Androgen receptor activity in prostate cancer dictates efficacy of bipolar androgen therapy through MYC.

Testosterone is the canonical growth factor of prostate cancer but can paradoxically suppress its growth when present at supraphysiological levels. We have previously demonstrated that the cyclical administration of supraphysiological androgen (SPA), termed bipolar androgen therapy (BAT), can result in tumor regression and clinical benefit for patients with castration-resistant prostate cancer. However, predictors and mechanisms of response and resistance have been ill defined. Here, we show that growth inhibition of prostate cancer models by SPA required high androgen receptor (AR) activity and were driven in part by downregulation of MYC. Using matched sequential patient biopsies, we show that high pretreatment AR activity predicted downregulation of MYC, improved clinical response, and prolonged progression-free and overall survival for patients on BAT. BAT induced strong downregulation of AR in all patients, which is shown to be a primary mechanism of acquired resistance to SPA. Acquired resistance was overcome by alternating SPA with the AR inhibitor enzalutamide, which induced adaptive upregulation of AR and resensitized prostate cancer to SPA. This work identifies high AR activity as a predictive biomarker of response to BAT and supports a treatment paradigm for prostate cancer involving alternating between AR inhibition and activation.

Specific Detection of Prostate Cancer Cells in Urine by RNA In Situ Hybridization.

PURPOSE: Noninvasive tests that can accurately detect prostate cancer are urgently needed for prostate cancer diagnosis, surveillance and prognosis. Exfoliated prostate cells captured in urine represent a promising resource for noninvasive detection of prostate cancer. We investigated performance of a novel cell-based urine test for detection of clinically significant prostate cancer. MATERIALS AND METHODS: We previously developed a multiplex RNA in situ hybridization assay targeting NKX3-1, PRAC1 and PCA3 that enables identification and quantification of malignant and benign prostate cells released into urine. We investigated application of the assay for prostate cancer detection in a cohort of 98 patients suspected of harboring prostate cancer. Urine was collected following digital rectal examination, and the sediment was isolated and evaluated by RNA in situ hybridization. Samples were scored based on cellular expression of RNA in situ hybridization targets. Cells of prostate origin were defined by positivity for NKX3-1 and/or PRAC1, and prostate cancer cells by positivity for PCA3. RESULTS: Prostate cells (NKX3-1/PRAC1+ cells) were detected in 69 samples, among which 20 were positive for PCA3 (ie positive for prostate cancer cells). Comparison of RNA in situ hybridization results with biopsy outcome and clinical variables revealed that positivity for cancer by RNA in situ hybridization significantly correlated with intermediate/high risk cancer (p=0.003), PSA density (p=0.022), significant disease (p <0.0001) and Gleason score (p=0.003). The test was 95% specific and 51% sensitive for detection of clinically significant prostate cancer. CONCLUSIONS: Identification of exfoliated prostate cancer cells in urine by RNA in situ hybridization provides a novel tool for highly specific and noninvasive detection of prostate cancer.

A novel method for detection of exfoliated prostate cancer cells in urine by RNA in situ hybridization.

BACKGROUND: In the current study, we explore the feasibility of detecting exfoliated prostate cancer cells in urine using an RNA in situ hybridization (RISH) assay. We hypothesized that robust and specific labeling of prostate cancer cells could be achieved in post-digital rectal examination (DRE) urine samples using RISH. METHODS: We focused on method development, optimization, and analytical evaluation of RISH-based detection of prostate cancer in urine. We optimized a sample collection, processing, and target detection workflow for urine cytology specimens in conjunction with RNA target detection by RISH. We screened a panel of 11 prostate-specific RNA targets, and selected NKX3-1 and PRAC1 as markers for cells of prostate origin and PCA3 as a marker of prostate malignancy. Following analytical validation of a multiplexed NKX3-1/PRAC1/PCA3 assay, we evaluated whether prostate cancer cells can be detected in a pilot cohort of 19 post-DRE specimens obtained from men diagnosed with prostate cancer. RESULTS: Using cytology specimens prepared from spiked urine samples, we established the analytical validity of the RISH assay for detection and visualization of prostate cells in urine. Cells of prostate origin could be readily and specifically identified and separated into benign and malignant cell populations based on the multiplex test that consisted of markers specific for prostate cells (NKX3-1, PRAC1) and prostate cancer cells (PCA3). Upon evaluation of post-DRE urine from a pilot cohort of prostate cancer patients, we identified 11 samples in which prostate cells were present, 6 of which were also positive for prostate cancer cells. CONCLUSIONS: Multiplex RISH enables the direct visualization and molecular characterization of individual exfoliated prostate cells in urine. This proof-of-principle study provides evidence supporting the application of RISH as a potential noninvasive tool for prostate cancer detection.

