AbstractPurpose: The progression of prostate cancer to metastatic and castration-resistant disease represents a
critical step. We previously showed that the transcription factor Twist1, which promotes epithelial–
mesenchymal transition, was involved in castration-resistant progression. Similarly, protein kinase C
(PKC) has been implicated in both metastatic progression and castration resistance in prostate cancer.
Experimental Design: In this study, we aimed to elucidate the role of PKC/Twist1 signaling in castration
resistance, and to apply this information to the development of a novel therapeutic concept using PKC
inhibitor Ro31-8220 against prostate cancer using various prostate cancer cell lines.
Results: Androgen deprivation and the next-generation antiandrogen enzalutamide induced PKC
activation and Twist1 expression, which were reversed by the PKC inhibitor Ro31-8220. Ro31-8220
suppressed cell proliferation in androgen-dependent prostate cancer LNCaP cells, which was augmented
by its combination with androgen deprivation or enzalutamide. The favorable anticancer effects of the
combination of Ro31-8220 and enzalutamide were also observed in castration-resistant C4-2 and 22Rv1
cells. Furthermore, PKC phosphorylation was elevated in castration-resistant and enzalutamide-resistant
cells compared with their parental cells, leading to persistent sensitivity to Ro-31-8220 in castration- and
enzalutamide-resistant cells.
Conclusions: Taken together, these findings indicate that PKC/Twist1 signaling contributes to castration
resistance as well as enzalutamide resistance in prostate cancer, and suggest that therapeutics targeting PKC/
Twist1 signaling, such as PKC inhibitors, represent a promising novel therapeutic strategy for prostate
cancer, especially castration-resistant prostate cancer, when combined with enzalutamide. Clin Cancer Res;
20(4); 951–61. �2013 AACR.
IntroductionProstate cancer is the most common noncutaneous can-
cer and the second leading cause of cancer-relatedmortalityamong men in developed countries. Androgen-deprivationtherapy is the gold standard treatment for recurrent oradvanced prostate cancer (1). Although most prostate can-cers are initially dependent on androgen receptor (AR)signaling for cell proliferation and cellular survival andrespond well to androgen-deprivation therapy, most even-tually relapse in a castration-resistant manner during such
therapy, and are defined as castration-resistant prostatecancer (CRPC; ref. 2). Several novel AR-targeting agentsagainst CRPC have recently appeared. For example, thecytochrome P17 inhibitor abiraterone acetate has beenaccepted by the U.S. Food and Drug Administration (FDA)for use in a postchemotherapy setting in 2011 (3) and in aprechemotherapy setting in 2013 (4). More recently, thenext-generation antiandrogen enzalutamide has beenapproved by the FDA (5). Enzalutamide (MDV3100) is oneof the most anticipated agents for CRPC treatment; how-ever, efficacy, represented by decline of prostate-specificantigen, is seen in only around 60% of patients with CRPC(5). Novel therapeutic strategies are therefore required toimprove the therapeutic effects of enzalutamide.
Aberrant activation of AR under low levels of circulatingandrogens is critical to the development of castration resis-tance (6). The mechanisms responsible for this include ARoverexpression (7, 8), AR mutations (7), AR coregulators(9), AR activation by intracellular signal-transduction path-ways (10), de novo androgen synthesis (11), and AR splicevariants (7). The transcription factor Twist1 is known topromote epithelial–mesenchymal transition (EMT) and
Authors' Affiliations: Departments of 1Urology and 2Clinical Chemistryand Laboratory Medicine, Graduate School of Medical Sciences, KyushuUniversity, Fukuoka, Japan
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Akira Yokomizo, Department of Urology, Gradu-ate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi,Higashi-ku, Fukuoka 812-8582, Japan. Phone: 81-92-642-5603; Fax:81-92-642-5618; E-mail: [email protected]
binds to the E-box (50-CANNTG-30) sequence to upregulatethe expression of its target genes (12). We previously sug-gested that Twist1 promoted castration resistance throughupregulation of AR expression (13). Twist1was upregulatedfollowing androgen ablation in a mouse xenograft model(14), as well as in human prostate cancer tissues (15).Targeting Twist1 in prostate cancer cells may represent apromising strategy against CRPC by increasing its vulner-ability to androgen-deprivation therapy. However, to thebest of our knowledge, no agents directly targeting Twist1have been identified to date.
