Prostate Cancer Patient Specimens Research Articles

Abstract 7645: Differentially expressed genes as prognostic markers in racial disparities of prostate cancer

Abstract Racial disparities in prostate cancer are prevalent, with African American (AA) men experiencing 60% greater incidence and 2 to 3 times higher mortality than Caucasian American (CA) men. Consequently, there is a pressing need for improved means of interrogating race/ethnicity-based prostate cancer biology in order to identify biomarkers to help stratify men at high-risk for prostate cancer-specific progression and death and offer treatment recommendation that would mostly benefit AA men undergoing early aggressive therapies. Current predictive models rely on clinical variables but do not account for these disparities. The overall goal is to construct a race-specific prostate cancer prognostic prediction model for risk assessment. Here we performed comprehensive bioinformatics analysis using the data from 17 pairs of gene expression array from AA and CA cohorts and identified 63 differentially expressed genes (DEGs) by race. Among them, chemokine receptor 4 (CXCR4), viz. p-Akt (Ser473), fatty acid synthase (FASN), interleukin-6 (IL-6) and matrix metallopeptidase 9 (MMP-9) gene expressions were significantly altered in AA groups. Immunohistochemistry was performed on 163 prostate cancer patient specimens to validate DEG expression and immunoreactive scores (IRS) were calculated. CXCR4 exhibited significantly higher expression in AA tumors, with > 85% of AA prostate tumors showing weak to strong CXCR4 expression, compared to < 5% in CA specimens. While immunostaining for IL-6, FASN, MMP9 and p-Akt did not provide a significant distinction between CA and AA cases. IRS analysis confirmed higher expression of CXCR4 in AA cancers, compared to CA prostate cancer (p < 0.001). MMP9 and IL6 expression were moderately higher in AA cancer compared to CA prostate cancer (p < 0.05). In contrast, FASN expression was significantly higher in CA cancer, compared to AA prostate cancer (p < 0.001). We further investigated the impact of these genes on biochemical recurrence-free survival (BCRFS) using Kaplan-Meier analysis to examine the differences between low-risk versus high-risk groups. BCRFS analysis demonstrated that CXCR4 is a determinant factor of recurrence risk in AA prostate cancer patients. The activation of the PI3K-Akt signaling pathway, particularly in AA patients, suggests its role in prostate cancer aggressiveness. Taken together, this study demonstrates the importance of differential expression of biomarkers which constitute the bulk tumors in driving cancer. The distinct gene expression patterns, especially the upregulation of CXCR4, in AA prostate cancer highlight its potential as a prognostic biomarker and a target for personalized treatment strategies. These findings encourage further study into the role of population-specific difference in adding CXCR4 in risk model construction, especially in machine learning models. Citation Format: Amel A. Ahmed, Shiv Verma, Ibrahim M. Atawia, Daniel L. Shen, Gregory T. MacLennan, Pingfu Fu, Sanjay Gupta. Differentially expressed genes as prognostic markers in racial disparities of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7645.

Genetic Signatures for Distinguishing Chemo-Sensitive from Chemo-Resistant Responders in Prostate Cancer Patients.

Prostate cancer remains a significant public health concern in sub-Saharan Africa, particularly impacting South Africa with high mortality rates. Despite many years of extensive research and significant financial expenditure, there has yet to be a definitive solution to prostate cancer. It is not just individuals who vary in their response to treatment, but even different nodules within the same tumor exhibit unique transcriptome patterns. These distinctions extend beyond mere differences in gene expression levels to encompass the control and networking of individual genes. Escalating chemotherapy resistance in prostate cancer patients has prompted increased research into its underlying mechanisms. The heterogeneous nature of transcriptomic organization among men makes the pursuit of universal biomarkers and one-size-fits-all treatments impractical. This study delves into the expression of drug resistance-associated genes, ABCB1 and CYP1B1, in cancer cells. Employing bioinformatics, we explored the molecular pathways and cascades linked to drug resistance following upregulation of these genes. Samples were obtained from archived prostate cancer patient specimens through pre-treatment biopsies of two categories: good vs. poor responders, with cDNAs synthesized from isolated RNAs subjected to qPCR analysis. The results revealed increased ABCB1 and CYP1B1 expression in tumor samples of the poor responders. Gene enrichment and network analysis associated ABCB1 with ABC transporters and LncRNA-mediated therapeutic resistance (WP3672), while CYP1B1 was linked to ovarian steroidogenesis, tryptophan metabolism, steroid hormone biosynthesis, benzo(a)pyrene metabolism, the sulindac metabolic pathway, and the estrogen receptor pathway, which are associated with drug resistance. Both ABCB1 and CYP1B1 correlated with microRNAs in cancer and the Nuclear Receptors Meta-Pathway. STRING analysis predicted protein-protein interactions of ABCB1 and CYP1B1 with Glutathione S-transferase Pi, Catechol O-methyltransferase, UDP-glucuronosyltransferase 1-6, Leucine-rich Transmembrane and O-methyltransferase (LRTOMT), and Epoxide hydrolase 1, with scores of 0.973, 0.971, 0.966, 0.966, and 0.966, respectively. Furthermore, molecular docking analysis of the chemotherapy drug, docetaxel, with CYP1B1 and ABCB1 revealed robust molecular interactions, with binding energies of -20.37 and -15.25 Kcal/mol, respectively. These findings underscore the susceptibility of cancer patients to drug resistance due to increased ABCB1 and CYP1B1 expression in tumor samples from patients in the poor-responders category that affects associated molecular pathways. The potent molecular interactions of ABCB1 and CYP1B1 with docetaxel further emphasize the potential basis for chemotherapy resistance.