Effects of Black Raspberries and Their Constituents on Rat Prostate Carcinogenesis and Human Prostate Cancer Cell Growth In Vitro.

Prostate cancer patients often use dietary supplements, such as black raspberries, which are a rich source of compounds with antioxidant and anticancer activity, particularly on gastrointestinal cancers. Feeding black raspberries inhibited mammary cancer induction in rats and growth of cancer cells in nude mice, indicating systemic bioavailability of bioactive compounds. We tested whether feeding black raspberries and its constituents would inhibit prostate cancer development. However, we did not find preventive effects in two rat prostate carcinogenesis models, even though the berry anthocyanin metabolite protocatechuic acid was detectable in their prostates. Black raspberry extract, the anthocyanin cyanidin-3-rutinoside and protocatechuic acid did not inhibit prostate cancer cell growth in vitro, but ellagic acid and its urolithin A metabolite did at high concentrations. Prostate cancer cell migration was not affected by these agents nor was growth in soft agar, except that ellagic acid reduced colony formation at physiological concentrations and protocatechuic acid at high concentrations. Low bioavailability of bioactive berry compounds and metabolites may limit exposure of tissues such as the prostate, since we found that cyanidin-3-rutinoside was not bioavailable to prostate cancer cells, but its aglycone cyanidin was and inhibited their growth. Thus, black raspberries are unlikely to prevent prostate cancer.

Effects of Black Raspberries and Their Ellagic Acid and Anthocyanin Constituents on Taxane Chemotherapy of Castration-Resistant Prostate Cancer Cells.

Cancer patients often use dietary supplements while on therapy, but little is known about interactions of supplements with cancer chemotherapy. Black raspberries (BRB) have anti-cancer effects, but have not been evaluated for interference with chemotherapy for castrate-resistant prostate cancer (CRPC). Here we studied whether BRB and some of their constituents interact with docetaxel and cabazitaxel on CRPC cells in culture and implanted into nude mice. Ellagic acid increased, but BRB extract inhibited, microtubule assembly. Ellagic acid decreased tubulin polymerization by cabazitaxel and bound to tubulin. Ellagic acid, its metabolite urolithin A, BRB extract, and the anthocyanin metabolite protocatechuic acid (PCA) did not alter cytotoxicity of taxanes. Ellagic acid inhibited drug efflux in CRPC cells, but BRB extract and PCA did not. None of these compounds altered CYP3A4 activity. Although dietary ellagic acid did not alter the tumor growth inhibition by docetaxel of xenografted 22Rv1 cells, ellagic acid has the potential to interfere with taxane chemotherapy by reducing tubulin polymerization while inhibiting P-glycoprotein drug efflux. These data are cause for concern of consuming ellagic acid during treatment for CRPC and indicate need for further research, but BRB consumption appears safe.

Approaches to urinary detection of prostate cancer.

BACKGROUND: Prostate cancer is the most common cancer in American men that ranges from low risk states amenable to active surveillance to high-risk states that can be lethal especially if untreated. There is a critical need to develop relatively non-invasive and clinically useful methods for screening, detection, prognosis, disease monitoring, and prediction of treatment efficacy. In this review, we focus on important advances as well as future efforts needed to drive clinical innovation in this area of urine biomarker research for prostate cancer detection and prognostication. METHODS: We provide a review of current literature on urinary biomarkers for prostate cancer. We evaluate the strengths and limitations of a variety of approaches that vary in sampling strategies and targets measured; discuss reported urine tests for prostate cancer with respect to their technical, analytical, and clinical parameters; and provide our perspectives on critical considerations in approaches to developing a urine-based test for prostate cancer. RESULTS: There has been an extensive history of exploring urine as a source of biomarkers for prostate cancer that has resulted in a variety of urine tests that are in current clinical use. Importantly, at least three tests have demonstrated high sensitivity (~90%) and negative predictive value (~95%) for clinically significant tumors; however, there has not been widespread adoption of these tests. CONCLUSIONS: Conceptual and methodological advances in the field will help to drive the development of novel urinary tests that in turn may lead to a shift in the clinical paradigm for prostate cancer diagnosis and management.

Lack of combination effects of soy isoflavones and taxane chemotherapy of castration-resistant prostate cancer.

