Search

Abstract N-Myc is a key driver of neuroblastoma and neuroendocrine prostate cancer (NEPC). One potential way to circumvent the challenge of undruggable N-Myc is to target the protein homeostasis (proteostasis) system that maintains N-Myc levels. Here, we identify heat shock protein 70 (HSP70) as a top partner of N-Myc, which binds a conserved “SELILKR” motif and prevents the access of E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), possibly through steric hindrance. When HSP70’s dwell time on N-Myc is increased by treatment with the HSP70 allosteric inhibitor, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites and forms polyubiquitination chains linked by the K11 and K63 sites. Notably, HSP70 inhibition significantly suppressed NEPC tumor growth, increased the efficacy of aurora kinase A (AURKA) inhibitors, and limited the expression of neuroendocrine-related pathways.

Abstract Recent advancements in single-cell RNA sequencing (scRNAseq) have facilitated the discovery of previously unrecognized subtypes within prostate cancer (PCa), offering new insights into cancer heterogeneity and progression. In this study, we integrated scRNAseq data from multiple studies, comprising publicly available cohorts and data generated by our research team, and established the Human Prostate Single cell Atlas (HuPSA) and Mouse Prostate Single cell Atlas (MoPSA) datasets. Through comprehensive analysis, we identified two novel double-negative PCa populations: KRT7 cells characterized by elevated KRT7 expression and progenitor-like cells marked by SOX2 and FOXA2 expression, distinct from NEPCa, and displaying stem/progenitor features. Furthermore, HuPSA-based deconvolution re-classified human PCa specimens, validating the presence of these novel subtypes. We then developed a user-friendly web application, “HuPSA–MoPSA” ( https://pcatools.shinyapps.io/HuPSA-MoPSA/ ), for visualizing gene expression across all newly established datasets. Our study provides comprehensive tools for PCa research and uncovers novel cancer subtypes that can inform clinical diagnosis and treatment strategies.

Abstract While better management of loco-regional prostate cancer (PC) has greatly improved survival, advanced PC remains a major cause of cancer deaths. Identification of novel targetable pathways that contribute to tumor progression in PC could open new therapeutic options. The di-ganglioside GD2 is a target of FDA-approved antibody therapies in neuroblastoma, but the role of GD2 in PC is unexplored. Here, we show that GD2 is expressed in a small subpopulation of PC cells in a subset of patients and a higher proportion of metastatic tumors. Variable levels of cell surface GD2 expression were seen on many PC cell lines, and the expression was highly upregulated by experimental induction of lineage progression or enzalutamide resistance in CRPC cell models. GD2high cell fraction was enriched upon growth of PC cells as tumorspheres and GD2high fraction was enriched in tumorsphere-forming ability. CRISPR-Cas9 knockout (KO) of the rate-limiting GD2 biosynthetic enzyme GD3 Synthase (GD3S) in GD2high CRPC cell models markedly impaired the in vitro oncogenic traits and growth as bone-implanted xenograft tumors and reduced the cancer stem cell and epithelial-mesenchymal transition marker expression. Our results support the potential role of GD3S and its product GD2 in promoting PC tumorigenesis by maintaining cancer stem cells and suggest the potential for GD2 targeting in advanced PC.

Abstract Therapeutic approaches targeting proteins on the surface of cancer cells have emerged as an important strategy for precision oncology. To capitalize on the potential impact of drugs targeting surface proteins, detailed knowledge about the expression patterns of the target proteins in tumor tissues is required. In castration-resistant prostate cancer (CRPC), agents targeting prostate-specific membrane antigen (PSMA) have demonstrated clinical activity. However, PSMA expression is lost in a significant number of CRPC tumors. The identification of additional cell surface targets is necessary to develop new therapeutic approaches. Here, we performed a comprehensive analysis of the expression heterogeneity and co-expression patterns of trophoblast cell-surface antigen 2 (TROP2), delta-like ligand 3 (DLL3), and carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in CRPC samples from a rapid autopsy cohort. We show that DLL3 and CEACAM5 exhibit the highest expression in neuroendocrine prostate cancer (NEPC), while TROP2 is expressed across different CRPC molecular subtypes, except for NEPC. We further demonstrated that AR alterations were associated with higher expression of PSMA and TROP2. Conversely, PSMA and TROP2 expression was lower in RB1-altered tumors. In addition to genomic alterations, we show a tight correlation between epigenetic states, particularly histone H3 lysine 27 methylation (H3K27me3) at the transcriptional start site and gene body of TACSTD2 (encoding TROP2), DLL3, and CEACAM5, and their respective protein expression in CRPC patient-derived xenografts. Collectively, these findings provide insights into patterns and determinants of expression of TROP2, DLL3, and CEACAM5 with implications for the clinical development of cell surface targeting agents in CRPC.