Upregulation of TLR5 indicates a favorable prognosis in prostate cancer.

Toll-like receptors (TLRs) are the key sensors of innate immunity for triggering immune responses against infections. TLRs are well known to be expressed and activated in innate immune cells, such as macrophage and dendritic cells, but we and others have found that some TLRs are also functional in epithelial cells. However, the role of an epithelial TLR in prostate cancer remains elusive. TLR5 expression in messenger RNA and protein level in prostate cancer was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). The activation of TLR5 signaling in epithelial cells was detected upon nuclear factor-κB activation by luciferase assay and western blot analysis, and proinflammatory cytokine activation by RT-qPCR. Distinguishing between the TLR5 and NLRC4 pathways, both recognizing flagellin, is determined by small interfering RNA and proinflammatory cytokine activation. The role of TLR5 in prostate cancer was analyzed by IHC and bioinformatics using a general and single-cell database. In the present study, we show that TLR5, among other TLRs, is exceedingly expressed in human prostate cancer cells. This cancer epithelial cell TLR5 functions to activate the TLR5 signaling pathway in human prostate cancer cells, as it does with innate immune cell TLR5. The bacterial protein flagellin induces a robust immune response in prostate cancer cells in a TLR5-dependent but NLRC4-independent manner. TLR5 is highly expressed in prostate cancer patient specimens, and high TLR5 expression in prostate cancer patients indicates a favorable prognosis. TLR5, as an innate immunity receptor, is a functional TLR in human prostate cancer epithelial cells. TLR5 plays an important role in prostate cancer development and is a new potential prognosis biomarker. TLR5 may represent a novel immunotherapy target against prostate cancer.

SETDB1 Modulates Degradation of Phosphorylated RB and Anticancer Efficacy of CDK4/6 Inhibitors.

The identification of a role for TRIM28 and SETDB1 in regulating CDK4/6-phosphorylated RB stability uncovers a combination strategy using CDK4/6 and SETDB1 inhibition to decrease RB degradation and inhibit cancer growth.

Clinical and molecular significance of the RNA m6A methyltransferase complex in prostate cancer.

N6-methyladenosine (m6A) is the most abundant internal mRNA modification and is dynamically regulated through distinct protein complexes that methylate, demethylate, and/or interpret the m6A modification. These proteins, and the m6A modification, are involved in the regulation of gene expression, RNA stability, splicing and translation. Given its role in these crucial processes, m6A has been implicated in many diseases, including in cancer development and progression. Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in men and recent studies support a role for m6A in PCa. Despite this, the literature currently lacks an integrated analysis of the expression of key components of the m6A RNA methyltransferase complex, both in PCa patients and in well-established cell line models. For this reason, this study used immunohistochemistry and functional studies to investigate the mechanistic and clinical significance of the METTL3, METTL14, WTAP and CBLL1 components of the m6A methyltransferase complex in PCa specimens and cell lines. Expression of METTL3 and CBLL1, but not METTL14 and WTAP, was associated with poorer PCa patient outcomes. Expression of METTL3, METTL14, WTAP and CBLL1 was higher in PCa cells compared with non-malignant prostate cells, with the highest expression seen in castrate-sensitive, androgen-responsive PCa cells. Moreover, in PCa cell lines, expression of METTL3 and WTAP was found to be androgen-regulated. To investigate the mechanistic role(s) of the m6A methyltransferase complex in PCa cells, short hairpin RNA (shRNA)-mediated knockdown coupled with next generation sequencing was used to determine the transcriptome-wide roles of METTL3, the catalytic subunit of the m6A methyltransferase complex. Functional depletion of METTL3 resulted in upregulation of the androgen receptor (AR), together with 134 AR-regulated genes. METTL3 knockdown also resulted in altered splicing, and enrichment of cell cycle, DNA repair and metabolic pathways. Collectively, this study identified the functional and clinical significance of four essential m6A complex components in PCa patient specimens and cell lines for the first time. Further studies are now warranted to determine the potential therapeutic relevance of METTL3 inhibitors in development to treat leukaemia to benefit patients with PCa.

