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Anaplastic lymphoma kinase (ALK) is a tyrosine kinase with genomic and expression changes in many solid tumors. ALK inhibition is the first-line therapy for lung cancers with ALK alterations, and an effective therapy in other tumor types, but has not been well-studied in prostate cancer. Here, we aim to delineate the role of ALK genomic and expression changes in primary and metastatic prostate cancer. We determined ALK expression by IHC and RNA sequencing, and genomic alterations by NGS. We assessed functional consequences of ALK overexpression and pharmacologic ALK inhibition by cell proliferation and cell viability assays. Among 372 primary prostate cancer cases, we identified one case with uniformly high ALK protein expression. Genomic analysis revealed a novel SLC45A3-ALK fusion which promoted oncogenesis in in vitro assays. We observed ALK protein expression in 5 of 52 (9%) of metastatic prostate cancer cases, of which 4 of 5 had neuroendocrine features. ALK-expressing neuroendocrine prostate cancer had a distinct transcriptional program, and earlier disease progression. An ALK-expressing neuroendocrine prostate cancer model was sensitive to pharmacologic ALK inhibition. In summary, we found that ALK overexpression is rare in primary prostate cancer, but more frequent in metastatic prostate cancers with neuroendocrine differentiation. Furthermore, ALK fusions similar to lung cancer are an occasional driver in prostate cancer. Our data suggest that ALK-directed therapies could be an option in selected patients with advanced prostate cancer. Significance: Anaplastic lymphoma kinase (ALK) is a validated drug target in cancer. Here we delineate the spectrum of ALK alterations in prostate cancer. We show that ALK overexpression is present in advanced prostate cancers, in particular in cases with features of neuroendocrine carcinoma. Furthermore, ALK expression is associated with responses to pharmacologic ALK inhibition. Our study demonstrates that ALK-directed therapies should be considered in selected prostate cancer cases.

Feature-region combination AUCs and benchmarking Sheet 1: Log2 fold-change, p-value, and q-value between ARPC and NEPC lines for central mean coverage in all queried (338) TFs (two tailed Mann-Whitney U test, Benjamini-Hochberg adjusted). Sheet 2: 100-fold cross-validation AUCs for all region and feature combinations subset by the ‘AR10’ overlapping features (see Methods). Sheet 3: 100-fold cross-validation AUCs for all region and feature combinations subset by the ‘Phenotype 47-defining’ overlapping features. Sheet 4: 100-fold cross-validation AUCs for all region and feature combinations (global). Sheet 5: Predictions scores, tumor fraction, depth of coverage, and subtype for benchmarking admixtures. Sheet 6: AUCs for unsupervised prediction of admixture subtypes grouped by tumor fraction and depth. Sheet 7: NEPC:ARPC ratio, tumor fraction, and ARPC and NEPC fraction predictions for mixed phenotype admixtures using Keraon (see Methods).

Supplementary Figures S1-S18 and Supplementary Table legends.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase with genomic and expression changes in many solid tumors. ALK inhibition is the first-line therapy for lung cancers with ALK alterations, and an effective therapy in other tumor types, but has not been well-studied in prostate cancer. Here, we aim to delineate the role of ALK genomic and expression changes in primary and metastatic prostate cancer. We determined ALK expression by IHC and RNA sequencing, and genomic alterations by NGS. We assessed functional consequences of ALK overexpression and pharmacologic ALK inhibition by cell proliferation and cell viability assays. Among 372 primary prostate cancer cases, we identified one case with uniformly high ALK protein expression. Genomic analysis revealed a novel SLC45A3-ALK fusion which promoted oncogenesis in in vitro assays. We observed ALK protein expression in 5 of 52 (9%) of metastatic prostate cancer cases, of which 4 of 5 had neuroendocrine features. ALK-expressing neuroendocrine prostate cancer had a distinct transcriptional program, and earlier disease progression. An ALK-expressing neuroendocrine prostate cancer model was sensitive to pharmacologic ALK inhibition. In summary, we found that ALK overexpression is rare in primary prostate cancer, but more frequent in metastatic prostate cancers with neuroendocrine differentiation. Furthermore, ALK fusions similar to lung cancer are an occasional driver in prostate cancer. Our data suggest that ALK-directed therapies could be an option in selected patients with advanced prostate cancer.Significance:Anaplastic lymphoma kinase (ALK) is a validated drug target in cancer. Here we delineate the spectrum of ALK alterations in prostate cancer. We show that ALK overexpression is present in advanced prostate cancers, in particular in cases with features of neuroendocrine carcinoma. Furthermore, ALK expression is associated with responses to pharmacologic ALK inhibition. Our study demonstrates that ALK-directed therapies should be considered in selected prostate cancer cases.

