Colm Morrissey

Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer

Prostate cancer is the second most common cancer in men worldwide and causes over 250,000 deaths each year. Overtreatment of indolent disease also results in significant morbidity. Common genetic alterations in prostate cancer include losses of NKX3.1 (8p21) and PTEN (10q23), gains of AR (the androgen receptor gene) and fusion of ETS family transcription factor genes with androgen-responsive promoters. Recurrent somatic base-pair substitutions are believed to be less contributory in prostate tumorigenesis but have not been systematically analyzed in large cohorts. Here, we sequenced the exomes of 112 prostate tumor and normal tissue pairs. New recurrent mutations were identified in multiple genes, including MED12 and FOXA1. SPOP was the most frequently mutated gene, with mutations involving the SPOP substrate-binding cleft in 6–15% of tumors across multiple independent cohorts. Prostate cancers with mutant SPOP lacked ETS family gene rearrangements and showed a distinct pattern of genomic alterations. Thus, SPOP mutations may define a new molecular subtype of prostate cancer.

EZH2 Oncogenic Activity in Castration-Resistant Prostate Cancer Cells Is Polycomb-Independent

Alternative Role for EZH2 Epigenetic regulators are implicated in cancer progression and proposed as therapeutic targets. Xu et al. (p. 1465; see the Perspective by Cavalli) report that EZH2 (Enhancer of zeste homolog 2), a factor previously thought to exert its oncogenic function primarily as part of the polycomb repressive complex, acts through a distinct mechanism in cells of castration-resistant prostate cancer. Rather than exclusively silencing gene expression through histone methylation, EZH2 acts as a transcriptional coactivator. The activation function of EZH2 plays a critical role in the growth of castration-resistant prostate cancer cells, which could be relevant in future drug development. An epigenetic regulator positively regulates gene expression in cell-based models of hormone-resistant prostate cancer. Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. We found that the oncogenic function of EZH2 in cells of castration-resistant prostate cancer is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a coactivator for critical transcription factors including the androgen receptor. This functional switch is dependent on phosphorylation of EZH2 and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.

Quantitative and stoichiometric analysis of the microRNA content of exosomes

Significance Exosomes have been a subject of great interest in recent years, especially in the context of the microRNAs (miRNAs) that they contain. Exosome-mediated miRNA transfer between cells has been proposed to be a mechanism for intercellular signaling and exosome-associated miRNAs in biofluids have been suggested as potential minimally invasive biomarkers for multiple human disease states. Remarkably, we show here that most exosomes derived from standard preparations do not harbor many copies of miRNA molecules. These findings suggest a reevaluation of current models of the mechanism of exosome-mediated miRNA communication and indicate that stoichiometric analysis will be valuable for the study of other populations of extracellular vesicles and their associated RNAs as well. Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.

Inherited DNA-Repair Gene Mutations in Men with Metastatic Prostate Cancer

BACKGROUND Inherited mutations in DNA-repair genes such as BRCA2 are associated with increased risks of lethal prostate cancer. Although the prevalence of germline mutations in DNA-repair genes among men with localized prostate cancer who are unselected for family predisposition is insufficient to warrant routine testing, the frequency of such mutations in patients with metastatic prostate cancer has not been established. METHODS We recruited 692 men with documented metastatic prostate cancer who were unselected for family history of cancer or age at diagnosis. We isolated germline DNA and used multiplex sequencing assays to assess mutations in 20 DNA-repair genes associated with autosomal dominant cancer-predisposition syndromes. RESULTS A total of 84 germline DNA-repair gene mutations that were presumed to be deleterious were identified in 82 men (11.8%); mutations were found in 16 genes, including BRCA2 (37 men [5.3%]), ATM (11 [1.6%]), CHEK2 (10 [1.9% of 534 men with data]), BRCA1 (6 [0.9%]), RAD51D (3 [0.4%]), and PALB2 (3 [0.4%]). Mutation frequencies did not differ according to whether a family history of prostate cancer was present or according to age at diagnosis. Overall, the frequency of germline mutations in DNA-repair genes among men with metastatic prostate cancer significantly exceeded the prevalence of 4.6% among 499 men with localized prostate cancer (P<0.001), including men with high-risk disease, and the prevalence of 2.7% in the Exome Aggregation Consortium, which includes 53,105 persons without a known cancer diagnosis (P<0.001). CONCLUSIONS In our multicenter study, the incidence of germline mutations in genes mediating DNA-repair processes among men with metastatic prostate cancer was 11.8%, which was significantly higher than the incidence among men with localized prostate cancer. The frequencies of germline mutations in DNA-repair genes among men with metastatic disease did not differ significantly according to age at diagnosis or family history of prostate cancer. (Funded by Stand Up To Cancer and others.).

