YAP1 regulates prostate cancer stem cell-like characteristics to promote castration resistant growth.
ABSTRACT: Castration resistant prostate cancer (CRPC) is a stage of relapse that arises after various forms of androgen ablation therapy (ADT) and causes significant morbidity and mortality. However, the mechanism underlying progression to CRPC remains poorly understood. Here, we report that YAP1, which is negatively regulated by AR, influences prostate cancer (PCa) cell self-renewal and CRPC development. Specifically, we found that AR directly regulates the methylation of YAP1 gene promoter via the formation of a complex with Polycomb group protein EZH2 and DNMT3a. In normal conditions, AR recruits EZH2 and DNMT3a to YAP1 promoter, thereby promoting DNA methylation and the repression of YAP1 gene transcription. Following ADT treatment or when AR activity is antagonized by Bicalutamide or Enzalutamide, YAP1 gene expression is switched on. In turn, YAP1 promotes SOX2 and Nanog expression and the de-differentiation of PCa cells to stem/progenitor-like cells (PCSC), which potentially contribute to disease recurrence. Finally, the knock down of YAP1 expression or the inhibition of YAP1 function by Verteporfin in TRAMP prostate cancer mice significantly suppresses tumor recurrence following castration. In conclusion, our data reveals that AR suppresses YAP1 gene expression through a novel epigenetic mechanism, which is critical for PCa cells self-renewal and the development of CRPC.
Project description:Prostate cancer (PCa) is the most commonly diagnosed cancer among men in western countries. Androgen receptor (AR) signaling plays key roles in the development of PCa. Androgen deprivation therapy (ADT) remains the standard therapy for advanced PCa. In addition to its ligand androgen, accumulating evidence indicates that posttranscriptional modification is another important mechanism to regulate AR activities during the progression of PCa, especially in castration resistant prostate cancer (CRPC). To date, a number of posttranscriptional modifications of AR have been identified, including phosphorylation (e.g. by CDK1), acetylation (e.g. by p300 and recognized by BRD4), methylation (e.g. by EZH2), ubiquitination (e.g. by SPOP), and SUMOylation (e.g. by PIAS1). These modifications are essential for the maintenance of protein stability, nuclear localization and transcriptional activity of AR. This review summarizes posttranslational modifications that influence androgen-dependent and -independent activities of AR, PCa progression and therapy resistance. We further emphasize that in addition to androgen, posttranslational modification is another important way to regulate AR activity, suggesting that targeting AR posttranslational modifications, such as proteolysis targeting chimeras (PROTACs) of AR, represents a potential and promising alternate for effective treatment of CRPC. Potential areas to be investigated in the future in the field of AR posttranslational modifications are also discussed.
Project description:Prostate cancer (PCa) growth is mainly driven by androgen receptor (AR), and tumors that initially respond to androgen deprivation therapy (ADT) or AR inhibition usually relapse into a more aggressive, castration-resistant PCa (CRPC) stage. Circulating growth hormone (GH) has a permissive role in PCa development in animal models and in human PCa xenograft growth. As GH and GH receptor (GHR) are both expressed in PCa cells, we assessed whether prostatic GH production is linked to AR activity and whether GH contributes to the castration-resistant phenotype. Using online datasets, we found that GH is highly expressed in human CRPC. We observed increased GH expression in castration-resistant C4-2 compared with castration-sensitive LNCaP cells as well as in enzalutamide (MDV3100)-resistant (MDVR) C4-2B (C4-2B MDVR) cells compared with parental C4-2B. We describe a negative regulation of locally produced GH by androgens/AR in PCa cells following treatment with AR agonists (R1881) and antagonists (enzalutamide, bicalutamide). We also show that GH enhances invasive behavior of CRPC 22Rv1 cells, as reflected by increased migration, invasion, and anchorage-independent growth, as well as expression of matrix metalloproteases. Moreover, GH induces expression of the AR splice variant 7, which correlates with antiandrogen resistance, and also induces insulinlike growth factor 1, which is implicated in PCa progression and ligand-independent AR activation. In contrast, blockade of GH action with the GHR antagonist pegvisomant reverses these effects both in vitro and in vivo. GH induction following ADT or AR inhibition may contribute to CRPC progression by bypassing androgen growth requirements.