Prostate cancer is a frequent malignancy in older men and has a very high 5‐year survival rate if diagnosed early. The prognosis is much less promising if the tumor has already spread outside the prostate gland. Targeted treatments mainly aim at blocking androgen receptor (AR) signaling and initially show good efficacy. However, tumor progression due to AR‐dependent and AR‐independent mechanisms is often observed after some time, and novel treatment strategies are urgently needed. Dysregulation of the PI3K/AKT/mTOR pathway in advanced prostate cancer and its implication in treatment resistance has been reported. We compared the impact of PI3K/AKT/mTOR pathway inhibitors with different selectivity profiles on in vitro cell proliferation and on caspase 3/7 activation as a marker for apoptosis induction, and observed the strongest effects in the androgen‐sensitive prostate cancer cell lines VCaP and LNCaP. Combination treatment with the AR inhibitor darolutamide led to enhanced apoptosis in these cell lines, the effects being most pronounced upon cotreatment with the pan‐PI3K inhibitor copanlisib. A subsequent transcriptomic analysis performed in VCaP cells revealed that combining darolutamide with copanlisib impacted gene expression much more than individual treatment. A comprehensive reversal of the androgen response and the mTORC1 transcriptional programs as well as a marked induction of DNA damage was observed. Next, an in vivo efficacy study was performed using the androgen‐sensitive patient‐derived prostate cancer (PDX) model LuCaP 35 and a superior efficacy was observed after the combined treatment with copanlisib and darolutamide. Importantly, immunohistochemistry analysis of these treated tumors showed increased apoptosis, as revealed by elevated levels of cleaved caspase 3 and Bcl‐2‐binding component 3 (BBC3). In conclusion, these data demonstrate that concurrent blockade of the PI3K/AKT/mTOR and AR pathways has superior antitumor efficacy and induces apoptosis in androgen‐sensitive prostate cancer cell lines and PDX models.

The αVβ6 integrin, an epithelial-specific cell surface receptor absent in normal prostate and expressed during prostate cancer (PrCa) progression, is a therapeutic target in many cancers. Here, we report that transcript levels of ITGB6 (encoding the β6 integrin subunit) are significantly increased in metastatic castrate-resistant androgen receptor-negative prostate tumors compared to androgen receptor-positive prostate tumors. In addition, the αVβ6 integrin protein levels are significantly elevated in androgen receptor-negative PrCa patient derived xenografts (PDXs) compared to androgen receptor-positive PDXs. In vitro, the androgen receptor-negative PrCa cells express high levels of the αVβ6 integrin compared to androgen receptor-positive PrCa cells. Additionally, expression of androgen receptor (wild type or variant 7) in androgen receptor-negative PrCa cells downregulates the expression of the β6 but not αV subunit compared to control cells. We demonstrate an efficient strategy to therapeutically target the αVβ6 integrin during PrCa progression by using short interfering RNA (siRNA) loaded into PrCa cell-derived small extracellular vesicles (sEVs). We first demonstrate that fluorescently-labeled siRNAs can be efficiently loaded into PrCa cell-derived sEVs by electroporation. By confocal microscopy, we show efficient internalization of these siRNA-loaded sEVs into PrCa cells. We show that sEV-mediated delivery of ITGB6-targeting siRNAs into PC3 cells specifically downregulates expression of the β6 subunit. Furthermore, treatment with sEVs encapsulating ITGB6 siRNA significantly reduces cell adhesion and migration of PrCa cells on an αVβ6-specific substrate, LAP-TGFβ1. Our results demonstrate an approach for specific targeting of the αVβ6 integrin in PrCa cells using sEVs encapsulating ITGB6-specific siRNAs.

Abstract Androgen deprivation therapies aimed to target prostate cancer (PrCa) are only partially successful given the occurrence of neuroendocrine PrCa (NEPrCa), a highly aggressive and highly metastatic form of PrCa, for which there is no effective therapeutic approach. Our group has demonstrated that while absent in prostate adenocarcinoma, the αVβ3 integrin expression is increased during PrCa progression toward NEPrCa. Here, we show a novel pathway activated by αVβ3 that promotes NE differentiation (NED). This novel pathway requires the expression of a GPI-linked surface molecule, NgR2, also known as Nogo-66 receptor homolog 1. We show here that NgR2 is upregulated by αVβ3, to which it associates; we also show that it promotes NED and anchorage-independent growth, as well as a motile phenotype of PrCa cells. Given our observations that high levels of αVβ3 and, as shown here, of NgR2 are detected in human and mouse NEPrCa, our findings appear to be highly relevant to this aggressive and metastatic subtype of PrCa. This study is novel because NgR2 role has only minimally been investigated in cancer and has instead predominantly been analyzed in neurons. These data thus pave new avenues toward a comprehensive mechanistic understanding of integrin-directed signaling during PrCa progression toward a NE phenotype.

Ning et al. report expression of a neural lineage gene signature in enzalutamide-resistant prostate cancer cells and datasets from patients with neuroendocrine prostate cancer. One of these genes, ARHGEF2, is associated with poor overall and disease-free survival in patients with castration-resistant disease and supports cell viability in vitro.