Exome sequencing identifies a spectrum of mutation frequencies in advanced and lethal prostate cancers

To catalog protein-altering mutations that may drive the development of prostate cancers and their progression to metastatic disease systematically, we performed whole-exome sequencing of 23 prostate cancers derived from 16 different lethal metastatic tumors and three high-grade primary carcinomas. All tumors were propagated in mice as xenografts, designated the LuCaP series, to model phenotypic variation, such as responses to cancer-directed therapeutics. Although corresponding normal tissue was not available for most tumors, we were able to take advantage of increasingly deep catalogs of human genetic variation to remove most germline variants. On average, each tumor genome contained ∼200 novel nonsynonymous variants, of which the vast majority was specific to individual carcinomas. A subset of genes was recurrently altered across tumors derived from different individuals, including TP53, DLK2, GPC6, and SDF4. Unexpectedly, three prostate cancer genomes exhibited substantially higher mutation frequencies, with 2,000–4,000 novel coding variants per exome. A comparison of castration-resistant and castration-sensitive pairs of tumor lines derived from the same prostate cancer highlights mutations in the Wnt pathway as potentially contributing to the development of castration resistance. Collectively, our results indicate that point mutations arising in coding regions of advanced prostate cancers are common but, with notable exceptions, very few genes are mutated in a substantial fraction of tumors. We also report a previously undescribed subtype of prostate cancers exhibiting “hypermutated” genomes, with potential implications for resistance to cancer therapeutics. Our results also suggest that increasingly deep catalogs of human germline variation may challenge the necessity of sequencing matched tumor-normal pairs.

Androgen Receptor Variants Occur Frequently in Castration Resistant Prostate Cancer Metastases

Background Although androgens are depleted in castration resistant prostate cancer (CRPC), metastases still express nuclear androgen receptor (AR) and androgen regulated genes. We recently reported that C-terminal truncated constitutively active AR splice variants contribute to CRPC development. Since specific antibodies detecting all C-terminal truncated AR variants are not available, our aim was to develop an approach to assess the prevalence and function of AR variants in prostate cancer (PCa). Methodology/Principal Findings Using 2 antibodies against different regions of AR protein (N- or C-terminus), we successfully showed the existence of AR variant in the LuCaP 86.2 xenograft. To evaluate the prevalence of AR variants in human PCa tissue, we used this method on tissue microarrays including 50 primary PCa and 162 metastatic CRPC tissues. RT-PCR was used to confirm AR variants. We observed a significant decrease in nuclear C-terminal AR staining in CRPC but no difference between N- and C-terminal AR nuclear staining in primary PCa. The expression of the AR regulated proteins PSA and PSMA were marginally affected by the decrease in C-terminal staining in CRPC samples. These data suggest that there is an increase in the prevalence of AR variants in CRPC based on our ability to differentiate nuclear AR expression using N- and C-terminal AR antibodies. These findings were validated using RT-PCR. Importantly, the loss of C-terminal immunoreactivity and the identification of AR variants were different depending on the site of metastasis in the same patient. Conclusions We successfully developed a novel immunohistochemical approach which was used to ascertain the prevalence of AR variants in a large number of primary PCa and metastatic CRPC. Our results showed a snapshot of overall high frequency of C-terminal truncated AR splice variants and site specific AR loss in CRPC, which could have utility in stratifying patients for AR targeted therapeutics.

The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression.

Retinoblastoma (RB; encoded by RB1) is a tumor suppressor that is frequently disrupted in tumorigenesis and acts in multiple cell types to suppress cell cycle progression. The role of RB in tumor progression, however, is poorly defined. Here, we have identified a critical role for RB in protecting against tumor progression through regulation of targets distinct from cell cycle control. In analyses of human prostate cancer samples, RB loss was infrequently observed in primary disease and was predominantly associated with transition to the incurable, castration-resistant state. Further analyses revealed that loss of the RB1 locus may be a major mechanism of RB disruption and that loss of RB function was associated with poor clinical outcome. Modeling of RB dysfunction in vitro and in vivo revealed that RB controlled nuclear receptor networks critical for tumor progression and that it did so via E2F transcription factor 1-mediated regulation of androgen receptor (AR) expression and output. Through this pathway, RB depletion induced unchecked AR activity that underpinned therapeutic bypass and tumor progression. In agreement with these findings, disruption of the RB/E2F/nuclear receptor axis was frequently observed in the transition to therapy resistance in human disease. Together, these data reveal what we believe to be a new paradigm for RB function in controlling prostate tumor progression and lethal tumor phenotypes.