Project description:The mechanism(s) by which androgen receptor (AR) splice variants contribute to castration-resistant prostate cancer (CRPC) is still lacking.Expressions of epithelial-to-mesenchymal transition (EMT) and stem cell markers were molecularly tested using prostate cancer (PCa) cells transfected with AR and AR3 (also known as AR-V7) plasmids or siRNA, and also cultured cells under androgen deprivation therapy (ADT) condition. Cell migration, clonogenicity, sphere-forming capacity was assessed using PCa cells under all experimental conditions and 3,3'-diindolylmethane (DIM; BR-DIM) treatment. Human PCa samples from BR-DIM untreated or treated patients were also used for assessing the expression of AR3 and stem cell markers.Overexpression of AR led to the induction of EMT phenotype, while overexpression of AR3 not only induced EMT but also led to the expression of stem cell signature genes. More importantly, ADT enhanced the expression of AR and AR3 concomitant with up-regulated expression of EMT and stem cell marker genes. Dihydrotestosterone (DHT) treatment decreased the expression of AR and AR3, and reversed the expression of these EMT and stem cell marker genes. BR-DIM administered to PCa patients prior to radical prostatectomy inhibited the expression of cancer stem cell markers consistent with inhibition of self-renewal of PCa cells after BR-DIM treatment.AR variants could contribute to PCa progression through induction of EMT and acquisition of stem cell characteristics, which could be attenuated by BR-DIM, suggesting that BR-DIM could become a promising agent for the prevention of CRPC and/or for the treatment of PCa.
Project description:Prostate cancer (PCa) progression is regulated by the androgen receptor (AR); however, patients undergoing androgen-deprivation therapy (ADT) for disseminated PCa eventually develop castration-resistant PCa (CRPC). Results of previous studies indicated that AR, a transcription factor, occupies distinct genomic loci in CRPC compared with hormone-naïve PCa; however, the cause of this distinction was unknown. The E3 ubiquitin ligase Nrdp1 is a model AR target modulated by androgens in hormone-naïve PCa but not in CRPC. Using Nrdp1, we investigated how AR switches transcription programs during CRPC progression. The proximal Nrdp1 promoter contains an androgen response element (ARE); we demonstrated AR binding to this ARE in androgen-sensitive PCa. Analysis of hormone-naive human prostatectomy specimens revealed correlation between Nrdp1 and AR expression, supporting AR regulation of NRDP1 levels in androgen-sensitive tissue. However, despite sustained AR levels, AR binding to the Nrdp1 promoter and Nrdp1 expression were suppressed in CRPC. Elucidation of the suppression mechanism demonstrated correlation of NRDP1 levels with nuclear localization of the scaffolding protein filamin A (FLNA) which, as we previously showed, is itself repressed following ADT in many CRPC tumors. Restoration of nuclear FLNA in CRPC stimulated AR binding to Nrdp1 ARE, increased its transcription, and augmented NRDP1 protein expression and responsiveness to ADT, indicating that nuclear FLNA controls AR-mediated androgen-sensitive Nrdp1 transcription. Expression of other AR-regulated genes lost in CRPC was also re-established by nuclear FLNA. Thus, our results indicate that nuclear FLNA promotes androgen-dependent AR-regulated transcription in PCa, while loss of nuclear FLNA in CRPC alters the AR-regulated transcription program.
Project description:Enzalutamide, approved by the United States Food and Drug Administration in 2018 for the management of metastatic castration-resistant prostate cancer (CRPC), is an androgen receptor (AR) inhibitor. It blocks androgen binding to the AR, AR nuclear translocation, and AR-mediated DNA binding. Unfortunately, a considerable proportion of tumors eventually develop resistance during the treatment. The molecular mechanisms underlying enzalutamide resistance are not completely understood. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of polycomb repressor complex 2, has been proposed as a prognostic marker for prostate cancer (PCa). With the goal to test whether EZH2 also plays a critical role in acquisition of enzalutamide resistance in CRPC, here we examined whether EZH2 inhibition/depletion enhances the efficacy of enzalutamide in enzalutamide-resistant PCa cells. We show that combining the EZH2 inhibitor GSK126 with enzalutamide synergistically inhibits cell proliferation and colony formation and promotes apoptosis in enzalutamide-resistant PCa cells. EZH2 depletion also overcomes enzalutamide resistance in both cultured cells and xenograft tumors. Mechanistically, we found that EZH2 directly binds to the promoter of prostate-specific antigen and inhibits its expression in enzalutamide-resistant PCa cells. In agreement, bioinformatics analysis of clinical RNA sequencing data involving GSEA indicated a strong correlation between AR and EZH2 gene expression during PCa progression. Our study provides critical insights into the mechanisms underlying enzalutamide resistance, which may offer new approaches to enhance the efficacy of enzalutamide in CRPC.
Project description:The major challenge in castration-resistant prostate cancer (CRPC) remains the ability to predict the clinical responses to improve patient selection for appropriate treatments. The finding that androgen deprivation therapy (ADT) induces alterations in the androgen receptor (AR) transcriptional program by AR coregulators activity in a context-dependent manner, offers the opportunity for identifying signatures discriminating different clinical states of prostate cancer (PCa) progression. Gel electrophoretic analyses combined with western blot showed that, in androgen-dependent PCa and CRPC in vitro models, the subcellular distribution of spliced and serine-phosphorylated heterogeneous nuclear ribonucleoprotein K (hnRNP K) isoforms can be associated with different AR activities. Using mass spectrometry and bioinformatic analyses, we showed that the protein sets of androgen-dependent (LNCaP) and ADT-resistant cell lines (PDB and MDB) co-immunoprecipitated with hnRNP K varied depending on the cell type, unravelling a dynamic relationship between hnRNP K and AR during PCa progression to CRPC. By comparing the interactome of LNCaP, PDB, and MDB cell lines, we identified 51 proteins differentially interacting with hnRNP K, among which KLK3, SORD, SPON2, IMPDH2, ACTN4, ATP1B1, HSPB1, and KHDRBS1 were associated with AR and differentially expressed in normal and tumor human prostate tissues. This hnRNP K?AR-related signature, associated with androgen sensitivity and PCa progression, may help clinicians to better manage patients with CRPC.