CXCR7 is an atypical chemokine receptor that recruits β-arrestin (ARRB2) and internalizes into clathrin-coated intracellular vesicles where the complex acts as a scaffold for cytoplasmic kinase assembly and signal transduction. Here, we report that CXCR7 was elevated in the majority of prostate cancer (PCa) cases with neuroendocrine features (NEPC). CXCR7 markedly induced mitotic spindle and cell cycle gene expression. Mechanistically, we identified Aurora Kinase A (AURKA), a key regulator of mitosis, as a novel target that was bound and activated by the CXCR7-ARRB2 complex. CXCR7 interacted with proteins associated with microtubules and golgi, and, as such, the CXCR7-ARRB2-containing vesicles trafficked along the microtubules to the pericentrosomal golgi apparatus, where the complex interacted with AURKA. Accordingly, CXCR7 promoted PCa cell proliferation and tumor growth, which was mitigated by AURKA inhibition. In summary, our study reveals a critical role of CXCR7-ARRB2 in interacting and activating AURKA, which can be targeted by AURKA inhibitors to benefit a subset of patients with NEPC.

Summary: 3′ untranslated region (3′ UTR) somatic mutations represent a largely unexplored avenue of alternative oncogenic gene dysregulation. To determine the significance of 3′ UTR mutations in disease, we identify 3′ UTR somatic variants across 185 advanced prostate tumors, discovering 14,497 single-nucleotide mutations enriched in oncogenic pathways and 3′ UTR regulatory elements. By developing two complementary massively parallel reporter assays, we measure how thousands of patient-based mutations affect mRNA translation and stability and identify hundreds of functional variants that allow us to define determinants of mutation significance. We demonstrate the clinical relevance of these mutations, observing that CRISPR-Cas9 endogenous editing of distinct variants increases cellular stress resistance and that patients harboring oncogenic 3′ UTR mutations have a particularly poor prognosis. This work represents an expansive view of the extent to which disease-relevant 3′ UTR mutations affect mRNA stability, translation, and cancer progression, uncovering principles of regulatory functionality and potential therapeutic targets in previously unexplored regulatory regions.

Chondroitin sulfate (CS) is one of the most abundant glycosaminoglycans in prostate cancers. Here the authors show that inhibition of the androgen receptor pathway leads to the upregulation of CS, which promotes prostate cancer growth and metastasis.

Prostate-specific membrane antigen (PSMA) is an important cell surface target in prostate cancer. There are limited data on the heterogeneity of PSMA tissue expression in metastatic castration-resistant prostate cancer (mCRPC). Furthermore, the mechanisms regulating PSMA expression (encoded by the FOLH1 gene) are not well understood. Here, we demonstrate that PSMA expression is heterogeneous across different metastatic sites and molecular subtypes of mCRPC. In a rapid autopsy cohort in which multiple metastatic sites per patient were sampled, we found that 13 of 52 (25%) cases had no detectable PSMA and 23 of 52 (44%) cases showed heterogeneous PSMA expression across individual metastases, with 33 (63%) cases harboring at least 1 PSMA-negative site. PSMA-negative tumors displayed distinct transcriptional profiles with expression of druggable targets such as MUC1. Loss of PSMA was associated with epigenetic changes of the FOLH1 locus, including gain of CpG methylation and loss of histone 3 lysine 27 (H3K27) acetylation. Treatment with histone deacetylase (HDAC) inhibitors reversed this epigenetic repression and restored PSMA expression in vitro and in vivo. Collectively, these data provide insights into the expression patterns and regulation of PSMA in mCRPC and suggest that epigenetic therapies — in particular, HDAC inhibitors — can be used to augment PSMA levels.

Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer. NEPC is characterized by the loss of androgen receptor (AR) signaling and transdifferentiation toward small-cell neuroendocrine (SCN) phenotypes, which results in resistance to AR-targeted therapy. NEPC resembles other SCN carcinomas clinically, histologically and in gene expression. Here, we leveraged SCN phenotype scores of various cancer cell lines and gene depletion screens from the Cancer Dependency Map (DepMap) to identify vulnerabilities in NEPC. We discovered ZBTB7A, a transcription factor, as a candidate promoting the progression of NEPC. Cancer cells with high SCN phenotype scores showed a strong dependency on RET kinase activity with a high correlation between RET and ZBTB7A dependencies in these cells. Utilizing informatic modeling of whole transcriptome sequencing data from patient samples, we identified distinct gene networking patterns of ZBTB7A in NEPC versus prostate adenocarcinoma. Specifically, we observed a robust association of ZBTB7A with genes promoting cell cycle progression, including apoptosis regulating genes. Silencing ZBTB7A in a NEPC cell line confirmed the dependency on ZBTB7A for cell growth via suppression of the G1/S transition in the cell cycle and induction of apoptosis. Collectively, our results highlight the oncogenic function of ZBTB7A in NEPC and emphasize the value of ZBTB7A as a promising therapeutic strategy for targeting NEPC tumors.

The role of MYC in transcriptional reprogramming in prostate cancer remains poorly characterized. Here, MYC overexpression antagonizes the canonical AR transcriptional program leading to prostate tumor initiation and progression by disrupting transcriptional pause release at AR-regulated genes.

Transcriptional changes during prostate cancer progression are not yet fully understood. Here, the authors integrate a transcriptomics atlas of prostate cancer and validate it with preclinical models and single-cell RNA-seq, revealing the role of EZH2 and macrophage polarisation in tumour progression.

