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.

Molecularly targeted therapies for advanced prostate cancer include castration modalities that suppress ligand-dependent transcriptional activity of the androgen receptor (AR). However, persistent AR signalling undermines therapeutic efficacy and promotes progression to lethal castration-resistant prostate cancer (CRPC), even when patients are treated with potent second-generation AR-targeted therapies abiraterone and enzalutamide. Here we define diverse AR genomic structural rearrangements (AR-GSRs) as a class of molecular alterations occurring in one third of CRPC-stage tumours. AR-GSRs occur in the context of copy-neutral and amplified AR and display heterogeneity in breakpoint location, rearrangement class and sub-clonal enrichment in tumours within and between patients. Despite this heterogeneity, one common outcome in tumours with high sub-clonal enrichment of AR-GSRs is outlier expression of diverse AR variant species lacking the ligand-binding domain and possessing ligand-independent transcriptional activity. Collectively, these findings reveal AR-GSRs as important drivers of persistent AR signalling in CRPC.

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.

Cancer dormancy refers to the prolonged clinical disease-free time between removal of the primary tumor and recurrence, which is common in prostate cancer (PCa), breast cancer, esophageal cancer, and other cancers. PCa disseminated tumor cells (DTC) are detected in both patients with no evidence of disease (NED) and advanced disease (ADV). However, the molecular and cellular nature of DTC is unknown. We performed a first-in-field study of single DTC transcriptomic analyses in cancer patients to identify a molecular signature associated with cancer dormancy. We profiled eighty-five individual EpCAM+/CD45− cells from the bone marrow of PCa patients with NED or ADV. We analyzed 44 DTC with high prostate-epithelial signatures, and eliminated 41 cells with high erythroid signatures and low prostate epithelial signatures. DTC were clustered into 3 groups: NED, ADV_1, and ADV_2, in which the ADV_1 group presented a distinct gene expression pattern associated with the p38 stress activated kinase pathway. Additionally, DTC from the NED group were enriched for a tumor dormancy signature associated with head and neck squamous carcinoma and breast cancer. This study provides the first clinical evidence of the p38 pathway as a potential biomarker for early recurrence and an attractive target for therapeutic intervention.