Protein kinase C (PKC) has also been suggested to beinvolved in both prostate cancer progression and castrationresistance. The PKC isozymes comprise a large family,which is divided into 3 subfamilies of conventional, novel,and atypical isoforms, according to their structures andregulation mechanisms. The conventional PKCs, includingPKC-a, PKC-bI, PKC-bII and PKC-g , are activated by calci-um, phosphatidylserine, and diacylglycerol (DAG). Thenovel PKCs, including PKC-d, PKC-e, PKC-h, and PKC-q,only require phosphatidylserine and DAG for activation,
whereas the atypical PKCs, including PKC-l/i and PKC-z,only require phosphatidylserine for activation (16). PKCserine/threonine kinases are involved in signal transductionregulating cell proliferation, differentiation, tumorigenesis,apoptosis, EMT, and cytoskeletal remodeling (16). Severalstudies have demonstrated the involvement of PKCs inprostate cancer development and progression. Overexpres-sion of PKC-e in the prostate in a transgenic mouse modelwas associated with the occurrence of prostatic intraepithe-lial neoplasia (17).Conversely, deletionof PKC-e in TRAMPmice inhibited prostate cancer development andmetastasis(18). Similarly, PKC-b is also shown to be augmented inprostate cancer and implicated in prostate cancer develop-
ment (19–21), as supported by numerous in vitro studiesshowing that inhibitionof PKC signaling attenuatedprostatecancer proliferation (21–23). Although, PKCshave alsobeenimplicated in castration resistance, and constitutively activePKC signaling promoted androgen-dependent, as well ascastration-resistant proliferation of prostate cancer cells(24).Aswell, inhumanprostate cancer tissues, PKCsignalingwas highly activated in CRPC specimens compared withhormone-na€�ve cancers (10). Similarly, PKC expression cor-related with poor clinical parameters such as biochemicalfailure after radical prostatectomy, Gleason score, and clin-ical stage (22, 23). These observations collectively implicatePKCs in the pathogenesis of prostate cancer, and suggest thatthe PKC signaling pathway, similar to Twist1, may be apromising therapeutic target in prostate cancer.
Considering such common features of PKC and Twist1 inprostate cancer pathogenesis, especially such as EMT andcastration resistance, a close link between PKC and Twist1has been suggested.We therefore aimed to elucidate the linkof PKC/Twist1 signaling as well as their roles in castration-and enzalutamide-resistant prostate cancer, and examine therelevance of therapeutics targeting PKC signaling, combinedwith androgen-deprivation and enzalutamide treatment.
Materials and MethodsCell culture
Human prostate cancer LNCaP, C4-2, and 22Rv1 cellswere cultured in RPMI 1640 (Invitrogen) containing 10%FBS. LNCaP and 22Rv1 cells were obtained from the Amer-ican Type Culture Collection and LNCaP cells were usedafter 10 to 40 rounds of propagation. C4-2 cells were kindlyprovided by Dr. M. Gleave (Vancouver Prostate Centre,Vancouver, BC, Canada). Enzalutamide-resistant deriva-tives of 22Rv1 cells (22Rv1/MDV cells) were establishedby long-term culture in the appropriatemedium containinggradually increasing concentrations of enzalutamide, andmaintained in media containing 50 mmol/L enzalutamide.The cell lines were maintained in a 5% CO2 atmosphere at37�C.
AntibodiesAntibodies against cyclin D1 (sc-718), AR (N-20, sc-816),
and Twist1 (sc-81417) were purchased from Santa CruzBiotechnology. Antibodies against retinoblastoma protein(pRB; #9309), phosphorylated pRBSer807/811 (p-pRB,#9308), cleaved caspase3 (#9664), caspase3 (#9662),cleaved PARP (#9541), PARP (#9542), and phosphorylatedPKCSer660 (bII) (p-PKC; #9371) were purchased from CellSignaling Technology. Antibodies against E-cadherin(#610181) and fibronectin (#610077)were purchased fromBD Biosciences. Anti-PKC (SAB4502356) and anti-b-actin(A3854) antibodies were obtained from Sigma. Anti-PSAantibody (#1984) was obtained from Epitomics.
Transfection with siRNAs and plasmidsThe double-strandedRNA25-base-pair oligonucleotides
were commercially generated (Invitrogen). The targetsequence was listed in Supplementary Table S1. The
Translational RelevanceThis study showed that androgen deprivation and
novel antiandrogen agent enzalutamide inducedproteinkinase C (PKC) activation and Twist1 induction. How-ever, PKC inhibition using small molecule inhibitorRo31-8220 suppressed Twist1 induction by androgen-deprivation therapy, suggesting the novel relationshipbetween PKC signaling and Twist1 expression. However,PKC inhibition combined with androgen ablation andantiandrogen agent enzalutamide exerted excellent syn-ergistic anticancer effects in androgen-dependent pros-tate cancer cells (LNCaP). Similarly, enzalutamide com-bined with PKC inhibitor Ro31-8220 suppressed cellgrowth in castration-resistant prostate cancer (CRPC)cells (C4-2 and 22Rv1). Finally, enzalutamide-resistantprostate cancer cells were vulnerable to PKC inhibitionby Ro31-8220. Taken together, this study indicated thatPKC inhibition using small molecule inhibitor Ro31-8220 might be a promising therapeutic strategy againstandrogen-dependent as well as CRPC.