Supplementary Figures 1-19, Tables 1-2

Supplementary Methods from The Androgen-Regulated Protease TMPRSS2 Activates a Proteolytic Cascade Involving Components of the Tumor Microenvironment and Promotes Prostate Cancer Metastasis

Supplementary Figure S1. TMPRSS2 expression is associated with androgen receptor (AR) levels and is regulated by androgens in vivo. Supplementary Figure S2. Characterization of the differentiation status, Tmprss2 and AR expression in Tmprss2+/+ TRAMP and Tmprss2 -/- TRAMP mice. Supplementary Figure S3. Suppression of TMPRSS2 inhibits cell proliferation and invasion. Supplementary Figure S4. TMPRSS2 influences tumor cell clearance and growth in metastatic sites. Supplementary Figure S5. Comparison of cleavage specificities for type II transmembrane serine proteases expressed in prostate epithelium. Supplementary Figure S6. Identification of putative TMPRSS2 substrates based on peptide cleavage specificities determined by PS-SCL. Supplementary Figure S7. Assessment of TMPRSS2 substrate activity. Supplementary Figure S8. Characterization of TMPRSS2 chemical inhibitors. Supplementary Figure S9. Bromhexine suppresses prostate cancer cell invasion but but does not influence proliferation in vitro.

Figure S2. Analyses of BCL2 family members in prostate cancer.

Supplementary Figure S3 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

A cluster analysis of adenocarcinoma and neuronal CRPC metastases (n=71). The CHGA+/SYP+ sites cluster together (highlighted in orange (AR+) and yellow (AR-)). Genes between CHGA- vs. CHGA+/SYP+ were compared and considered differentially expressed when SAM score was >3 or

AgilentTM oligo array expression analysis of thirty-seven genes with >2.5-fold differential expression common to 5 CHGA+/SYP+/AR- and 5 CHGA+/SYP+/AR+ CRPC metastases when compared to 61 CHGA- or CHGA+/SYP- CRPC metastases. Mean-centered ratios of genes are colored according to scale (Supplemental Figure 2).

Supplementary Data from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Supplementary Table S2 S4 S6 S7 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

(A) Hypothetical interactions between NE-associated transcription factors upregulated in CHGA+/SYP+/AR- tumors that promote the AR- NE phenotype. (B) A Venn diagram comparing genes with a SAM score >2.5 between CHGA- and CHGA+/SYP- metastases and CHGA+/SYP+/AR- metastases (598 genes) or CHGA+/SYP+/AR+ metastases (61 genes). Thirty-seven genes with a SAM score >2.5 were common to CHGA+/SYP+/AR- and CHGA+/SYP+/AR+ metastases.

Supplementary Table S3 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Purpose: The neuroendocrine phenotype is associated with the development of metastatic castration-resistant prostate cancer (CRPC). Our objective was to characterize the molecular features of the neuroendocrine phenotype in CRPC.Experimental Design: Expression of chromogranin A (CHGA), synaptophysin (SYP), androgen receptor (AR), and prostate-specific antigen (PSA) was analyzed by 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: Coexpression 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 neuroendocrine transcript signature. The neuronal transcriptional regulator SRRM4 transcript was associated with the neuroendocrine signature in CHGA+/SYP+ metastases and all CHGA+/SYP+ LuCaP xenografts. In addition, expression of SRRM4 in LuCaP neuroendocrine xenografts correlated with a splice variant of REST that lacks the transcriptional repressor domain.Conclusions: (i) Metastatic neuroendocrine status can be heterogeneous in the same patient, (ii) the CRPC neuroendocrine molecular phenotype can be defined by CHGA+/SYP+ dual positivity, (iii) the neuroendocrine phenotype is not necessarily associated with the loss of AR activity, and (iv) the splicing of REST by SRRM4 could promote the neuroendocrine phenotype in CRPC. Clin Cancer Res; 21(20); 4698–708. ©2015 AACR.

Purpose:Lineage plasticity in prostate cancer—most commonly exemplified by loss of androgen receptor (AR) signaling and a switch from a luminal to alternate differentiation program—is now recognized as a treatment resistance mechanism. Lineage plasticity is a spectrum, but neuroendocrine prostate cancer (NEPC) is the most virulent example. Currently, there are limited treatments for NEPC. Moreover, the incidence of treatment-emergent NEPC (t-NEPC) is increasing in the era of novel AR inhibitors. In contradistinction to de novo NEPC, t-NEPC tumors often express the AR, but AR's functional role in t-NEPC is unknown. Furthermore, targetable factors that promote t-NEPC lineage plasticity are also unclear.Experimental Design:Using an integrative systems biology approach, we investigated enzalutamide-resistant t-NEPC cell lines and their parental, enzalutamide-sensitive adenocarcinoma cell lines. The AR is still expressed in these t-NEPC cells, enabling us to determine the role of the AR and other key factors in regulating t-NEPC lineage plasticity.Results:AR inhibition accentuates lineage plasticity in t-NEPC cells—an effect not observed in parental, enzalutamide-sensitive adenocarcinoma cells. Induction of an AR-repressed, lineage plasticity program is dependent on activation of the transcription factor E2F1 in concert with the BET bromodomain chromatin reader BRD4. BET inhibition (BETi) blocks this E2F1/BRD4-regulated program and decreases growth of t-NEPC tumor models and a subset of t-NEPC patient tumors with high activity of this program in a BETi clinical trial.Conclusions:E2F1 and BRD4 are critical for activating an AR-repressed, t-NEPC lineage plasticity program. BETi is a promising approach to block this program.

(A) Representative examples of marker response by IHC for CHGA, SYP, and AR in CRPC bone metastases. 05-114: CHGA+/SYP+/AR-, 05-221: CHGA+/SYP+/AR- (white arrow), CHGA-/SYP-AR+ (black arrow). 07-042: CHGA+/SYP+AR+. Percent of (B) CHGA and (C) SYP positive cells in 22 CRPC CHGA+/SYP+ metastases. Virtually all cells in CHGA+/SYP+ metastases had a neuroendocrine phenotype.

