chromatin, gene, and rna regulation · chromatin immunoprecipitation chromatin immunoprecipitation...

Chromatin, Gene, and RNA Regulation

Role of WNT7B-induced Noncanonical Pathway in AdvancedProstate Cancer

Dali Zheng1,3, Keith F. Decker1,3, Tianhua Zhou1,3, Jianquan Chen3,4, Zongtai Qi1,3, Kathryn Jacobs1,3,Katherine N. Weilbaecher2,3, Eva Corey5, Fanxin Long3,4, and Li Jia1,3

AbstractAdvanced prostate cancer is characterized by incurable castration-resistant progression and osteoblastic bone

metastasis. While androgen deprivation therapy remains the primary treatment for advanced prostate cancer,resistance inevitably develops. Importantly, mounting evidence indicates that androgen receptor (AR) signalingcontinues to play a critical role in the growth of advanced prostate cancer despite androgen deprivation. While themechanisms of aberrant AR activation in advanced prostate cancer have been extensively studied, the downstreamAR target genes involved in the progression of castration resistance are largely unknown.Here, we identifyWNT7Bas a direct AR target gene highly expressed in castration-resistant prostate cancer (CRPC) cells.Our results show thatexpression of WNT7B is necessary for the growth of prostate cancer cells and that this effect is enhanced underandrogen-deprived conditions. Further analyses reveal that WNT7B promotes androgen-independent growth ofCRPC cells likely through the activation of protein kinase C isozymes. Our results also show that prostate cancer-produced WNT7B induces osteoblast differentiation in vitro through a direct cell–cell interaction, and thatWNT7B is upregulated in human prostate cancer xenografts that cause an osteoblastic reaction when grownin bone. Taken together, these results suggest that AR-regulated WNT7B signaling is critical for the growth ofCRPC and development of the osteoblastic bone response characteristic of advanced prostate cancer. Mol CancerRes; 11(5); 482–93. �2013 AACR.

IntroductionProstate cancer is dependent on androgens for growth.

While androgen deprivation therapy for advanced prostatecancer effectively reduces tumor burden, the disease typicallyrecurs as incurable castration-resistant prostate cancer(CRPC). The progression of CRPC is often associated withmetastases that occur primarily in bone (1). While bothosteolytic (bone lysing) and osteoblastic (bone forming)processes occur during prostate cancer bone metastasis,prostate cancer predominantly yields osteoblastic lesions,in contrast to the mostly osteolytic lesions observed in othercancers (lung, kidney, and breast). Studies suggest thattumor-induced osteoblastic and osteolytic activity may playdifferent roles in promoting tumor growth (2). While anti-osteolytic therapies have been developed, they show limited

effectiveness against osteoblastic bone disease in prostatecancer. Antiosteoblastic approaches still lack therapeutictargets and remain restricted to palliative radiotherapy andsystemic chemotherapy (3). Increased understanding of themechanisms underlying castration resistance and osteoblas-tic bone metastases will provide opportunities to developmore effective therapies for advanced prostate cancer.WNT signaling plays a central role in both developmental

and oncogenic processes. There are 19 closely relatedWNTgenes identified in humans. SecretedWNT proteins bind tothe Frizzled receptors and other coreceptors at the plasmamembrane and initiate canonical or noncanonical intracel-lular signaling cascades (4). Canonical WNT signalingstabilizes intracellular b-catenin by preventing its phosphor-ylation-dependent degradation, resulting in transcriptionalactivation of WNT target genes. Noncanonical WNT sig-naling activates intracellular kinases such as c-Jun-NH2kinase (JNK), Ca2þ/calmodulin-dependent protein kinaseII (CaMKII), and protein kinase C (PKC). Aberrant WNTsignaling has been linked to a number of cancers. In prostatecancer, nuclear b-catenin was frequently detected inadvanced prostate cancer despite the infrequency of b-cate-nin mutations (�5%) in tumors (5, 6). Gene expressionprofiles of prostate cancer often show overexpression ofWNT ligands or underexpression of secreted WNT inhi-bitors (7–10). These changes are likely to be involved inenhancement of nuclear b-catenin and prostate cancerdevelopment and progression.

Authors' Affiliations: 1Center for Pharmacogenomics, 2Division of Oncol-ogy, 3Department of Medicine, 4Department of Developmental Biology,Washington University School of Medicine, St. Louis, Missouri; and5Department of Urology, University of Washington School of Medicine,Seattle, Washington

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Li Jia, Center for Pharmacogenomics, Depart-ment of Medicine,Washington University School of Medicine, 660 S EuclidAvenue, Campus Box 8220, St. Louis, MO 63110. Phone: 314-362-6968;Fax: 314-362-8844; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-12-0520

�2013 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 11(5) May 2013482

on April 25, 2021. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst February 5, 2013; DOI: 10.1158/1541-7786.MCR-12-0520