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pCMV-AR plasmid expressing wild-type AR was kindlyprovided by Dr. C. Chang (University of Rochester, Roche-ster, NY). Prostate cancer cells were transfected with siRNAor plasmids using Lipofectamine 2000 (Invitrogen)according to the manufacturer’s protocol.
RNA isolation, reverse transcription, and quantitativereal-time PCRRNA isolationand reverse transcriptionwereperformedas
described previously (25, 26). Quantitative real-time PCRwas performed using TaqMan Gene Expression Assays forTwist1 (Hs00361186_m1), full-length AR (Hs00171172_m1), AR V7 (made to order), and glyceraldehyde 3-phosphatedehydrogenase (GAPDH; Hs02758991_g1; Applied Biosys-tems) and TaqMan Gene Expression Master Mix (AppliedBiosystems) with a 7900HT PCR system (Applied Biosys-tems). The transcript levels of the target genes were correctedaccording to the correspondingGAPDH transcript levels. Allvalues represent the results of at least 3 independent experi-
ments. Representative Ct values are listed in SupplementaryTable S2.
Western blotting analysisWhole-cell, nuclear, and cytoplasmic extracts were pre-
pared as described previously (25). Briefly, the concentra-tions of the prepared protein extracts were quantified usinga protein assay (Bio-Rad) based on the Bradford method.Aliquots (30 mg protein) were separated by 4% to 20% SDS-PAGE and transferred to polyvinylidene difluoride micro-porous membranes (GE Healthcare Bio-Science) using asemi-dry blotter. Themembranes were then incubated withthe primary antibodies described above for 1 hour atroom temperature, followed by incubation with peroxi-dase-conjugated secondary antibodies for 40 minutes atroom temperature. The bound antibodies were visualizedusing an ECL Kit (GE Healthcare Bio-Science), and imageswere obtained using an image analyzer (LAS-3000 mini;Fujifilm).
Figure 1. Blocking AR signaling induces PKC phosphorylation and Twist1 expression. A, LNCaP cells were incubated in charcoal-stripped serum (CSS)-supplemented medium with or without 10 nmol/L DHT for 3 hours. Quantitative real-time PCR was performed using the primers and probes for Twist1 andGAPDH. Twist1 transcript levels were corrected according to the corresponding GAPDH transcript levels. All values represent the results of at least 3independent experiments. The Twist1 transcript level in untreated cells was defined as 1. Boxes, mean; bars, � SD. �, P < 0.05 (compared with no treatment).B, LNCaP cellswere incubated inCSS-supplementedmediumwith or without 10 nmol/L DHT for the indicated durations.Whole-cell extracts were subjected toSDS-PAGE, followed by Western blotting analyses of the indicated proteins. C, LNCaP cells were incubated with 10 mmol/L enzalutamide for theindicated durations. Quantitative real-time PCR was performed using the indicated primers and probes for Twist1 and GAPDH. Twist1 transcript levels werecorrected according to the corresponding GAPDH transcript level. All values represent the results of at least 3 independent experiments. The Twist1transcript level in untreated cells was defined as 1. Boxes, mean; bars, � SD. �, P < 0.05 (compared with no treatment) D, LNCaP cells were incubatedwith 10 mmol/L enzalutamide for the indicated durations. Whole-cell extracts were subjected to SDS-PAGE, followed by Western blotting analyses of theindicated proteins.
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Figure2. PKCinhibitorsuppresses inductionofTwist1andAR.A (left), LNCaPcellswere incubated inFBS-supplementedmediumwith2.5mmol/LRo31-8220 forthe indicateddurations.Quantitative real-timePCRwasperformedusing the indicatedprimers andprobes forTwist1,AR, andGAPDH.Twist1 andAR transcriptlevels were corrected according to the corresponding GAPDH transcript level. All values represent the results of at least 3 independent experiments. Thelevel of each transcript in untreated cellswasdefinedas1.Boxes,mean; bars,�SD. �,P< 0.05 (comparedwith no treatment); (right) LNCaPcellswere incubatedwith 2.5mmol/LRo31-8220 for the indicateddurations.Whole-cell extractswere subjected toSDS-PAGE, followedbyWesternblottinganalysesof the indicatedproteins. B (left), LNCaP cells were incubated in CSS-supplemented medium with or without 2.5 mmol/L Ro31-8220 for 0, 3 (Twist1), and 12 hours (AR).Quantitative real-timePCRwasperformedusing theprimersandprobes forTwist1,AR, andGAPDH.Twist1andAR transcript levelswerecorrectedaccording tothe corresponding GAPDH transcript level. All values represent the results of at least 3 independent experiments. The level of each transcript inuntreated cells without Ro31-8220 was defined as 1. Boxes, mean; bars,� SD. �, P < 0.05; (right) LNCaP cells were incubated in CSS-supplemented mediumwith or without 2.5 mmol/L Ro31-8220 for the indicated durations. Whole-cell extracts were subjected to SDS-PAGE, followed byWestern blotting analyses ofthe indicated proteins. C (left), LNCaP cells were incubated with 10 mmol/L enzalutamide and/or 2.5 mmol/L Ro31-8220 for 72 hours. Quantitative real-timePCR was performed using the indicated primers and probes for Twist1, AR, and GAPDH. Twist1 and AR transcript levels were corrected according to thecorresponding GAPDH transcript level. All values represent the results of at least 3 independent experiments. (Continued on the following page.)