Neuroendocrine carcinomas (NECs) arise from different anatomic sites, but have similar histological and clinical features. Here, the authors show that the epigenetic landscape of a range of NECs converges towards a common epigenetic state, while distinct subtypes occur within neuroendocrine prostate cancer contributing to intratumor heterogeneity in clinical samples.

Mutations in 5’ untranslated regions (UTRs) have a functional role in gene expression in cancer. Here, the authors develop a sequencing-based high throughput functional assay named PLUMAGE and show the effects of these mutations on gene expression and their association with clinical outcomes in prostate cancer.

Abstract Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer that rarely develops de novo in primary tumors and is commonly acquired during the development of treatment resistance. NEPC is characterized by gain of neuroendocrine markers and loss of androgen receptor (AR), making it resistant to current therapeutic strategies targeting the AR signaling axis. Here, we report that MCM2, MCM3, MCM4, and MCM6 (MCM2/3/4/6) are elevated in human NEPC and high levels of MCM2/3/4/6 are associated with liver metastasis and poor survival in prostate cancer patients. MCM2/3/4/6 are four out of six proteins that form a core DNA helicase (MCM2-7) responsible for unwinding DNA forks during DNA replication. Inhibition of MCM2-7 by treatment with ciprofloxacin inhibits NEPC cell proliferation and migration in vitro, significantly delays NEPC tumor xenograft growth, and partially reverses the neuroendocrine phenotype in vivo. Our study reveals the clinical relevance of MCM2/3/4/6 proteins in NEPC and suggests that inhibition of MCM2-7 may represent a new therapeutic strategy for NEPC.

The molecular processes that lead to neuroendocrine prostate cancer after treating prostate adenocarcinoma (PRAD) are not well understood. Here the authors show that regulation by FOXA1 and changes in the epigenomic profile drive the transition from PRAD to a neuroendocrine phenotype.

Abstract Bladder cancer is among the most prevalent cancers worldwide. Currently, few bladder cancer models have undergone thorough characterization to assess their fidelity to patient tumors, especially upon propagation in the laboratory. Here, we establish and molecularly characterize CoCaB 1, an aggressive cisplatin-resistant muscle-invasive bladder cancer patient-derived xenograft (PDX) and companion organoid system. CoCaB 1 was a subcutaneous PDX model reliably transplanted in vivo and demonstrated an acceleration in growth upon serial transplantation, which was reflected in organoid and 2D cell culture systems. Transcriptome analysis revealed progression towards an increasingly proliferative and stem-like expression profile. Gene expression differences between organoid and PDX models reflected expected differences in cellular composition, with organoids enriched in lipid biosynthesis and metabolism genes and deprived of extracellular components observed in PDXs. Both PDX and organoid models maintained the histological fidelity and mutational heterogeneity of their parental tumor. This study establishes the CoCaB 1 PDX and organoid system as companion representative tumor models for the development of novel bladder cancer therapies.

The androgen receptor (AR) is a master transcription factor that regulates prostate cancer (PC) development and progression. Inhibition of AR signaling by androgen deprivation is the first-line therapy with initial efficacy for advanced and recurrent PC. Paradoxically, supraphysiological levels of testosterone (SPT) also inhibit PC progression. However, as with any therapy, not all patients show a therapeutic benefit, and responses differ widely in magnitude and duration. In this study, we evaluated whether differences in the AR cistrome before treatment can distinguish between SPT-responding (R) and -nonresponding (NR) tumors. We provide the first preclinical evidence to our knowledge that SPT-R tumors exhibit a distinct AR cistrome when compared with SPT-NR tumors, indicating a differential biological role of the AR. We applied an integrated analysis of ChIP-Seq and RNA-Seq to the pretreatment tumors and identified an SPT-R signature that distinguishes R and NR tumors. Because transcriptomes of SPT-treated clinical specimens are not available, we interrogated available castration-resistant PC (CRPC) transcriptomes and showed that the SPT-R signature is associated with improved survival and has the potential to identify patients who would respond to SPT. These findings provide an opportunity to identify the subset of patients with CRPC who would benefit from SPT therapy.

Neuroendocrine prostate cancer (NEPC) is the most virulent form of prostate cancer. Importantly, our recent work examining metastatic biopsy samples demonstrates NEPC is increasing in frequency. In contrast to prostate adenocarcinomas that express a luminal gene expression program, NEPC tumors express a neuronal gene expression program. Despite this distinction, the diagnosis of NEPC is often challenging, demonstrating an urgent need to identify new biomarkers and therapeutic targets. Our prior work demonstrated that the histone demethylase LSD1 (KDM1A) is important for survival of prostate adenocarcinomas, but little was known about LSD1’s role in NEPC. Recently, a neural-specific transcript variant of LSD1—LSD1+8a—was discovered and demonstrated to activate neuronal gene expression in neural cells. The splicing factor SRRM4 was previously shown to promote LSD1+8a splicing in neuronal cells, and SRRM4 promotes NEPC differentiation and cell survival. Therefore, we sought to determine if LSD1+8a might play a role in NEPC and whether LSD1+8a splicing was linked to SRRM4. To investigate a potential role for LSD1+8a in NEPC, we examined a panel of prostate adenocarcinoma and NEPC patient-derived xenografts and metastatic biopsies. LSD1+8a was expressed exclusively in NEPC samples and correlated significantly with elevated expression of SRRM4. Using SRRM4-overexpressing cell lines, we determined that SRRM4 mediates alternative splicing of LSD1+8a. Finally, using gain of function studies, we confirmed that LSD1+8a and SRRM4 co-regulate target genes distinct from canonical LSD1. Our findings suggest further study of the interplay between SRRM4 and LSD1+8a and mechanisms by which LSD1+8a regulates gene expression in NEPC is warranted.