(A) IHC and AgilentTM oligo array expression analysis of prostate and neuroendocrine-associated genes in 24 LuCaP xenograft lines. Mean-centered ratios of genes are colored according to scale (Supplemental Figure 2). LuCaP 49, 93, 145.1 and 145.2 are characterized as neuronal lines while the other 20 lines are adenocarcinoma. Representative IHC images are shown in B.

Supplementary Table S8-S12 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Purpose:Small-cell neuroendocrine prostate cancer (SCNPC) exhibits an aggressive clinical course and incidence rates seem to be increasing following resistance to potent androgen receptor (AR) antagonists. Currently, treatment options are limited and few model systems are available to identify new approaches for treatment. We sought to evaluate commonalities between SCNPC and other aggressive neuroendocrine carcinomas to identify therapeutic targets.Experimental Design:We generated whole transcriptome RNA-sequencing data from AR-active prostate cancers (ARPCs) and SCNPCs from tumors collected at rapid autopsy and two other neuroendocrine carcinomas, Merkel cell carcinoma (MCC), and small-cell lung cancer. We performed cross-tumor comparisons to identify conserved patterns of expression of druggable targets. We tested inhibitors to highly upregulated drug targets in a panel of prostate cancer cell lines and in vivo patient-derived xenograft (PDX) models.Results:We identified BCL2 as highly upregulated in SCNPC compared with ARPC. Inhibitors targeting BCL2 induced apoptotic cell death in SCNPC cell lines at nanomolar concentrations while ARPC cell lines were resistant. Treatment with the BCL2 inhibitor navitoclax leads to a reduction of growth of SCNPC PDX tumors in vivo, whereas ARPC PDX models were more resistant. We identified Wee1 as a second druggable target upregulated in SCNPC. Treatment with the combination of navitoclax and the Wee1 inhibitor AZD-1775 repressed the growth of SCNPC PDX resistant to single-agent BCL2 inhibitors.Conclusions:The combination of BCL2 and Wee1 inhibition presents a novel therapeutic strategy for the treatment of SCNPC.

Supplemental tables 1-5

Advanced prostate cancers comprise distinct phenotypes, but tumor classification remains clinically challenging. Here, we harnessed circulating tumor DNA (ctDNA) to study tumor phenotypes by ascertaining nucleosome positioning patterns associated with transcription regulation. We sequenced plasma ctDNA whole genomes from patient-derived xenografts representing a spectrum of androgen receptor active (ARPC) and neuroendocrine (NEPC) prostate cancers. Nucleosome patterns associated with transcriptional activity were reflected in ctDNA at regions of genes, promoters, histone modifications, transcription factor binding, and accessible chromatin. We identified the activity of key phenotype-defining transcriptional regulators from ctDNA, including AR, ASCL1, HOXB13, HNF4G, and GATA2. To distinguish NEPC and ARPC in patient plasma samples, we developed prediction models that achieved accuracies of 97% for dominant phenotypes and 87% for mixed clinical phenotypes. Although phenotype classification is typically assessed by IHC or transcriptome profiling from tumor biopsies, we demonstrate that ctDNA provides comparable results with diagnostic advantages for precision oncology. Significance: This study provides insights into the dynamics of nucleosome positioning and gene regulation associated with cancer phenotypes that can be ascertained from ctDNA. New methods for classification in phenotype mixtures extend the utility of ctDNA beyond assessments of somatic DNA alterations with important implications for molecular classification and precision oncology. This article is highlighted in the In This Issue feature, p. 517

Purpose: Small-cell neuroendocrine prostate cancer (SCNPC) exhibits an aggressive clinical course and incidence rates seem to be increasing following resistance to potent androgen receptor (AR) antagonists. Currently, treatment options are limited and few model systems are available to identify new approaches for treatment. We sought to evaluate commonalities between SCNPC and other aggressive neuroendocrine carcinomas to identify therapeutic targets. Experimental Design: We generated whole transcriptome RNA-sequencing data from AR-active prostate cancers (ARPCs) and SCNPCs from tumors collected at rapid autopsy and two other neuroendocrine carcinomas, Merkel cell carcinoma (MCC), and small-cell lung cancer. We performed cross-tumor comparisons to identify conserved patterns of expression of druggable targets. We tested inhibitors to highly upregulated drug targets in a panel of prostate cancer cell lines and in vivo patient-derived xenograft (PDX) models. Results: We identified BCL2 as highly upregulated in SCNPC compared with ARPC. Inhibitors targeting BCL2 induced apoptotic cell death in SCNPC cell lines at nanomolar concentrations while ARPC cell lines were resistant. Treatment with the BCL2 inhibitor navitoclax leads to a reduction of growth of SCNPC PDX tumors in vivo, whereas ARPC PDX models were more resistant. We identified Wee1 as a second druggable target upregulated in SCNPC. Treatment with the combination of navitoclax and the Wee1 inhibitor AZD-1775 repressed the growth of SCNPC PDX resistant to single-agent BCL2 inhibitors. Conclusions: The combination of BCL2 and Wee1 inhibition presents a novel therapeutic strategy for the treatment of SCNPC.

