A Multivalent Peptoid Conjugate Modulates Androgen Receptor Transcriptional Activity to Inhibit Therapy-resistant Prostate Cancer.

Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance due to ongoing AR expression and function. To counter this, we developed a new approach to modulate the AR and inhibit castration-resistant prostate cancer (CRPC) using multivalent peptoid conjugates (MPC) that contain multiple copies of the AR-targeting ligand ethisterone attached to a peptidomimetic scaffold. Here, we investigated the antitumor effects of compound MPC309, a trivalent display of ethisterone conjugated to a peptoid oligomer backbone that binds to the AR with nanomolar affinity. MPC309 exhibited potent antiproliferative effects on various enzalutamide-resistant prostate cancer models, including those with AR splice variants, ligand-binding mutations, and noncanonical AR gene expression programs, as well as mouse prostate organoids harboring defined genetic alterations that mimic lethal human prostate cancer subtypes. MPC309 is taken up by cells through macropinocytosis, an endocytic process more prevalent in cancer cells than in normal ones, thus providing an opportunity to target tumors selectively. MPC309 triggers a distinct AR transcriptome compared with DHT and enzalutamide, a clinically used antiandrogen. Specifically, MPC309 enhances the expression of differentiation genes while reducing the expression of genes needed for cell division and metabolism. Mechanistically, MPC309 increases AR chromatin occupancy and alters AR interactions with coregulatory proteins in a pattern distinct from DHT. In xenograft studies, MPC309 produced significantly greater tumor suppression than enzalutamide. Altogether, MPC309 represents a promising new AR modulator that can combat resistant disease by promoting an AR antiproliferative gene expression program.

PIM1 phosphorylation of the androgen receptor and 14-3-3 ζ regulates gene transcription in prostate cancer.

PIM1 is a serine/threonine kinase over-expressed in prostate cancer. We have previously shown that PIM1 phosphorylates the androgen receptor (AR), the primary therapeutic target in prostate cancer, at serine 213 (pS213), which alters expression of select AR target genes. Therefore, we sought to investigate the mechanism whereby PIM1 phosphorylation of AR alters its transcriptional activity. We previously identified the AR co-activator, 14-3-3 ζ, as an endogenous PIM1 substrate in LNCaP cells. Here, we show that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, and that they extensively occupy the same sites on chromatin in an AR-dependent manner. Their occupancy at a number of genes involved in cell migration and invasion results in a PIM1-dependent increase in the expression of these genes. We also use rapid immunoprecipitation and mass spectrometry of endogenous proteins on chromatin (RIME), to find that select AR co-regulators, such as hnRNPK and TRIM28, interact with both AR and 14-3-3 ζ in PIM1 over-expressing cells. We conclude that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, which in turn recruits additional co-regulatory proteins to alter AR transcriptional activity.

Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9.

BACKGROUND: The autonomous retroelement Long Interspersed Element-1 (LINE-1) mobilizes though a copy and paste mechanism using an RNA intermediate (retrotransposition). Throughout human evolution, around 500,000 LINE-1 sequences have accumulated in the genome. Most of these sequences belong to ancestral LINE-1 subfamilies, including L1PA2-L1PA7, and can no longer mobilize. Only a small fraction of LINE-1 sequences, approximately 80 to 100 copies belonging to the L1Hs subfamily, are complete and still capable of retrotransposition. While silenced in most cells, many questions remain regarding LINE-1 dysregulation in cancer cells. RESULTS: Here, we optimized CRISPR Cas9 gRNAs to specifically target the regulatory sequence of the L1Hs 5'UTR promoter. We identified three gRNAs that were more specific to L1Hs, with limited binding to older LINE-1 sequences (L1PA2-L1PA7). We also adapted the C-BERST method (dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging) to identify LINE-1 transcriptional regulators in cancer cells. Our LINE-1 C-BERST screen revealed both known and novel LINE-1 transcriptional regulators, including CTCF, YY1 and DUSP1. CONCLUSION: Our optimization and evaluation of gRNA specificity and application of the C-BERST method creates a tool for studying the regulatory mechanisms of LINE-1 in cancer. Further, we identified the dual specificity protein phosphatase, DUSP1, as a novel regulator of LINE-1 transcription.

RIP-seq reveals LINE-1 ORF1p association with p-body enriched mRNAs.