Abstract 401: Single-cell transcriptome and chromatin sequencing uncover gene expression and gene regulatory patterns associated with enzalutamide resistance

Abstract Resistance to androgen receptor-targeted therapy due to tumor heterogeneity and clonal evolution is a key challenge for improving prostate cancer outcomes. Despite this, the transcriptomic and chromatin accessibility changes contributing to the emergence of resistance remain incompletely understood at the level of individual cells. Using single-cell assays for transposase-accessible chromatin (ATAC) and RNA sequencing in models of early treatment response and resistance to enzalutamide, we previously identified pre-existing and persistent cell subpopulations that possess regenerative potential when subjected to treatment. Here we analyze the chromatin and transcriptomes of these single cells to characterize their gene regulation and gene expression trajectories. We present evidence of a model of enzalutamide resistance emergence in which the pre-existing and treatment-persistent cells regenerate the bulk of resistant cells. This process is underpinned by chromatin reprogramming that increases the overall relaxation of chromatin upon resistance. We show that the reprogramming of the chromatin further differentially contributes to transcription factor-mediated transcriptional reprogramming via DNA motif exposure in different cell subpopulations. For example, in the treatment-persistent cells, we identify chromatin configurations characterized by the exposure of DNA motifs for GATA2, RELA (a NFkB subunit), CREB1, and E2F1. Pre-existing and treatment-persistent cells consistently display transcriptional features of high developmental potential and RNA velocity analysis identifies them as precursors of cell populations that arise from enzalutamide treatment. We also analyze the pre-existing and treatment-persistent cells in spatial transcriptomics of prostate cancer patient specimens based on their characteristic gene expression profiles. We find these cells to be enriched in cancerous regions of the tissue but also detect them within apparent benign regions, which has potential implications for treatment choice. In summary, we show patterns of gene expression regulation in preclinical models and patient samples that uncover mechanisms of resistance to androgen receptor-targeted therapy in prostate cancer. Citation Format: Sinja Taavitsainen, Nikolai Engedal, Shaolong Cao, Florian Handle, Andrew Erickson, Stefan Prekovic, Daniel Wetterskog, Teemu Tolonen, Elisa M. Vuorinen, Antti Kiviaho, Reetta Nätkin, Tomi Häkkinen, Wout Devlies, Sallamari Henttinen, Roosa Kaarijärvi, Mari Lahnalampi, Heidi Kaljunen, Karolina Nowakowska, Heimo Syvälä, Merja Bläuer, Paolo Cremaschi, Frank Claessens, Tapio Visakorpi, Teuvo L. Tammela, Teemu Murtola, Kirsi J. Granberg, Alastair D. Lamb, Kirsi Ketola, Ian G. Mills, Gerhardt Attard, Wenyi Wang, Matti Nykter, Alfonso Urbanucci. Single-cell transcriptome and chromatin sequencing uncover gene expression and gene regulatory patterns associated with enzalutamide resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 401.

Peroxiredoxin IV plays a critical role in cancer cell growth and radioresistance through the activation of the Akt/GSK3 signaling pathways

High levels of redox enzymes have been commonly observed in various types of human cancer, although whether and how the enzymes contribute to cancer malignancy and therapeutic resistance have yet to be understood. Peroxiredoxin IV (Prx4) is an antioxidant with bona fide peroxidase and molecular chaperone functions. Here, we report that Prx4 is highly expressed in prostate cancer patient specimens, as well as established prostate cancer cell lines, and that its levels can be further stimulated through the activation of androgen receptor signaling. We used lentivirus-mediated shRNA knockdown and CRISPR-Cas9 based KO techniques to establish Prx4-depleted prostate cancer cells, which showed delayed cell cycle progression, reduced rate of cell proliferation, migration, and invasion compared to control cells. In addition, we used proteome profiler phosphokinase arrays to identify signaling changes in Prx4-depleted cells; we found that loss of Prx4 results in insufficient phosphorylation of both Akt and its downstream kinase GSK3α/β. Moreover, we demonstrate that Prx4-depleted cells are more sensitive to ionizing radiation as they display compromised ability to scavenge reactive oxygen species and increased accumulation of DNA damage. In mouse xenograft models, we show depletion of Prx4 leads to significant suppression of tumor growth, and tumors formed by Prx4-depleted cells respond more effectively to radiation therapy. Our findings suggest that increased levels of Prx4 contribute to the malignancy and radioresistance of prostate cancer through the activation of Akt/GSK3 signaling pathways. Therefore, strategies targeting Prx4 may be utilized to potentially inhibit tumor growth and overcome radioresistance in prostate cancer.