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It is well known that AR signaling is essential during allphases of prostate cancer development and progression.While b-catenin is a coactivator for TCF/LEF transcriptionfactors in the canonical WNT pathway, it also interacts withAR and enhances AR-mediated transcription in the presenceof androgen (11–13). Although cooperative gene regulationby AR and TCF/LEF transcription factors has beenreported, these 2 transcription factors often compete forb-catenin, with both signaling pathways promoting cellgrowth (14, 15). The relative importance of AR andTCF/LEF factors in prostate cancer growth depends on theexpression levels of each component at any given time, aswell as the local androgen levels. In the presence of androgen,activation ofWNT/b-catenin signaling in AR-positive pros-tate cancer cells often acts through AR-dependent mechan-isms rather than classical TCF/LEF-dependent mecha-nisms (16).Emerging evidence suggests that WNT signaling plays an

important role during progression to CRPC. WNT signal-ing supports the survival and growth of prostate epithelialcells after androgen deprivation (17, 18). Enhancement ofthe WNT/b-catenin pathway modulates AR signaling andaugments AR transcriptional activity under androgen-deprived conditions (19). Interaction between AR andWNT signaling pathways in CRPC is also significantlyincreased in vivo (20). Recent exome sequencing furtherrevealed a significant enrichment of mutations in WNTsignaling components in castration-resistant compared withpaired castration-sensitive tumors (21). While these resultssupport the importance ofWNT signaling in prostate cancerprogression, the WNT members critical for CRPC growthand the mechanisms of WNT regulation and signaling inCRPC remain unclear.Given the critical role of WNT signaling in the differen-

tiation and activity of osteoblasts (22), WNT signaling islikely to contribute to the development of prostate cancerosteoblastic bone metastasis. This is supported by evidencethat the WNT antagonist DKK1 strongly inhibits boneformation in osteoblastic lesions (9). It is unclear, however,which WNT ligands induce osteoblastic activity in prostatecancer bonemetastasis and why osteoblastic lesions are oftenobserved in prostate cancer given that WNT signaling isactivated in other cancers as well.In the present study, we have established a new connection

between AR and WNT signaling with important implica-tions for prostate cancer progression and bone metastasis.Our data show that WNT7B expression is regulated by AR,WNT7B remains at a high level in CRPC cells afterandrogen deprivation, and that WNT7B activates a nonca-nonical WNT signaling pathway promoting prostate cancergrowth and survival after androgen deprivation. Further-more, we show that prostate cancer-produced WNT7B isassociated with osteoblastic responses in the bone. Giventhat AR is expressed in almost all prostate cancer cells andremains active even under castrated conditions, AR-regu-lated WNT7B signaling is likely one of the critical mechan-isms underlying osteoblastic bone metastasis in advancedprostate cancer.

Materials and MethodsCell lines and materialsLNCaP,C4-2B, 22RV1,HCT116, ST2, andC3H10T1/2

cells were maintained in cell culture medium with 10%FBS as previously described (23–25). LAPC4 cells (fromDr. Charles Sawyers, University of California, Los Angeles;ref. 26) were maintained in RPMI-1640 with 20% FBS. Allcell lines were frozen within 2 passages after receipt fromthe cell bank and used within less than 6 months after resusc-itation. All prostate cancer cell lines were authenticated on thebasis of growth, morphology, and RNA-sequencing results.Recombinant human WNT3A (R&D Systems) and 5a-dihydrotestosterone (DHT, Sigma-Aldrich) were purchased.

Stable prostate cancer cell lines with WNT7Bknockdown or overexpressionFor stable WNT7B knockdown, C4-2B cells were infected

with lentiviruses encoding short hairpinRNA(shRNA) againsthuman WNT7B or LacZ control for 24 hours, and selectedwith puromycin (1.5 mg/mL). Pooled populations were main-tained in RPMI-1640 containing 10% FBS and puromycin.WNT7B knockdown efficiency was examined by quantitativereverse transcription PCR (qRT-PCR). The shRNA lentiviralvectors were obtained from the RNAi Core at WashingtonUniversity (St. Louis, MO). The shRNA sequences are listedin Supplementary Table S1. The pLKO.1 vector system wasused to produce lentiviral particles. For stable WNT7B over-expression, viruses expressing mouse WNT7B and coexpres-sing GFP via an internal ribosome entry site were generated aspreviously described (25, 27). LNCaP, C4-2B, and LAPC4cells were infected with viruses expressing WNT7B or emptyvector, andmore than 90% infection efficiencywas achieved asdetermined by GFP detection.

Western blot analysisWhole-cell lysates were prepared from prostate cancer cells as

indicated, proteins were separated on 4%–15% SDS-PAGEgels (Bio-Rad), and transferred onto polyvinylidene difluoridemembranes. Blotted proteins were detected by the followingantibodies: anti-WNT7B (AF3460; 1:1,000, R&D Systems),anti-AR (ab74272; 1:1,000,Abcam), anti-b-tubulin (sc-80011;1:4,000, Santa Cruz Technology), anti-phospho-b-catenin(T41/S45; 9565, 1:1,000, Cell Signaling), anti-b-catenin(9582; 1:1,000, Cell Signaling), anti-phospho-MARCKS(2741; 1:250, Cell Signaling), anti-MARCKS (5607;1:1,000, Cell Signaling), anti-lamin B1 (ab16048; 1:1,000,Abcam), anti-PKCa (sc-208; 1:2,000, Santa Cruz Technolo-gy), anti-PKCd (sc-937; 1:1,000, Santa Cruz Technology).Nuclear proteins were extracted using NE-PER Nuclear andCytoplasmic Extraction Reagent Kit (Thermo Scientific).Membraneproteinswere extractedusingMem-PEREukaryoticMembraneProtein ExtractionReagentKit (ThermoScientific).

Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) analyses were

conducted as described previously (23). The primers forquantitative real-time PCR (qPCR) are listed in Supplemen-tary Table S1.

WNT7B in Advanced Prostate Cancer

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qRT-PCRqRT-PCR was conducted as described previously (23).

Values are means � SDs of triplicate wells. The primers arelisted in Supplementary Table S1.

RNA interferenceA predesigned pool of 4 siRNA duplexes (Thermo Sci-

entific) or single siRNA duplexes (Qiagen or IDT) againstWNT7B, AR, b-catenin, PKCa, PKCd, and MARCKSwere purchased. A nonspecific pool of 4 siRNA duplexes(siNS1) and 2 different nonspecific single siRNA duplexes(siNS2 and siNS3) were used as controls. All siRNAsequences are listed in Supplementary Table S1. Cells weretransfected with siRNA as indicated at a final concentrationof 15 nmol/L using Lipofectamine RNAiMAXTransfectionReagent (Life Technologies) according to themanufacturer'sinstruction.

Cell proliferationLNCaP (1 � 104 cells/well), C4-2B (6 � 103 cells/well),

or 22RV1 (1 � 104 cells/well) cells were plated in 96-wellplates and transfected with gene-specific siRNA. Cells weremaintained in phenol red-free RPMI-1640 containing 5%charcoal-stripped serum (CSS) with ethanol or 10 nmol/LDHT for 3 or 5 days. The number of viable cells wasanalyzed using the CCK-8 kit (Dojindo MolecularTechnologies).

Soft agar colony formation assayC4-2B cells with stable WNT7B knockdown or over-

expression were plated in 24-well plate (2,000 cells/well).Cells were suspended in RPMI-1640 containing 10% FBSand 0.5% agar, and overlaid onto a solid layer of the samemedium containing 1.0% agar. After 2-week incubation,colonies were counted and photographed at � 40magnification.

Apoptosis assayLNCaP, C4-2B, or 22RV1 cells were grown in phenol

red-free RPMI-1640 containing 5%CSS with ethanol or 10nmol/L DHT for 3 days after WNT7B or nonspecificsiRNA transfection. Cell apoptosis was determined bycaspase-3 and -7 activities using Caspase-Glo 3/7 assay kit(Promega) or by DNA fragmentation using terminal deox-ynucleotidyl transferase–mediated dUTP nick end labeling(TUNEL) assay kit (Promega) according to the manufac-turer's instruction.

Luciferase assayC4-2B (3 � 104 cells/well) and HCT116 cells (1 � 104

cells/well) were plated in 48-well plates and grown in RPMI-1640 containing 5% FBS for 24 hours. Cells were trans-fected with TOPFlash or FOPFlash luciferase reporterplasmids (200 ng/well, Addgene) using Lipofectamine LTXReagent (Life Technologies). pRL-TK Renilla luciferasereporter (10 ng/well, Promega) was cotransfected as aninternal control. After plasmid transfection, cells were trea-ted with or without recombinant WNT3A (50 ng/mL,

R&D systems) for 24 hours. Firefly and Renilla luciferaseactivities were measured using Dual-Glo Luciferase AssaySystem (Promega). The results are represented as Firefly/Renilla ratio.

Osteoblast differentiation assayST2 and C3H10T1/2 cells (1 � 105 cells/well) trans-

duced to overexpress WNT7B or vector control were seededin a 12-well plate in a-minimum essential medium(a-MEM) and basal medium eagle (BME), respectively,with 10%FBS in the presence of 50mg/mL ascorbic acid and50 mmol/L b-glycerophosphate. After 1 week, the expres-sion levels of osteoblast differentiation markers, alkalinephosphatase (ALP), and bone sialoprotein (BSP) were mea-sured by qRT-PCR. ALP enzymatic activity was also mea-sured as previously described (25, 27). After 3 weeks ofincubation, mineralization was examined using von Kossastaining as previously described (25, 27).In coculture experiments, ST2 or C3H10T1/2 cells (1�

105 cells/well) were seeded in a 12-well plate together withprostate cancer cells at 1:1 ratio. Prostate cancer cells werestably overexpressingWNT7B or transfected withWNT7BsiRNA 1 day before coculture. The cells were grown ina-MEM or BMEmedium with 10% FBS in the presence of50 mg/mL ascorbic acid and 50 mmol/L b-glyceropho-sphate. ST2 or C3H10T1/2 osteoblast differentiation wasexamined as described above.

Microarray data analysisFor gene expression from prostate cancer xenograft

tumors, RNA was extracted from 24 LuCaP prostate cancerxenografts as described previously (28) and amplified andhybridized to Agilent 44K whole human genome expressionoligonucleotide microarrays (Agilent Technologies). Probelabeling and hybridization were conducted following theAgilent suggested protocols, and fluorescent array imageswere collected using the Agilent DNA microarray scannerG2565BA. Agilent Feature Extraction Software was used togrid, extract, and normalize data. Differences in gene expres-sion were analyzed via Welch test with the Benjamini–Hochberg correction for multiple testing. WNT7B expres-sion data from clinical prostate cancer tumors were obtainedfrom the NCBI GEO database GSE2443 (29) andGSE3325 (30), and analyzed using limma (31).