BACKGROUND: Long INterspersed Element-1 (LINE-1) is an autonomous retroelement able to "copy-and-paste" itself into new loci of the host genome through a process called retrotransposition. The LINE-1 bicistronic mRNA codes for two proteins, ORF1p, a nucleic acid chaperone, and ORF2p, a protein with endonuclease and reverse transcriptase activity. Both proteins bind LINE-1 mRNA in cis and are necessary for retrotransposition. While LINE-1 transcription is usually repressed in most healthy somatic cells through a plethora of mechanisms, ORF1p expression has been observed in nearly 50% of tumors, and new LINE-1 insertions have been documented in a similar fraction of tumors, including prostate cancer. RESULTS: Here, we utilized RNA ImmunoPrecipitation (RIP) and the L1EM analysis software to identify ORF1p bound RNA in prostate cancer cells. We identified LINE-1 loci that were expressed in parental androgen sensitive and androgen independent clonal derivatives. In all androgen independent cells, we found higher levels of LINE-1 RNA, as well as unique expression patterns of LINE-1 loci. Interestingly, we observed that ORF1p bound many non-LINE-1 mRNA in all prostate cancer cell lines evaluated, and polyA RNA, and RNA localized in p-bodies were especially enriched. Furthermore, the expression levels of RNAs identified in our ORF1p RIP correlated with RNAs expressed in LINE-1 positive tumors from The Cancer Genome Atlas (TCGA). CONCLUSION: Our results show a significant remodeling of LINE-1 loci expression in androgen independent cell lines when compared to parental androgen dependent cells. Additionally, we found that ORF1p bound a significant amount of non-LINE-1 mRNA, and that the enriched ORF1p bound mRNAs are also amplified in LINE-1 expressing TCGA prostate tumors, indicating the biological relevance of our findings to prostate cancer.

Identification of PIM1 substrates reveals a role for NDRG1 phosphorylation in prostate cancer cellular migration and invasion.

PIM1 is a serine/threonine kinase that promotes and maintains prostate tumorigenesis. While PIM1 protein levels are elevated in prostate cancer relative to local disease, the mechanisms by which PIM1 contributes to oncogenesis have not been fully elucidated. Here, we performed a direct, unbiased chemical genetic screen to identify PIM1 substrates in prostate cancer cells. The PIM1 substrates we identified were involved in a variety of oncogenic processes, and included N-Myc Downstream-Regulated Gene 1 (NDRG1), which has reported roles in suppressing cancer cell invasion and metastasis. NDRG1 is phosphorylated by PIM1 at serine 330 (pS330), and the level of NDRG1 pS330 is associated higher grade prostate tumors. We have shown that PIM1 phosphorylation of NDRG1 at S330 reduced its stability, nuclear localization, and interaction with AR, resulting in enhanced cell migration and invasion.

Dickkopf-1 Can Lead to Immune Evasion in Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) with low androgen receptor (AR) and without neuroendocrine signaling, termed double-negative prostate cancer (DNPC), is increasingly prevalent in patients treated with AR signaling inhibitors and is in need of new biomarkers and therapeutic targets. METHODS: Candidate genes enriched in DNPC were determined using differential gene expression analysis of discovery and validation cohorts of mCRPC biopsies. Laboratory studies were carried out in human mCRPC organoid cultures, prostate cancer (PCa) cell lines, and mouse xenograft models. Epigenetic studies were carried out in a rapid autopsy cohort. RESULTS: Dickkopf-1 (DKK1) expression is increased in DNPC relative to prostate-specific antigen (PSA)-expressing mCRPC in the Stand Up to Cancer/Prostate Cancer Foundation discovery cohort (11.2 v 0.28 reads per kilobase per million mapped reads; q < 0.05; n = 117) and in the University of Washington/Fred Hutchinson Cancer Research Center cohort (9.2 v 0.99 fragments per kilobase of transcript per million mapped reads; P < .0001). DKK1 expression can be regulated by activated Wnt signaling in vitro and correlates with activating canonical Wnt signaling mutations and low PSA mRNA in mCRPC biopsies (P < .05). DKK1 hypomethylation was associated with increased DKK1 mRNA expression (Pearson r = -0.66; P < .0001) in a rapid autopsy cohort (n = 7). DKK1-high mCRPC biopsies are infiltrated with significantly higher numbers of quiescent natural killer (NK) cells (P < .005) and lower numbers of activated NK cells (P < .0005). Growth inhibition of the human PCa model PC3 by the anti-DKK1 monoclonal antibody DKN-01 depends on the presence of NK cells in a severe combined immunodeficient xenograft mouse model. CONCLUSION: These results support DKK1 as a contributor to the immunosuppressive tumor microenvironment of DNPC. These data have provided the rationale for a clinical trial targeting DKK1 in mCRPC (ClinicalTrials.gov identifier: NCT03837353).

