The Mutational Landscape of Lethal Castrate Resistant Prostate Cancer
ABSTRACT: 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: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: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: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:The androgen receptor (AR) is a ligand-inducible transcription factor that mediates androgen action in target tissues. Upon ligand binding, the AR binds to thousands of genomic loci and activates a cell-type specific gene program. Prostate cancer growth and progression depend on androgen-induced AR signaling. Treatment of advanced prostate cancer through medical or surgical castration leads to initial response and durable remission, but resistance inevitably develops. In castration-resistant prostate cancer (CRPC), AR activity remains critical for tumor growth despite androgen deprivation. Although previous studies have focused on ligand-dependent AR signaling, in this study we explore AR function under the androgen-deprived conditions characteristic of CRPC. Our data demonstrate that AR persistently occupies a distinct set of genomic loci after androgen deprivation in CRPC. These androgen-independent AR occupied regions have constitutively open chromatin structures that lack the canonical androgen response element and are independent of FoxA1, a transcription factor involved in ligand-dependent AR targeting. Many AR binding events occur at proximal promoters, which can act as enhancers to augment transcriptional activities of other promoters through DNA looping. We further show that androgen-independent AR binding directs a gene expression program in CRPC, which is necessary for the growth of CRPC after androgen withdrawal.
Project description:FoxA1 (FOXA1) is a pioneering transcription factor of the androgen receptor (AR) that is indispensible for the lineage-specific gene expression of the prostate. To date, there have been conflicting reports on the role of FoxA1 in prostate cancer progression and prognosis. With recent discoveries of recurrent FoxA1 mutations in human prostate tumors, comprehensive understanding of FoxA1 function has become very important. Here, through genomic analysis, we reveal that FoxA1 regulates two distinct oncogenic processes via disparate mechanisms. FoxA1 induces cell growth requiring the AR pathway. On the other hand, FoxA1 inhibits cell motility and epithelial-to-mesenchymal transition (EMT) through AR-independent mechanism directly opposing the action of AR signaling. Using orthotopic mouse models, we further show that FoxA1 inhibits prostate tumor metastasis in vivo. Concordant with these contradictory effects on tumor progression, FoxA1 expression is slightly upregulated in localized prostate cancer wherein cell proliferation is the main feature, but is remarkably downregulated when the disease progresses to metastatic stage for which cell motility and EMT are essential. Importantly, recently identified FoxA1 mutants have drastically attenuated ability in suppressing cell motility. Taken together, our findings illustrate an AR-independent function of FoxA1 as a metastasis inhibitor and provide a mechanism by which recurrent FoxA1 mutations contribute to prostate cancer progression.
Project description:The inevitable progression of advanced prostate cancer to castration resistance, and ultimately to lethal metastatic disease, depends on primary or acquired resistance to conventional androgen deprivation therapy (ADT) and accumulated resistance strategies to evade androgen receptor (AR) suppression. In prostate cancer cells, AR adaptations that arise in response to ADT are not singular, but diverse, and include gene amplification, mutation, and even complete loss of receptor expression. Collectively, each of these AR adaptations contributes to a complex, heterogeneous, ADT-resistant tumor. Here, we examined prostate cancer cell lines that model common castration-resistant prostate cancer (CRPC) subtypes, each with different AR composition, and focused on novel regulators of tumor progression, the Bromodomain and Extraterminal (BET) family of proteins. We found that BRD4 regulates cell migration across all models of CRPC, regardless of aggressiveness and AR status, whereas BRD2 and BRD3 only regulate migration and invasion in less aggressive models that retain AR expression or signaling. BRD4, a coregulator of gene transcription, controls migration and invasion through transcription of AHNAK, a large scaffolding protein linked to promotion of metastasis in a diverse set of cancers. Furthermore, treatment of CRPC cell lines with low doses of MZ1, a small-molecule, BRD4-selective degrader, inhibits metastatic potential. Overall, these results reveal a novel BRD4-AHNAK pathway that may be targetable to treat metastatic CRPC (mCRPC). IMPLICATIONS: BRD4 functions as the dominant regulator of CRPC cell migration and invasion through direct transcriptional regulation of AHNAK, which together offer a novel targetable pathway to treat metastatic CRPC.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/8/1627/F1.large.jpg.
Project description:Emerging studies have shown that the expression of AR splice variants (ARv) lacking ligand-binding domain is associated with castrate-resistant prostate cancer (CRPC) and higher risk of tumor metastasis and recurrence. Nuclear export protein XPO1 regulates the nuclear localization of many proteins including tumor suppressor proteins. Increased XPO1 in prostate cancer is associated with a high Gleason score and bone metastasis. In this study, we found that high expression of AR splice variant 7 (AR-v7) was correlated with increased XPO1 expression. Silencing of XPO1 by RNAi or treatment with Selective Inhibitor of Nuclear Export (SINE) compounds selinexor and eltanexor (KPT-8602) down-regulated the expression of AR, AR-v7 and ARv567es at mRNA and protein levels. XPO1 silencing also inhibited the expression of AR and ARv regulators including FOXA1, Src, Vav3, MED1 and Sam68, leading to the suppression of ARv and AR target genes, UBE2C and PSA. By targeting XPO1/ARv signaling, SINE suppressed prostate cancer (PCa) growth in vitro and in vivo and potentiated the anti-cancer activity of anti-AR agents, enzalutamide and abiraterone. Therefore, XPO1 inhibition could be a novel promising agent used in combination with conventional chemotherapeutics and AR-targeted therapy for the better treatment of PCa, especially CRPC.
