Sustained androgen receptor (AR) signaling during relapse is a central driver of metastatic castration- resistant prostate cancer (mCRPC). Current AR antagonists, such as enzalutamide, fail to provide long- term benefit for the mCRPC patients who have dramatic increases in AR expression. Here, we report AR antagonists with efficacy in AR- overexpressing models. These molecules bind to the ligand- binding domain of the AR, promote AR localization to the nucleus, yet potently and selectively down- regulate AR- target genes. The molecules BG-15a and the pharmacokinetically optimized BG-15n elicit a decrease in cell and tumor growth in vitro and in vivo in models of mCRPC. BG-15a/n treatment causes the collapse of chromatin loops between enhancers and promoters at key genes in the AR- driven epigenome. AR binding in the promoter, as well as 3D chromatin clustering, is needed for genes to respond. BG-15a/n represent promising agents for treating patients with relapsed AR- driven mCRPC tumors.

Flavanols are metabolized by the gut microbiota to bioavailable metabolites, and the absorbed fraction is excreted primarily via urine. Uroepithelial cells are thus a potential site of activity due to exposure to high concentrations of these compounds. Chemoprevention by flavanols may be partly due to these metabolites. In Vitro work in this area relies on a limited pool of commercially available microbial metabolites, and little has been done in bladder cancer. The impact of physiologically relevant mixtures of flavanols and their metabolites remains unknown. Rats were fed various flavanols and urine samples, approximating the bioavailable metabolome, were collected. Urines were profiled by UPLC-MS/MS, and their anti-proliferative activities were assayed In Vitro in four bladder cancer models. Significant interindividual variability was observed for composition and proliferation. Microbial metabolite concentrations (valerolactones, phenylalkyl acids and hippuric acids) were positively associated with reduced bladder cancer proliferation In Vitro, while native flavanols were poorly correlated with activity. These results suggest that microbial metabolites may be responsible for chemoprevention in uroepithelial cells following flavanol consumption. This highlights the potential to use individual genetics and microbial metabotyping to design personalized dietary interventions for cancer prevention and/or adjuvant therapy to reduce bladder cancer incidence and improve outcomes.

Castration-resistant prostate cancer represents a continuum of phenotypes, including tumors with high levels of androgen receptor (AR) expression and activity and those which do not express AR and rely on alternative pathways for survival. The process by which AR-positive prostate cancer cells and tumors lose AR expression and acquire neuroendocrine features is referred to as neuroendocrine differentiation. Numerous therapies and exposures have been demonstrated to induce neuroendocrine differentiation in vitro , including the pro-inflammatory cytokine, interleukin 1 beta (IL-1β), encoded by the gene IL1B . The purpose of our studies was to determine the relationship between the expression and activity of AR in relationship to IL-1β and IL1B in prostate cancer. We performed analysis of de-identified human clinical data and generated prostate cancer cell lines with overexpression or knockout of IL1B . In primary prostate cancer, higher expression of IL1B predicts longer time to biochemical recurrence. In metastatic castration-resistant prostate cancer, IL1B expression is decreased and inversely correlates with AR and AR-target gene expression and AR activity, while positively correlating with the neuroendocrine prostate cancer (NEPC) score and neuroendocrine marker gene expression. In vitro , we report that AR-positive castration-resistant prostate cancer cells (C4-2B, 22Rv1) secrete IL-1β, and knockout of IL1B in these cells results in increased AR activity, in the presence and absence of dihydrotestosterone (DHT). Importantly, knockout of IL1B prevented AR attrition during androgen-deprivation. Taken together, our studies demonstrate that loss of IL1B in AR-positive castration-resistant prostate cancer cells can increase and maintain AR activity in the absence of androgens, suggesting another potential mechanism of high AR activity in castration-resistant prostate cancer.

Abstract Castration-resistant prostate cancer can be treated with the antiandrogen enzalutamide, but responses and duration of response are variable. To identify genes that support enzalutamide resistance, we performed a short hairpin RNA (shRNA) screen in the bone-homing, castration-resistant prostate cancer cell line, C4-2B. We identified 11 genes (TFAP2C, CAD, SPDEF, EIF6, GABRG2, CDC37, PSMD12, COL5A2, AR, MAP3K11, and ACAT1) whose loss resulted in decreased cell survival in response to enzalutamide. To validate our screen, we performed transient knockdowns in C4-2B and 22Rv1 cells and evaluated cell survival in response to enzalutamide. Through these studies, we validated three genes (ACAT1, MAP3K11, and PSMD12) as supporters of enzalutamide resistance in vitro. Although ACAT1 expression is lower in metastatic castration-resistant prostate cancer samples versus primary prostate cancer samples, knockdown of ACAT1 was sufficient to reduce cell survival in C4-2B and 22Rv1 cells. MAP3K11 expression increases with Gleason grade, and the highest expression is observed in metastatic castration-resistant disease. Knockdown of MAP3K11 reduced cell survival, and pharmacologic inhibition of MAP3K11 with CEP-1347 in combination with enzalutamide resulted in a dramatic increase in cell death. This was associated with decreased phosphorylation of AR-Serine650, which is required for maximal AR activation. Finally, although PSMD12 expression did not change during disease progression, knockdown of PSMD12 resulted in decreased AR and AR splice variant expression, likely contributing to the C4-2B and 22Rv1 decrease in cell survival. Our study has therefore identified at least three new supporters of enzalutamide resistance in castration-resistant prostate cancer cells in vitro.

