Identification of alternative protein targets of glutamate-ureido-lysine associated with PSMA tracer uptake in prostate cancer cells.

Prostate-specific membrane antigen (PSMA) is highly overexpressed in most prostate cancers and is clinically visualized using PSMA-specific probes incorporating glutamate-ureido-lysine (GUL). PSMA is effectively absent from certain high-mortality, treatment-resistant subsets of prostate cancers, such as neuroendocrine prostate cancer (NEPC); however, GUL-based PSMA tracers are still reported to have the potential to identify NEPC metastatic tumors. These probes may bind unknown proteins associated with PSMA-suppressed cancers. We have identified the up-regulation of PSMA-like aminopeptidase NAALADaseL and the metabotropic glutamate receptors (mGluRs) in PSMA-suppressed prostate cancers and find that their expression levels inversely correlate with PSMA expression and are associated with GUL-based radiotracer uptake. Furthermore, we identify that NAALADaseL and mGluR expression correlates with a unique cell cycle signature. This provides an opportunity for the future study of the biology of NEPC and potential therapeutic directions. Computationally predicting that GUL-based probes bind well to these targets, we designed and synthesized a fluorescent PSMA tracer to investigate these proteins in vitro, where it shows excellent affinity for PSMA, NAALADaseL, and specific mGluRs associated with poor prognosis.

Polymeric black tea polyphenols (PBPs) inhibit benzo(a)pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone-induced lung carcinogenesis potentially through down-regulation of p38 and Akt phosphorylation in A/J mice.

The aim of our study was to evaluate chemopreventive efficacy and possible mechanism of most abundant polyphenolic fraction in black tea, polymeric black tea polyphenols (PBPs), in experimental lung carcinogenesis model. Effect of 1.5% black tea derived PBPs on benzo(a)pyrene [B(a)P] and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced lung lesions were studied over 28 wks. Chemopreventive efficacy was studied using decrease in tumor incidence and/or multiplicity and/or delay in the latency period in A/J mice. Histopathological analysis of lung was carried out post-carcinogen treatment weeks to analyze the microscopic lung lesions. Inflammation, cell proliferation, and apoptosis markers along with signaling kinases like p38, Akt, and their phosphorylated forms were studied using immunoblotting and immunohistochemistry at 4th, 10th, and 18th wk post-carcinogen treatment. Administration of PBPs throughout the treatment period significantly decreased the multiplicity of surface tumors as well as microscopic lung lesions, including adenomas. Although tumor incidence and latency period remains unaffected, histopathological evaluation of lung at 6, 10, and 18 wks post- carcinogen treatment period showed decrease in tumor multiplicity which was also correlated with different molecular markers. Anti- inflammatory action of PBPs was demonstrated by reduced Cox-2 expression. PBPs down-regulated the B(a)P and NNK-induced cell proliferation (diminished PCNA expression, proliferation index, and Bcl-2 expression) and enhanced apoptosis (increased Bax expression and apoptotic index) potentially through phosphorylation of p38 and Akt. PBPs, most abundant polyphenolic component in the black tea, have chemopreventive effect through inhibition of inflammation, cellular proliferation, and induction of apoptosis possibly via modulation of signaling kinases. © 2016 Wiley Periodicals, Inc.

Notch signaling suppresses neuroendocrine differentiation and alters the immune microenvironment in advanced prostate cancer.

Notch signaling can have either an oncogenic or tumor-suppressive function in cancer depending on the cancer type and cellular context. While Notch can be oncogenic in early prostate cancer, we identified significant downregulation of the Notch pathway during prostate cancer progression from adenocarcinoma to neuroendocrine (NE) prostate cancer, where it functions as a tumor suppressor. Activation of Notch in NE and Rb1/Trp53-deficient prostate cancer models led to phenotypic conversion toward a more indolent, non-NE state with glandular features and expression of luminal lineage markers. This was accompanied by upregulation of MHC and type I IFN and immune cell infiltration. Overall, these data support Notch signaling as a suppressor of NE differentiation in advanced prostate cancer and provide insights into how Notch signaling influences lineage plasticity and the tumor microenvironment (TME).

Lineage-specific canonical and non-canonical activity of EZH2 in advanced prostate cancer subtypes.

