The mutational landscape of lethal castration-resistant prostate cancer.
ABSTRACT: Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains and losses, including ETS gene family fusions, PTEN loss and androgen receptor (AR) amplification, which drive prostate cancer development and progression to lethal, metastatic castration-resistant prostate cancer (CRPC). However, less is known about the role of mutations. Here we sequenced the exomes of 50 lethal, heavily pre-treated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment-naive, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPCs (2.00 per megabase) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1 that define a subtype of ETS gene family fusion-negative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in approximately one-third of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Furthermore, we identified recurrent mutations in multiple chromatin- and histone-modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with the AR, which is required for AR-mediated signalling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signalling and increases tumour growth. Proteins that physically interact with the AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX (also known as KDM6A) and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signalling deregulated in prostate cancer, and prioritize candidates for future study.
Project description:Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains/losses, including ETS gene fusions, PTEN loss and androgen receptor (AR) amplification, that drive prostate cancer development and progression to lethal, metastatic castrate resistant prostate cancer (CRPC)1. As less is known about the role of mutations2-4, here we sequenced the exomes of 50 lethal, heavily-pretreated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment naïve, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPC (2.00/Mb) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1, which define a subtype of ETS‑ prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in ~1/3 of CRPCs (commonly through TMPRSS2:ERG fusions), is a prostate cancer tumor suppressor that can also be deregulated through mutation. Further, we identified recurrent mutations in multiple chromatin/histone modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with AR, which is required for AR mediated signaling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC) , and showed that mutated FOXA1 represses androgen signaling and increases tumor growth in vitro and in vivo. Proteins that physically interact with AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX, and ASXL1 were found to be mutated in CRPC, suggesting novel drivers of prostate cancer progression and potential resistance mechanisms to anti-androgen therapies. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signaling deregulated in prostate cancer, and prioritize candidates for future study. Gene expression profiling and array CGH (aCGH) was performed on matched benign prostate tissues (n=28), localized prostate cancer (n=59), and metastatic castrate resistant prostate cancer (CRPC, n=35). For gene expression profiling, frozen prostate tissue samples (channel 2), were hybridized against a commercial pool of benign prostate tissue (Clontech, channel 1). For aCGH, frozen prostate tissue samples (channel 2) were hybridized against a commerical sample of Human Male Genomic DNA (Promega, channel 1).
Project description:Castration-resistant prostate cancer (CRPC) continues to pose a significant clinical challenge with new generation second-line hormonal therapies affording limited improvement in disease outcome. As the androgen receptor (AR) remains a critical driver in CRPC, understanding the determinants of its transcriptional activity is important for developing new AR-targeted therapies. FOXA1 is a key component of the AR transcriptional complex yet its role in prostate cancer progression and the relationship between AR and FOXA1 are not completely resolved. It is well established that FOXA1 levels are elevated in advanced prostate cancer and metastases. We mimicked these conditions by overexpressing FOXA1 in the androgen-responsive LNCaP prostate cancer cell line and observed a significant increase in AR genomic binding at novel regions that possess increased chromatin accessibility. High levels of FOXA1 resulted in increased proliferation at both sub-optimal and high 5?-dihydrotestosterone (DHT) concentrations. Immunohistochemical staining for FOXA1 in a clinical prostate cancer cohort revealed that high FOXA1 expression is associated with shorter time to biochemical recurrence after radical prostatectomy (hazard ratio (HR) 5.0, 95% confidence interval (CI) 1.2-21.1, P=0.028), positive surgical margins and higher stage disease at diagnosis. The gene expression program that results from FOXA1 overexpression is enriched for PTEN, Wnt and other pathways typically represented in CRPC gene signatures. Together, these results suggest that in an androgen-depleted state, elevated levels of FOXA1 enhance AR binding at genomic regions not normally occupied by AR, which in turn facilitates prostate cancer cell growth.
