer growth, in vivo.
avb6 integrin promotes prostate cancer cell proliferation andactivates the AR pathway
To determine the potential basis for avb6-mediated aggressivetumor behavior in vivo, we next measured the proliferation kinet-ics of integrin-expressing cells in the presence or absence ofandrogen. Mock-LNCaP and avb3-LNCaP cells exhibit increasedproliferation in response to androgen (R1881; Fig. 3A). Converse-ly, two independent clones of avb6-LNCaP cells show increasedrates of proliferation compared to mock-LNCaP or avb3-LNCaPcells in the absence of androgen, and this response is unaffected
Figure 2.
Inhibitory effect of mAb 6.3G9 on tumor growth in noncastrated Ptenpc�/� mice. A and B, Ptenpc�/� mice (5–8 weeks) were intraperitoneally injected with6.3G9 or 1E6 as described in Fig. 1, then mice were sacrificed and the differences in tumor weight were quantified. A, representative prostate specimens (normalgland or adenocarcinoma) of Ptenpc�/� mice injected with mAb 6.3G9 or control mAb 1E6 were analyzed histologically by hematoxylin and eosin staining.Arrow, cancer epithelial cells. Arrowhead, apoptotic cancer cells in the lumen; none, untreated. Original magnification, left column, �200; right column,�400. B, individual tumor weight is plotted (n ¼ 8). Statistical significance was observed in tumor weight, P ¼ 0.0042 (Wilcoxon rank-sum two-sided test).C, avb6-LNCaP (filled circle) or avb3-LNCaP (open square) cells were injected subcutaneously into male CB17/SCID mice (16 mice/group), and differences intumor volume were quantified. Statistical analysis shows significant differences in tumor growth between two cell lines from day 54 to 85 (�� , P < 0.01); data areshown as mean � SEM.
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by androgen (Fig. 3A, left). The avb6-LNCaP, mock-LNCaP andavb3-LNCaP cells do not showmorphologic differences (Fig. 3B).In an anchorage-independent assay, we downregulated b6 in ARþ
RWPE cells using two independent siRNAs, D1 and D2 (Fig. 3C,right). b6 silencing by two independent siRNAs, under theseconditions, suppresses anchorage-independent tumor growthwhereas nontargeting siRNA (NS) or oligofectamine (OF) alonehas no effect (Fig. 3C, left).
To analyze the mechanism by which avb6 affects prostatecancer growth, we tested AR activity in the presence or absenceof androgen in avb6-LNCaP (Fig. 4A, left) or avb6-C4-2B cells(Fig. 4B, left); the results show that avb6-LNCaP exhibit consti-tutively high levels of PSA, an established AR target gene, without
addition of exogenous androgen, and PSA levels are only mini-mally affected in the presence of androgen. In control experi-ments, mock-transfectants, avb3-LNCaP or avb3-C4-2B cellsshow negligible basal levels of PSA, whereas androgen (R1881)stimulation elevates PSA expression in these cells (Fig. 4A and B,left; also shown in Supplementary Fig. S4). Silencing AR by siRNAin avb6-LNCaP cells (Fig. 4A, right) or avb6-C4-2B cells (Fig. 4B,right) abolishes avb6-induced PSA, confirming AR specificity ofthese findings. Similar results are obtained in the analysis of ARpromoter activity; avb6-LNCaP cells exhibit constitutive highlevels of androgen responsive elements (ARE)4-luciferase activity,irrespective of androgen stimulation, as compared with mock-transfected cultures (Supplementary Fig. S5). In addition, neither
Figure 3.
avb6 integrin expression enhancesprostate cancer cell proliferation. A,mock-, avb6-LNCaP (clones 1 and 2) oravb3-LNCaP cells were stimulatedwithR1881 (1 nmol/L) or vehicle afterstarvation, and quantified for cellproliferation by SRB staining. B,mock-,avb6- and avb3-LNCaP cells werecultured in either tissue culture platesor FN (5 mg/mL)-coated plates. After72 hours, cellular morphology wasvisualized. C, RWPE cells transfectedwith b6-directed (D1 or D2) or non-silencing (NS) siRNA were analyzed foranchorage-independent cell growth,and the total number of colonies in 20fields was scored 14 days after seeding(left). RWPEcellswere transfectedwithb6 integrin-directed siRNA andanalyzed by FACSwith anmAb toavb6or nonbinding IgG (mIgG; right); OF,oligofectamine.A and C, data are mean� SEM; �� , P < 0.01.