Purpose: Lineage plasticity in prostate cancer—most commonly exemplified by loss of androgen receptor (AR) signaling and a switch from a luminal to alternate differentiation program—is now recognized as a treatment resistance mechanism. Lineage plasticity is a spectrum, but neuroendocrine prostate cancer (NEPC) is the most virulent example. Currently, there are limited treatments for NEPC. Moreover, the incidence of treatment-emergent NEPC (t-NEPC) is increasing in the era of novel AR inhibitors. In contradistinction to de novo NEPC, t-NEPC tumors often express the AR, but AR's functional role in t-NEPC is unknown. Furthermore, targetable factors that promote t-NEPC lineage plasticity are also unclear. Experimental Design: Using an integrative systems biology approach, we investigated enzalutamide-resistant t-NEPC cell lines and their parental, enzalutamide-sensitive adenocarcinoma cell lines. The AR is still expressed in these t-NEPC cells, enabling us to determine the role of the AR and other key factors in regulating t-NEPC lineage plasticity. Results: AR inhibition accentuates lineage plasticity in t-NEPC cells—an effect not observed in parental, enzalutamide-sensitive adenocarcinoma cells. Induction of an AR-repressed, lineage plasticity program is dependent on activation of the transcription factor E2F1 in concert with the BET bromodomain chromatin reader BRD4. BET inhibition (BETi) blocks this E2F1/BRD4-regulated program and decreases growth of t-NEPC tumor models and a subset of t-NEPC patient tumors with high activity of this program in a BETi clinical trial. Conclusions: E2F1 and BRD4 are critical for activating an AR-repressed, t-NEPC lineage plasticity program. BETi is a promising approach to block this program.

Clinical trials of high-dose androgen (HDA) therapy for prostate cancer (PC) have shown promising efficacy but are limited by lack of criteria to identify likely responders. To elucidate factors that govern the growth-repressive effects of HDAs, we applied an unbiased integrative approach using genetic screens and transcriptional profiling of PC cells with or without demonstrated phenotypic sensitivity to androgen-mediated growth repression. Through this comprehensive analysis, we identified genetic events and related signaling networks that determine the response to both HDA and androgen withdrawal. We applied these findings to develop a gene signature that may serve as an early indicator of treatment response and identify men with tumors that are amenable to HDA therapy.

Prostate cancer (PC) initially depends on androgen receptor (AR) signaling for survival and growth. Therapeutics designed to suppress AR activity serve as the primary intervention for advanced disease. However, supraphysiological androgen (SPA) concentrations can produce paradoxical responses leading to PC growth inhibition. We sought to discern the mechanisms by which SPA inhibits PC and to determine if molecular context associates with antitumor activity. SPA produced an AR-mediated, dose-dependent induction of DNA double-strand breaks, G(0)/G(1) cell-cycle arrest, and cellular senescence. SPA repressed genes involved in DNA repair and delayed the restoration of damaged DNA, which was augmented by poly (ADP-ribose) polymerase 1 inhibition. SPA-induced double-strand breaks were accentuated in BRCA2-deficient patients with PC, and combining SPA with poly (ADP-ribose) polymerase or DNA-dependent protein kinase inhibition further repressed growth. Next-generation sequencing was performed on biospecimens from patients with PC receiving SPA as part of ongoing phase II clinical trials. Patients with mutations in genes mediating homology-directed DNA repair were more likely to exhibit clinical responses to SPA. These results provide a mechanistic rationale for directing SPA therapy to patients with PC who have AR amplification or DNA repair deficiency and for combining SPA therapy with poly (ADP-ribose) polymerase inhibition.

BACKGROUND: Testosterone is a driver of prostate cancer (PC) growth via ligand-mediated activation of the androgen receptor (AR). Tumors that have escaped systemic androgen deprivation, castration resistant prostate cancers (CRPC), have measurable intratumoral levels of testosterone, suggesting that a resistance mechanism still depends on androgen-simulated growth. However, AR activation requires an optimal intracellular concentration of androgens, a situation challenged by low circulating testosterone concentrations. Notably, PC cells may optimize their androgen levels by regulating the expression of steroid metabolism enzymes that convert androgen precursors into androgens. Here we propose that testosterone entry into the cell could be another control point. METHODS: To determine whether testosterone enters cells via a transporter, we performed in vitro (3)H-testosterone uptake assays in androgen-dependent LNCaP and androgen and AR-independent PC3 cells. To determine if the uptake mechanism depended on a concentration gradient, we modified UGT2B17 levels in LNCaP cells and measured androgen levels by LLE-MS (liquid-liquid extraction mass spectrometry). We also analyzed CRPC metastases for expression of AKR1C3 to determine whether this enzyme that converts adrenal androgens to testosterone was present in the tumor stroma (microenvironment) in addition to its expression in the tumor epithelium. RESULTS: Testosterone uptake followed a concentration gradient but unlike in passive diffusion, was saturable and temperature-dependent, thus suggesting facilitated transport. Suppression of UGT2B17 to abrogate a testosterone gradient reduced testosterone transport while overexpression of the enzyme enhanced it. The facilitated transport suggests a paracrine route of testosterone uptake for maintaining optimal intracellular levels. We found that AKR1C3 was expressed in the tumor microenvironment of CRPC metastases in addition to epithelial cells and the pattern of relative abundance of the enzyme in epithelium versus stroma varied substantially between the metastatic sites. CONCLUSIONS: Our findings suggest that in addition to testosterone transport and metabolism by tumor epithelium, testosterone could also be produced by components of the tumor microenvironment. Facilitated testosterone uptake by tumor cells supports a cell non-autonomous mechanism for testosterone signaling in CRPC.