ResultsWNT7B expression is regulated by the AR in CRPC cellsUsing RNA-seq, we examined WNT gene expression in

androgen-dependent LNCaP and LNCaP-derived castra-tion-resistant C4-2B cells in the presence and absence of10 nmol/L DHT (23). Of 19WNT genes,WNT7B was theonly WNT member found to be upregulated after DHTstimulation in both LNCaP and C4-2B cells. RNA-seqresults for all WNT genes are presented in SupplementaryTable S2. WNT7B expression results were confirmed byqRT-PCR and Western blot analysis (Fig. 1A and B).Notably, the high level of WNT7B expression in C4-2B

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cells in the absence of androgen is comparable with theDHT-induced WNT7B expression levels in LNCaP cells,implying that WNT7B may play a role in androgen-inde-pendent growth of C4-2B cells.ChIP-seq analyses of LNCaP andC4-2B cells revealed one

strong AR binding site approximately 1.7 kb downstream ofthe transcription start site (within the first intron) and oneweak AR binding site approximately 700 bp downstream ofthe 30 end of theWNT7B gene (Fig. 1C). Both binding siteswere validated by an independent site-specific ChIP-qPCRanalysis (Fig. 1D). Interestingly, we observed AR occupancy(5-fold enrichment over the control) at the weak AR-bindingsite (Site 1) in the absence of androgen inC4-2B cells but notin LNCaP. This is consistent with the fact that AR protein ismore active, stable, and constitutively nuclear in CRPC cells(32) andmay explain the higher basal expression ofWNT7Bin C4-2B cells under androgen-deprived conditions. Fur-thermore, the basal expression level of WNT7B in C4-2Bcells was decreased more than 70% after knockdown of ARusing 2 different AR siRNA (Fig. 1E), indicating thatWNT7B expression depends on AR even in androgen-

deprived conditions. We further examined AR-mediatedWNT7B expression in an additional CRPC 22RV1 cellline. Notably, the AR was constitutively bound to theWNT7B locus (Site 2) in 22RV1 cells (5-fold enrichmentover the control, Supplementary Fig. S1A), which may bedue to the predominant expression of an AR splice variantlacking the ligand-binding domain in this cell line (33).WNT7B expression levels in the absence of androgen weresimilar to those in the presence of androgen (SupplementaryFig. S1B) but decreased about 40% after RNA interferenceagainst AR (Supplementary Fig. S1C and S1D). Takentogether, these results show that WNT7B is an AR targetgene in both androgen-dependent and CRPC cells, and thatWNT7B remains at a high level inCRPC cells in the absenceof androgen due to distinct AR-binding events.

WNT7B is required for the growth of both androgen-dependent and castration-resistant prostate cancer cellsTo investigate the potential oncogenic role of WNT7B in

prostate cancer cells, we examined LNCaP and C4-2B cellproliferation after knockdown of WNT7B. A high

Figure 1. WNT7B is anAR target gene in prostate cancer cells. A, qRT-PCR results showing expression ofWNT7B in LNCaPandC4-2B cells. Cells were growninRPMI-1640mediumwith 5%CSS for 3days followedby the treatmentwith ethanol or 10 nmol/LDHT for 16hours. B,Western blot analysis showingWNT7Bprotein levels under the same conditions as in (A). C, ChIP-seq results showing 2 AR-binding sites at theWNT7B locus in LNCaP and C4-2B cells. D, ChIP-qPCR analyses were conducted to confirm the ChIP-seq results. The prostate specific antigen (PSA) enhancer site was used as a positive control. ARoccupancy at the negative control region was defined as 1. E, C4-2B cells were grown in RPMI-1640 medium with 5%CSS for 2 days followed by AR siRNAtransfection. qRT-PCRanalysesofWNT7BmRNA levelswere conducted 3days afterARsiRNA transfection.ARprotein levelswere examinedbyWesternblotanalysis in parallel (inset). �, P < 0.05; ��, P < 0.01, determined by a 2-tailed Student t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.