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and emerging therapeutic target that is overexpressed in most castration-resistant prostate cancers and implicated as a driver of disease progression and resistance to hormonal therapies. Here we define the lineage-specific action and differential activity of EZH2 in both prostate adenocarcinoma and neuroendocrine prostate cancer (NEPC) subtypes of advanced prostate cancer to better understand the role of EZH2 in modulating differentiation, lineage plasticity, and to identify mediators of response and resistance to EZH2 inhibitor therapy. Mechanistically, EZH2 modulates bivalent genes that results in upregulation of NEPC-associated transcriptional drivers (e.g., ASCL1) and neuronal gene programs in NEPC, and leads to forward differentiation after targeting EZH2 in NEPC. Subtype-specific downstream effects of EZH2 inhibition on cell cycle genes support the potential rationale for co-targeting cyclin/CDK to overcome resistance to EZH2 inhibition.

Lineage-specific canonical and non-canonical activity of EZH2 in advanced prostate cancer subtypes.

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and emerging therapeutic target that is overexpressed in most castration-resistant prostate cancers and implicated as a driver of disease progression and resistance to hormonal therapies. Here we define the lineage-specific action and differential activity of EZH2 in both prostate adenocarcinoma (PRAD) and neuroendocrine prostate cancer (NEPC) subtypes of advanced prostate cancer to better understand the role of EZH2 in modulating differentiation, lineage plasticity, and to identify mediators of response and resistance to EZH2 inhibitor therapy. Mechanistically, EZH2 modulates bivalent genes that results in upregulation of NEPC-associated transcriptional drivers (e.g., ASCL1) and neuronal gene programs, and leads to forward differentiation after targeting EZH2 in NEPC. Subtype-specific downstream effects of EZH2 inhibition on cell cycle genes support the potential rationale for co-targeting cyclin/CDK to overcome resistance to EZH2 inhibition.

Significance of RB Loss in Unlocking Phenotypic Plasticity in Advanced Cancers.

Cancer cells can undergo plasticity in response to environmental stimuli or under selective therapeutic pressures that result in changes in phenotype. This complex phenomenon of phenotypic plasticity is now recognized as a hallmark of cancer. Lineage plasticity is often associated with loss of dependence on the original oncogenic driver and is facilitated, in part, by underlying genomic and epigenetic alterations. Understanding the molecular drivers of cancer plasticity is critical for the development of novel therapeutic strategies. The retinoblastoma gene RB1 (encoding RB) is the first tumor suppressor gene to be discovered and has a well-described role in cell-cycle regulation. RB is also involved in diverse cellular functions beyond cell cycle including differentiation. Here, we describe the emerging role of RB loss in unlocking cancer phenotypic plasticity and driving therapy resistance across cancer types. We highlight parallels in cancer with the noncanonical role of RB that is critical for normal development and lineage specification, and the downstream consequences of RB loss including epigenetic reprogramming and chromatin reorganization that can lead to changes in lineage program. Finally, we discuss potential therapeutic approaches geared toward RB loss cancers undergoing lineage reprogramming.

Targeting DNA methylation and B7-H3 in RB1-deficient and neuroendocrine prostate cancer.

Aberrant DNA methylation has been implicated as a key driver of prostate cancer lineage plasticity and histologic transformation to neuroendocrine prostate cancer (NEPC). DNA methyltransferases (DNMTs) are highly expressed, and global DNA methylation is dysregulated in NEPC. We identified that deletion of DNMT genes decreases expression of neuroendocrine lineage markers and substantially reduced NEPC tumor development and metastasis in vivo. Decitabine, a pan-DNMT inhibitor, attenuated tumor growth in NEPC patient-derived xenograft models, as well as retinoblastoma gene (RB1)-deficient castration-resistant prostate adenocarcinoma (CRPC) models compared with RB1-proficient CRPC. We further found that DNMT inhibition increased expression of B7 homolog 3 (B7-H3), an emerging druggable target, via demethylation of B7-H3. We tested DS-7300a (i-DXd), an antibody-drug conjugate targeting B7-H3, alone and in combination with decitabine in models of advanced prostate cancer. There was potent single-agent antitumor activity of DS-7300a in both CRPC and NEPC bearing high expression of B7-H3. In B7-H3-low models, combination therapy of decitabine plus DS-7300a resulted in enhanced response. DNMT inhibition may therefore be a promising therapeutic target for NEPC and RB1-deficient CRPC and may sensitize B7-H3-low prostate cancer to DS-7300a through increasing target expression. NEPC and RB1-deficient CRPC represent prostate cancer subgroups with poor prognosis, and the development of biomarker-driven therapeutic strategies for these populations may ultimately help improve patient outcomes.

Landscape of prostate-specific membrane antigen heterogeneity and regulation in AR-positive and AR-negative metastatic prostate cancer.