Roles for MDC1 in cancer development and treatment.

The DNA damage response (DDR) is necessary to maintain genome integrity and prevent the accumulation of oncogenic mutations. Consequently, proteins involved in the DDR often serve as tumor suppressors, carrying out the crucial task of keeping DNA fidelity intact. Mediator of DNA damage checkpoint 1 (MDC1) is a scaffold protein involved in the early steps of the DDR. MDC1 interacts directly with γ-H2AX, the phosphorylated form of H2AX, a commonly used marker for DNA damage. It then propagates the phosphorylation of H2AX by recruiting ATM kinase. While the function of MDC1 in the DDR has been reviewed previously, its role in cancer has not been reviewed, and numerous studies have recently identified a link between MDC1 and carcinogenesis. This includes MDC1 functioning as a tumor suppressor, with its loss serving as a biomarker for cancer and contributor to drug sensitivity. Studies also indicate that MDC1 operates outside of its traditional role in DDR, and functions as a co-regulator of nuclear receptor transcriptional activity, and that mutations in MDC1 are present in tumors and can also cause germline predisposition to cancer. This review will discuss reports that link MDC1 to cancer and identify MDC1 as an important player in tumor formation, progression, and treatment. We also discuss mechanisms by which MDC1 levels are regulated and how this contributes to tumor formation.

UXT in Sertoli cells is required for blood-testis barrier integrity†.

Spermatogenesis is a complex process that establishes male fertility and involves proper communication between the germline (spermatozoa) and the somatic tissue (Sertoli cells). Many factors that are important for spermatozoa production are also required for Sertoli cell function. Recently, we showed that the transcriptional cofactor ubiquitously expressed transcript (UXT) encodes a protein that is essential in germ cells for spermatogenesis and fertility. However, the role of UXT within Sertoli cells and how it affects Sertoli cell function was still unclear. Here we describe a novel role for UXT in the Sertoli cell's ability to support spermatogenesis. We find that the conditional deletion of Uxt in Sertoli cells results in smaller testis size and weight, which coincided with a loss of germ cells in a subset of seminiferous tubules. In addition, the deletion of Uxt has no impact on Sertoli cell abundance or maturity, as they express markers of mature Sertoli cells. Gene expression analysis reveals that the deletion of Uxt in Sertoli cells reduces the transcription of genes involved in the tight junctions of the blood-testis barrier (BTB). Furthermore, tracer experiments and electron microscopy reveal that the BTB is permeable in UXT KO animals. These findings broaden our understanding of UXT's role in Sertoli cells and its contribution to the structural integrity of the BTB.

Prostate-specific loss of UXT promotes cancer progression.

Ubiquitously-expressed, prefoldin-like chaperone (UXT) also called Androgen Receptor Trapped clone-27 (ART-27) is widely expressed in human tissues. Our previous studies showed that UXT regulates transcription repression including androgen receptor (AR) signaling in prostate cancer. Here we analyzed a tissue microarray consisting of normal prostate, benign prostatic hyperplasia, high grade prostatic intraepithelial neoplasia (HGPIN) and primary prostate cancer cases for UXT protein expression. We found that HGPIN and malignant tumors have significantly decreased UXT expression compared to the normal prostate. Loss of UXT expression in primary prostate cancer is positively associated with high Gleason grade and poor relapse-free survival. We engineered prostate-specific Uxt KO mice that developed a hyperplastic phenotype with apparent prostate secretion fluid blockage as well as PIN by 4-6 months. Doubly mutant Uxt KO /Pten KO mice developed a more aggressive PIN phenotype. UXT depletion in prostate cancer cells also increased retroelements expression, including LINE-1 and Alu. Consistent with this finding Uxt KO mice have increased LINE-1 protein levels in the prostate compared to control mice. In addition, cancer cells with UXT depletion have increased retrotransposition activity and accumulated DNA damage. Our findings demonstrate that loss of UXT is an early event during prostate cancer progression, which may contribute to genome instability.