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Cell-proliferation assayThe cell-proliferation assay was performed as described
previously (25). Briefly, 2.5 � 104 LNCaP cells seeded into12-well plates were transfected with 1.0 mg/mL of theindicated plasmid and incubated for 24 hours, followedby treatment with or without 5 mmol/L Ro31-8220 for 48hours. Cells were harvested with trypsin and counted dailyusing a cell counter (Invitrogen). The results presented arerepresentative of at least 3 independent experiments.
Cytotoxicity analysisCytotoxicity analyses were performed as described pre-
viously (26). Briefly, prostate cancer cells (2.5 � 103) wereseeded in 96-well plates. Various concentrations of enzalu-tamide with or without Ro31-8220 were applied the fol-lowing day. After 48 hours, surviving cells were stainedusing the alamarBlue assay (TREK Diagnostic Systems) at37�C for 180 minutes. The absorbance of each well wasmeasured using the ARVO MX (Perkin Elmer Inc.) platereader. The results presented are representative of at least 3independent experiments. Synergistic effects of enzaluta-mide and Ro31-8220 were calculated by analyzing the datausing CalcuSyn software (BIOSOFT). A dose-effect curvewas drawn for each treatment and the combination indexwas calculated at several effective doses (combination index¼ 1; additive effect, combination index < 1; synergy effect,combination index > 1; antagonistic effect).
Statistical analysisAll data were assessed using the Student t test. Levels of
statistical significance were set at P < 0.05.
ResultsBlocking AR signaling induces PKC phosphorylationand Twist1 expressionThe responses of PKC and Twist1 to androgen-depriva-
tion therapy by castration and/or an antiandrogen agentwere examined by measuring Twist1 transcript levels afterandrogen depletion. As shown in Fig. 1A, androgen deple-tion increased Twist1 transcription in androgen-dependentLNCaP cells, although itwas unaffectedwhen the cells wereincubated with dihydrotestosterone (DHT). PKC phos-phorylation and Twist1 protein were also induced byandrogen depletion in LNCaP cells, which effects wereabolished by incubation with androgen (Fig. 1B). Similar-ly, blocking AR signaling using the novel antiandrogenenzalutamide upregulated Twist1 transcription (Fig. 1C),PKC phosphorylation, and Twist1 protein expression,which was accompanied by a decreased E-cadherin and
an increased fibronectin expression (Fig. 1D), consistentlywith the previous reports on EMT promotion by enzalu-tamide (27, 28).
PKC inhibitor suppresses Twist1 and AR inductionWe used the small molecule inhibitor of PKC, Ro31-
8220, to elucidate the relationship between PKC and theTwist1/AR pathway. Ro31-8220 reduced Twist1 and ARtranscript levels (Fig. 2A) and Twist1 and AR protein levelsin LNCaP cells (Fig. 2A). Accordingly, we investigated theeffects of Ro31-8220 on Twist1 and AR inductions byblockade of AR signaling. As shown in Fig. 2B, Ro31-8220 suppressed the induction of Twist1 and AR transcrip-tion and Twist1 and AR protein expression under andro-gen-depleted conditions. Similarly, Ro31-8220 abolishedthe induction of Twist1 and AR mRNA by enzalutamide,accompanied by similar effects on Twist1 and AR proteinlevels (Fig. 2C), although AR protein expression wasdecreased at 72 hours possibly because of an increased ARinstability (29).
To further elucidatewhat PKC isoformsmediate the effectof Ro31-8220, we examined Twist1 and AR transcript levelsafter knockdown of PKC isoforms. As shown in Fig. 2D,amongvarious PKC isoforms, knockdownofPKC-borPKC-e reduced Twist1 and AR transcript levels. Consistently,PKC-b or PKC-e shutdown suppressed Twist1 and AR pro-tein expression (Fig. 2D).