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expression of avb6 or avb3 integrin affects endogenous ARprotein levels, in the presence or absence of androgen (Fig.4C), indicating that the increase in AR activity observed in avb6þcells does not reflect changes in AR protein expression. Immu-nofluorescence (IF) analysis shows increased nuclear localizationof AR in the absence of androgen in avb6-LNCaP cells (Fig. 4D).In contrast, AR reactivity is detected largely in the cytoplasm ofunstimulated avb3-LNCaP or mock-LNCaP cells, and becomesintense in the nuclei only in response to androgen stimulation.
The ability of avb6 to activate AR, however, does not promoteresistance to enzalutamide, an AR antagonist developed for thetreatment of metastatic CRPC patients. Mechanistically, enzalu-tamide inhibits translocation of AR to the nucleus and prevents
binding of AR toDNAor to coactivator proteins (34).On the basisof this knowledge, we investigated whether this compound mayalso inhibit ligand-independent AR activation mediated by avb6.Wefind thatavb6 integrin expressiondoes not promote resistanceto enzalutamide at concentrations up to 100 mmol/L and thatenzalutamide is effective in suppressing proliferation of avb6-,avb3- and mock- LNCaP cell transfectants in a comparablefashion (Fig. 4E).
avb6 activation of JNK1 regulates AR activityTo determine the mechanism by which avb6 activates AR, we
analyzed the kinase cascades typically activated by integrin ligandbinding. Adhesion of avb6-LNCaP cells to the avb6 ligand
Figure 4.
avb6 integrin regulates AR activityand nuclear trafficking. A and B, left,LNCaP and C4-2B transfectants werestarved in 2% CS serum for 24 hoursand stimulated with R1881 (1 nmol/L)or ethanol for 24 hours. PSA levelwas analyzed by immunoblotting (IB).Right, avb6-LNCaP and avb6-C4-2Bcells were transfected withAR-directed siRNA and the PSA levelwas evaluated by immunoblotting.Original blots are shown inSupplementary Fig. S4. C, mock-,avb3-, avb6- and parental (�) LNCaPcells were analyzed for AR expressionwith or without R1881 byimmunoblotting. A–C, ERK1/2 or Aktwas used as a loading control. OF,oligofectamine; NS, non-silencing.D, cellular distribution ofAR in avb6-, avb3-, and mock-LNCaPcells. Indicated cells were seededon poly–L-lysine (1 mg/mL)-coatedcoverslips for 2 days, starved,and treated with or withoutR1881 (1 nmol/L). E, LNCaP celltransfectants were treated with theindicated concentrations ofenzalutamide in culture medium for72 hours and cell proliferation ratewascalculated by cell counting.Proliferation is valued as thepercentage of the control groupstreated with DMSO only. Data aremean � SD.
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LAP-TGFb1 or FN results in JNK1 activation as evaluated by itsphosphorylation pattern (Fig. 5A, top). In parallel, we observethat PSA is increased inavb6-transfectants attached to LAP-TGFb1or FN whereas avb3-transfectants show barely detectable PSAexpression (Fig. 5A, middle). avb3-LNCaP cells adhere to FN orLAP-TGFb1 but fail to induce JNK1 phosphorylation (Fig. 5A,top). PC3 cells stably transfected with b6 (avb6-PC3) also exhibitincreased JNK1 phosphorylation upon adhesion to FN or LAP-TGFb1, whereas mock-transfected cells do not (Fig. 5A, bottom).The function-blocking Ab 6.3G9 inhibits attachment to LAP-TGFb1 in a concentration-dependent manner, thus identifyingavb6 as the predominant LAP-TGFb1 receptor in prostate cancercells (Fig. 5A, graph; ref. 35). Conversely, ligand binding to avb6does not activate related signaling pathways, including ERK, p38(Supplementary Fig. S6A and S6B), or JNK2 (data not shown) asdetermined by IB with phosphorylation specific Abs. BecauseJNK1 activation has been implicated in controlling AR activityvia nuclear translocation (36), we infected avb6-C4-2B cells witha retrovirus encoding a JBD, which functions as a dominantnegative JNK (27, 37). avb6-C4-2B cells exhibit high PSA levels,
which is not furthermodulated by androgen (R1881) stimulation(Fig. 5B; also shown in Supplementary Fig. S7), in agreement withthe data presented above. In contrast, JBD inhibits the basalexpression of PSA in avb6-C4-2B cells in a concentration-depen-dent manner, and completely abolishes PSA levels after androgenstimulation (Fig. 5B), whereas infection of WT-JNK1 does notaffect PSA levels inavb6-C4-2B cells, in the presence or absence ofandrogen. Androgenmodulation of PSA inmock- or avb3-C4-2Bcells is unaffected by WT-JNK1 or JBD (Fig. 5B). To identify apotential link between JNK1 activity and AR-dependent geneexpression, we next looked at potential changes in AR localiza-tion. IF staining shows that infection of JBD in avb6-LNCaP cellssignificantly impairs AR nuclear localization (Fig. 5C). Overall,this study shows that upon ligand binding, avb6 enhances ARtransactivation via JNK1 stimulation, which in turn controls ARnuclear trafficking.