Cancers with homology-directed DNA repair (HRR) deficiency exhibit high response rates to poly(ADP-ribose) polymerase inhibitors (PARPi) and platinum chemotherapy. Though mutations disrupting BRCA1 and BRCA2 associate with HRR deficiency (HRRd), patterns of genomic aberrations and mutation signatures may be more sensitive and specific indicators of compromised repair. Here, we evaluated whole-exome sequences from 418 metastatic prostate cancers (mPCs) and determined that one-fifth exhibited genomic characteristics of HRRd that included Catalogue Of Somatic Mutations In Cancer mutation signature 3. Notably, a substantial fraction of tumors with genomic features of HRRd lacked biallelic loss of a core HRR-associated gene, such as BRCA2. In this subset, HRRd associated with loss of chromodomain helicase DNA binding protein 1 but not with mutations in serine-protein kinase ATM, cyclin dependent kinase 12, or checkpoint kinase 2. HRRd genomic status was strongly correlated with responses to PARPi and platinum chemotherapy, a finding that supports evaluating biomarkers reflecting functional HRRd for treatment allocation.

The alpha particle-emitting radionuclide astatine-211 (211At) is of interest for targeted radiotherapy; however, low in vivo stability of many 211At-labeled cancer-targeting molecules has limited its potential. As an alternative labeling method, we evaluated whether a specific type of astatinated aryl compound that has the At atom in a higher oxidation state might be stable to in vivo deastatination. In the research effort, para-iodobenzoic acid methyl ester and dPEG4-amino acid methyl ester derivatives were prepared as HPLC standards. The corresponding para-stannylbenzoic acid derivatives were also prepared and labeled with 125I and 211At. Oxidization of the [125I]iodo- and [211At]astato-benzamidyl-dPEG4-acid methyl ester derivatives provided materials for in vivo evaluation. A biodistribution was conducted in mice with coinjected oxidized 125I- and 211At-labeled compounds. The oxidized radioiodinated derivative was stable to in vivo deiodination, but unfortunately the oxidized [211At]astatinated benzamide derivative was found to be unstable under the conditions of isolation by radio-HPLC (post animal injection). Another biodistribution study in mice evaluated the tissue concentrations of coinjected [211At]NaAtO3 and [125I]NaIO3. Comparison of the tissue concentrations of the isolated material from the oxidized [211At]benzamide derivative with those of [211At]astatate indicated the species obtained after isolation was likely [211At]astatate.

Abstract Background Prostate cancer displays different morphologies which, in turn, affect patient outcome. This fact prompted questions about the lineage relationship between differentiated, more treatable prostate adenocarcinoma and poorly differentiated, less treatable non-adenocarcinoma including small cell carcinoma, and the molecular mechanism underlying prostate cancer differentiation. Methods Newly available non-adenocarcinoma/small cell carcinoma PDX LuCaP lines were analyzed for expression of stem cell transcription factors (scTF) LIN28A, NANOG, POU5F1, SOX2, which are responsible for reprogramming or de-differentiation. cDNA of these genes were cloned from small cell carcinoma LuCaP 145.1 into expression vectors to determine if they could function in reprogramming. Results Expression of scTF was detected in small cell carcinoma LuCaP 93, 145.1, 145.2, and non-adenocarcinoma LuCaP 173.1, 173.2A. Transfection of scTF from LuCaP 145.1 altered the gene expression of prostate non-small cell carcinoma cells, as well as fibroblasts. The resultant cells grew in stem-like colonies. Of note was a 10-fold lower expression of B2M in the transfected cells. Low B2M was also characteristic of LuCaP 145.1. Conversely, B2M was increased when stem cells were induced to differentiate. Conclusions This work suggested a pathway in the emergence of non-adenocarcinoma/small cell carcinoma from adenocarcinoma through activation of scTF genes that produced cancer de-differentiation.