A re-evaluation of LINE-1 ORF2 expression in LNCaP prostate cancer cells.

BACKGROUND: We previously examined expression of Long Interspersed Element-1 (LINE-1) in a variety of prostate cancer cells including hormone-dependent LNCaP cells. These studies demonstrated expression and sub-cellular localization of LINE-1 proteins, ORF1p, with antibody 4H1, and ORF2p, with antibody chA1-L1. RESULTS: Here we conduct immunoprecipitation/mass spectrometry analysis using chA1-L1 antibody against ORF2p in LNCaP cells. Our results indicate that antigens detected by the antibody include the transcriptional regulator BCLAF1. We show that chA1-L1 recognizes BCLAF1 using siRNA knockdown and overexpression of a tagged BCLAF1. We also show that chA1-L1 antibody recognizes ORF2p in HEK293 cells overexpressing LINE-1. Further, analysis of ORF2p (chA1-L1) and BCLAF1 foci using immunofluorescence in LNCaP cells showed significant colocalization. CONCLUSIONS: Overall, our findings indicate that chA1-L1 antibody recognizes both BCLAF1 and ORF2p but the majority of antigen recognized in LNCaP cells is BCLAF1.

Role of the Unconventional Prefoldin Proteins URI and UXT in Transcription Regulation.

The Unconventional prefoldin RPB5 interacting protein (URI), also known as RPB5-Mediating Protein (RMP) has been shown to play several regulatory roles in different cellular compartments including the mitochondria, as a phosphatase binding protein; in the cytoplasm, as a chaperone-like protein; and in the nucleus, as a transcriptional regulator through binding to RPB5 and RNA polymerase II (polII). This chapter focuses on the role URI plays in transcriptional regulation in the prostate cell. In prostate cells, URI is tightly bound to another prefoldin-like protein called UXT, a known androgen receptor (AR) cofactor. Part of a multiprotein complex, URI and UXT act as transcriptional repressors, and URI regulates KAP1 through PP2A phosphatase activity. The discovery of the interaction of URI and UXT with KAP1, AR, and PP2A, as well as the numerous interactions between URI and components of the R2TP/prefoldin-like complex, RPB5, and nuclear proteins involved in DNA damage response, chromatin remodeling and gene transcription, reveal a pleiotropic effect of the URI/UXT complex on nuclear processes. The mechanisms by which URI/UXT affect transcription, chromatin structure and regulation, and genome stability, remain to be elucidated but will be of fundamental importance considering the many processes affected by alterations of URI/UXT and other prefoldins and prefoldin-like proteins.

Design of Peptoid-peptide Macrocycles to Inhibit the ß-catenin TCF Interaction in Prostate Cancer.

New chemical inhibitors of protein-protein interactions are needed to propel advances in molecular pharmacology. Peptoids are peptidomimetic oligomers with the capability to inhibit protein-protein interactions by mimicking protein secondary structure motifs. Here we report the in silico design of a macrocycle primarily composed of peptoid subunits that targets the ß-catenin:TCF interaction. The ß-catenin:TCF interaction plays a critical role in the Wnt signaling pathway which is over-activated in multiple cancers, including prostate cancer. Using the Rosetta suite of protein design algorithms, we evaluate how different macrocycle structures can bind a pocket on ß-catenin that associates with TCF. The in silico designed macrocycles are screened in vitro using luciferase reporters to identify promising compounds. The most active macrocycle inhibits both Wnt and AR-signaling in prostate cancer cell lines, and markedly diminishes their proliferation. In vivo potential is demonstrated through a zebrafish model, in which Wnt signaling is potently inhibited.

UXT is required for spermatogenesis in mice.

Male mammals must simultaneously produce prodigious numbers of sperm and maintain an adequate reserve of stem cells to ensure continuous production of gametes throughout life. Failures in the mechanisms responsible for balancing germ cell differentiation and spermatogonial stem cell (SSC) self-renewal can result in infertility. We discovered a novel requirement for Ubiquitous Expressed Transcript (UXT) in spermatogenesis by developing the first knockout mouse model for this gene. Constitutive deletion of Uxt is embryonic lethal, while conditional knockout in the male germline results in a Sertoli cell-only phenotype during the first wave of spermatogenesis that does not recover in the adult. This phenotype begins to manifest between 6 and 7 days post-partum, just before meiotic entry. Gene expression analysis revealed that Uxt deletion downregulates the transcription of genes governing SSC self-renewal, differentiation, and meiosis, consistent with its previously defined role as a transcriptional co-factor. Our study has revealed the first in vivo function for UXT in the mammalian germline as a regulator of distinct transcriptional programs in SSCs and differentiating spermatogonia.