SU086, an inhibitor of HSP90, impairs glycolysis and represents a treatment strategy for advanced prostate cancer

SummaryAmong men, prostate cancer is the second leading cause of cancer-associated mortality, with advanced disease remaining a major clinical challenge. We describe a small molecule, SU086, as a therapeutic strategy for advanced prostate cancer. We demonstrate that SU086 inhibits the growth of prostate cancer cells in vitro, cell-line and patient-derived xenografts in vivo, and ex vivo prostate cancer patient specimens. Furthermore, SU086 in combination with standard of care second-generation anti-androgen therapies displays increased impairment of prostate cancer cell and tumor growth in vitro and in vivo. Cellular thermal shift assay reveals that SU086 binds to heat shock protein 90 (HSP90) and leads to a decrease in HSP90 levels. Proteomic profiling demonstrates that SU086 binds to and decreases HSP90. Metabolomic profiling reveals that SU086 leads to perturbation of glycolysis. Our study identifies SU086 as a treatment for advanced prostate cancer as a single agent or when combined with second-generation anti-androgens.

MAP3K7-IKK Inflammatory Signaling Modulates AR Protein Degradation and Prostate Cancer Progression

Androgen receptor (AR) is a major survival factor for prostate cancer. Inflammation is implicated in many cancer types, including prostate cancer. Activation of MAP3K7 (also termed TAK1) and downstream IκB kinase β (IKKβ) by proinflammatory cytokines such as TNFα stimulates NF-κB survival pathways. Paradoxically, MAP3K7 is often deleted in human prostate cancer. Here, we demonstrate that AR protein expression is lower in inflammatory tumor areas compared with non-inflammatory tissues in patients with prostate cancer. Map3k7 knockout increased AR protein levels and activity in the mouse prostate, and MAP3K7 and AR protein levels were inversely correlated in prostate cancer patient specimens. TNFα treatment increased AR protein ubiquitination and proteasomal degradation. Mechanistically, activation of IKKβ by TNFα induced phosphorylation and TRCP1/2 E3 ligase-mediated polyubiquitination and degradation of AR protein. TNFα suppressed prostate cancer proliferation, which could be rescued by blockade of AR degradation. These findings reveal a previously unrecognized tumor suppressive function of the inflammation-activated MAP3K7-IKKβ axis in degrading AR protein. Moreover, they suggest that aberrant elevation of AR protein could be a prognostic biomarker and therapeutic target for MAP3K7-deficient prostate cancer. SIGNIFICANCE: This study identifies that MAP3K7-IKKβ signaling plays a tumor-suppressive role in prostate cancer by degrading AR, revealing potential prognostic and therapeutic strategies for MAP3K7-deficient tumors.

Mast Cell-Derived SAMD14 Is a Novel Regulator of the Human Prostate Tumor Microenvironment.

Mast cells (MCs) are important cellular components of the tumor microenvironment and are significantly associated with poor patient outcomes in prostate cancer and other solid cancers. The promotion of tumor progression partly involves heterotypic interactions between MCs and cancer-associated fibroblasts (CAFs), which combine to potentiate a pro-tumor extracellular matrix and promote epithelial cell invasion and migration. Thus far, the interactions between MCs and CAFs remain poorly understood. To identify molecular changes that may alter resident MC function in the prostate tumor microenvironment, we profiled the transcriptome of human prostate MCs isolated from patient-matched non-tumor and tumor-associated regions of fresh radical prostatectomy tissue. Transcriptomic profiling revealed a distinct gene expression profile of MCs isolated from prostate tumor regions, including the downregulation of SAMD14, a putative tumor suppressor gene. Proteomic profiling revealed that overexpression of SAMD14 in HMC-1 altered the secretion of proteins associated with immune regulation and extracellular matrix processes. To assess MC biological function within a model of the prostate tumor microenvironment, HMC-1-SAMD14+ conditioned media was added to co-cultures of primary prostatic CAFs and prostate epithelium. HMC-1-SAMD14+ secretions were shown to reduce the deposition and alignment of matrix produced by CAFs and suppress pro-tumorigenic prostate epithelial morphology. Overall, our data present the first profile of human MCs derived from prostate cancer patient specimens and identifies MC-derived SAMD14 as an important mediator of MC phenotype and function within the prostate tumor microenvironment.

