SUMMARY Androgen receptor (AR) signaling is a distinctive feature of prostate carcinoma (PC) and represents the major therapeutic target for treating metastatic prostate cancer (mPC). Though highly effective, AR antagonism can produce tumors that bypass a functional requirement for AR, often through neuroendocrine (NE) transdifferentiation. Through the molecular assessment of mPCs over two decades, we find a phenotypic shift has occurred in mPC with the emergence of an AR-null NE-null phenotype. These “double-negative” PCs are notable for elevated FGF and MAPK pathway activity, which can bypass AR dependence. Pharmacological inhibitors of MAPK or FGFR repressed the growth of double-negative PCs in vitro and in vivo. Our results indicate that FGF/MAPK blockade may be particularly efficacious against mPCs with an AR-null phenotype.

Intra-individual 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 within an individual, we analyzed multiple tumors from men with disseminated prostate cancer by whole exome sequencing, array CGH and RNA transcript profiling and compared the genomic diversity within and between individuals. In contrast to substantial heterogeneity between men, there was limited diversity comparing metastases within an individual. Numbers of somatic mutations, the burden of genomic copy number alterations, and aberrations in known oncogenic drivers were highly concordant as were metrics of androgen receptor (AR) activity and cell cycle activity. AR activity inversely associated with cell proliferation, whereas the expression of Fanconi anemia (FA) complex genes correlated with elevated cell cycle progression, E2F1 expression and RB1 loss. Men with somatic aberrations in FA complex genes or ATM exhibited significantly longer treatment response durations to carboplatin compared to men without defects in genes encoding DNA repair proteins. Collectively, these data indicate that though exceptions exist, evaluating a single metastasis provides a reasonable assessment of the major oncogenic driver alterations present in disseminated tumors within an individual, and may be useful for selecting treatments based on predicted molecular vulnerabilities.

resulting in the suppression of AR target genes and clinical remissions that generally last several years (1). However, ADT is not curative. PC recurs as castration-resistant prostate cancer (CRPC), typically with reactivated AR signaling. Second-generation AR pathway inhibitors (ARIs), such as enzalutamide (ENZ) and abiraterone (ABI), were designed to further repress AR signaling and are primarily used to treat CRPC. Although these agents extend survival, durable complete responses are rare and these therapies also eventually fail (2, 3). Typically, the vast majority of metastatic CRPC (mCRPC) tumors progress with rising prostate-specific antigen (PSA/KLK3) levels despite standard of care treatment. Moreover, most mCRPC tumors are adenocarcinomas, which have robust AR program activity (4). Though rigorous epidemiological data are lacking, recent studies report that a substantial number of mCRPC tumors progressing on ARIs have lost AR signaling (5). Paralleling increased use of ARIs has been an increase in the proportion of treatment-resistant CRPC metastases that have AR-null phenotypes, i.e. tumors with diffuse small cell or neuroendocrine (NE) characteristics (SCNPC) or the recently described double-negative (DNPC) phenotype that lacks both NE and AR activity (5). A contemporary study evaluating the histology and molecular characteristics of 202 men with mCRPC found that 17% of the evaluable tumors were classified as SCNPC and this phenotype was associated with short-Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease with diverse drivers of disease progression and mechanisms of therapeutic resistance. We conducted deep phenotypic characterization of CRPC metastases and patient-derived xenograft (PDX) lines using whole-genome RNA sequencing, gene set enrichment analysis, and immunohistochemistry. Our analyses revealed 5 mCRPC phenotypes based on the expression of well-characterized androgen receptor (AR) or neuroendocrine (NE) genes: AR-high tumors (ARPC), AR-low tumors (ARLPC), amphicrine tumors composed of cells coexpressing AR and NE genes (AMPC), double-negative tumors (i.e., AR-/NE-; DNPC), and tumors with small cell or NE gene expression without AR activity (SCNPC). RE1 silencing transcription factor (REST) activity, which suppresses NE gene expression, was lost in AMPC and SCNPC PDX models. However, knockdown of REST in cell lines revealed that attenuated REST activity drives the AMPC phenotype but is not sufficient for SCNPC conversion. We also identified a subtype of DNPC tumors with squamous differentiation and generated an encompassing 26-gene transcriptional signature that distinguished the 5 mCRPC phenotypes. Together, our data highlight the central role of AR and REST in classifying treatment-resistant mCRPC phenotypes. These molecular classifications could potentially guide future therapeutic studies and clinical trial design.

Background Metastatic prostate cancer is a common and lethal disease for which there are no therapies that produce cures or long-term durable remissions. Clinically relevant preclinical models are needed to increase our understanding of the biology of this malignancy and to evaluate new agents that might provide effective treatment. Our objective was to establish and characterize patient-derived xenografts (PDXs) from advanced prostate cancer (PC) to investigation of biology and evaluation of new treatment modalities. Methods Samples of advanced PC obtained from surgery or from metastases collected at time of death were implanted into immunocompromised mice to establish PDXs. Established LuCaP PDXs were propagated in vivo. Genomic, transcriptomic and STR profiles were generated. Responses to androgen deprivation and docetaxel in vivo were characterized. Results We established multiple PDXs (LuCaP series), which represent the major genomic and phenotypic features of the disease in humans, including amplification of androgen receptor, PTEN deletion, TP53 deletion and mutation, RB1 loss, and TMPRSS2-ERG rearrangements, SPOP mutation, hypermutation due to MSH2/MSH6 genomic aberrations, and BRCA2 loss. The PDX models also exhibited variation in intra-tumoral androgen levels. Our in vivo results show heterogeneity of response to androgen deprivation and docetaxel, standard therapies for advanced PC, similar to the responses of patients to these treatments. Conclusions The LuCaP PDX series reflects the diverse molecular composition of human castration-resistant PC and allows for hypothesis-driven cause-and-effect studies of mechanisms underlying treatment response and resistance.

Purpose The neuroendocrine (NE) phenotype is associated with the development of metastatic castration-resistant prostate cancer (CRPC). Our objective was to characterize the molecular features of the NE phenotype in CRPC. Experimental Design Expression of chromogranin A (CHGA), synaptophysin (SYP), androgen receptor (AR), and prostate-specific antigen (PSA) was analyzed by immunohistochemistry (IHC) in 155 CRPC metastases from 50 patients and in 24 LuCaP prostate cancer patient-derived xenografts (PDX). Seventy-one of 155 metastases and the 24 LuCaP xenograft lines were analyzed by whole genome microarrays. REST splicing was verified by PCR. Results Co-expression of CHGA and SYP in >30% of cells was observed in 22 of 155 metastases (9 patients); 11 of the 22 metastases were AR+/PSA+ (6 patients), 11/22 were AR−/PSA− (4 patients), and 4/24 LuCaP PDXs were AR−/PSA−. By IHC, of the 71 metastases analyzed by whole genome microarrays, 5 metastases were CHGA+/SYP+/AR− and 5 were CHGA+/SYP+/AR+. Only CHGA+/SYP+ metastases had a NE transcript signature. The neuronal transcriptional regulator SRRM4 transcript was associated with the NE signature in CHGA+/SYP+ metastases and all CHGA+/SYP+ LuCaP xenografts. Additionally, expression of SRRM4 in LuCaP NE xenografts correlated with a splice variant of REST that lacks the transcriptional repressor domain. Conclusions (a) metastatic NE status can be heterogeneous in the same patient, (b) the CRPC NE molecular phenotype can be defined by CHGA+/SYP+ dual positivity, (c) the NE phenotype is not necessarily associated with the loss of AR activity, and (d) the splicing of REST by SRRM4 could promote the NE phenotype in CRPC.