Epidemiologic and in vitro studies suggest that SLCO-encoded organic anion transporting polypeptide (OATP) transporters influence the response of prostate cancer (PCa) to androgen deprivation by altering intratumor androgens. We have previously shown that castration-resistant metastases express multiple SLCO transporters at significantly higher levels than primary PCa, suggesting that OATP-mediated steroid transport is biologically relevant in advanced disease. However, whether OATP-mediated steroid transport can actually modify prostate tumor androgen levels in vivo has never been demonstrated. We sought to determine whether OATP-mediated steroid transport can measurably alter PCa androgen levels in vivo. We evaluated the uptake of dehydroepiandrosterone (DHEAS), E1S and testosterone in LNCaP cells engineered to express OATP1B1, 1B3, 2B1 or 4A1. We measured the uptake via administration of tritiated steroids to castrate mice bearing vector control or OATP1B1-, 2B1- or 4A1-expressing xenografts. We treated tumor-bearing mice with DHEAS and testosterone at physiologically relevant levels and measured intratumor accumulation of administered steroids by mass spectrometry. OATP1B1- and 2B-expressing xenografts each showed a threefold increase in tritiated-DHEAS uptake vs vector controls (P=0.002 and P=0.036, respectively). At circulating DHEAS levels similar to those in abiraterone-treated men (~15 μg dl−1), OATP1B1- and 2B1-expressing xenografts showed a 3.9-fold (P=0.057) and 1.9-fold (P=0.048) increase in tumor accumulation of DHEAS and a 1.6-fold (P=0.057) and 2.7-fold (P=0.095) increase in DHEA, respectively. At the substantial circulating testosterone levels found in eugonadal men, a consistent effect of OATP1B1, 2B1 or 4A1 on testosterone uptake in vivo was not detected. OATP transporters measurably alter DHEAS uptake and intratumor androgen levels in prostate tumors in vivo, even at circulating androgen levels achieved in abiraterone-treated patients. These novel data emphasize the continued need to inhibit ligand-mediated androgen receptor signaling in PCa tumors, and support prospective evaluation of studies designed to test inhibition of OATP-mediated DHEAS uptake and utilization.

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.

Summary: Prostate cancers (PCs) with loss of the potent tumor suppressors TP53 and RB1 exhibit poor outcomes. TP53 and RB1 also influence cell plasticity and are frequently lost in PCs with neuroendocrine (NE) differentiation. Therapeutic strategies that address these aggressive variant PCs are urgently needed. Using deep genomic profiling of 410 metastatic biopsies, we determine the relationships between combined TP53 and RB1 loss and PC phenotypes. Notably, 40% of TP53/RB1-deficient tumors are classified as AR-active adenocarcinomas, indicating that NE differentiation is not an obligate consequence of TP53/RB1 inactivation. A gene expression signature reflecting TP53/RB1 loss is associated with diminished responses to AR antagonists and reduced survival. These tumors exhibit high proliferation rates and evidence of elevated DNA repair processes. While tumor cells lacking TP53/RB1 are highly resistant to all single-agent therapeutics tested, the combination of PARP and ATR inhibition is found to produce significant responses, reflecting a clinically exploitable vulnerability resulting from replication stress.

The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2) in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host. peerReviewed

Purpose: The neuroendocrine phenotype is associated with the development of metastatic castration-resistant prostate cancer (CRPC). Our objective was to characterize the molecular features of the neuroendocrine phenotype in CRPC. Experimental Design: Expression of chromogranin A (CHGA), synaptophysin (SYP), androgen receptor (AR), and prostate-specific antigen (PSA) was analyzed by 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: Coexpression 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 neuroendocrine transcript signature. The neuronal transcriptional regulator SRRM4 transcript was associated with the neuroendocrine signature in CHGA+/SYP+ metastases and all CHGA+/SYP+ LuCaP xenografts. In addition, expression of SRRM4 in LuCaP neuroendocrine xenografts correlated with a splice variant of REST that lacks the transcriptional repressor domain. Conclusions: (i) Metastatic neuroendocrine status can be heterogeneous in the same patient, (ii) the CRPC neuroendocrine molecular phenotype can be defined by CHGA+/SYP+ dual positivity, (iii) the neuroendocrine phenotype is not necessarily associated with the loss of AR activity, and (iv) the splicing of REST by SRRM4 could promote the neuroendocrine phenotype in CRPC. Clin Cancer Res; 21(20); 4698–708. ©2015 AACR.

SummaryThe significance of ERG in human prostate cancer is unclear because mouse prostate is resistant to ERG-mediated transformation. We determined that ERG activates the transcriptional program regulated by YAP1 of the Hippo signaling pathway and found that prostate-specific activation of either ERG or YAP1 in mice induces similar transcriptional changes and results in age-related prostate tumors. ERG binds to chromatin regions occupied by TEAD/YAP1 and transactivates Hippo target genes. In addition, in human luminal-type prostate cancer cells, ERG binds to the promoter of YAP1 and is necessary for YAP1 expression. These results provide direct genetic evidence of a causal role for ERG in prostate cancer and reveal a connection between ERG and the Hippo signaling pathway.