Tumor expression of prostate-specific membrane antigen (PSMA) is lost in 15-20% of men with castration-resistant prostate cancer (CRPC), yet the underlying mechanisms remain poorly defined. In androgen receptor (AR)-positive CRPC, we observed lower PSMA expression in liver lesions versus other sites, suggesting a role of the microenvironment in modulating PSMA. PSMA suppression was associated with promoter histone 3 lysine 27 methylation and higher levels of neutral amino acid transporters, correlating with 18F-fluciclovine uptake on positron emission tomography imaging. While PSMA is regulated by AR, we identified a subset of AR-negative CRPC with high PSMA. HOXB13 and AR co-occupancy at the PSMA enhancer and knockout models point to HOXB13 as an upstream regulator of PSMA in AR-positive and AR-negative prostate cancer. These data demonstrate how PSMA expression is differentially regulated across metastatic lesions and in the context of the AR, which may inform selection for PSMA-targeted therapies and development of complementary biomarkers.

Prostate Cancer Progression Relies on the Mitotic Kinase Citron Kinase.

Prostate cancer remains the second leading cause of cancer death in men in Western cultures. A deeper understanding of the mechanisms by which prostate cancer cells divide to support tumor growth could help devise strategies to overcome treatment resistance and improve survival. Here, we identified that the mitotic AGC family protein kinase citron kinase (CIT) is a pivotal regulator of prostate cancer growth that mediates prostate cancer cell interphase progression. Increased CIT expression correlated with prostate cancer growth induction and aggressive prostate cancer progression, and CIT was overexpressed in prostate cancer compared with benign prostate tissue. CIT overexpression was controlled by an E2F2-Skp2-p27 signaling axis and conferred resistance to androgen-targeted treatment strategies. The effects of CIT relied entirely on its kinase activity. Conversely, CIT silencing inhibited the growth of cell lines and xenografts representing different stages of prostate cancer progression and treatment resistance but did not affect benign epithelial prostate cells or nonprostatic normal cells, indicating a potential therapeutic window for CIT inhibition. CIT kinase activity was identified as druggable and was potently inhibited by the multikinase inhibitor OTS-167, which decreased the proliferation of treatment-resistant prostate cancer cells and patient-derived organoids. Isolation of the in vivo CIT substrates identified proteins involved in diverse cellular functions ranging from proliferation to alternative splicing events that are enriched in treatment-resistant prostate cancer. These findings provide insights into the regulation of aggressive prostate cancer cell behavior by CIT and identify CIT as a functionally diverse and druggable driver of prostate cancer progression. SIGNIFICANCE: The poorly characterized protein kinase citron kinase is a therapeutic target in prostate cancer that drives tumor growth by regulating diverse substrates, which control several hallmarks of aggressive prostate cancer progression. See related commentary by Mishra et al., p. 4008.

CHD1 Promotes Sensitivity to Aurora Kinase Inhibitors by Suppressing Interaction of AURKA with Its Coactivator TPX2.

Clinical studies have shown that subsets of patients with cancer achieve a significant benefit from Aurora kinase inhibitors, suggesting an urgent need to identify biomarkers for predicting drug response. Chromodomain helicase DNA binding protein 1 (CHD1) is involved in chromatin remodeling, DNA repair, and transcriptional plasticity. Prior studies have demonstrated that CHD1 has distinct expression patterns in cancers with different molecular features, but its impact on drug responsiveness remains understudied. Here, we show that CHD1 promotes the susceptibility of prostate cancer cells to inhibitors targeting Aurora kinases, while depletion of CHD1 impairs their efficacy in vitro and in vivo. Pan-cancer drug sensitivity analyses revealed that high expression of CHD1 was associated with increased sensitivity to Aurora kinase A (AURKA) inhibitors. Mechanistically, KPNA2 served as a direct target of CHD1 and suppressed the interaction of AURKA with the coactivator TPX2, thereby rendering cancer cells more vulnerable to AURKA inhibitors. Consistent with previous research reporting that loss of PTEN elevates CHD1 levels, studies in a genetically engineered mouse model, patient-derived organoids, and patient samples showed that PTEN defects are associated with a better response to AURKA inhibition in advanced prostate cancer. These observations demonstrate that CHD1 plays an important role in modulating Aurora kinases and drug sensitivities, providing new insights into biomarker-driven therapies targeting Aurora kinases for future clinical studies. SIGNIFICANCE: CHD1 plays a critical role in controlling AURKA activation and promoting Aurora kinase inhibitor sensitivity, providing a potential clinical biomarker to guide cancer treatment.

Genomic alterations impact cell cycle-related genes during prostate cancer progression.