Project description:Continued androgen receptor (AR) signaling is an established mechanism underlying castration-resistant prostate cancer (CRPC), and suppression of androgen receptor signaling remains a therapeutic goal of CRPC therapy. Constitutively active androgen receptor splice variants (AR-Vs) lack the androgen receptor ligand-binding domain (AR-LBD), the intended target of androgen deprivation therapies including CRPC therapies such as abiraterone and MDV3100. While the canonical full-length androgen receptor (AR-FL) and AR-Vs are both increased in CRPCs, their expression regulation, associated transcriptional programs, and functional relationships have not been dissected. In this study, we show that suppression of ligand-mediated AR-FL signaling by targeting AR-LBD leads to increased AR-V expression in two cell line models of CRPCs. Importantly, treatment-induced AR-Vs activated a distinct expression signature enriched for cell-cycle genes without requiring the presence of AR-FL. Conversely, activation of AR-FL signaling suppressed the AR-Vs signature and activated expression programs mainly associated with macromolecular synthesis, metabolism, and differentiation. In prostate cancer cells and CRPC xenografts treated with MDV3100 or abiraterone, increased expression of two constitutively active AR-Vs, AR-V7 and ARV567ES, but not AR-FL, paralleled increased expression of the androgen receptor-driven cell-cycle gene UBE2C. Expression of AR-V7, but not AR-FL, was positively correlated with UBE2C in clinical CRPC specimens. Together, our findings support an adaptive shift toward AR-V-mediated signaling in a subset of CRPC tumors as the AR-LBD is rendered inactive, suggesting an important mechanism contributing to drug resistance to CRPC therapy.
Project description:The androgen receptor (AR) is critical for the progression of prostate cancer to a castration-resistant (CRPC) state. AR antagonists are ineffective due to their inability to repress the expression of AR or its splice variant, AR-V7. Here, we report that the tyrosine kinase ACK1 (TNK2) phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site. The WDR5/MLL2 complex reads the H4-Y88-phosphorylation marks and deposits the transcriptionally activating H3K4-trimethyl marks promoting AR transcription. Reversal of the pY88-H4 epigenetic marks by the ACK1 inhibitor (R)-9bMS-sensitized naive and enzalutamide-resistant prostate cancer cells and reduced AR and AR-V7 levels to mitigate CRPC tumor growth. Thus, a feedforward ACK1/pY88-H4/WDR5/MLL2/AR epigenetic circuit drives CRPC and is necessary for maintenance of the malignant state.
Project description:Androgen receptor (AR) is critical for the progression of prostate cancer to the castration resistant (CRPC) state. Despite initial response, AR antagonists are ineffective long term in stifling AR activity due to their inability to repress expression of the AR or its splice variant, AR-V7. Here, we report that ACK1 tyrosine kinase, frequently deregulated in CRPCs, phosphorylates histone H4 at tyrosine 88, upstream of the AR transcription start site. WDR5/MLL2 complex reads the H4Y88 marks and modifies H3 in trans to deposit the transcriptionally activating H3K4 trimethyl marks, promoting AR transcription. Reversal of the pY88-H4 epigenetic marks by ACK1 small molecule inhibitor, (R)-9bMS, sensitized naïve and enzalutamide-resistant cancer cells, repressed AR and AR-V7 levels to mitigate in vivo CRPC tumor growth. Thus, a feed-forward ACK1-H4-WDR5/MLL2-AR/AR-V7 epigenetic circuit drives CRPC and is necessary for maintenance of the malignant state. Overall design: Two-condition experiment, DMSO versus inhibitor treated cells. 5 prostate cancer cell lines, LNCaP-C4-2B, VCaP, LAPC4, PC3 and DU145. Cells were treated with 3.5 uM of inhibitor for 24 hours.
Project description:Understanding the mechanism underlying the regulation of the androgen receptor (AR), a central player in the development of castration-resistant prostate cancer (CRPC), holds promise for overcoming the challenge of treating CRPC. We demonstrate that the ubiquitin ligase Siah2 targets a select pool of NCOR1-bound, transcriptionally-inactive AR for ubiquitin-dependent degradation, thereby promoting expression of select AR target genes implicated in lipid metabolism, cell motility, and proliferation. Siah2 is required for prostate cancer cell growth under androgen-deprivation conditions in vitro and in vivo, and Siah2 inhibition promotes prostate cancer regression upon castration. Notably, Siah2 expression is markedly increased in human CRPCs. Collectively, we find that selective regulation of AR transcriptional activity by the ubiquitin ligase Siah2 is important for CRPC development.