Project description:TMPRSS2/ERG rearrangement, PTEN gene deletion, and androgen receptor (AR) gene amplification have been observed in various stages of human prostate cancer. We hypothesized that using these markers as a combined panel would allow better differentiation between low-risk and high-risk prostate cancer. We analyzed 110 primary prostate cancer samples, 70 metastatic tumor samples from 11 patients, and 27 xenograft tissues derived from 22 advanced prostate cancer patients using fluorescence in situ hybridization (FISH) analysis with probes targeting the TMPRSS2/ERG, PTEN, and AR gene loci. Heterogeneity of the aberrations detected was evaluated. Genetic patterns were also correlated with transcript levels. Among samples with complete data available, the three-marker FISH panel detected chromosomal abnormalities in 53% of primary prostate cancers and 87% of metastatic (Met) or castration-resistant (CRPC) tumors. The number of markers with abnormal FISH result had a different distribution between the two groups (P<0.001). At the patient level, Met/CRPC tumors are 4.5 times more likely to show abnormalities than primary cancer patients (P<0.05). Heterogeneity among Met/CRPC tumors is mostly inter-patient. Intra-patient heterogeneity is primarily due to differences between the primary prostate tumor and the metastases while multiple metastatic sites show consistent abnormalities. Intra-tumor variability is most prominent with the AR copy number in primary tumors. AR copy number correlated well with the AR mRNA expression (rho?=?0.52, P<0.001). Especially among TMPRSS2:ERG fusion-positive CRPC tumors, AR mRNA and ERG mRNA levels are strongly correlated (rho?=?0.64, P<0.001). Overall, the three-marker FISH panel may represent a useful tool for risk stratification of prostate cancer patients.
Project description:The enhancer pioneer transcription factor FoxA1 is a global mediator of steroid receptor (SR) action in hormone-dependent cancers. In castration-resistant prostate cancer (CRPC), FoxA1 acts as an androgen receptor cofactor to drive G? to M-phase cell-cycle transit. Here, we describe a mechanistically distinct SR-independent role for FoxA1 in driving G? to S-phase cell-cycle transit in CRPC. By comparing FoxA1 binding sites in prostate cancer cell genomes, we defined a codependent set of FoxA1-MYBL2 and FoxA1-CREB1 binding sites within the regulatory regions of the Cyclin E2 and E2F1 genes that are critical for CRPC growth. Binding at these sites upregulate the Cyclin E2 and Cyclin A2 genes in CRPC but not in earlier stage androgen-dependent prostate cancer, establishing a stage-specific role for this pathway in CRPC growth. Mechanistic investigations indicated that FoxA1, MYBL2, or CREB1 induction of histone H3 acetylation facilitated nucleosome disruption as the basis for codependent transcriptional activation and G? to S-phase cell-cycle transit. Our findings establish FoxA1 as a pivotal driver of the cell-cycle in CRPC which promotes G? to S-phase transit as well as G? to M-phase transit through two distinct mechanisms.
Project description:Androgen receptor (AR) is a ligand-activated transcription factor and a key driver of prostate cancer (PCa) growth and progression. Understanding the factors influencing AR-mediated gene expression provides new opportunities for therapeutic intervention. Poly(ADP-ribose) Polymerase (PARP) is a family of enzymes, which posttranslationally modify a range of proteins and regulate many different cellular processes. PARP-1 and PARP-2 are two well-characterized PARP members, whose catalytic activity is induced by DNA-strand breaks and responsible for multiple DNA damage repair pathways. PARP inhibitors are promising therapeutic agents that show synthetic lethality against many types of cancer (including PCa) with homologous recombination (HR) DNA-repair deficiency. Here, we show that, beyond DNA damage repair function, PARP-2, but not PARP-1, is a critical component in AR transcriptional machinery through interacting with the pioneer factor FOXA1 and facilitating AR recruitment to genome-wide prostate-specific enhancer regions. Analyses of PARP-2 expression at both mRNA and protein levels show significantly higher expression of PARP-2 in primary PCa tumors than in benign prostate tissues, and even more so in castration-resistant prostate cancer (CRPC) tumors. Selective targeting of PARP-2 by genetic or pharmacological means blocks interaction between PARP-2 and FOXA1, which in turn attenuates AR-mediated gene expression and inhibits AR-positive PCa growth. Next-generation antiandrogens act through inhibiting androgen synthesis (abiraterone) or blocking ligand binding (enzalutamide). Selective targeting of PARP-2, however, may provide an alternative therapeutic approach for AR inhibition by disruption of FOXA1 function, which may be beneficial to patients, irrespective of their DNA-repair deficiency status.