The recent genomic characterization of patient specimens has started to reveal the landscape of somatic alterations in clinical prostate cancer (CaP) and its association with disease progression and treatment resistance. The extent to which such alterations impact hallmarks of cancer is still unclear. Here, we interrogate genomic data from thousands of clinical CaP specimens that reflect progression from treatment-naïve, to castration-recurrent, and in some cases, neuroendocrine CaP for alterations in cell cycle-associated and -regulated genes, which are central to cancer initiation and progression. We evaluate gene signatures previously curated to evaluate G1-S and G2-M phase transitions or to represent the cell cycle-dependent proteome. The resulting CaP (stage)-specific overview confirmed the presence of well-known driver alterations impacting, for instance, the genes encoding p53 and MYC, and uncovered novel previously unrecognized mutations that affect others such as the PKMYT1 and MTBP genes. The cancer dependency and drugability of representative genomically altered cell cycle determinants were verified also. Taken together, these analyses on hundreds of often less-characterized cell cycle regulators expand considerably the scope of genomic alterations associated with CaP cell proliferation and cell cycle and isolate such regulatory proteins as putative drivers of CaP treatment resistance and entirely novel therapeutic targets for CaP therapy.

Novel insights in cell cycle dysregulation during prostate cancer progression.

Prostate cancer (CaP) remains the second leading cause of cancer deaths in Western men. These deaths occur because metastatic CaP acquires resistance to available treatments. The novel and functionally diverse treatment options that have been introduced in the clinic over the past decade each eventually induce resistance for which the molecular basis is diverse. Both initiation and progression of CaP have been associated with enhanced cell proliferation and cell cycle dysregulation. A better understanding of the specific pro-proliferative molecular shifts that control cell division and proliferation during CaP progression may ultimately overcome treatment resistance. Here, we examine literature for support of this possibility. We start by reviewing recently renewed insights in prostate cell types and their proliferative and oncogenic potential. We then provide an overview of the basic knowledge on the molecular machinery in charge of cell cycle progression and its regulation by well-recognized drivers of CaP progression such as androgen receptor and retinoblastoma protein. In this respect, we pay particular attention to interactions and reciprocal interplay between cell cycle regulators and androgen receptor. Somatic alterations that impact the cell cycle-associated and -regulated genes encoding p53, PTEN and MYC during progression from treatment-naïve, to castration-recurrent, and in some cases, neuroendocrine CaP are discussed. We considered also non-genomic events that impact cell cycle determinants, including transcriptional, epigenetic and micro-environmental switches that occur during CaP progression. Finally, we evaluate the therapeutic potential of cell cycle regulators and address challenges and limitations in the approaches modulating their action for CaP treatment.

AR-dependent phosphorylation and phospho-proteome targets in prostate cancer.

Prostate cancer (CaP) is the second leading cause of cancer-related deaths in Western men. Because androgens drive CaP by activating the androgen receptor (AR), blocking AR's ligand activation, known as androgen deprivation therapy (ADT), is the default treatment for metastatic CaP. Despite an initial remission, CaP eventually develops resistance to ADT and progresses to castration-recurrent CaP (CRPC). CRPC continues to rely on aberrantly activated AR that is no longer inhibited effectively by available therapeutics. Interference with signaling pathways downstream of activated AR that mediate aggressive CRPC behavior may lead to alternative CaP treatments. Developing such therapeutic strategies requires a thorough mechanistic understanding of the most clinically relevant and druggable AR-dependent signaling events. Recent proteomics analyses of CRPC clinical specimens indicate a shift in the phosphoproteome during CaP progression. Kinases and phosphatases represent druggable entities, for which clinically tested inhibitors are available, some of which are incorporated already in treatment plans for other human malignancies. Here, we reviewed the AR-associated transcriptome and translational regulon, and AR interactome involved in CaP phosphorylation events. Novel and for the most part mutually exclusive AR-dependent transcriptional and post-transcriptional control over kinase and phosphatase expression was found, with yet other phospho-regulators interacting with AR. The multiple mechanisms by which AR can shape and fine-tune the CaP phosphoproteome were reflected in diverse aspects of CaP biology such as cell cycle progression and cell migration. Furthermore, we examined the potential, limitations and challenges of interfering with AR-mediated phosphorylation events as alternative strategy to block AR function during CaP progression.

Diversity in Androgen Receptor Action Among Treatment-naïve Prostate Cancers Is Reflected in Treatment Response Predictions and Molecular Subtypes.