Resistance to AR signaling inhibitors (ARSis) in a subset of metastatic castration-resistant prostate cancers (mCRPCs) occurs with the emergence of AR– neuroendocrine prostate cancer (NEPC) coupled with mutations/deletions in PTEN, TP53, and RB1 and the overexpression of DNMTs, EZH2, and/or SOX2. To resolve whether the lack of AR is the driving factor for the emergence of the NE phenotype, molecular, cell, and tumor biology analyses were performed on 23 xenografts derived from patients with PC, recapitulating the full spectrum of genetic alterations proposed to drive NE differentiation. Additionally, phenotypic response to CRISPR/Cas9-mediated AR KO in AR+ CRPC cells was evaluated. These analyses document that (a) ARSi-resistant NEPC developed without androgen deprivation treatment; (b) ARS in ARSi-resistant AR+/NE+ double-positive “amphicrine” mCRPCs did not suppress NE differentiation; (c) the lack of AR expression did not necessitate acquiring a NE phenotype, despite concomitant mutations/deletions in PTEN and TP53, and the loss of RB1 but occurred via emergence of an AR–/NE– double-negative PC (DNPC); (d) despite DNPC cells having homogeneous genetic driver mutations, they were phenotypically heterogeneous, expressing basal lineage markers alone or in combination with luminal lineage markers; and (e) AR loss was associated with AR promoter hypermethylation in NEPCs but not in DNPCs.

These data include secondary analysis of publicly available RNA-seq data from castration-resistant prostate cancer (CRPC) patients as well as RT-qPCR and Western blotting analyses of patient-derived xenograft models and a CRPC cell line. We applied Spearman correlation analysis to assess the relationship between canonical androgen receptor (AR) splicing and alternative AR splicing. We also assessed the ratio of AR splice variants (AR-Vs) to the full-length AR (AR-FL) at the RNA and protein levels by absolute RT-qPCR and Western blotting, respectively. These data are critical for studying the mechanisms underlying upregulated expression of AR-Vs after AR-directed therapies and the importance of AR-Vs to castration-resistant progression of prostate cancer. Data presented here are related to the research article by Ma et al., “Increased transcription and high translation efficiency lead to accumulation of androgen receptor splice variant after androgen deprivation therapy”, Cancer Lett. In Press [1].

With the advent of potent second-line anti-androgen therapy, we and others have observed an increased incidence of androgen receptor (AR)-null small cell or neuroendocrine prostate cancer (SCNPC) in metastatic castration-resistant prostate cancer (mCRPC). Our study was designed to determine the effect of cabozantinib, a multi-targeted tyrosine kinase inhibitor that inhibits VEGFR2, MET and RET on SCNPC. Transcriptome analysis of the University of Washington rapid autopsy and SU2C mCRPC datasets revealed upregulated MET and RET expression in SCNPCs relative to adenocarcinomas. Additionally, increased MET expression correlated with attenuated AR expression and activity. In vitro treatment of SCNPC patient-derived xenograft (PDX) cells with the MET inhibitor AMG-337 had no impact on cell viability in LuCaP 93 (MET+/RET+) and LuCaP 173.1 (MET-/RET-), whereas cabozantinib decreased cell viability of LuCaP 93, but not LuCaP 173.1. Notably, MET+/RET+ LuCaP 93 and MET-/RET- LuCaP 173.1 tumor volumes were significantly decreased with cabozantinib treatment in vivo, and this activity was independent of MET or RET expression in LuCaP 173.1. Tissue analysis indicated that cabozantinib did not inhibit tumor cell proliferation (Ki67), but significantly decreased microvessel density (CD31) and increased hypoxic stress and glycolysis (HK2) in LuCaP 93 and LuCaP 173.1 tumors. RNA-Seq and gene set enrichment analysis revealed that hypoxia and glycolysis pathways were increased in cabozantinib-treated tumors relative to control tumors. Our data suggest that the most likely mechanism of cabozantinib-mediated tumor growth suppression in SCNPC PDX models is through disruption of the tumor vasculature. Thus, cabozantinib may represent a potential therapy for patients with metastatic disease in tumor phenotypes that have a significant dependence on the tumor vasculature for survival and proliferation.

Neuroendocrine prostate cancer (NEPrCa) arises de novo or after accumulation of genomic alterations in pre-existing adenocarcinoma tumors in response to androgen deprivation therapies. We have provided evidence that small extracellular vesicles released by PrCa cells and containing the αVβ3 integrin promote neuroendocrine differentiation of PrCa in vivo and in vitro. Here, we examined αVβ3 integrin expression in three murine models carrying a deletion of PTEN (SKO), PTEN and RB1 (DKO), or PTEN, RB1 and TRP53 (TKO) genes in the prostatic epithelium; of these three models, the DKO and TKO tumors develop NEPrCa with a gene signature comparable to those of human NEPrCa. Immunostaining analysis of SKO, DKO and TKO tumors shows that αVβ3 integrin expression is increased in DKO and TKO primary tumors and metastatic lesions, but absent in SKO primary tumors. On the other hand, SKO tumors show higher levels of a different αV integrin, αVβ6, as compared to DKO and TKO tumors. These results are confirmed by RNA-sequencing analysis. Moreover, TRAMP mice, which carry NEPrCa and adenocarcinoma of the prostate, also have increased levels of αVβ3 in their NEPrCa primary tumors. In contrast, the αVβ6 integrin is only detectable in the adenocarcinoma areas. Finally, analysis of 42 LuCaP patient-derived xenografts and primary adenocarcinoma samples shows a positive correlation between αVβ3, but not αVβ6, and the neuronal marker synaptophysin; it also demonstrates that αVβ3 is absent in prostatic adenocarcinomas. In summary, we demonstrate that αVβ3 integrin is upregulated in NEPrCa primary and metastatic lesions; in contrast, the αVβ6 integrin is confined to adenocarcinoma of the prostate. Our findings suggest that the αVβ3 integrin, but not αVβ6, may promote a shift in lineage plasticity towards a NE phenotype and might serve as an informative biomarker for the early detection of NE differentiation in prostate cancer.