HOXD13 suppresses prostate cancer metastasis and BMP4-induced epithelial-mesenchymal transition by inhibiting SMAD1.

The HOX genes are a group of highly conserved Homeobox-containing genes that control the body plan organization during development. However, their contributions to tumorigenesis and tumor progression remain uncertain and controversial. Here we provided evidence of tumor-suppressive activity of HOXD13 in prostate cancer. HOXD13 depletion contributes to more aggressiveness of prostate cancer cells in vitro and in vivo. These effects were corroborated in a metastatic mice model, where we observed more bone metastatic lesions formed by prostate cancer cells with HOXD13 ablation. Mechanistically, HOXD13 prevents BMP4-induced epithelial-mesenchymal transition (EMT) by inhibiting mothers against decapentaplegic homolog 1 (SMAD1) transcription. Both bioinformation and our tissue microarray cohort data show that HOXD13 expression inversely correlated in advanced prostate cancer patient specimens. Our findings establish HOXD13 as a negative regulator of prostate cancer progression and metastasis by preventing BMP4/SMAD1 signaling, and potentially suggest new strategies for targeting metastatic prostate cancer.

SU086, a New Inhibitor of HSP90, Impairs Glycolysis and Represents a New Treatment Strategy for Advanced Prostate Cancer

Among men, prostate cancer is the second leading cause of cancer-associated mortality, with advanced disease remaining a major clinical challenge. We describe a small molecule, SU086, as a new therapeutic strategy for advanced prostate cancer. We demonstrate that SU086 inhibits the growth of prostate cancer cells in vitro, cell line and patient-derived xenografts in vivo, and ex vivo prostate cancer patient specimens. Furthermore, SU086 in combination with standard of care second-generation anti-androgen therapies displayed increased impairment of prostate cancer cell and tumor growth in vitro and in vivo. Cellular thermal shift assay (CETSA) revealed that SU086 binds to heat shock protein 90 (HSP90) and leads to a decrease in HSP90 levels. Proteomic and metabolomic profiling further revealed that the SU086 decreases HSP90 and leads to perturbation of glycolysis. Our study identifies SU086 as a new treatment for advanced prostate cancer as a single agent or when combined with second-generation anti-androgens.

Abstract 761: Up-regulation of miR-10a affect on prostate cancer racial disparity

Abstract Purpose: Prostate cancer (PC) is the second most diagnosed cancer in men. African-American (AfA) men have higher incidence and twice the PC mortality rates compared to Caucasian-American (CaA) men. In this study, we investigated the biochemical role of miR-10a in prostate cancer racial disparity. Methods: We used PCa cell lines from CaA (DU145, LNCaP, PC3), AfA (MDA-PCa-2b, E006AA-hT) and a normal epithelial cell line (PWR1E) to examine miR-10a expression levels. Total RNA was extracted from total 138 human prostate cancer clinical samples (AfA: n=75, CaA: n=63) and cell lines. Gene expression levels were determined by quantitative real-time PCR (qPCR). A miRNA target database (miRWalk that integrates mainly three databases, miRDB, TargetScan and miRTarBase) was used for miR-10a target prediction. Cell proliferation and apoptosis were monitored after miR-10a knockdown by using anti-miR-10a. Results: miR-10a was significantly up-regulated in AfA prostate cancer cell line, E006AA-hT compared to CaA cells. Utilizing SFVAMC and NDRI patient cohorts, we confirmed that miR-10a expression was linked to a racial difference between AfA/CaA PCa patients. Knockdown of miR-10a showed decreased growth and apoptosis, though the effect was less in CaA compared to AfA cells. We found NCOR2 which is frequently deleted in castration- resistant prostate cancer patient specimens and tumor-suppressor PTEN as miR-10a targets that overlapped in multiple miRNA target prediction databases. Conclusion: These results demonstrate that miR-10a may be a central regulator of crucial events that contribute to racial differences in prostate cancer. Regulation of miR-10a expression may be a new therapeutic strategy for AfA prostate cancer patients. Citation Format: Yutaka Hashimoto, Marisa Shiina, Yuichiro Tanaka, Pritha Dasgupta, Priyanka Kulkarni, Taku Kato, Ryan K. Wong, Varahram Shahryari, Shigekatsu Maekawa, Soichiro Yamamura, Divya Bhagiratha, Sharanjot Saini, Guoren Deng, Laura Tabatabai, Shahana Majid, Rajvir Dahiya, Rajvir Dahiya. Up-regulation of miR-10a affect on prostate cancer racial disparity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 761.

Overcoming EZH2 Inhibitor Resistance by Taxane in PTEN-Mutated Cancer.