BACKGROUND: Metastatic prostate cancer (CaP) treatments are evolving rapidly but without evidence-based biomarkers to predict responses, and to maximize remissions and survival. OBJECTIVE: To determine the activity of androgen receptor (AR), the target for default first-line systemic treatment, in localized treatment-naïve CaP and its association with clinical risk factors, molecular markers, CaP subtypes, and predictors of treatment response. DESIGN SETTING AND PARTICIPANTS: We examined 452 bona fide AR target genes in clinical-grade expression profiles from 6532 such CaPs collected between 2013 and 2017 by US physicians ordering the Decipher RP test. Results were validated in three independent smaller cohorts (n = 73, 90, and 127) and clinical CaP AR ChIP-Seq data. Association with CaP differentiation and progression was analyzed in independent datasets. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Unsupervised clustering of CaPs based on AR target gene expression was aligned with clinical variables, differentiation scores, molecular subtypes, and predictors of response to hormonal therapy, radiotherapy, and chemotherapy. AR target gene sets were analyzed via Gene Set Enrichment Analysis for differentiation and treatment resistance, Ingenuity Pathway Analysis for associated biology, and Cistrome for genomic AR binding site (ARBS) composition. RESULTS AND LIMITATIONS: Expression of eight AR target gene subsignatures gave rise to five CaP clusters, which were preferentially associated with CaP molecular subtypes, differentiation, and predictors of treatment response rather than with clinical variables. Subsignatures differed in contribution to CaP progression, luminal/basal differentiation, CaP biology, and ARBS composition. Validation in prospective trials and optimized quantitation are needed for clinical implementation. CONCLUSIONS: Measurement of AR activity patterns in treatment-naïve CaP may serve as a first branch of an evidence-based decision tree to optimize personalized treatment plans. PATIENT SUMMARY: Treatment options for metastatic prostate cancer are increasing without information needed to choose the right treatment for the right patient. We found variation in the behavior of the target for the default first-line therapy before treatment, which may help optimize treatment plans.

Protein Kinase N1 control of androgen-responsive serum response factor action provides rationale for novel prostate cancer treatment strategy.

Sustained reliance on androgen receptor (AR) after failure of AR-targeting androgen deprivation therapy (ADT) prevents effective treatment of castration-recurrent (CR) prostate cancer (CaP). Interfering with the molecular machinery by which AR drives CaP progression may be an alternative therapeutic strategy but its feasibility remains to be tested. Here, we explore targeting the mechanism by which AR, via RhoA, conveys androgen-responsiveness to serum response factor (SRF), which controls aggressive CaP behavior and is maintained in CR-CaP. Following a siRNA screen and candidate gene approach, RNA-Seq studies confirmed that the RhoA effector Protein Kinase N1 (PKN1) transduces androgen-responsiveness to SRF. Androgen treatment induced SRF-PKN1 interaction, and PKN1 knockdown or overexpression severely impaired or stimulated, respectively, androgen regulation of SRF target genes. PKN1 overexpression occurred during clinical CR-CaP progression, and hastened CaP growth and shortened CR-CaP survival in orthotopic CaP xenografts. PKN1's effects on SRF relied on its kinase domain. The multikinase inhibitor lestaurtinib inhibited PKN1 action and preferentially affected androgen regulation of SRF over direct AR target genes. In a CR-CaP patient-derived xenograft, expression of SRF target genes was maintained while AR target gene expression declined and proliferative gene expression increased. PKN1 inhibition decreased viability of CaP cells before and after ADT. In patient-derived CaP explants, lestaurtinib increased AR target gene expression but did not significantly alter SRF target gene or proliferative gene expression. These results provide proof-of-principle for selective forms of ADT that preferentially target different fractions of AR's transcriptional output to inhibit CaP growth.

A comprehensive analysis of coregulator recruitment, androgen receptor function and gene expression in prostate cancer.

Standard treatment for metastatic prostate cancer (CaP) prevents ligand-activation of androgen receptor (AR). Despite initial remission, CaP progresses while relying on AR. AR transcriptional output controls CaP behavior and is an alternative therapeutic target, but its molecular regulation is poorly understood. Here, we show that action of activated AR partitions into fractions that are controlled preferentially by different coregulators. In a 452-AR-target gene panel, each of 18 clinically relevant coregulators mediates androgen-responsiveness of 0-57% genes and acts as a coactivator or corepressor in a gene-specific manner. Selectivity in coregulator-dependent AR action is reflected in differential AR binding site composition and involvement with CaP biology and progression. Isolation of a novel transcriptional mechanism in which WDR77 unites the actions of AR and p53, the major genomic drivers of lethal CaP, to control cell cycle progression provides proof-of-principle for treatment via selective interference with AR action by exploiting AR dependence on coregulators.