Project description:Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.
Project description:Retention of androgen receptor (AR) signalling in castrate-resistant prostate cancer (CRPC) highlights the requirement for the development of more effective AR targeting therapies. A key mechanism of resistance to anti-androgens is through expression of constitutively active AR variants (AR-Vs) that are refractory to next-generation therapies, including Enzalutamide and Abiraterone. By maintaining an androgenic gene signature, AR-Vs drive tumour survival and progression in castrate conditions. Critically, however, our understanding of the mechanics of AR-V-driven transcription is limited, particularly with respect to dependency on pioneer factor function. Here we show that depletion of FOXA1 in the CWR22Rv1 CRPC cell line abrogates the oncogenic potential of AR-Vs. Gene expression profiling reveals that approximately 41% of the AR-V transcriptome requires FOXA1 and that depletion of FOXA1 attenuates AR-V binding at a sub-set of analysed co-regulated genes. Interestingly, AR-V levels are elevated in cells depleted of FOXA1 as a consequence of attenuated negative feedback on the AR gene, but is insufficient to maintain cell growth as evidenced by marked anti-proliferative effects in FOXA1 knockdown cells. In all, our data suggests that AR-Vs are dependent on FOXA1 for sustaining a pro-proliferative gene signature and agents targeting FOXA1 may represent novel therapeutic options for CRPC patients.
Project description:To study Wnt/?-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the ?-catenin-androgen receptor (AR) interaction.We carried out ?-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of ?-catenin-mediated transcription), and sequenced the ?-catenin gene in MDA prostate cancer 118a, MDA prostate cancer 118b, MDA prostate cancer 2b, and PC-3 prostate cancer cells. We knocked down ?-catenin in AR-negative MDA prostate cancer 118b cells and carried out comparative gene-array analysis. We also immunohistochemically analyzed ?-catenin and AR in 27 bone metastases of human CRPCs.?-Catenin nuclear accumulation and TOP-flash reporter activity were high in MDA prostate cancer 118b but not in MDA prostate cancer 2b or PC-3 cells. MDA prostate cancer 118a and MDA prostate cancer 118b cells carry a mutated ?-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA prostate cancer 118b cells with downregulated ?-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant ?-catenin. Finally, we found nuclear localization of ?-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher's exact test), suggesting that reduced AR expression enables Wnt/?-catenin signaling.We identified a previously unknown downstream target of ?-catenin, HAS2, in prostate cancer, and found that high ?-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone metastatic prostate cancer. These findings may guide physicians in managing these patients.
Project description:While androgen-deprivation therapy can induce dramatic clinical responses in advanced and metastatic prostate cancer, refractory disease (castration-resistant prostate cancer [CRPC]) eventually emerges. In recent years, several studies have demonstrated the importance of residual intratumoral androgens in maintaining androgen receptor (AR) transcriptional activity in CRPC. The cytochrome P450 enzyme CYP17 is an obligatory step in androgen synthesis, and therefore a critical therapeutic target in CRPC. Abiraterone acetate is a selective, irreversible inhibitor of CYP17 and can suppress adrenal synthesis of androgen precursors, and possibly in situ steroidogenesis in the tumor microenvironment. In a phase III multicenter study, abiraterone in combination with prednisone improved median overall survival of men with docetaxel-refractory CRPC by 3.9 months compared to placebo plus prednisone, and also resulted in higher objective prostate-specific antigen and radiographic response rates. The study led to the FDA approval in April 2011 of abiraterone for treatment of chemotherapy-refractory CRPC patients, validating steroidogenesis and the AR axis in general as therapeutic targets in CRPC. The FDA indication for abiraterone was expanded to all CRPCs in December 2012, while evaluation in even earlier disease states is ongoing. We propose a comprehensive AR axis-targeting approach via simultaneous, frontline enzymatic blockade of several steroidogenic enzymes (eg, CYP17 and AKR1C3) in combination with gonadotropin-releasing hormone analogs and potent, second-generation AR antagonists (eg, enzalutamide) in order to improve outcomes in patients with prostate cancer.