Though metastatic cancers often initially respond to genotoxic therapeutics, acquired resistance is common. In addition to cytotoxic effects on tumor cells, DNA damaging agents such as ionizing radiation and chemotherapy induce injury in benign cells of the tumor microenvironment resulting in the production of paracrine-acting factors capable of promoting tumor resistance phenotypes. In studies designed to characterize the responses of prostate and bone stromal cells to genotoxic stress, we found that transcripts encoding glial cell line-derived neurotrophic factor (GDNF) increased several fold following exposures to cytotoxic agents including radiation, the topoisomerase inhibitor mitoxantrone and the microtubule poison docetaxel. Fibroblast GDNF exerted paracrine effects toward prostate cancer cells resulting in enhanced tumor cell proliferation and invasion, and these effects were concordant with the expression of known GDNF receptors GFRA1 and RET. Exposure to GDNF also induced tumor cell resistance to mitoxantrone and docetaxel chemotherapy. Together, these findings support an important role for tumor microenvironment damage responses in modulating treatment resistance and identify the GDNF signaling pathway as a potential target for improving responses to conventional genotoxic therapeutics.

Apoptosis inhibitor of macrophage (AIM, encoded by cd5l) is a multi-functional circulating protein that has a beneficial role in the regulation of a broad range of diseases, some of which are ameliorated by AIM administration in mice. In blood, AIM is stabilized by association with IgM pentamers and maintains its high circulating levels. The mechanism regulating the excessive accumulation of blood AIM remains unknown, although it is important, since a constitutive increase in AIM levels promotes chronic inflammation. Here we found a physiological AIM-cleavage process that induces destabilization of AIM and its excretion in urine. In blood, IgM-free AIM appeared to be cleaved and reduced in size approximately 10 kDa. Cleaved AIM was unable to bind to IgM and was selectively filtered by the glomerulus, thereby excreted in urine. Amino acid substitution at the cleavage site resulted in no renal excretion of AIM. Interestingly, cleaved AIM retained a comparable potency with full-length AIM in facilitating the clearance of dead cell debris in injured kidney, which is a key response in the recovery of acute kidney injury. Identification of AIM-cleavage and resulting functional modification could be the basis for designing safe and efficient AIM therapy for various diseases.

BACKGROUND ETS-related gene (ERG) protein is present in 40–70% of prostate cancer and is correlated with TMPRSS2-ERG gene rearrangements. This study evaluated ERG expression at radical prostatectomy to determine whether it was predictive of earlier relapse or prostate cancer-specific mortality (PCSM).

Amine catabolism by monoamine oxidase A (MAOA) contributes to oxidative stress, which plays a role in prostate cancer (PCa) development and progression. An upstream variable-number tandem repeat (uVNTR) in the MAOA promoter influences gene expression and activity, and may thereby affect PCa susceptibility.Caucasian (n = 2,572) men from two population-based case-control studies of PCa were genotyped for the MAOA-VNTR. Logistic regression was used to assess PCa risk in relation to genotype.Common alleles of the MAOA-VNTR were not associated with the relative risk of PCa, nor did the relationship differ by clinical features of the disease. The rare 5-copy variant (frequency: 0.5% in cases; 1.8% in controls), however, was associated with a reduced PCa risk (odds ratio, OR = 0.30, 95% CI 0.13-0.71).A rare polymorphism of the MAOA promoter previously shown to confer low expression was associated with a reduced risk of developing PCa. This novel finding awaits confirmation in other study populations.

Purpose: Prostate cancer cells are dependent on AR for growth and survival, but recent reports have shown a paradoxical role of supraphysiologic androgen in growth inhibition, known as hormone interference. Androgen stimulation in AR-overexpressing prostate cancer cells leads to formation of double-strand breaks (DSBs). Here, we investigate the potential mechanism of high androgen induced DSBs and growth inhibition and its effect on survival following androgen stimulation in combination with DNA damage repair inhibitors or defects. Experimental Design: AR-dependent LNCaP, AR overexpressing LNCaP and PC3, AR-independent PC3, and APIPC were used for this study. High levels of androgen (R1881)/ DHT and olaparib-induced DNA damage were evaluated by gH2AX and 53BP1 foci formation after 6 or 24h. For cell death assays the drug was administered continuously for 3 days following the plating day. DNA PKcs activity was determined by Ser2056foci formation and dissociation from chromatin was measured by Thr2609 foci. AR activity or DNA binding was evaluated by Ser81 AR phosphorylation. An ex vivo PDX model (LuCaP 96CR) was utilized to mimic the BRCA2 defect condition. Results: We demonstrated that supraphysiologic androgen treatment on androgen-dependent and AR-overexpressing prostate cancer cell lines resulted in a dose-dependent induction of widespread DSBs, which was augmented after PARP inhibition (olaparib). AR expression correlated with persistent DNA damage as AR-independent cells lacked DSBs. High androgen treatment significantly induced caspase activity and reduced survival in AR-overexpressing cells. Hyperactivated DNA PKcs was retained on chromatin following high androgen exposure in these cells, leading to impaired dissociation of DNA PKcs from chromatin and inducing DNA damage. AR phosphorylation at Ser81 was also in concordance with hyperphosphorylated DNA PKcs, particularly in AR-overexpressing cells following high androgen and olaparib treatment. HR-deficient, BRCA2-null cells induced more DSBs after high androgen treatment with or without olaparib and were susceptible to DNA PKcs inhibition following androgen treatment. In contrast to this, AR-overexpressing cells did not exhibit any DNA damage following DNA PKcs inhibition after high androgen exposure. Ex vivo PDX models demonstrated a synergistic interaction between olaparib and high levels of androgen-induced DSB formation as well as hyperactive DNA PKcs. Conclusions: These results suggest a potential mechanism of supraphysiologic androgen stimulated AR-induced DNA damage and proliferation inhibition. AR binds to DNA PKcs following high androgen and until AR levels become saturated DNA PKcs and AR stay on the chromatin, which leads to inefficient DNA damage repair. HR-deficient cells were particularly susceptible to high androgen. Our data provide a mechanistic rationale for the response of AR upregulated cells as well as DNA repair deficient cells to supraphysiologic androgen therapy in combination with PARP inhibition. Citation Format: Payel Chatterjee, Jared M. Lucas, Michael D. Nyquist, Eva Corey, Peter S. Nelson. DNA damage echoes are key to prostate cancer susceptibility to supraphysiologic androgen levels [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B049.