Rationale: The Polycomb group (PcG) protein EZH2 is implicated in cancer progression due to its frequent overexpression in many cancer types and therefore is a promising therapeutic target. Forkhead box transcription factor-1 (FOXO1) is a tumor suppressor that is often transcriptionally downregulated in human cancers such as prostate cancer although the underlying regulatory mechanisms remain elusive.Methods: Analysis of EZH2 ChIP-seq and ChIP-on-chip data in various cell types was performed. ChIP-qPCR, RT-qPCR, and western blot analyses were conducted to determine the mechanism by which EZH2 represses FOXO1 expression. Immunohistochemistry was employed to assess the correlation between EZH2 and FOXO1 protein expression in prostate cancer patient specimens. In vitro MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and animal experiments were performed to determine the anti-cancer efficacy of EZH2 inhibitor alone or in combination of docetaxel, a chemotherapy agent of the taxane family, and dependency of the efficacy on FOXO1 expression.Results: We demonstrated that EZH2 binds to the FOXO1 gene promoter. EZH2 represses FOXO1 gene expression at the transcriptional level. EZH2 protein level inversely correlated with FOXO1 protein expression in prostate cancer patient specimens. This repression requires the methyltransferase activity and the functional PRC2 complex. While effectively inducing loss of viability of PTEN-positive 22Rv1 prostate cancer cells, EZH2 inhibitor failed to inhibit growth of PTEN-negative C4-2 prostate cancer cells. Co-treatment with docetaxel overcame EZH2 inhibitor resistance in PTEN-negative cancer cells in vitro and in mice. This effect was largely mediated by docetaxel-induced nuclear localization and activation of FOXO1.Conclusions: This study identifies FOXO1 as a bona fide repression target of EZH2 and an essential mediator of EZH2 inhibition-induced cell death. Our findings suggest that EZH2 repression of FOXO1 can be targeted by EZH2 inhibitor as a monotherapy for PTEN-proficient cancers or in combination with taxane for treatment of cancers with PTEN mutation or deletion.

RUNX2 overexpression and PTEN haploinsufficiency cooperate to promote CXCR7 expression and cellular trafficking, AKT hyperactivation and prostate tumorigenesis.

Rationale: The overall success rate of prostate cancer (PCa) diagnosis and therapy has been improved over the years. However, genomic and phenotypic heterogeneity remains a major challenge for effective detection and treatment of PCa. Efforts to better classify PCa into functional subtypes and elucidate the molecular mechanisms underlying prostate tumorigenesis and therapy resistance are warranted for further improvement of PCa outcomes.Methods: We generated Cre+;Runx2-cTg;Ptenp/+ (Runx2-Pten double mutant) mice by crossbreeding Cre+;Runx2-cTg males with Pten conditional (Ptenp/p) females. By using Hematoxylin and Eosin (H&E) staining, SMA and Masson's Trichrome staining, we investigated the effect of PTEN haploinsufficiency in combination with Runx2 overexpression on prostate tumorigenesis. Moreover, we employed immunohistochemistry (IHC) to stain Ki67 for cell proliferation, cleaved caspase 3 for apoptosis and AKT phosphorylation for signaling pathway in prostate tissues. Chromatin immunoprecipitation coupled quantitative PCR (ChIP-qPCR), reverse transcription coupled quantitative PCR (RT-qPCR), western blot (WB) analyses and immunofluorescence (IF) were conducted to determine the underlying mechanism by which RUNX2 regulates CXCR7 and AKT phosphorylation in PCa cells.Results: We demonstrated that mice with prostate-specific Pten heterozygous deletion and Runx2 overexpression developed high-grade prostatic intraepithelial neoplasia (HGPIN) and cancerous lesions at age younger than one year, with concomitant high level expression of Akt phosphorylation and the chemokine receptor Cxcr7 in malignant glands. RUNX2 overexpression induced CXCR7 transcription and membrane location and AKT phosphorylation in PTEN-deficient human PCa cell lines. Increased expression of RUNX2 also promoted growth of PCa cells and this effect was largely mediated by CXCR7. CXCR7 expression also positively correlated with AKT phosphorylation in PCa patient specimens.Conclusions: Our results reveal a previously unidentified cooperative role of RUNX2 overexpression and PTEN haploinsufficiency in prostate tumorigenesis, suggesting that the defined RUNX2-CXCR7-AKT axis can be a viable target for effective treatment of PCa.

The Role of Alcohol-Induced Golgi Fragmentation for Androgen Receptor Signaling in Prostate Cancer.