Survivin is a downstream effector of the avb6 integrinTo identify potential downstream effectors of integrin-medi-
ated prostate cancer progression, we focused on survivin, that
Figure 5.
JNK1 mediates transactivation of AR in avb6 integrin–expressing cells. A, avb6-LNCaP or avb3-LNCaP cells (top) were seeded on FN or LAP-TGFb1 (LAP)–coated plates, and analyzed for JNK1 phosphorylation by immunoblotting (IB). White lines indicate that intervening lanes were spliced out. PSA levels were alsoanalyzed by immunoblotting. c-src was used as a loading control. PC3 cells stably transfected with pBabe-b6 (avb6) or pBabe vector (mock) were seededon FN-, LAP-, or BSA-coated plates and analyzed for JNK1 activation by detection of phospho-JNK1 (p-JNK1). p-JNK1 levels in parental cells (�) exposed to UVirradiation were used as a positive control for JNK1 activation. Total JNK1 was used as a loading control. PC3 cells (bottom) were incubated in the presenceof the indicated concentrations ofmAb6.3G9 toavb6, and adhesion to LAP-coated plateswas quantified. Data aremean� SEM; �� ,P<0.01.B,avb6-,avb3- ormockC4-2B cells were infected with WT-JNK or decreasing concentrations of JBD and analyzed for PSA expression by immunoblotting. Akt or b-actin wasused as a loading control. Uninfected cells (�) were used as controls. Original blots are shown in Supplementary Fig. S7.C,avb6-LNCaP cells transiently infectedwithWT-JNK (WT) or JBD were seeded on a poly–L-lysine–coated coverslip for 24 hours, starved, and analyzed with an Ab to AR by IF (left). DAPI (right).
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has been implicated in prostate cancer maintenance and resis-tance to therapy (19, 38). avb6-C4-2B cells exhibit higher levelsof survivin, compared with avb3-C4-2B (Fig. 6A), whereas XIAPor Bcl2, two other molecules known to inhibit apoptosis, arenot affected (Fig. 6A). Similar results were obtained withLNCaP cells stably transfected with a b3/6 integrin chimera(Fig. 6B and Supplementary Fig. S8), containing the b6 intra-cellular domain and the b3 extracellular domain expressed atthe cell surface, suggesting that the b6 cytoplasmic domain isrequired for survivin induction.
In this context, silencing of AR significantly reduces survivinlevels in avb6-LNCaP cells, whereas a nontargeting siRNA isineffective (Fig. 6C). Inhibition of JNK activity in these cells viainfection with JBD also decreases survivin levels independently ofandrogen, whereas expression of WT JNK1 moderately up-regu-lates survivin in avb6-LNCaP cells (Fig. 6D). Silencing of survivinby siRNA, or interference with survivin function via adenoviraltransduction of a survivin Thr34Ala dominant–negative mutant
(Fig. 6E, left), abolishes prostate cancer colony formation in softagar, that is, anchorage-independent growth. In contrast, a non-targeting siRNA or adenoviral transduction of WT survivin doesnot affect anchorage-independent growth under the same condi-tions (Fig. 6E, right). We also analyzed the expression of avb6integrin in human primary specimens of prostatic adenocarcino-ma or normal prostate. Using an Ab that specifically recognizesavb6, 6.2A1, we found by IHC that this integrin is expressed inprostate cancer (35/47 specimens, 74%; Fig. 7A), consistent withits epithelial-specific expression, although in a highly heteroge-neous pattern with 5%�31% of the glands being avb6 positive.Identical results were obtained with an independent mAb (B1) toavb6 (Supplementary Fig. S9). In contrast, avb6 is undetectablein 8 out of 8 cases of normal prostate (Fig. 7A), and does notcorrelate with Gleason Score (5–10), or patient age (range, 41–83years). In addition, patient-derived primary prostate cancer cells,isolated as described (30) and reactive for cytokeratin (CK) 8 and18 (Fig. 7B, middle), express high levels of avb6, as detected by
Figure 6.