Androgens and the androgen receptor (AR) influence prostate carcinogenesis. Lowering intraprostatic dihydrotestosterone (DHT) by inhibiting 5-alpha-reductase (SRD5A) reduces prostate cancer (PCa) incidence, but is not uniformly effective. Mechanisms by which SRD5A inhibition influences PCa initiation and/or progression among different individuals have not been established. We sought to identify molecular alterations underlying the differential chemo-preventive activity of SRD5A inhibition. Men with clinically-localized PCa were randomized to prostatectomy alone (n=25) or 4 months treatment with the SRD5A-inhibitor dutasteride (0.5mg (n=26) or 3.5mg (n=24)) preceding prostatectomy. Serum and prostate androgens were measured using mass spectrometry. We evaluated benign epithelial gene expression using expression profiling and immunohistochemistry, and characterized tumor TMPRSS2-ERG fusion status using FISH. Dutasteride at 0.5 or 3.5mg decreased prostatic DHT by 93% (0.23ng/g; p

TMPRSS2 is an androgen-regulated cell-surface serine protease expressed predominantly in prostate epithelium. TMPRSS2 is expressed highly in localized high-grade prostate cancers and in the majority of human prostate cancer metastases. Through the generation of mouse models with a targeted deletion of Tmprss2, we demonstrate that the activity of this protease regulates cancer cell invasion and metastasis to distant organs. By screening combinatorial peptide libraries, we identified a spectrum of TMPRSS2 substrates that include pro-hepatocyte growth factor (HGF). HGF activated by TMPRSS2 promoted c-MET receptor tyrosine kinase signaling, and initiated a proinvasive epithelial-to-mesenchymal transition phenotype. Chemical library screens identified a potent bioavailable TMPRSS2 inhibitor that suppressed prostate cancer metastasis in vivo. Together, these findings provide a mechanistic link between androgen-regulated signaling programs and prostate cancer metastasis that operate via context-dependent interactions with extracellular constituents of the tumor microenvironment. Significance: The vast majority of prostate cancer deaths are due to metastasis. Loss of TMPRSS2 activity dramatically attenuated the metastatic phenotype through mechanisms involving the HGF–c-MET axis. Therapeutic approaches directed toward inhibiting TMPRSS2 may reduce the incidence or progression of metastasis in patients with prostate cancer. Cancer Discov; 4(11); 1310–25. ©2014 AACR. See related commentary by Rubin, p. 1262 This article is highlighted in the In This Issue feature, p. 1243

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.

ETS-related gene (ERG) protein is present in 40-70% of prostate cancer and is correlated with TMPRSS2-ERG gene rearrangements. This study evaluated ERG expression at radical prostatectomy to determine whether it was predictive of earlier relapse or prostate cancer-specific mortality (PCSM).One hundred patients who underwent radical prostatectomy at Virginia Mason in Seattle between 1991 and 1997 were identified. Recurrence was confirmed by tissue diagnosis or radiographic signs. PCSM was confirmed by death certificates. Thirty-three patients with metastases or PCSM were matched to patients without recurrence at a 1:2 ratio. Paraffin embedded tissue was stained with two anti-ERG monoclonal antibodies, EPR3864 and 9FY. Nuclear expression intensity was evaluated as present/absent, on a 4-point relative intensity scale, and as a composite score (0-300).Mean follow-up was 10.26 years. The two antibodies were highly correlated (P 0.0001). Patients with higher ERG expression intensity and composite scores were significantly more likely to develop biochemical relapse, metastases, and PCSM. Kaplan-Meier survival curve analysis for the composite score of ERG expression revealed a significant association between higher ERG expression (EPR3864) and shorter PCa-specific survival (P = 0.047).While the presence of ERG expression at the time of surgery was not predictive of earlier relapse or PCSM, the relative intensity and composite score for ERG expression was prognostic for the development of biochemical relapse, metastases, and PCSM. Quantitative ERG scoring may be useful to identify patients who would benefit from adjuvant treatment or closer follow-up, allowing more accurate individual patient treatment plans.