Project description:Loss of androgen receptor (AR) dependency in prostate cancer (PCa) cells is associated with progression to castration-resistant prostate cancer (CRPC). The tumor stroma is enriched in fibroblasts that secrete AR-activating factors. To investigate the roles of fibroblasts in AR activation under androgen deprivation, we used three sublines of androgen-sensitive LNCaP cells (E9 and F10 cells: low androgen sensitivity; and AIDL cells: androgen insensitivity) and original fibroblasts derived from patients with PCa. We performed in vivo experiments using three sublines of LNCaP cells and original fibroblasts to form homotypic tumors. The volume of tumors derived from E9 cells plus fibroblasts was reduced following androgen deprivation therapy (ADT), whereas that of F10 or AIDL cells plus fibroblasts was increased even after ADT. In tumors derived from E9 cells plus fibroblasts, serum prostate-specific antigen (PSA) decreased rapidly after ADT, but was still detectable. In contrast, serum PSA was increased even in F10 cells inoculated alone. In indirect cocultures with fibroblasts, PSA production was increased in E9 cells. Epidermal growth factor treatment stimulated Akt and p44/42 mitogen-activated protein kinase phosphorylation in E9 cells. Notably, AR splice variant 7 was detected in F10 cells. Overall, we found that fibroblast-secreted AR-activating factors modulated AR signaling in E9 cells after ADT and loss of fibroblast-dependent AR activation in F10 cells may be responsible for CRPC progression.
Project description:Prostate cancer (PCa) is the second leading cause of cancer-related death in males in the United States. Majority of prostate cancers are originally androgen-dependent and sensitive to androgen-deprivation therapy (ADT), however, most of them eventually relapse and progress into incurable castration-resistant prostate cancer (CRPC). Of note, the activity of androgen receptor (AR) is still required in CRPC stage. The mitotic kinase polo-like kinase 1 (Plk1) is significantly elevated in PCa and its expression correlates with tumor grade. In this study, we assess the effects of Plk1 on AR signaling in both androgen-dependent and androgen-independent PCa cells. We demonstrate that the expression level of Plk1 correlated with tumorigenicity and that inhibition of Plk1 caused reduction of AR expression and AR activity. Furthermore, Plk1 inhibitor BI2536 down-regulated SREBP-dependent expression of enzymes involved in androgen biosynthesis. Of interest, Plk1 level was also reduced when AR activity was inhibited by the antagonist MDV3100. Finally, we show that BI2536 treatment significantly inhibited tumor growth in LNCaP CRPC xenografts. Overall, our data support the concept that Plk1 inhibitor such as BI2536 prevents AR signaling pathway and might have therapeutic potential for CRPC patients.
Project description:<h4>Background</h4>Androgen deprivation therapy (ADT) is the first-line treatment to metastatic prostate cancer (PCa). However, sustained expression and function of the androgen receptor (AR) gene contribute to the progression of castration resistant prostate cancers (CRPC). Additionally, tumors can adapt the PI3K/AKT survival pathway to escape ADT. Co-targeting AR and PI3K/AKT signaling has been proposed to be a more effective therapeutic means for CRPC patients. Many clinical trials are ongoing to test whether PI3K/AKT inhibitors are beneficial to PCa patients. However whether these inhibitors have any impacts on the expressions of full length AR (AR-FL) and its splice variant (AR-V7) remains unclear.<h4>Methods</h4>Four human prostate cancer cell lines (LNCaP, LNCaP95, VCaP and 22Rv1) with different genetic backgrounds were treated with five PI3K/AKT inhibitors (LY294002, Wortmannin, BKM120, AKTi and AZD5363) and or AKT siRNA. AR and AR-V7 protein and mRNA levels were measured by immunoblotting and real-time PCR assays. AR gene transcription initiation, alternative RNA splicing and AR mRNA degradation rates were also determined.<h4>Results</h4>PI3K/AKT inhibitors had various impacts on AR protein expressions primarily through alterations of AR gene transcription initiation and RNA splicing. However, these effects remained unchanged in the presence RNA silencing of the AKT genes.<h4>Conclusion</h4>PI3K/AKT inhibitors have off-target effects on AR gene expression in prostate cancer cells, which shall be considered when applying these inhibitors to PCa patients, particularly patients under ADT treatment.