Background Most prostate cancers express androgen receptor (AR), and our previous studies have focused on identifying transcription factors that modify AR function. We have shown that nuclear factor I/B (NFIB) regulates AR activity in androgen-dependent prostate cancer cells in vitro. However, the status of NFIB in prostate cancer was unknown. Methods We immunostained a tissue microarray including normal, hyperplastic, prostatic intraepithelial neoplasia, primary prostatic adenocarcinoma, and castration-resistant prostate cancer tissue samples for NFIB, AR, and synaptophysin, a marker of neuroendocrine differentiation. We interrogated publically available data sets in cBioPortal to correlateNFIBexpression and AR and neuroendocrine prostate cancer (NEPCa) activity scores. We analyzed prostate cancer cell lines for NFIB expression via Western blot analysis and used nuclear and cytoplasmic fractionation to assess where NFIB is localized. We performed co-immunoprecipitation studies to determine if NFIB and AR interact. Results NFIB increased in the nucleus and cytoplasm of prostate cancer samples versus matched normal controls, independent of Gleason score. Similarly, cytoplasmic AR and synaptophysin increased in primary prostate cancer. We observed strong NFIB staining in primary small cell prostate cancer. The ratio of cytoplasmic-to-nuclear NFIB staining was predictive of earlier biochemical recurrence in prostate cancer, once adjusted for tumor margin status. Cytoplasmic AR was an independent predictor of biochemical recurrence. There was no statistically significant difference between NFIB and synaptophysin expression in primary and castration-resistant prostate cancer, but cytoplasmic AR expression was increased in castration-resistant samples. In primary prostate cancer, nuclear NFIB expression correlated with cytoplasmic NFIB and nuclear AR, while cytoplasmic NFIB correlated with synaptophysin, and nuclear and cytoplasmic AR. In castration-resistant prostate cancer samples,NFIBexpression correlated positively with an AR activity score, and negatively with the NEPCa score. In prostate cancer cell lines, NFIB exists in several isoforms. We observed NFIB predominantly in the nuclear fraction of prostate cancer cells with increased cytoplasmic expression seen in castration-resistant cell lines. We observed an interaction between AR and NFIB through co-immunoprecipitation experiments. Conclusion We have described the expression pattern of NFIB in primary and castration-resistant prostate cancer and its positive correlation with AR. We have also demonstrated AR interacts with NFIB.