AR regulates survivin expression and anchorage-independent-growth in avb6 integrin–expressing cells. A, avb3- and avb6-C4-2B cells were analyzed for survivinand XIAP expression by immunoblotting (IB; top). avb3- and avb6-C4-2B (clone 1 and 2) cells were stimulated with or without R1881 (1 nmol/L) andanalyzed for Bcl-2 levels by immunoblotting (bottom). B, mock-LNCaP (lane 1, 3), avb6-LNCaP (lane 2), or chimera avb3/6-LNCaP (lane 4) cells were analyzedfor survivin expression by immunoblotting. The white line indicates that intervening lanes were spliced out. C, avb6-LNCaP cells were transfected withnonsilencing (NS) or AR-directed siRNA for 24 hours, starved, and analyzed for expression of AR or survivin by immunoblotting. A–C, Akt or ERK1/2 was used as aloading control. OF, oligofectamine. D, avb6-LNCaP cells were transiently infected with WT-JNK or JBD and analyzed for survivin expression by immunoblotting.Akt was used as a loading control. Uninfected cells (�) were used as controls. E, avb6-LNCaP cells were infected with adenovirus containing survivinwild-type (WT) or a survivin mutant (T34A) and analyzed for anchorage-independent growth. The average of colony diameters (left) of the threegroups � SEM are plotted. avb6-LNCaP cells were transiently transfected with nonsilencing or survivin-directed (SV) siRNA for 48 hours and analyzed foranchorage-independent growth. The average number of the colonies (right) in the three groups � SEM are plotted. �� , P < 0.01.
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FACS analysis (Fig. 7B, top). This result was confirmed using 15fresh prostatic adenocarcinoma tissue lysates by IB using an Abto avb6 (6.2A1), which detects a 110 kDa protein consistentwith b6 reactivity (Fig. 7B, bottom, two representative speci-mens are shown) albeit with variable levels of expression.Survivin expression was also studied in 34 specimens and isfound to be expressed in 97% of adenocarcinoma specimens(Fig. 7C, 1–4); the association between avb6 and survivin isstatistically significant in 34 cases of adenocarcinoma (P <0.028) and is independent of Gleason scores (5–10) or disease
stage (T2-4; Supplementary Table S1). Overall, our in vitro andIHC analysis show that survivin is a downstream effector of theavb6 integrin.
DiscussionIn this study, we show that expression of an epithelial-specific
integrin, avb6, in prostate cancer as well as in castrated tumors, issufficient to promote aggressive tumor growth and castrate-resis-tance disease. The molecular requirements of this pathway are
Figure 7.
avb6 integrin and survivin arecoexpressed in human prostaticlesions. A, human adenocarcinoma(1–4) and normal prostate (5)specimens were analyzed for avb6expression by IHC using mAb 6.2A1(1–3) or mIgG was used as a negativecontrol (4). Representative casesare shown. Arrows, avb6-expressinggland; arrowheads, avb6-negativeglands (1 and 2). Originalmagnification, �200. The percentageof avb6-expressing adenocarcinomatissue specimens at different Gleasonscores is shown. B, primary prostaticepithelial cells were isolated fromthree cancer patients and analyzed forexpression of avb6 by FACS analysiswith mAb 10D5 (thick line; left). IFstaining of primary prostatic epithelialcells was carried out using Abs to CK 8or 18. Ab 1C10 against an endothelialmarker was used as a negative control(top right). Primary tumor extractswere analyzed for expression of b6integrin using mAb 6.2A1 (bottom).Akt was used as loading control.C, representative images ofadenocarcinoma (1–4) showingco-expression of avb6 integrin andsurvivin in the same gland. D,schematic diagram for integrinregulation of AR-mediated prostatetumorigenesis independently ofandrogen.
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centered on avb6 activation of JNK1, which in turn promotes ARnuclear shuttling, concomitant ligand-independent AR transcrip-tion and subsequent survivin-regulated tumor growth (Fig. 7D).