PKC inhibitor Ro31-8220 augments therapeutic effectsof androgen depletion and enzalutamide in androgen-dependent LNCaP cells
Given the above results, we examined the therapeuticpotential of Ro31-8220 for prostate cancer. Cellular sen-sitivities of LNCaP cells to Ro31-8220 were examinedwith or without androgen. As shown in Fig. 3A, androgendepletion augmented the anticancer effect of Ro31-8220.This enhancing effect of androgen depletion on Ro31-8220 cytotoxicity was supported by reduced phosphory-lation of pRB and cyclin D expression, which regulate cell-cycle transition from G1 to S phase (Fig. 3B).
We also revealed that Ro31-8220 augmented the ther-apeutic effect of enzalutamide. The combination indexdemonstrated synergism between enzalutamide andRo31-8220 (ED50 ¼ 0.43498, ED75 ¼ 0.30484, andED90 ¼ 0.22206; Fig. 3C). This effect was confirmed bydecreases in pRB phosphorylation and cyclin D1 expres-sion (Fig. 3D). Inversely, AR overexpression partiallyrescued the suppression of cell proliferation by Ro31-8220 (Fig. 3E).
(Continued.) The level of each transcript in untreated cells without Ro31-8220 was defined as 1. Boxes, mean; bars,� SD. �, P < 0.05; (right) LNCaP cells wereincubated with 10 mmol/L enzalutamide and/or 2.5 mmol/L Ro31-8220 for the indicated durations. Whole-cell extracts were subjected to SDS-PAGE,followed byWestern blotting analyses of the indicated proteins. D (left), LNCaP cells were transfected with 40 nmol/L of the indicated siRNA and incubated for48 hours. Quantitative real-time PCR was performed using the indicated primers and probes. The transcript level of the target transcript was correctedaccording to the corresponding GAPDH transcript level. All values represent the results of at least 3 independent experiments. The level of each transcript incontrol siRNA-transfected cells was defined as 1. Boxes, mean; bars, � SD. �, P < 0.05 (compared with control siRNA); (right) LNCaP cells weretransfected with 40 nmol/L of the indicated siRNA and incubated for 72 hours. Whole-cell extracts were subjected to SDS-PAGE, followed by Westernblotting analyses of the indicated proteins.
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PKC phosphorylation is upregulated, and Ro31-8220augments the therapeutic effect of enzalutamide incastration-resistant C4-2 cells
We compared the PKC activation statuses of androgen-dependent prostate cancer and CRPC. C4-2 cells arewell-known castration-resistant derivatives of andro-gen-dependent LNCaP cells (30). PKC phosphorylationlevel was increased in C4-2 cells compared with LNCaPcells (Fig. 4A). Similarly, in another castration-resistantCxR cell line, which we established previously (13), PKCphosphorylation was also increased (Fig. 4A). Becauseenzalutamide has shown efficacy even in CRPC (5), weinvestigated the combined effect of Ro31-8220 andenzalutamide in C4-2 cells. As shown in Fig. 5B,
Ro31-8220 exerted a modest suppressive effect on cel-lular viability in C4-2 cells, indicated by combinationindex values (ED50 ¼ 1.72601, ED75 ¼ 0.99113, andED90 ¼ 0.65574; Fig. 4B). This synergistic effect of enza-lutamide and Ro31-8220 was confirmed by decreasedpRB phosphorylation and cyclin D1 expression (Fig. 4C),as in LNCaP cells.
Ro31-8220 downregulates full-length AR and AR splicevariant, and augments the therapeutic effect ofenzalutamide in 22Rv1 cells
We subsequently examined the effects of Ro31-8220 onTwist1 and AR expression in another castration-resistant22Rv1 cell line, which expresses the usual full-length AR, as
Figure 3. PKC inhibitor Ro31-8220 augments therapeutic effect of androgen deletion and enzalutamide. A, LNCaP cells were seeded in 96-well plates. On thefollowing day, 5 or 10 mmol/L Ro31-8220 was applied to CSS-supplemented medium with or without 10 nmol/L DHT. After 48 hours, the cell survivalrateswere analyzed by cytotoxicity analyses. Boxes,mean; bars,�SD. �,P < 0.05 (comparedwith no treatment). B, LNCaP cells were treatedwith 2.5 mmol/LRo31-8220 in CSS-supplemented medium with or without 10 nmol/L DHT for 48 hours. Whole-cell extracts were subjected to SDS-PAGE, followed byWestern blotting analyses of the indicated proteins. C, LNCaP cells were seeded in 96-well plates. On the following day, various concentrations ofenzalutamide and Ro31-8220 were applied in a ratio of 20:1. After 48 hours, the cell survival rates were analyzed by cytotoxicity analyses. Boxes, mean;bars, � SD. D, LNCaP cells were treated with or without 50 mmol/L enzalutamide and 2.5 mmol/L Ro31-8220. After incubation for 48 hours, whole-cellextracts were subjected to SDS-PAGE, followed by Western blotting analyses of the indicated proteins. E, LNCaP cells seeded into 12-well plates weretransfectedwith 1.0 mg/mLof the indicatedplasmid and incubated for 24 hours, followed by treatmentwith or without 5mmol/L Ro31-8220. After 48 hours, thecell proliferations were analyzed by cytotoxicity analyses. Boxes, mean; bars, � SD.