Long interspersed nuclear element-1 expression and retrotransposition in prostate cancer cells.

BACKGROUND: Long Interspersed Nuclear Element-1 (LINE-1) is an autonomous retrotransposon that generates new genomic insertions through the retrotransposition of a RNA intermediate. Expression of LINE-1 is tightly repressed in most somatic tissues to prevent DNA damage and ensure genomic integrity. However, the reactivation of LINE-1 has been documented in cancer and the role of LINE-1 protein expression and retrotransposition has become of interest in the development, progression, and adaptation of many epithelial neoplasms, including prostate cancer. RESULTS: Here, we examined endogenous LINE-1 protein expression and localization in a panel of prostate cancer cells and observed a diverse range of LINE-1 expression patterns between cell lines. Subcellular localization of LINE-1 proteins, ORF1p and ORF2p, revealed distinct expression patterns. ORF1p, a nucleic acid chaperone that binds LINE-1 mRNA, was predominantly expressed in the cytoplasm, with minor localization in the nucleus. ORF2p, containing endonuclease and reverse transcriptase domains, exhibited punctate foci in the nucleus and also displayed co-localization with PCNA and γH2AX. Using a retrotransposition reporter assay, we found variations in LINE-1 retrotransposition between cell lines. CONCLUSIONS: Overall, our findings reveal new insight into the expression and retrotransposition of LINE-1 in prostate cancer. The prostate cancer cells we investigated provide a unique model for investigating endogenous LINE-1 activity and provide a functional model for studying LINE-1 mechanisms in prostate cancer.

Revisiting the role of Wnt/ß-catenin signaling in prostate cancer.

The androgen receptor (AR) is a widely accepted therapeutic target in prostate cancer and multiple studies indicate that the AR and Wnt/ß-catenin pathways intersect. Recent genome-wide analysis of prostate cancer metastases illustrate the importance of the Wnt/ß-catenin pathway in prostate cancer and compel us to reexamine the interaction of the AR and Wnt/ß-catenin signaling pathways. This review includes newer areas of interest such as non-canonical Wnt signaling and the role of Wnts in prostate cancer stem cells. The effort to develop Wnt modulating therapeutics, both biologics and small molecules, is also discussed.

Chromatin-Associated Protein SIN3B Prevents Prostate Cancer Progression by Inducing Senescence.

Distinguishing between indolent and aggressive prostate adenocarcinoma remains a priority to accurately identify patients who need therapeutic intervention. SIN3B has been implicated in the initiation of senescence in vitro Here we show that in a mouse model of prostate cancer, SIN3B provides a barrier to malignant progression. SIN3B was required for PTEN-induced cellular senescence and prevented progression to invasive prostate adenocarcinoma. Furthermore, SIN3B was downregulated in human prostate adenocarcinoma correlating with upregulation of its target genes. Our results suggest a tumor suppressor function for SIN3B that limits prostate adenocarcinoma progression, with potential implications for the use of SIN3B and its target genes as candidate diagnostic markers to distinguish indolent from aggressive disease. Cancer Res; 77(19); 5339-48. ©2017 AACR.

URI Regulates KAP1 Phosphorylation and Transcriptional Repression via PP2A Phosphatase in Prostate Cancer Cells.

URI (unconventional prefoldin RPB5 interactor protein) is an unconventional prefoldin, RNA polymerase II interactor that functions as a transcriptional repressor and is part of a larger nuclear protein complex. The components of this complex and the mechanism of transcriptional repression have not been characterized. Here we show that KAP1 (KRAB-associated protein 1) and the protein phosphatase PP2A interact with URI. Mechanistically, we show that KAP1 phosphorylation is decreased following recruitment of PP2A by URI. We functionally characterize the novel URI-KAP1-PP2A complex, demonstrating a role of URI in retrotransposon repression, a key function previously demonstrated for the KAP1-SETDB1 complex. Microarray analysis of annotated transposons revealed a selective increase in the transcription of LINE-1 and L1PA2 retroelements upon knockdown of URI. These data unveil a new nuclear function of URI and identify a novel post-transcriptional regulation of KAP1 protein that may have important implications in reactivation of transposable elements in prostate cancer cells.