At themolecular level, this transition to castrate resistance is anas yet poorly understood process (4). Our data unravel a novelpathway of castrate-resistant aggressive prostate cancer that ori-ginates in the tumor via avb6 integrin ligand binding, andultimately promotes AR-modulated gene expression, indepen-dent of androgen.
There is ample precedent for the role of integrins in controllingsteroid hormone receptor expression and function. In a prostatecancer in vitro model, our group recently found that avb6 isefficiently transferred via exosomes to avb6-negative recipientcells and localizes to the prostate cancer cell surface, promotingcell adhesion and migration on LAP-TGFb, suggesting a systemicrole of avb6 in tumor progression (39) in addition to its localeffect. These effects, however, do not require transfer of JNK viaexosomes, because recent data show that JNK is not detected inexosomes (L.R. Languino; unpublished data). In this study, ourdata indicate that expression of avb6 in prostate cancer cells issufficient to confer a "sub-optimal" androgen-independent phe-notype, characterized by proliferation and PSA expression in theabsence of exogenous androgen. This is further supported by ourin vivo studies, showing a significant increase in the growth ofavb6-expressing tumors in castrated as well as noncastratedmice.Finally, because avb6 effect does not promote resistance toenzalutamide, targeting this integrin may thus be a potentialadjuvant in enzalutamide treatment.
Themolecular requirements of signal integration from avb6 toAR-dependent gene expression in prostate cancer remain to befully elucidated. However, one of the key requirements in thispathway is avb6 integrin-mediated activation of the MAP kinasefamily member, JNK1. Despite its conflicting, and likely cell-typespecific, roles in both cell survival and apoptosis (37), the JNKpathway has recently emerged as an important regulator ofprostate cancer growth via AR (36). The JNK1 downstream target,c-Jun, strongly potentiates AR-mediated gene transcription(40, 41), and silencing of JNK1 inhibits prostate cancer tumorgrowth (42). The data presented here suggest that downstreamkinase activation by avb6 may be specific for JNK1, with noinvolvement of p38 or ERK kinase. In addition, differentialphosphorylation of Akt is not observed under these conditionsconsistentwith a context-dependent role of Akt inAR activation asdescribed by the following studies. Although PI3K/Akt signalingpromotes AR activity (43), examples of PTEN regulation of JNKsignaling independently of Akt have been reported (44); further-more, JNK deficiency promotes androgen-independent progeni-tor cell growth and metastatic prostate cancer more rapidly thanPTENdeficiency alone (33). Our data from several prostate cancercell lines (LNCaP, C4-2B, PC3) show that JNK1 promotes ARnuclear translocation and activation. With respect to how activeJNK1modulates AR function inavb6 integrin-expressing cells, thedata presented here are in line with several reports linking ARphosphorylation to its nuclear-cytoplasmic shuttling. AR can bephosphorylated at tyrosine, serine, and threonine residues by awide range of kinases, such as cyclin-dependent kinases (CDK),Akt, PIM1, Fer, Aurora-A, Src, Etk, PKC, PAK6, JNK and p38 (45).Akt, for example, can phosphorylate AR at S210/S790 and inhibitandrogen-induced apoptosis (46); CDK1 phosphorylates AR S81and regulates AR stability (47); in addition, soluble factorsderived from stroma activate ERK, which phosphorylates AR at
S81, promote AR-dependent transcription and anchorage-inde-pendent growth (48). The data presented in our study show thatavb6 predominantly promotes AR nuclear translocation andactivation through JNK1.
It should be stressed that AR is already activated in castrate-resistant cells, such as C4-2B, but the activity and/or nucleardistribution are not saturated or irreversible. Upon proper stim-ulation (49), the fraction of AR in the nucleus can be furtherincreased, as shown in avb6 transfectants in this study. Our datasuggest that integrins promote a novel mechanism for castrateresistance, other than the mechanisms dependent on AR muta-tions.Webelieve the use of the castrate-resistant cells in this articlesupports the major conclusion that avb6 is one of the key factorspromoting prostate cancer progression in castrate settings, inaddition to its protumor effect in androgen-sensitive cells suchas RWPE cells.