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well as the AR splice variant ARV7 (31). Ro31-8220 reducedexpression levels of Twist1, as well as full-length AR and ARV7 at both the transcript and protein levels (Fig. 5A).Furthermore, Ro31-8220 augmented the suppressive effect
of enzalutamide in 22Rv1 cells, as indicated by the combi-nation index values (ED50¼ 0.37048, ED75¼ 0.41529, andED90¼ 0.51479), as in LNCaP andC4-2 cells (Fig. 5B). Thissynergistic effect of enzalutamide and Ro31-8220 was con-firmed by decreased pRB phosphorylation and cyclin D1expression with combined treatment (Fig. 5C).
To delineate the link between Twist1 and AR in 22Rv1cells, we examined AR level after Twist1 knockdown. Asshown in Fig. 5D, Twist1 knockdown reduced the bothtranscript and protein expression of full-length AR aswell asAR V7. Furthermore, Twist1 knockdown also augmentedantiproliferative effect of enzalutamide similarly to Ro31-8220 (Fig. 5E).
PKC phosphorylation is upregulated, and Ro31-8220exerts enhanced cytotoxic effect in enzalutamide-resistant 22Rv1 cells
We finally aimed to clarify the effects of Ro31-8220 inenzalutamide-resistant cells. As noted above, enzalutamideis effective even in CRPC. Indeed, enzalutamide was alsoeffective in 22Rv1 cells at a relatively high concentration,comparedwith that in LNCaP andC4-2 cells (Figs. 4 and 5).We established an enzalutamide-resistant 22Rv1 cell line(22Rv1/MDV cells) by long-term culture in graduallyincreasing concentrations of enzalutamide. As shownin Fig. 6A, 22Rv1/MDV cells were about twice as resistantto enzalutamide as their parental 22Rv1 cells, although nomutation was detected in the AR-coding region (data notshown). PKC phosphorylation was increased in 22Rv1/MDV cells, accompanied by increased transcript and pro-tein expression of Twist1, full-length AR, and AR V7 com-pared with parental cells (Fig. 6B). The PKC inhibitor Ro31-8220 was therefore more cytotoxic to 22Rv1/MDV cellsthan to 22Rv1 cells (Fig. 6C).
DiscussionThe mechanism responsible for resistance to androgen-
deprivation therapy is thought to involve activation of theprosurvival and antiapoptotic pathways, including AR sig-naling (32). PKCandTwist1 signalinghavebeen consideredto promote castration resistance, possibly through AR sig-naling (13, 14, 22). Both PKC and Twist1 are consistentlyactivated by blocking AR function, suggesting that andro-gen-deprivation therapy itself evokes treatment resistance,while exerting an excellent therapeutic effect on androgen-dependent prostate cancer. The rationale of this study wastherefore that blocking activation of prosurvival and anti-apoptotic pathways might augment the therapeutic effectsof androgen-deprivation therapy.
Because no direct Twist1 inhibitor is available, we usedthe small molecule PKC inhibitor Ro31-8220 to suppressthe unfavorable effects of androgen-deprivation therapy.Indeed, blocking AR signaling induced Twist1 expression,resulting in activation of the AR pathway and contributingto prosurvival and antiapoptotic effects in prostate cancer.The PKC inhibitor Ro31-8220 thus blunted the inductionofTwist1 and AR by shutdown of AR signaling, indicatingdirect or indirect regulation of Twist1 by PKC, especially
Figure 4. PKC phosphorylation is upregulated, and Ro31-8220 augmentsthe therapeutic effect of enzalutamide inC4-2 cells. A,whole-cell extractsfrom LNCaP, C4-2, and CxR cells were subjected to SDS-PAGE,followed by Western blotting analyses of the indicated proteins. B, C4-2cells were seeded in 96-well plates. On the following day, variousconcentrations of enzalutamide and Ro31-8220 were applied in a ratio of20:1. After 48 hours, the cell survival rates were analyzed by cytotoxicityanalyses. Boxes, mean; bars, � SD. C, C4-2 cells were treated with orwithout 50 mmol/L enzalutamide and 2.5 mmol/L Ro31-8220. Afterincubation for 48 hours, whole-cell extracts were subjected to SDS-PAGE, followed by Western blotting analyses of the indicated proteins.