knockdown efficiency of WNT7B after RNA interferencewas confirmed by Western blot analysis (Fig. 2A). Knock-down ofWNT7B significantly inhibited LNCaP andC4-2Bgrowth (Fig. 2B). Notably, WNT7B RNA interferenceabolished prostate cancer cell growth and resulted in celldeath in the absence of androgen. Further analyses revealedthat prostate cancer cell apoptosis substantially increased 3days after WNT7B siRNA transfection as determined bycaspase-3 and -7 activity assays (Fig. 2C) as well as TUNELassays (Fig. 2D; Supplementary Fig. S2). In line with the cellproliferation results, the most prominent apoptosis wasobserved in androgen-deprived conditions. To test whetheroverexpression of WNT7B promotes C4-2B proliferation,we establishedWNT7B-overexpressing LNCaP and C4-2Bstable cell lines (Fig. 2E). Overexpression of WNT7B hadlittle effect on LNCaP andC4-2B cell proliferation (Fig. 2F).To further assess the role of WNT7B in prostate cancerprogression, we conducted anchorage-independent colonyformation assays on soft agar using C4-2B cells. Stableknockdown of WNT7B through lentiviral shRNA trans-duction (Supplementary Fig. S3) significantly inhibited C4-2B cell anchorage-independent growth as indicated by bothcolony number and size (Fig. 2G), whereas overexpression ofWNT7B significantly increased C4-2B cell anchorage-inde-pendent growth (Fig. 2H), suggestive of a potential role ofWNT7B in prostate cancer progression. In addition, wefound that WNT7B is also required for CRPC 22RV1 cellgrowth in both the presence and absence of androgen(Supplementary Fig. S4). Taken together, these resultssupport the notion that WNT7B is required for the growthof both androgen-dependent prostate cancer cells andCRPCcells under androgen-deprived conditions.Previous studies have shown that WNT7B is expressed in

a subset of high-grade prostate cancer and bone metastasisspecimens but not in normal prostate tissues (34). To furtherinvestigate whether WNT7B is critical for prostate cancerprogression, we analyzed 2 publicly available prostate cancermicroarray datasets (29, 30). Comparison between 10untreated androgen-dependent primary tumors versus 10androgen-independent primary tumors revealed thatWNT7B is significantly upregulated at the primary site ofpatients with metastatic CRPC (Supplementary Fig. S5A).WNT7B is also expressed at a higher level in hormone-refractory metastatic tumors compared with localized pros-tate tumors (Supplementary Fig. S5B). These results areconsistent with a role for WNT7B in promoting CRPCtumor growth.

WNT7B promotes CRPC growth after androgendeprivation through a noncanonical PKC pathwayWe next sought to investigate mechanisms of the

WNT7B-induced intracellular cascades responsible forC4-2B cell growth under androgen-deprived conditions.b-catenin is an important cofactor in the regulation ofcanonicalWNT signaling as well as AR signaling. Therefore,wefirst investigatedwhetherWNT7B altersb-catenin levels,phosphorylation, and nuclear translocation. Our resultsshow that knockdown of endogenous WNT7B in C4-2B

cells in the absence of androgen resulted in increased Thr41/Ser45 phosphorylation of b-catenin and decreased totalprotein levels of b-catenin (Fig. 3A). In contrast, overexpres-sion of WNT7B slightly decreased Thr41/Ser45 phosphor-ylation of b-catenin, yet had little effect on total b-cateninprotein levels. Importantly, while WNT7B had a modesteffect on total b-catenin levels, no differences in nuclearb-catenin protein levels were detected. Confocal microscopyconfirmed these results; b-catenin staining was presentpredominately at the cell membrane and diffused in thecytoplasm, and weak nuclear b-catenin staining was unal-tered after knockdown or overexpression of WNT7B (Sup-plementary Fig. S6). We were unable to detect significantTCF/LEF activity in C4-2B cells using TOPFlash reporterassays (Fig. 3B), and knockdown ofb-catenin did not inhibitC4-2B cell growth in the absence of androgen (Fig. 3C).These results indicate that b-catenin/TCF transcriptionalsignaling is unlikely to be involved in WNT7B-inducedandrogen-independent growth of C4-2B cells.We have previously reported that WNT7B induces oste-

oblast differentiation through the PKCd-mediated nonca-nonical WNT signaling pathway (25). In this study, wetested whether WNT7B stimulates androgen-independentC4-2B growth through PKC signaling, as shown in osteo-blasts. We observed that phosphorylation of MARCKS, aprototypic substrate of PKC, was significantly reduced afterknockdown of WNT7B and enhanced after overexpressionof WNT7B in C4-2B cells in the absence of androgen (Fig.3A). The total protein levels of MARCKS were slightlydecreased after WNT7B knockdown but not increased afterWNT7B overexpression, indicating that alterations of phos-phorylated MARCKS were unlikely to be attributable tochanges in total protein levels. In fact, the change in totalMARCKS levels may be secondary to altered phosphoryla-tion, as phosphorylated MARCKS is more protected fromproteolytic cleavage than the dephosphorylated form (35).Previous studies have shown that PKCa and PKCd areabundantly expressed in prostate cancer cells and exhibitdistinct functional properties (36). Phosphorylation ofMARCKS was effectively reduced by knockdown of eitherPKCa or PKCd (Fig. 4A and 4B), implicating both PKCisozymes in regulation of MARCKS phosphorylation. Fur-thermore, knockdown of PKCa or PKCd inhibited C4-2Bcell growth in the absence of androgen (Fig. 4C and 4D),suggesting that both PKC isozymes are required for andro-gen-independent C4-2B cell proliferation. Interestingly, ourRNA-seq and ChIP-seq results suggest that PKCa andPKCd are also direct AR target genes in C4-2B cells(Supplementary Fig. S7 and Supplementary Table S2). Asa result, ARmay bypassWNT7B and upregulate PKCa andPKCd directly in the presence of androgen, leading tofunctional compensation. This mechanismmay explain whyknockdown ofWNT7B has less inhibitory effects on andro-gen-dependent prostate cancer growth (Fig. 2B). Finally,knockdown of MARCKS also substantially affected C4-2Bcell growth (Fig. 4E), suggesting an important function ofthis gene in cell proliferation. Taken together, our resultshave suggested a previously undescribed PKC-mediated