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.

Global changes in chromatin accessibility may drive cancer progression by reprogramming transcription factor (TF) binding. In addition, histone acetylation readers such as bromodomain-containing protein 4 (BRD4) have been shown to associate with these TFs and contribute to aggressive cancers including prostate cancer (PC). Here, we show that chromatin accessibility defines castration-resistant prostate cancer (CRPC). We show that the deregulation of androgen receptor (AR) expression is a driver of chromatin relaxation and that AR/androgen-regulated bromodomain-containing proteins (BRDs) mediate this effect. We also report that BRDs are overexpressed in CRPCs and that ATAD2 and BRD2 have prognostic value. Finally, we developed gene stratification signature (BROMO-10) for bromodomain response and PC prognostication, to inform current and future trials with drugs targeting these processes. Our findings provide a compelling rational for combination therapy targeting bromodomains in selected patients in which BRD-mediated TF binding is enhanced or modified as cancer progresses.

The C-C chemokine ligand 2 (CCL2) stimulates migration, proliferation, and invasion of prostate cancer (PCa) cells, and its signaling also plays a role in the activation of osteoclasts. Therefore targeting CCL2 signaling in regulation of tumor progression in bone metastases is an area of intense research. The objective of our study was to investigate the efficacy of CCL2 blockade by neutralizing antibodies to inhibit the growth of PCa in bone. We used a preclinical model of cancer growth in the bone in which PCa C4-2B cells were injected directly into murine tibiae. Animals were treated for ten weeks with neutralizing anti-CCL2 antibodies, docetaxel, or a combination of both, and then followed an additional nine weeks. CCL2 blockade inhibited the growth of PCa in bone, with even more pronounced inhibition in combination with docetaxel. CCL2 blockade also resulted in increases in bone mineral density. Furthermore, our results showed that the tumor inhibition lasted even after discontinuation of the treatment. Our data provide compelling evidence that CCL2 blockade slows PCa growth in bone, both alone and in combination with docetaxel. These results support the continued investigations of CCL2 blockade as a treatment for advanced metastatic PCa.

Bone morphogenetic protein (BMP) signaling is important in prostate development and prostate cancer (PCa) progression. However, because of the multiple effects of different BMPs, no final conclusions have been made as to the role of BMPs in PCa. In our studies, we have focused on BMP-7 because it is involved in prostate morphogenesis, and its expression is regulated by androgens. The objective of our study was to determine BMP-7 expression in PCa metastases and investigate the effects of BMP-7 on PCa cells. Our results show that BMP-7 is expressed in metastatic PCa and its levels are increased in castration-resistant PCa versus androgen-dependent PCa, whereas the expression of BMP-7 is decreased in primary PCa versus normal prostate. Our in vitro results show that BMP-7 inhibits proliferation of androgen-sensitive LNCaP cells, stimulates androgen receptor signaling, increases the expression of differentiation-associated genes, and decreases the levels of some wingless-regulated transcripts. Interestingly, these effects were not detected in C4-2 castration-resistant PCa cells. In vivo expression of BMP-7 in castration-resistant C4-2 cells did not alter proliferation when these cells were grown subcutaneously, but their growth was inhibited in the bone environment. In summary, our results show that BMP-7 is expressed at the highest level in advanced castration-resistant PCa cells and that the inhibitory effects of BMP-7 are dependent on the differentiation status of PCa cells and the tumor microenvironment. Further studies are needed to identify changes in BMP-7 signaling that lead to the loss of its control of proliferation during PCa progression.

Treatments for advanced prostate cancer (CaP) typically involve androgen deprivation therapy. However, most patients eventually develop castration-resistant CaP (CRPC) for which highly effective therapies are limited. We explored the efficacy of a novel agent, HE3235, in inhibiting growth of CRPC in preclinical models. Castrated male mice were implanted subcutaneously with LuCaP35V CaP xenografts in the presence and absence of 5′-androstenediol (AED) and treated with HE3235. To investigate the effect of HE3235 on CaP tumor in the bone, castrated mice were injected intratibially with C4-2B CaP cells and treated with HE3235. Serum prostate-specific antigen (PSA) levels, tumor volume, immunohistochemistry, gene expression, and levels of intratumoral androgens were analyzed. HE3235 significantly prolonged the tumor doubling time of LuCaP35V, decreased androgen receptor expression, and lowered levels of intratumoral testosterone by ∼89% and dihydrotestosterone by ∼63% in both the presence and the absence of AED. HE3235 inhibited tumor growth in the bone environment. Weights of tumored tibiae of HE3235-treated animals were lower than those of control (P = .031), and normalized PSA levels were also significantly decreased at the end of study by HE3235 treatment (P = .0076). HE3235 inhibits the growth of subcutaneous CRPC as well as CRPC in the bone environment. Our data show that HE3235 exhibits a wide range of effects, including alteration of androgen receptor signaling and reductions in levels of intratumoral androgens. Our results support ongoing clinical investigations into the effectiveness of HE3235 in the setting of CRPC and warrants further studies into the mechanisms behind the effects of HE3235.