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Figure 5. Ro31-8220 downregulates full-length AR and AR splice variant, and augments the therapeutic effect of enzalutamide. A (left), 22Rv1 cells wereincubated with 1 mmol/L Ro31-8220 for the indicated durations. Quantitative real-time PCR was performed using the indicated primers and probes. Thetranscript level of the target transcript was corrected according to the corresponding GAPDH transcript level. All values represent the results ofat least 3 independent experiments. The level of each transcript from mock-transfected cells was defined as 1. Boxes, mean; bars, � SD; (right) 22Rv1cells were incubated with 1 mmol/L Ro31-8220 for the indicated durations. Whole-cell extracts were subjected to SDS-PAGE, followed by Westernblotting analyses of the indicated proteins. B, 22Rv1 cells were seeded in 96-well plates. On the following day, various concentrations ofenzalutamide and Ro31-8220 were applied in a ratio of 20:1. After 48 hours, the cell survival rates were analyzed by cytotoxicity analyses. Boxes, mean;bars, � SD. C, 22Rv1 cells were treated with or without 50 mmol/L enzalutamide and 2.5 mmol/L Ro31-8220. After incubation for 48 hours,whole-cell extracts were subjected to SDS-PAGE, followed by Western blotting analyses of the indicated proteins. D (left), 22Rv1 cells were transfectedwith 40 nmol/L of the indicated siRNA and incubated for 48 hours. Quantitative real-time PCR was performed using the indicated primers andprobes. The transcript level of the target transcript was corrected according to the corresponding GAPDH transcript level. All values represent theresults of at least 3 independent experiments. The level of each transcript in control siRNA-transfected cells was defined as 1. Boxes, mean; bars,� SD. �, P < 0.05 (compared with control siRNA); (right) 22Rv1 cells were transfected with 40 nmol/L of the indicated siRNA and incubated for72 hours. Whole-cell extracts were subjected to SDS-PAGE, followed by Western blotting analyses of the indicated proteins. E, 22Rv1 cells transfectedwith 40 nmol/L of the indicated siRNA were seeded in 96-well plates. On the following day, vehicle or 50 mmol/L enzalutamide were applied.After 48 hours, the cell survival rates were analyzed by cytotoxicity analyses. Boxes, mean; bars, � SD.
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PKC-b and PKC-e. To the best of our knowledge, no pre-vious studies have reported on the regulation of Twist1signaling by PKC, although PKC is known to be involved inthe regulation of EMT (33), suggesting that PKC mayregulate EMT through Twist1 signaling. In contrast, Twist1has been shown to regulate AR expression as a transcriptionfactor in prostate cancer cells (13). In line with thesefindings, PKC inhibition suppressed Twist1 activity, result-ing in suppression of AR induction by androgen-depriva-tion therapy. Taken together, PKC seemed to regulateTwist1/AR signaling in response to androgen-deprivationtherapy, functioning as an inhibitor of castration resistance,at least in an in vitro.
When the PKC inhibitor Ro31-8220 was used in com-bination with androgen deprivation therapy, it augment-ed the therapeutic effects of androgen depletion, as wellas the effects of the novel antiandrogen enzalutamide inandrogen-dependent prostate cancer cells. Androgen-deprivation therapy is known to induce cell-cycle arrest atG1–S phase (13, 15). The addition of a PKC inhibitorenhanced the decreases in phosphorylated pRB and cyclinD1 expression induced by androgen-deprivation therapy,suggesting augmentation of cell-cycle arrest at G1–S phaseby PKC inhibition. Ro31-8220 also demonstrated anexcellent synergistic therapeutic effect with enzalutamidein 22Rv1 cells, whereas the synergistic effect was modestin C4-2 cells; this difference may be related to the lowerTwist1 expression in C4-2 cells compared with 22Rv1cells (data not shown). Enzalutamide has recently beenshown to have a remarkable therapeutic effect on CRPC,even after chemotherapy (5). Based on the results of aphase III clinical trial, the FDA has approved enzaluta-mide for CRPC in a post-docetaxel setting. PKC inhibitorscould thus be applied to prostate cancer from the andro-gen-dependent to the castration-resistant stages.