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Figure 2. WNT7B is necessary for androgen-dependent and CRPC cell growth. A, WNT7B protein levels were examined by Western blot analysis 3 daysafter WNT7B siRNA transfection. B, LNCaP and C4-2B cell proliferation was examined using CCK8 assays 3 and 5 days after WNT7B siRNAtransfection in the presence or absence of 10 nmol/L DHT. C, Apoptosis assay with LNCaP and C4-2B cells showing caspase-3 and -7 activity 3 daysafter WNT7B siRNA transfection in the presence or absence of 10 nmol/L DHT. The results are presented as mean � SD of 2 independentexperiments. D, TUNEL assays were conducted under the same conditions as in (C). A total of 10 fields were photographed. Total number of cells andthe number of TUNEL-positive cells in each field were counted and percentage of apoptotic cells was calculated. E, WNT7B-overexpressing stableprostate cancer cell lines were confirmed by Western blot analysis. F, LNCaP and C4-2B cells overexpressing WNT7B or vector control weregrown in the presence or absence of 10 nmol/L DHT for 5 days before CCK8 assays. G, C4-2B cells with stable WNT7B knockdown (shWNT7B-1 and -2)or control knockdown (shLacZ) were examined in soft agar colony formation assays. The assays were conducted in RPMI-1640 mediumcontaining 10% FBS. The colony number is presented as mean � SD of 3 independent wells. The experiment was conducted for at least 3independent times. H, WNT7B-overexpressing or vector control C4-2B cells were used in soft agar colony formation assays as in (G). �, P < 0.05;��, P < 0.01; ��, P < 0.001; NS, not significant, determined by a 2-tailed Student t test.






noncanonical pathway in CRPC by which WNT7B pro-motes C4-2B cell growth after androgen deprivation.

WNT7B promotes the development of osteoblastic bonereactionsThe importance of WNT signaling in bone development

has been well established (27). Consistent with our previousresults (25), we observed WNT7B-induced osteoblast dif-ferentiation in mouse bone marrow–derived stromal ST2cells andmouse pluripotentmesenchymal C3H10T1/2 cells(Supplementary Fig. S8A and S8B). Increased osteoblasticactivity after WNT7B overexpression was shown byincreases in ALP enzymatic activity and mineralization. Itshould be noted that overexpression of WNT7B in C4-2Bcells did not increase ALP activity and mineralization of C4-2B cells (Supplementary Fig. S8C).We next asked whether prostate cancer-produced

WNT7B promotes osteoblast differentiation. Studies havesuggested that WNT7B is highly insoluble after posttrans-lational modification and that WNT7B activation is likelymediated through a direct cell–cell interaction (37). Weobserved enriched WNT7B protein on C4-2B cell mem-brane using immunofluorescence andWestern blot analyses(Supplementary Fig. S9A and S9B). Conditioned mediumfrom LNCaP and C4-2B cell culture had little effect on ST2

cell differentiation despite overexpression of WNT7B bythese cells (Supplementary Fig. S9C), suggesting that thelipid-modified WNT7B is likely linked to plasma mem-brane. We then cocultured C4-2B with ST2 cells in oste-ogenic medium and observed moderate increases in ALPactivity and mineralization of ST2 cells. Both effects werediminished after WNT7B knockdown in C4-2B cells (Fig.5A). The mRNA levels of osteoblast differentiation markers,ALP and BSP, were decreasedmore than 50% as determinedby qRT-PCR. Because we used primers specific for mousegenes, the mRNA levels of ALP and BSP reflected theexpression of these 2 genes in ST2 cells. The expression ofALP and BSP were barely detectable in C4-2B cells usingthese primers (Supplementary Fig. S10). In addition, cocul-ture of C4-2B cells overexpressing WNT7B with ST2 cellsmarkedly enhanced ALP activity, mineralization, and ALPand BSP mRNA levels in ST2 cells (Fig. 5B). Similar resultswere obtained with 2 additional prostate cancer cell lines.Overexpression of WNT7B in LNCaP or LAPC4 cellsmarkedly promoted ST2 osteoblast differentiation in thecoculture system (Supplementary Fig. S11). To examinewhether these effects are specific for ST2 cells, we conductedsimilar assays on C3H10T1/2 cells. Coculture of WNT7B-overexpressing C4-2B cells with C3H10T1/2 cells dramat-ically enhanced ALP activity, mineralization, and ALP and

Figure 3. WNT7B promotesandrogen-independent C4-2B cellgrowth in a b-catenin–independentmanner. A, Western blot analysisresults showing b-catenin,phosphorylated b-catenin(p-b-catenin), MARCKS andphosphorylated MARCKS(p-MARCKS) protein levels inC4-2B cells after knockdown(siNS1 vs. siWNT7B) oroverexpression (Vector vs.WNT7B) of WNT7B in the absenceof androgen. B, luciferase assays inHCT116 and C4-2B (with orwithout WNT7B overexpression)cells after transient transfection ofTOPFlash and FOPFalsh reporters.Cells were treated with or withoutrecombinant WNT3A. C, C4-2Bcell proliferation was examinedusing CCK8 assays 3 and 5 daysafter b-catenin siRNA (sib-catenin)transfection in the absence of DHT.Two different sib-catenin wereused and b-catenin protein levelswere examined by Western blotanalysis 3 days after sib-catenintransfection in C4-2B cells (inset).