Substantial interindividual and limited intraindividual genomic diversity among tumors from men with metastatic prostate cancer

Tumor heterogeneity may reduce the efficacy of molecularly guided systemic therapy for cancers that have metastasized. To determine whether the genomic alterations in a single metastasis provide a reasonable assessment of the major oncogenic drivers of other dispersed metastases in an individual, we analyzed multiple tumors from men with disseminated prostate cancer through whole-exome sequencing, array comparative genomic hybridization (CGH) and RNA transcript profiling, and we compared the genomic diversity within and between individuals. In contrast to the substantial heterogeneity between men, there was limited diversity among metastases within an individual. The number of somatic mutations, the burden of genomic copy number alterations and aberrations in known oncogenic drivers were all highly concordant, as were metrics of androgen receptor (AR) activity and cell cycle activity. AR activity was inversely associated with cell proliferation, whereas the expression of Fanconi anemia (FA)-complex genes was correlated with elevated cell cycle progression, expression of the E2F transcription factor 1 (E2F1) and loss of retinoblastoma 1 (RB1). Men with somatic aberrations in FA-complex genes or in ATM serine/threonine kinase (ATM) exhibited significantly longer treatment-response durations to carboplatin than did men without defects in genes encoding DNA-repair proteins. Collectively, these data indicate that although exceptions exist, evaluating a single metastasis provides a reasonable assessment of the major oncogenic driver alterations that are present in disseminated tumors within an individual, and thus may be useful for selecting treatments on the basis of predicted molecular vulnerabilities.

Clusterin Biogenesis Is Altered during Apoptosis in the Regressing Rat Ventral Prostate

Clusterin was first characterized as an apoptosis-associated transcript after it was identified as testosterone-repressed prostate message (TRPM-2) that is expressed in the epithelial cells of the regressing rat ventral prostate. Increases in clusterin mRNA and protein have been consistently detected in apoptotic cell death paradigms, establishing clusterin gene expression as a prominent marker of apoptotic cell loss. However, enhanced protein expression has also been reported in surviving cells. This ambiguity makes it difficult to define the contribution of clusterin to apoptosis. To address this problem, a panel of polyclonal and monoclonal antibodies were raised against the clusterin α-chain, β-chain, and mixed α/β epitopes. These antibodies detect changes in the biogenesis of clusterin during apoptosis by Western analysis and immunohistochemistry. A 42-kDa glyco/isoform of clusterin appears to be up-regulated in dying epithelial cells. This glyco/isoform is apparently generated as a result of apoptosis-induced stimulation of a normal but under-utilized, synthetic pathway. These data demonstrate that clusterin synthesized by apoptotic cells can be immunologically distinguished from clusterin synthesized by surviving cells in damaged tissue.

Circulating microRNA Profiling Identifies a Subset of Metastatic Prostate Cancer Patients with Evidence of Cancer-Associated Hypoxia

MicroRNAs (miRNAs) are small (∼22 nucleotide) non-coding RNAs that regulate a myriad of biological processes and are frequently dysregulated in cancer. Cancer-associated microRNAs have been detected in serum and plasma and hold promise as minimally invasive cancer biomarkers, potentially for assessing disease characteristics in patients with metastatic disease that is difficult to biopsy. Here we used miRNA profiling to identify cancer-associated miRNAs that are differentially expressed in sera from patients with metastatic castration resistant prostate cancer (mCRPC) as compared to healthy controls. Of 365 miRNAs profiled, we identified five serum miRNAs (miR-141, miR-200a, miR-200c, miR-210 and miR-375) that were elevated in cases compared to controls across two independent cohorts. One of these, miR-210, is a known transcriptional target of the hypoxia-responsive HIF-1α signaling pathway. Exposure of cultured prostate cancer cells to hypoxia led to induction of miR-210 and its release into the extracellular environment. Moreover, we found that serum miR-210 levels varied widely amongst mCRPC patients undergoing therapy, and correlated with treatment response as assessed by change in PSA. Our results suggest that (i) cancer-associated hypoxia is a frequent, previously under-appreciated characteristic of mCRPC, and (ii) serum miR-210 may be further developed as a predictive biomarker in patients with this distinct disease biology.