So far, several inhibitors targeting PKC have shownsignificant antitumor effects in various cancers in preclin-ical and clinical studies. Midostaurin (34) was the firstPKC inhibitor evaluated in clinical trials; however, it hasfailed to demonstrate significant activity in clinical trialsto date (35). ISIS 3521, a phosphorothioate antisenseoligonucleotide targeting the 30-untranslated region ofPKC-a mRNA (36,37), demonstrated anticancer activityin various types of cancers, including tumors refractory toconventional chemotherapy (38, 39), but has so far failedto exert clinically significant efficacy when combined withchemotherapy against advanced lung cancer in phase IIItrials (40). In contrast to these disappointing results,PKC-b inhibitor enzastaurin as a single agent showedpromising results in phase II trials against diffuse largeB-cell lymphoma (41), although one phase III trial inpatients with recurrent glioblastoma found no superioreffect to conventional therapy (42). A further phase IIIstudy in patients with diffuse large B-cell lymphoma iscurrently underway (40). Moreover, a phase I studycombining enzastaurin with gemcitabine and cisplatinhas suggested a promising effect in combination withchemotherapy (43). Several PKC inhibitors are now
Figure 6. PKC phosphorylation is upregulated, and Ro31-8220 exertsenhanced cytotoxic effect in enzalutamide-resistant 22Rv1cells. A, 22Rv1and 22Rv1/MDVcellswere seeded in 96-well plates. On the following day,various concentrations of enzalutamide were applied. After 48 hours, thecell survival rates were analyzed by cytotoxicity analyses. Boxes, mean;bars, � SD. B, left, after extraction of total RNA from 22Rv1 and 22Rv1/MDV cells and synthesis of cDNA, quantitative real-time PCR wasperformed using the indicated primers and probes. The transcript level ofthe target transcript was corrected according to the correspondingGAPDH transcript level. All values represent the results of at least 3independent experiments. The level of each transcript from 22Rv1 cellswas defined as 1. Boxes, mean; bars, � SD. �, P < 0.05 (compared with22Rv1 cells); right, whole-cell extracts from 22Rv1 and 22Rv1/MDV cellswere subjected to SDS-PAGE, followed by Western blotting analyses ofthe indicated proteins. C, 22Rv1 and 22Rv1/MDV cells were seeded in96-well plates. On the following day, various concentrations ofRo31-8220were applied. After 48 hours, the cell survival rates were analyzed bycytotoxicity analyses. Boxes, mean; bars, � SD.
PKC and Twist1 in Androgen-Deprivation Therapy
www.aacrjournals.org Clin Cancer Res; 20(4) February 15, 2014 959
under development for clinical use, and are expected to beused in the future, although the route to clinical use is noteasy. About prostate cancer, enzastaurin has not yetshown promising results as a single agent, althoughthe possibility of combining enzastaurin with docetaxelhas been suggested (44). The results of this study indi-cated that PKC inhibition, combined with blocking ARsignaling, augmented the therapeutic effect of each indi-vidual component, suggesting the possible therapeuticvalue of a PKC inhibitor administered concurrently withandrogen-deprivation therapy. The limited therapeuticoptions for CRPC suggest that this option should beexplored further.
In conclusion, PKC/Twist1 signaling upregulated ARtranscription in response to AR inhibition. In addition,inhibition of PKC augmented the anticancer effect ofandrogen-deprivation therapy, including that achievedwith enzalutamide. These results suggest that PKC/Twist1signaling may contribute to cellular survival duringandrogen-deprivation therapy through regulation of ARtranscription. PKC seems to represent a promising targetfor prostate cancer treatment, especially in combinationwith androgen-deprivation therapy, although additionalexperiments using an in vivo model would be requiredbefore clinical application.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConceptionanddesign:M.Shiota, A. Yokomizo, A. Takeuchi, E. Kashiwagi,J. InokuchiDevelopment of methodology: K. TatsugamiAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M. Shiota, A. YokomizoAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): M. Shiota, A. Yokomizo, A. TakeuchiWriting, review, and/or revision of the manuscript: M. Shiota, A. Yoko-mizo, A. TakeuchiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. Imada, Y. Song, K. Tatsugami,T. UchiumiStudy supervision: J. Inokuchi, S. Naito
AcknowledgmentsThe authors thank Dr. M. Gleave (Vancouver Prostate Centre, Vancouver,
BC, Canada) for providing the C4-2 cells and Dr. C. Chang (University ofRochester, Rochester, NY) for providing the pCMV-AR plasmid. The authorsalso thank Dr. D. Kang (Kyushu University, Fukuoka, Japan) for assistancewith quantitative real-time PCR, Edanz Group Japan for editorial assistance,and N. Hakoda and E. Gunshima for technical assistance.
Grant SupportThis work was supported by Kakenhi grants (25462484 and 24890160)
from the Ministry of Education, Culture, Sports, Science and Technology ofJapan (MEXT), Japan, a Medical Research Promotion Grant from TakedaScience Foundation, Japan, a Research Promotion Grant from the UeharaMemorial Foundation, Japan, Cancer Research Promotion Grant for YoungResearcher from The Yasuda Medical Foundation, Japan, and a ResearchPromotion Grant from The Sagawa Foundation for Promotion of CancerResearch, Japan.
The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received July 2, 2013; revised October 18, 2013; accepted November 19,2013; published OnlineFirst December 18, 2013.
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2014;20:951-961. Published OnlineFirst December 18, 2013.Clin Cancer Res Masaki Shiota, Akira Yokomizo, Ario Takeuchi, et al. Prostate CancerAnticancer Effects of Androgen Deprivation and Enzalutamide in Inhibition of Protein Kinase C/Twist1 Signaling Augments
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