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BSP mRNA levels in C3H10T1/2 cells (Fig. 5C). Similareffects were observed when C3H10T1/2 cells were cocul-tured with WNT7B-overexpressing LAPC4 cells (Supple-mentary Fig. S12). Thus, WNT7B produced from andro-gen-dependent and -independent prostate cancer cell linescan induce osteoblast differentiation.

WNT7B is upregulated in human prostate cancer tumorsthat cause osteoblastic lesions in bone metastasisTo further investigate the involvement of WNT7B in the

osteoblastic reaction elicited by human prostate cancertumor growth in the bone, we queried the levels of WNT7Bin LuCaP prostate cancer xenografts (Supplementary TableS3). LuCaP xenografts were established from primary andmetastatic human prostate tumors. Twenty-one androgen-dependent LuCaP xenografts were grown in intact mice,whereas 3 castration-resistant variants (LuCaP 23.1V,LuCaP 35V, and LuCaP 96V) were grown in castratedmice. Of 24 xenograft tumors, 4 were AR-negative neuro-endocrine tumors and expressed very low levels of WNT7B,consistent with the fact that WNT7B is an AR target gene.

These 4 samples were excluded from further analyses. Thexenograft tumors were grouped on the basis of whether theydid or did not elicit an osteoblastic reaction when growing inthe tibia of intact mice. Xenografts that elicit a large volumeof new bone formation were designated as osteoblastictumors, whereas the remaining tumors were classified asnonosteoblastic. Comparison of themRNA expression levelsof 8 WNT ligands in osteoblastic (n ¼ 5) and nonosteo-blastic (osteolytic, mixed, and no reaction, n ¼ 15) xeno-grafts showed that only WNT7B was overexpressed inprostate cancer xenografts that caused an osteoblastic reac-tion (Fig. 6). No differences were detected for WNTs3, 4,5A, 5B, 6, 10B, and 11. Notably, the tumor with the highestWNT7B expression level (LuCaP 23.1) induces a robustbone reaction in the bone [see Fig. 2 in ref. 38]. A LuCaP23.1-derived CRPC xenograft tumor (LuCaP 23.1V) con-tinued to express WNT7B at a very high level and elicitosteoblastic lesions (Supplementary Table S3). These resultsindicate that WNT7B may play an important role in theosteoblastic reaction resulting from growth of prostate can-cer in the bone.

Figure 4. WNT7B promotes androgen-independent C4-2B cell growth through the PKC signaling pathway. A and B, PKCa, PKCd, phosphorylated MARCKS(p-MARCKS), and MARCKS protein levels were examined by Western blot analysis 3 days after PKCa (A) or PKCd (B) RNA interference in C4-2B cells in theabsence of androgen. C–E, C4-2B cell proliferation was examined using CCK8 assays 3 and 5 days after transfection with siPKCa (C), siPKCd (D), orsiMARCKS (E) in the absence of androgen.MARCKS protein levels were examined byWestern blot analysis 3 days afterMARCKSRNA interference in C4-2Bcells (inset). ��, P < 0.001, between nonspecific siRNA and gene-specific siRNA as determined by a 2-tailed Student t test.






DiscussionDespite androgen deprivation, AR signaling persists in

CRPC through a variety of mechanisms. While it is believedthat a subset of AR target genes remain transcriptionallyactive, major efforts have been made to understand whichAR target genes are responsible for the growth of CRPC.Here, we report thatWNT7B is an AR-regulated gene that ispersistently upregulated in CRPC cells and promotes CRPCcell proliferation under androgen-deprived conditions. Ourdata also show that WNT7B produced by prostate cancercells induces osteoblastic bone lesions, and that theWNT7Bparacrine signal is mediated through a direct cell–cell inter-action (Fig. 7).

WNT-induced signaling pathways are cell- and context-dependent. We have shown that WNT7B promotes andro-gen-independent CRPC cell proliferation likely through aPKC-mediated noncanonical WNT pathway. A similarpathway was observed in WNT7B-induced osteoblast dif-ferentiation (25). PKC proteins are a family of serine/threonine kinases involved in the transduction of signalsfor cell proliferation, differentiation, and apoptosis. Theseisozymes may exhibit overlapping or opposing functions.While PKCa and PKCd generally function as proapoptoticproteins, they can also act as antiapoptotic proteins (39, 40).Whether pro- or antiapoptotic functions are exhibiteddepends on cell type, stimulus, and stage of cancer