The stalk, a characteristic structure of the large ribosomal subunit, is directly involved in the interaction with the soluble factors during translation. In the Mediterranean mussel Mytilus galloprovincialis, the stalk consists of one 32 kDa protein, MgP0, and two smaller, 12 kDa acidic proteins, MgP1 and MgP2, of pI 3.0 and 4.0, respectively, as revealed by analysis of purified ribosomes with electrophoresis and Western blot with a specific monoclonal antibody. Treatment of the ribosomes with alkaline phosphatase showed movement of the bands corresponding to the acidic MgP1 and MgP2 proteins to more basic pH after isoelectrofocusing, implying phosphorylation. The cDNA molecules of M. galloprovincialis ribosomal proteins MgP0, MgP1 and MgP2 and superoxide dismutase (MgSOD) were isolated from a cDNA library or constructed by RT-PCR, cloned in expression vectors and expressed in Escherichia coli. The recombinant proteins were purified with immobilized metal ion affinity chromatography (IMAC) and identified with immunoblotting. Exposure of mussels at cadmium and sorbitol and analysis of gill tissue extracts showed over expression of MgP0 protein.

A significant proportion of human prostate cancers carry a chromosomal rearrangement resulting in the overexpression of the ETS transcription factor, ERG; however, the functional significance of this event is poorly understood. We report here that up-regulation of ERG transcript is sufficient for the initiation of prostate neoplasia. In agreement with measurements of ERG transcripts, we found that ERG protein is expressed in neoplastic human prostate epithelium. Overexpression of ERG in prostate cell lines increased cell invasion. Moreover, targeted expression of this transcript in vivo in luminal prostate epithelial cells of transgenic mice results in initiation of prostate neoplasia observed as the development of focal precancerous prostatic intraepithelial neoplasia (PIN). Similar to human cancers, luminal epithelial cells in these PIN lesions displace diminishing in numbers basal epithelial cells and establish direct contact with the stromal cell compartment. Loss of basal cells is considered to be one of the critical hallmarks of human prostate cancer; however, the mechanisms responsible for this event were unknown. We propose that up-regulation of ERG in human prostate cancer activates cell invasion programs that subsequently displace basal cells by neoplastic epithelium. Our data demonstrate that ERG plays an important causal role in the transformation of prostate epithelium and should be considered as a target for prevention or early therapeutic intervention.

Hepatocyte activator inhibitor-1 (HAI-1) is a transmembrane serine protease inhibitor that regulates the conversion of latent to active hepatocyte growth factor (HGF). Studies supporting a role for the HGF pathway in prostate carcinogenesis prompted an analysis of HAI-1 expression in the prostate. Here we analyze the regulation of HAI-1 expression by androgen, oncogenic transformation, and cancer progression. Immunohistochemical analysis revealed that HAI-1 expression was restricted to prostate epithelium, where staining occurred primarily in basal and atrophic luminal epithelial cells. Compared to normal glands, HAI-1 expression was significantly increased in localized prostate cancer and was present in most prostate cancer metastases. HAI-1 protein expression levels were sensitive to androgen in normal epithelium but not in cancer. Although androgen did not increase HAI-1 protein expression levels in LNCaP cells, it decreased HAI-1 surface expression, consistent with previous data from our group (Martin DB, Gifford DR, Wright ME, Keller A, Yi E, Goodlett DR, Aebersold R, Nelson PS: Quantitative proteomic analysis of proteins released by neoplastic prostate epithelium. Cancer Res 2004, 64:347–355). HAI-1 overexpression in cancer was predictive of prostate-specific antigen recurrence (relative risk, 1.24). These results suggest that HAI-1 regulates the HGF Met axis on prostate epithelial cells and influences HGF mediated tumor invasion and metastasis.

SUMMARYMarine mussels are renowned for their ability to produce an extra-organismic tendon-like structure that can withstand the wave forces associated with the intertidal habitat. Initial characterization of byssal properties has focused on Mytilus edulis, with few detailed comparisons with other mussels. M. galloprovincialis, a closely related species, provides an opportunity for a thorough comparison. Three full-length cDNA clones encoding the byssal collagens, precollagen D(preCol-D), preCol-NG and preCol-P, were isolated from M. galloprovincialis. Comparisons with M. edulis preCol-D,preCol-NG and preCol-P reveal a 91.3 %, 88.6 % and 90.1 % identity with the cDNA and an 89.0 %, 88.1 % and 89.0 % identity with the deduced protein sequences, respectively. Key elements are maintained between the species: in particular, modeled bends in the collagen helix due to breaks in the Gly-X-Y pattern and the location of cysteine and putative 3,4-dihydroxyphenylalanine (DOPA) residues. A potentially important difference between the two is that, in all cases, M. galloprovincialis byssal collagens contain additional histidine residues in their flanking domains. The significance of this may lie in the ability of M. galloprovincialisto utilize more metal chelate cross-links, which have been implicated in byssal thread stability.M. edulis threads are typically twice the length and diameter of M. galloprovincialis threads and appear to contain nearly 10 % more collagen. These differences are maintained even when the different thread portions are compared. Despite differences in a number of parameters, most notably that whole M. galloprovincialis threads are stiffer, threads whether whole or separated into proximal and distal portions, have similar mechanical behaviors. It is apparent from this comparison that M. galloprovincialis and M. edulis are seemingly interchangeable models for byssal research.