Dissemination of prostate cancer (PCa) cells to the bone marrow is an early event in the disease process. In some patients, disseminated tumor cells (DTC) proliferate to form active metastases after a prolonged period of undetectable disease known as tumor dormancy. Identifying mechanisms of PCa dormancy and reactivation remain a challenge partly due to the lack of in vitro models. Here, we characterized in vitro PCa dormancy-reactivation by inducing cells from three patient-derived xenograft (PDX) lines to proliferate through tumor cell contact with each other and with bone marrow stroma. Proliferating PCa cells demonstrated tumor cell-cell contact and integrin clustering by immunofluorescence. Global gene expression analyses on proliferating cells cultured on bone marrow stroma revealed a downregulation of TGFB2 in all of the three proliferating PCa PDX lines when compared to their non-proliferating counterparts. Furthermore, constitutive activation of myosin light chain kinase (MLCK), a downstream effector of integrin-beta1 and TGF-beta2, in non-proliferating cells promoted cell proliferation. This cell proliferation was associated with an upregulation of CDK6 and a downregulation of E2F4. Taken together, our data provide the first clinically relevant in vitro model to support cellular adhesion and downregulation of TGFB2 as a potential mechanism by which PCa cells may escape from dormancy. Targeting the TGF-beta2-associated mechanism could provide novel opportunities to prevent lethal PCa metastasis.

The clinical utility of estrogens for treating prostate cancer (CaP) was established in the 1940s by Huggins. The classic model of the anti-CaP activity of estrogens postulates an indirect mechanism involving the suppression of androgen production. However, clinical, preclinical studies have shown that estrogens exert growth-inhibitory effects on CaP under low-androgen conditions, suggesting additional modes whereby estrogens affect CaP cells and/or the microenvironment. Here we have investigated the activity of 17β estradiol (E2) against androgen-independent CaP, identified molecular alterations in tumors exposed to E2. E2 treatment inhibited the growth of all four androgen-independent CaP xenografts studied (LuCaP 35V, LuCaP 23.1AI, LuCaP 49, LuCaP 58) in castrated male mice. The molecular basis of growth suppression was studied by cDNA microarray analysis, which indicated that multiple pathways are altered by E2 treatment. Of particular interest are changes in transcripts encoding proteins that mediate immune responses, regulate androgen receptor signaling. In conclusion, our data show that estrogens have powerful inhibitory effects on CaP in vivo in androgendepleted environments, suggest novel mechanisms of estrogen-mediated antitumor activity. These results indicate that incorporating estrogens into CaP treatment protocols could enhance therapeutic efficacy even in cases of advanced disease.

Factors influencing differential responses of prostate tumors to androgen receptor (AR) axis-directed therapeutics are poorly understood, and predictors of treatment efficacy are needed. We hypothesized that the efficacy of inhibiting DHT ligand synthesis would associate with intra-tumoral androgen ratios indicative of relative dependence on DHT-mediated growth.We characterized two androgen-sensitive prostate cancer xenograft models after androgen suppression by castration in combination with the SRD5A inhibitor, dutasteride, as well as a panel of castration resistant metastases obtained via rapid autopsy.In LuCaP35 tumors (intra-tumoral T:DHT ratio 2:1) dutasteride suppressed DHT to 0.02 ng/gm and prolonged survival vs. castration alone (337 vs.152 days, HR 2.8, p = 0.0015). In LuCaP96 tumors (T:DHT 10:1), survival was not improved despite similar DHT reduction (0.02 ng/gm). LuCaP35 demonstrated higher expression of steroid biosynthetic enzymes maintaining DHT levels (5-fold higher SRD5A1, 41 fold higher, 99-fold higher RL-HSD, p

Cabozantinib is an inhibitor of multiple receptor tyrosine kinases, including MET and VEGFR2. In a phase II clinical trial in advanced prostate cancer (PCa), cabozantinib treatment improved bone scans in 68% of evaluable patients. Our studies aimed to determine the expression of cabozantinib targets during PCa progression and to evaluate its efficacy in hormone-sensitive and castration-resistant PCa in preclinical models while delineating its effects on tumor and bone. Using immunohistochemistry and tissue microarrays containing normal prostate, primary PCa, and soft tissue and bone metastases, our data show that levels of MET, P-MET, and VEGFR2 are increasing during PCa progression. Our data also show that the expression of cabozantinib targets are particularly pronounced in bone metastases. To evaluate cabozantinib efficacy on PCa growth in the bone environment and in soft tissues we used androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B PCa tumors. In vivo, cabozantinib inhibited the growth of PCa in bone as well as growth of subcutaneous tumors. Furthermore, cabozantinib treatment attenuated the bone response to the tumor and resulted in increased normal bone volume. In summary, the expression pattern of cabozantinib targets in primary and castration-resistant metastatic PCa, and its efficacy in two different models of PCa suggest that this agent has a strong potential for the effective treatment of PCa at different stages of the disease.