Multiple epidemiologic observations and meta-analysis clearly indicate the link between alcohol abuse and the incidence and progression of prostate cancer; however, the mechanism remains enigmatic. Recently, it was found that ethanol (EtOH) induces disorganization of the Golgi complex caused by impaired function of the largest Golgi matrix protein, giantin (GOLGB1), which, in turn, alters the Golgi docking of resident Golgi proteins. Here, it is determined that in normal prostate cells, histone deacetylase 6 (HDAC6), the known regulator of androgen receptor (AR) signaling, localizes in the cytoplasm and nucleus, while its kinase, glycogen synthase kinase β (GSK3β), primarily resides in the Golgi. Progression of prostate cancer is accompanied by Golgi scattering, translocation of GSK3β from the Golgi to the cytoplasm, and the cytoplasmic shift in HDAC6 localization. Alcohol dehydrogenase-generated metabolites induces Golgi disorganization in androgen-responsive LNCaP and 22Rv1 cells, facilitates tumor growth in a mouse xenograft model and activates anchorage-independent proliferation, migration, and cell adhesion. EtOH-treated cells demonstrate reduced giantin and subsequent cytoplasmic GSK3β; this phenomenon was validated in giantin-depleted cells. Redistribution of GSK3β to the cytoplasm results in phosphorylation of HDAC6 and its retention in the cytoplasm, which, in turn, stimulates deacetylation of HSP90, AR import into the nucleus, and secretion of prostate-specific antigen (PSA). Finally, the relationship between Golgi morphology, HDAC6 cytoplasmic content, and clinicopathologic features was assessed in human prostate cancer patient specimens with and without a history of alcohol dependence. IMPLICATIONS: This study demonstrates the importance of alcohol-induced Golgi fragmentation in the activation of AR-mediated proliferation.

Abstract A081: Activation of sphingosine kinase 1 by lipopolysaccharide promotes prostate cancer cell invasion via matriptase

Abstract A 15-year cohort study indicates that bacterial-induced prostatitis is correlated to prostate cancer (PCa) incidence. Several gram-negative bacteria have been identified in PCa patient specimens and inflammatory cytokines derived from bacterial infection have been involved in PCa tumorigenesis. However, the direct effect of lipopolysaccharide (LPS), a potent inflammatory substance from gram-negative bacteria, on PCa development is not well studied. In this study, we examine the effect of LPS on the growth (MTT assay) and invasion (Transwell assay) of several PCa cells lines including LNCaP, PC3, and DU145. Although LPS does not change the cell growth, our results reveal that LPS can enhance the PCa cell invasiveness and increase pericellular proteolytic activities. Using protease inhibitor screening and proteolytic analyses, we have identified that matriptase (MTX), a member of the membrane-anchored serine protease family, plays a key role in LPS-induced PCa cell invasiveness. The activated levels of MTX can be detected in the cells and the conditioned medium (CM) from LPS-treated cells. Indeed, the elevation of MTX expression is associated with high-grade PCa in archival specimens. Knowing LPS as a ligand for toll-like receptor 4 (TLR4), specific antagonists and shRNA knockdown methodology were used to delineate the mechanism by which TLR4/sphingosine kinase 1 (SphK1) signaling cascade plays a role in LPS-induced PCa cell invasion. The results show that SphK1 was important for MTX activation and PCa cell invasion. Moreover, we found that LPS could induce the activation of SphK1 based on an elevated phosphorylation level and SphK1 translocation to plasma membrane for the production of sphingosine 1-phosphate (S1P). S1P can subsequently induce MTX activation and PCa cell invasion through S1P receptor 4 (S1PR4) in a paracrine or autocrine manner. S1PR4 is further identified as an important receptor for S1P to mediate MAPK signaling. Furthermore, there is a significant correlation between the elevated expression of SphK1 and the Gleason scores of PCa specimens. The phenomenon was further corroborated by the fact that LPS could induce SphK1 activation in patient-derived explant model (PDeX). Taken together, we have unveiled a novel LPS-elicited pathway leading to PCa cell invasion. Our results provide not only the potential impact of bacterial infection on PCa progression but also several therapeutic target(s) against PCa metastasis. Citation Format: Cheng-Fan Lee, Benjamin Chen, Jer-Tsong Hsieh, Ming-Shyue Lee. Activation of sphingosine kinase 1 by lipopolysaccharide promotes prostate cancer cell invasion via matriptase [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 A081.