Multivalent Peptoid Conjugates Which Overcome Enzalutamide Resistance in Prostate Cancer Cells.

Development of resistance to antiandrogens for treating advanced prostate cancer is a growing concern and extends to recently developed therapeutics, including enzalutamide. Therefore, new strategies to block androgen receptor (AR) function in prostate cancer are required. Here, we report the characterization of a multivalent conjugate presenting two bioactive ethisterone ligands arrayed as spatially defined pendant groups on a peptoid oligomer. The conjugate, named Multivalent Peptoid Conjugate 6 (MPC6), suppressed the proliferation of multiple AR-expressing prostate cancer cell lines including those that failed to respond to enzalutamide and ARN509. The structure-activity relationships of MPC6 variants were evaluated, revealing that increased spacing between ethisterone moieties and changes in peptoid topology eliminated its antiproliferative effect, suggesting that both ethisterone ligand presentation and scaffold characteristics contribute to MPC6 activity. Mechanistically, MPC6 blocked AR coactivator-peptide interaction and prevented AR intermolecular interactions. Protease sensitivity assays suggested that the MPC6-bound AR induced a receptor conformation distinct from that of dihydrotestosterone- or enzalutamide-bound AR. Pharmacologic studies revealed that MPC6 was metabolically stable and displayed a low plasma clearance rate. Notably, MPC6 treatment reduced tumor growth and decreased Ki67 and AR expression in mouse xenograft models of enzalutamide-resistant LNCaP-abl cells. Thus, MPC6 represents a new class of compounds with the potential to combat treatment-resistant prostate cancer. Cancer Res; 76(17); 5124-32. ©2016 AACR.

Dynein axonemal heavy chain 8 promotes androgen receptor activity and associates with prostate cancer progression.

To gain insight into cellular factors regulating AR action that could promote castration resistant prostate cancer (CRPC), we performed a genome-wide RNAi screen for factors that promote ligand-independent AR transcriptional activity and integrated clinical databases for candidate genes that are positively associated with prostate cancer metastasis and recurrence. From this analysis, we identified Dynein Axonemal Heavy Chain 8 (DNAH8) as an AR regulator that displayed higher mRNA expression in metastatic than in primary tumors, and showed high expression in patients with poor prognosis. Axonemal dyneins function in cellular motility, but the function of DNAH8 in prostate cancer or other cell types has not been reported. DNAH8 is on chromosome 6q21.2, a cancer-associated amplicon, and is primarily expressed in prostate and testis. Its expression is higher in primary tumors compared to normal prostate, and is further increased in metastatic prostate cancers. Patients expressing high levels of DNAH8 have a greater risk of relapse and a poor prognosis after prostatectomy. Depletion of DNAH8 in prostate cancer cells suppressed AR transcriptional activity and proliferation. Androgen treatment increased DNAH8 mRNA expression, and AR bound the DNAH8 promoter sequence indicating DNAH8 is an AR target gene. Thus, DNAH8 is a new regulator of AR associated with metastatic tumors and poor prognosis.

Divergent Androgen Receptor and Beta-Catenin Signaling in Prostate Cancer Cells.

Despite decades of effort to develop effective therapy and to identify promising new drugs, prostate cancer is lethal once it progresses to castration-resistant disease. Studies show mis-regulation of multiple pathways in castration-resistant prostate cancer (CRPC), reflecting the heterogeneity of the tumors and also hinting that targeting androgen receptor (AR) pathway alone might not be sufficient to treat CRPC. In this study, we present evidence that the Wnt/ß-catenin pathway might be activated in prostate cancer cells after androgen-deprivation to promote androgen-independent growth, partly through enhanced interaction of ß-catenin with TCF4. Androgen-independent prostate cancer cells were more prone to activate a Wnt-reporter, and inhibition of the Wnt/ß-catenin pathway increased sensitivity of these cells to the second-generation antiandrogen, enzalutamide. Combined treatment of enzalutamide and Wnt/ß-catenin inhibitor showed increased growth repression in both androgen-dependent and -independent prostate cancer cells, suggesting therapeutic potential for this approach.