Figure 5. C4-2B-producedWNT7B promotes osteoblastdifferentiation. A, C4-2B cells weretransfected with WNT7B siRNA(siWNT7B) or nonspecific siRNA(siNS1). After 24 hours, C4-2B cellswere cocultured with ST2 cells inosteogenic medium. To determineosteoblastic activity of ST2 cells,alkaline phosphatase staining wasconducted on day 7 and von Kossastaining for calcium deposits wasconducted on day 21 (left). MouseALP and BSP mRNA levels wereexamined by qRT-PCR on day 7(right). B, WNT7B-overexpressingC4-2B cells or vector control cellswere cocultured with ST2 cells inosteogenic medium. The sameassays were conducted as in (A).C, WNT7B-overexpressing C4-2Bcells or vector control cells werecocultured with C3H10T1/2 cells inosteogenic medium. The sameassays were conducted as in (A).�, P < 0.05; ��, P < 0.01, determinedby a 2-tailed Student t test.

Zheng et al.





development and progression. The specific role of each PKCisozyme in different types of cancers is not completelyunderstood (36). It is well documented that PKCa andPKCd mediate phorbol ester-induced apoptosis in prostatecancer cells (41). Our data, however, suggest a novel onco-genic function of these 2 PKC isozymes through their role inWNT7B-induced proliferation of CRPC cells. MARCKS isa ubiquitously expressed substrate of PKC involved in cell

adhesion, spreading,motility, andmitogenesis through actincytoskeletal regulation (42, 43). Upon phosphorylation byPKC, MARCKS translocates from the cell membrane to thecytosol, leading to actin rearrangement and enhanced cellmigration and invasion.While our data show thatMARCKSis required for the growth of CRPC cells, it is conceivablethat WNT7B-induced phosphorylation of MARCKS mayincrease metastatic activity as observed in other cancer cells(44, 45).Prostate cancer cells secrete a number of osteogenic factors,

such as endothlin-1 (ET-1), bone morphogenetic proteins(BMP), and WNTs. These factors promote differentiation ofosteoblasts that, in turn, release growth factors that facilitateprostate cancer growth inbone.As a result, prostate cancer cells,capable of producing factors that induce bone formation, tendto survive and proliferate in bone. Several lines of evidenceindicate thatWNT signaling is likely the primary determinantof tumor-induced bone remodeling. It is well known thatincreasing DKK1 expression may convert prostate cancer cellsfrom an osteoblastic to an osteolytic type. ET-1 may alsoincrease bone formation by suppression ofDKK1 (46).WNTspromote BMP expression andmodulate osteoblastic activity inprostate cancer bonemetastasis (47). The present study furthersupports the notion that AR-mediated upregulation of WNTsignalingmight play amajor role in osteoblastic bonemetastasisin advanced prostate cancer. Interestingly, WNT7B has beenidentified as an AR target gene in ER-negative breast cancercells (48), and approximately 10%–15% of breast cancer bonemetastases display predominantly osteoblastic lesions (49).Whether these bone lesions are associatedwith highARactivityand WNT7B expression is unknown.Current antiandrogen therapies for advanced prostate

cancer have focused on blocking AR ligand binding andandrogen synthesis. While these novel therapies have been amajor breakthrough, increases in survival have been mea-sured in months. Our findings suggest a unique opportunityfor development of more effective therapies by targeting theAR/WNT7B/PKC signaling cascade, which would inhibit

Figure 6. WNT7B is overexpressedin prostate cancer xenograft tissuescausing osteoblastic lesions. WNTgene expression levels frommicroarray studies were analyzedbetween prostate cancer LuCaPxenograft tissues with osteoblasticlesions (n ¼ 5) versusnonosteoblastic lesions (n ¼ 15).

Figure 7. Schematic model of WNT7B signaling in CRPC cells andosteoblasts.






prostate cancer proliferation and prevent the formation of ametastatic reservoir in the bone.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: D. Zheng, T. Zhou, L. JiaDevelopment of methodology: D. Zheng, K.N. Weilbaecher, L. JiaAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): D. Zheng, J. Chen, Z. Qi, E. Corey, L. JiaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): D. Zheng, K.F. Decker, T. Zhou, E. Corey, L. JiaWriting, review, and/or revision of the manuscript: D. Zheng, K.F. Decker, T.Zhou, Z. Qi, K.N. Weilbaecher, E. Corey, F. Long, L. JiaAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): T. Zhou, J. Chen, L. JiaStudy supervision: L. Jia

AcknowledgmentsThe authors thank Drs. David R. Piwnica-Worms, John R. Edwards, Scot

Matkovich (Washington University in St. Louis), and Robert L. Vessella (Universityof Washington, Seattle) for valuable discussions.

Grant SupportThis work was supported by the Concern Foundation, the Siteman Cancer Center

Developmental Research Award in Prostate Cancer Research, and the BRIGHTInstitute at Washington University School of Medicine/P50 Molecular ImagingCenter Pilot Research Fund.

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 August 31, 2012; revised January 16, 2013; accepted January 16, 2013;published OnlineFirst February 5, 2013.

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2013;11:482-493. Published OnlineFirst February 5, 2013.Mol Cancer Res Dali Zheng, Keith F. Decker, Tianhua Zhou, et al. Prostate CancerRole of WNT7B-induced Noncanonical Pathway in Advanced

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