Background Male lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often use LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a pro-inflammatory and pro-fibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to pro-inflammatory stimuli and identified downstream targets of OPN in prostate stromal cells. Methods Immunohistochemistry was performed on prostate sections obtained from the transition zone (TZ) of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS i.e. surgical BPH (S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging system and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot. The ability of prostate cells to secrete osteopontin in response to IL-1β and TGF-β1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by ELISA. qPCR was used to measure gene expression changes in these cells in response to OPN. Results OPN immunostaining (p=0.0107) and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-β1 stimulated OPN secretion by NHPrE-1 cells and both IL-1β and TGF-β1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression of CXCL1 , CXCL2 , CXCL8 , PTGS2 and IL6 in BHPrS-1, but not in epithelial cell lines. Conclusions OPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by pro-inflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of pro-inflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (MP06)1 Apr 2020MP06-06 INCREASED EXPRESSION OF OSTEOPONTIN IN THE PROSTATE IS ASSOCIATED WITH THE CLINICAL PROGRESSION OF BPH Petra Popovics*, Wisam N. Awadallah, Sarah Kohrt, Thomas C. Case, Nicole L. Miller, Emily Ricke, Wei Huang, Marisol Ramirez-Solano, Qi Liu, Robert J. Matusik, William A. Ricke, and Magdalena M. Grabowska Petra Popovics*Petra Popovics* More articles by this author , Wisam N. AwadallahWisam N. Awadallah More articles by this author , Sarah KohrtSarah Kohrt More articles by this author , Thomas C. CaseThomas C. Case More articles by this author , Nicole L. MillerNicole L. Miller More articles by this author , Emily RickeEmily Ricke More articles by this author , Wei HuangWei Huang More articles by this author , Marisol Ramirez-SolanoMarisol Ramirez-Solano More articles by this author , Qi LiuQi Liu More articles by this author , Robert J. MatusikRobert J. Matusik More articles by this author , William A. RickeWilliam A. Ricke More articles by this author , and Magdalena M. GrabowskaMagdalena M. Grabowska More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000820.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Approximately 30% of men with lower urinary tract symptoms (LUTS) are resistant to medical therapies and progress to surgical intervention. The pathological changes producing LUTS include prostatic proliferation identified histologically as benign prostatic hyperplasia (BPH). Fibrogenic and inflammatory processes are also implicated in LUTS. Identification of new molecular pathways that provoke inflammatory responses and fibrosis, which are not targeted by current therapies, are vital to improve treatments of LUTS. Our study compared the expression of osteopontin (OPN), a pro-inflammatory and fibrotic molecule, in the prostate of patients with BPH characterized as incidental BPH, or as progressed to surgery. We also identified stimulatory signals contributing to the prostatic expression of OPN, as well as, genes that are activated in response to its elevated levels in vitro. METHODS: Immunohistochemistry was performed on prostate sections obtained from patients who had either undergone surgery to relieve LUTS (S-BPH, n=30) or had incidental BPH (I-BPH). I-BPH refers to specimens isolated from the transition zone of prostates after radical prostatectomy for low grade prostate cancer (n=8). Stained slides were captured with a Nuance Multispectral Imaging system and scored with inForm software. Protein level of OPN was determined by Western blot. The ability of prostate cells to secrete osteopontin in response to IL-1β and TGF-β1 and the expressional changes in genes regulated by OPN were determined in stromal (BHPrS-1) and epithelial (NHPrE-1) cell lines by ELISA and qPCR, respectively. RESULTS: The S-BPH group had significantly elevated H-score (p=0.0107, Mann-Whitney U test) and increased protein levels of OPN. OPN expression showed a widespread tissue distribution with a visibly higher level in glandular cells. We found that multiple splice variants of OPN are expressed in stromal and epithelial cells and its secretion is stimulated by TGF-β1 in NHPrE-1 and IL-1β and TGF-β1 in BHPrS-1 cells. Interestingly, we observed an increase in the expression of inflammatory genes in response to OPN including CXCL1, CXCL2, CXCL8, PTGS2 and IL6 in BHPrS-1, but this effect was not replicated in epithelial cell lines. CONCLUSIONS: Increased OPN levels are associated with the progression of BPH which appears to be related to inflammatory processes since OPN secretion by prostate cells is stimulated by cytokines. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting inflammatory and fibrotic pathways. Source of Funding: NIDDK K12 DK100022 to PP, CWRU Start-up fund to MMG, 5R01 DK111554-03 to RJM, U54 DK104310 to WAR © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e53-e53 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Petra Popovics* More articles by this author Wisam N. Awadallah More articles by this author Sarah Kohrt More articles by this author Thomas C. Case More articles by this author Nicole L. Miller More articles by this author Emily Ricke More articles by this author Wei Huang More articles by this author Marisol Ramirez-Solano More articles by this author Qi Liu More articles by this author Robert J. Matusik More articles by this author William A. Ricke More articles by this author Magdalena M. Grabowska More articles by this author Expand All Advertisement PDF downloadLoading ...

Most prostate cancers express androgen receptor (AR), and our previous studies have focused on identifying transcription factors that interact with and modify AR function. We have shown that transcription factor nuclear factor I/B (NFIB) regulates androgen receptor (AR) activity and that its expression is decreased in the luminal cells of severe benign prostatic hyperplasia. To assess whether changes in NFIB expression are associated with prostate cancer progression, we immunostained a tissue microarray including normal, hyperplastic, prostatic intraepithelial neoplasia, primary prostatic adenocarcinoma, and castration-resistant prostate cancer tissue samples for NFIB, AR, and synaptophysin, a marker of neuroendocrine differentiation. We observed increased NFIB in the nucleus and cytoplasm of prostate cancer samples independent of Gleason score. We also observed strong NFIB staining in primary small cell prostate cancer. While increased NFIB nuclear and cytoplasmic staining were not predictive of biochemical recurrence, the ratio of cytoplasmic-to-nuclear NFIB staining was predictive of earlier biochemical recurrence once the analysis was adjusted for tumor margin status. We next assessed expression of NFIB in prostate cancer cell lines and detected several isoforms (62 kDa, 57 kDa, 49 kDa, and 39 kDa). Using nuclear and cytoplasmic fractionation, we observed NFIB predominantly in the nuclear fraction of prostate cancer cells with increased cytoplasmic expression seen in castration-resistant prostate cancer cell lines. Through transient transfection of AR and NFIB into the AR and NFIB low JEG-3 cells, followed by co-immunoprecipitation, we also observed a physical interaction between AR and NFIB. In order to understand the consequences of NFIB over-expression in prostate cancer, we generated vector or 3X-FLAG-NFIB-expressing androgen-dependent LNCaP and castration-resistant C4-2B cells (a LNCaP derivative cell line). While over-expression of NFIB did not increase AR expression, it did increase prostate specific antigen (PSA) production and PSA promoter activity. In summary, we have described the expression pattern of NFIB in prostate cancer and propose that one consequence of NFIB over-expression in AR-dependent prostate cancers is increased AR activity as measured by PSA induction.