DUB3 Promotes BET Inhibitor Resistance and Cancer Progression by Deubiquitinating BRD4

The bromodomain and extra-terminal domain (BET) protein BRD4 is emerging as a promising anticancer therapeutic target. However, resistance to BET inhibitors often occurs, and it has been linked to aberrant degradation of BRD4 protein in cancer. Here, we demonstrate that the deubiquitinase DUB3 binds toBRD4 and promotes its deubiquitination and stabilization. Expression of DUB3 is transcriptionally repressed by the NCOR2-HDAC10 complex. The NCOR2 gene is frequently deleted in castration-resistant prostate cancer patient specimens, and loss of NCOR2 induces elevation of DUB3 and BRD4 proteins in cancer cells. DUB3-proficient prostate cancer cells are resistant to the BET inhibitor JQ1 invitro and in mice, but this effect is diminished by DUB3 inhibitory agents such as CDK4/6 inhibitorin a RB-independent manner. Our findings identify a previously unrecognized mechanism causing BRD4 upregulation and drug resistance, suggesting that DUB3 is a viable therapeutic target to overcome BET inhibitor resistance in cancer.

Abstract C42: Alternative splicing of FGFR3 as a mechanism for African American prostate cancer health disparities

Abstract Introduction: In this study, we investigate the role of differential expression of splicing factors and exon skipping in the receptor tyrosine kinase family on prostate cancer health disparities in African American men. Background: Prostate cancer (PCa) is the most diagnosed cancer in men and the second leading cause of male-cancer related deaths in the U.S. Dramatic ethnic disparities have been observed in PCa patients, as African American (AA) men are 60% more likely to be diagnosed with PCa and have a 2.4 fold higher mortality rate compared to European American (EA) men. Increasing evidence suggests that, after accounting for epidemiological factors, a remaining component of this disparity is due to intrinsic genetic and biological factors. Interestingly, recent exon array data from our lab suggest that differential expression of splicing factors (SFs) and differential alternative splicing may be occurring in AA PCa. We hypothesize that differential alternative splicing involving exon 14 of the FGFR3 gene is generating a shorter, more oncogenic variant in AA PCa, which is absent or weakly expressed in EA PCa. Differential splicing of FGFR3 and increased expression of SFs in AA patients may be mechanisms contributing to AA PCa health disparities. Results: Exon array data suggested FGFR3 as a candidate for differential alternative splicing. Exon profiling and RT-PCR validated enriched expression of a short variant of FGFR3 due to skipping of exon 14 in AA patient samples and cell lines. Cloning confirmed the presence of the FGFR3-L variant (containing exon 14) and the FGFR3-S variant (without exon 14) from an EA and AA PCa cell line, respectively. Enrichment of FGFR3-S resulted in increased cell proliferation in an AA cell line. RNA-seq data analysis suggests decreased survival of PCa patients with high FGFR3-S/-L expression ratios. Additionally, our exon array data predicted increased expression of seven SFs in AA patients. RT-PCR and IHC analysis validated increased expression in AA specimens. Knockdown of these SFs resulted in decreased invasion and FGFR3 splice switching in an AA cell line. Conclusions: We have identified an oncogene of interest, FGFR3, which undergoes exon skipping that is specific to AA PCa. In cell lines, this shorter isoform of FGFR3 leads to an increased oncogenic phenotype based on proliferation assays. We have shown that AA PCa patient specimens have increased expression of specific SFs compared to EA specimens and knockdown of SFs reduces AA PCa cell invasion and causes splice switching of FGFR3. Thus, differential expression of SFs and exon skipping in FGFR3 may be one mechanism contributing to the increased aggressiveness of PCa in AA patients. Citation Format: Jacqueline Olender, Bi-Dar Wang, Norman H. Lee. Alternative splicing of FGFR3 as a mechanism for African American prostate cancer health disparities. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr C42.

The lncRNA PCAT29 inhibits oncogenic phenotypes in prostate cancer.

Long noncoding RNAs (lncRNA) have recently been associated with the development and progression of a variety of human cancers. However, to date, the interplay between known oncogenic or tumor-suppressive events and lncRNAs has not been well described. Here, the novel lncRNA, prostate cancer-associated transcript 29 (PCAT29), is characterized along with its relationship to the androgen receptor. PCAT29 is suppressed by DHT and upregulated upon castration therapy in a prostate cancer xenograft model. PCAT29 knockdown significantly increased proliferation and migration of prostate cancer cells, whereas PCAT29 overexpression conferred the opposite effect and suppressed growth and metastases of prostate tumors in chick chorioallantoic membrane assays. Finally, in prostate cancer patient specimens, low PCAT29 expression correlated with poor prognostic outcomes. Taken together, these data expose PCAT29 as an androgen-regulated tumor suppressor in prostate cancer. This study identifies PCAT29 as the first androgen receptor-repressed lncRNA that functions as a tumor suppressor and that its loss may identify a subset of patients at higher risk for disease recurrence. Visual Overview: http://mcr.aacrjournals.org/content/early/2014/07/31/1541-7786.MCR-14-0257/F1.large.jpg.