An additional downstream effector of aggressive prostatecancer phenotype of avb6 integrin-expressing cells is identifiedhere as survivin, a critical prostate cancer–promoting molecule(18) regulated by PI3K/Akt signaling, as a mediator of drugresistance (19), and as an effector of tumor progression.Although Interleukin-4–induced survivin is independent ofJNK (32), in our model JNK is required for upregulation ofsurvivin by avb6 integrin, suggesting a different mechanism inthis context. Against this backdrop, survivin expression corre-lates here with avb6 integrin levels in primary prostate cancerspecimens, is directly regulated by AR, and is required foranchorage-independent growth, in agreement with an emerg-ing role of survivin in this disease (50).
In summary, we have identified a novel signaling circuit linkingintegrins with modulation of nuclear receptor gene expression,critically important to confer a castrate-resistant phenotype andaggressive behavior of prostate cancer in humans. The identifica-tion ofmechanisms of castrate resistance amenable to therapeuticintervention is an urgent and as yet unmet need in advancedprostate cancer, a disease stage where therapeutic options are fewand only minimally effective. The translational relevance of ourfindings is highlighted by the recognized role of integrins as bonafide therapeutic targets for drug development. The preclinicalability of an mAb to avb6 integrin to inhibit tumor growth anddisrupt the epithelial architecture of prostate cancer, withouttoxicity to normal prostate, as presented here, bodes well forfurther clinical development of avb6 antagonists.
Disclosure of Potential Conflicts of InterestS.M. Violette and P.H. Weinreb have ownership interest (including patents)
in Biogen, Inc. No potential conflicts of interest were disclosed by the otherauthors.
DisclaimerThe Pennsylvania Department of Health specifically disclaims responsibility
for any analyses, interpretations, or conclusions.
Authors' ContributionsConception and design: H. Lu, T. Wang, J. Li, R.V. Iozzo, S.M. Violette,R.J. Davis, T.J. FitzGerald, L.R. LanguinoDevelopment of methodology: H. Lu, T. Wang, J. Li, S.M. Violette, R.J. Davis,T.J. FitzGeraldAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Lu, T. Wang, J. Li, C. Fedele, J. Zhang, Z. Jiang,T.J. FitzGerald
Lu et al.
Cancer Res; 76(17) September 1, 2016 Cancer Research5172
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):H.Lu, T.Wang, J. Li, C. Fedele,Q. Liu, Z. Jiang,D. Jain,R.V. Iozzo, S.M. Violette, P.H. Weinreb, D. Gioeli, T.J. FitzGerald, L.R. LanguinoWriting, review, and/or revision of the manuscript: H. Lu, T. Wang, J. Li,C. Fedele, Q. Liu, Z. Jiang, D. Jain, S.M. Violette, P.H. Weinreb, R.J. Davis,D. Gioeli, T.J. FitzGerald, D.C. Altieri, L.R. LanguinoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Wang, S.M. Violette, D. Gioeli, T.J. FitzGeraldStudy supervision: L.R. Languino
AcknowledgmentsWe thank Drs. Dean Sheppard for B1, rabbit mAb to b6; Michael Lu for ARE
constructs; Lan Hart for b6 integrin constructs; Shutsing Liao for retroviruscontaining pLNCX2-humanAR;HongWu for Ptenloxp/loxpmice; TakehikoDohifor preparation of adenovirus constructs; David Garlick and Irwin Leav forevaluation of Ptenpc�/� prostate specimens and Rebecca Galanti for technicalhelpwith the 6.3G9Ab in vivo analysis.We are grateful toDr. ChungC.Hsieh forbiostatistical analysis. For this study, Sidney Kimmel Cancer Center Bioimagingand Histology Core Facilities, which is supported in part by NCI Cancer Center-
Support Grant P30 CA56036, were used. We are grateful toMrs. Cecilia Deemerfor administrative support with this manuscript.
Grant SupportNIH CA89720 and CA109874 (L.R. Languino), CA78810 and CA90917
(D.C. Altieri), CA140043 (L.R. Languino and D.C. Altieri), CA65861 (R.J.Davis), CA39481 (R.V. Iozzo), Prostate Cancer Foundation Challenge Award(L.R. Languino and D.C. Altieri), Danny Cancer Funds P000100033(T. Wang), Postdoctoral Research Fellowship from the American ItalianCancer Foundation (C. Fedele). This project is also funded, in part, undera Commonwealth University Research Enhancement Program grant with thePennsylvania Department of Health (H.R.).
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
Received February 19, 2016; revised May 27, 2016; accepted June 20, 2016;published OnlineFirst July 22, 2016.
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