BACKGROUND: Patients with cancer, including prostate cancer, often use dietary supplements, such as soy or isoflavones, before, during, or after therapy. There is little information about possible interactions between supplements and cancer chemotherapy. There are some reports suggesting enhancement by genistein of taxane chemotherapy for castrate-resistant prostate cancer (CRPC). METHODS: We investigated whether physiologically attainable concentrations of soy isoflavones (≤10 µM) interact with taxanes on growth inhibition of CRPC cells in vitro and in vivo in nude mice exposed via the diet, on microtubule disassembly in vitro, and on P-glycoprotein-mediated drug efflux in 22Rv1 cells and CYP3A4 activity in microsomes. RESULTS: Genistein, daidzein, and equol did not affect growth of VCaP, 22Rv1, C4-2, and PC-3 CRPC cells or growth inhibition of these cells by docetaxel and cabazitaxel. These isoflavones did not inhibit microtubule disassembly in vitro or inhibit the microtubule effects of taxanes and genistein did not bind substantially to microtubules. Genistein considerably inhibited P-glycoprotein-mediated drug efflux in 22Rv1 cells and CYP3A4 activity in microsomes. However, dietary supplementation with genistein at 250 and 500 ppm did not affect the tumor growth inhibiting effect of docetaxel on 22Rv1 cells xenografted in nude mice. CONCLUSIONS: Our results with relevant cell models and clinically achievable concentrations of soy isoflavones do not support the notion that genistein or other soy isoflavones can enhance the effects of taxane chemotherapy in CRPC cell and xenograft models. Yet, the inhibitory effects of genistein on drug efflux in 22Rv1 cells and on microsomal CYP3A4 activity raise the possibility that genistein can affect taxane effects on CRPC cells in other circumstances than those we studied, which merits further research.

Interactive effects of 9-cis-retinoic acid and androgen on proliferation, differentiation, and apoptosis of LNCaP prostate cancer cells.

9-cis-Retinoic acid (9cRA), which binds to both retinoic acid receptors and retinoic X receptors, inhibits prostate cancer induction in rats and reduces growth of prostate cancer cells. However, the nature of this growth inhibition and the interactive influence of androgens are not well defined and are the subject of this report. LNCaP and PC-3 cells were cultured and treated with a range of 9cRA concentrations for 3-6 days in the absence or presence of 5α-dehydrotestosterone. 9cRA inhibited cell proliferation in a dose-dependent manner, plateauing at 10 mol/l. Treatment of cells with 10 mol/l 9cRA inhibited 5α-dihydroxytestosterone (DHT)-stimulated proliferation, the effect of which was maximal at 10 mol/l DHT. Treatment of DHT (10 mol/l)-exposed cells with 9cRA caused a dose-dependent increase in prostate-specific antigen in the medium after 6 days, but not 3 days. 9cRA caused a dose-dependent increase in apoptotic cells stained with H33258 after 3 days, but not 6 days; however, on using flow cytometry, apoptosis was apparent at both 3 and 6 days. Flow cytometry also revealed interference of G0/G1 to S phase transition by 9cRA. Inhibition by 9cRA of anchorage-independent growth of PC-3 cells was also found; LNCaP cells did not grow colonies in soft agar. 9cRA inhibited growth and induced differentiation of human LNCaP prostate cancer cells in vitro and inhibited anchorage-independent growth of PC-3 cells. Because 9cRA and 13-cis-retinoic acid, which is retinoic acid receptor-selective, prevent prostate carcinogenesis in rats, and 13-cis-retinoic acid also inhibits growth of human prostate cancer cells, the RAR is a potential molecular target for prostate cancer prevention and therapy.

A Perspective on Prostate Carcinogenesis and Chemoprevention.

In this perspective, modifiable carcinogenic factors for the prostate are summarized. This is followed by a discussion of how current knowledge about causation of prostate cancer and chemoprevention of prostate cancer can be used to develop preventive strategies. Prostate cancer is a slowly developing cancer which offers opportunities for preventive interventions. Only a few randomized clinical trials of prostate cancer prevention have been completed. The SELECT study with selenium and vitamin E did not find protective effects, but in two trials with 5α-reductase inhibitors risk was reduced about 25%, showing that chemoprevention is possible and indicating that the androgen receptor is a suitable target. Besides smoking cessation and reduction of obesity, there are no known dietary or life style interventions that will have a major impact on prostate cancer risk. Inflammation of the prostate is an attractive target and aspirin may be a promising candidate agent, but has not been addressed yet in preclinical and clinical studies. Antioxidants other than selenium and vitamin E are unlikely to be very effective and data on several dietary supplements are not encouraging. More candidate agents need to be identified and tested in relevant and adequate preclinical models and Phase II trials that have predictive value for outcome of Phase III randomized studies. Doing this will require a systematic approach comparing preclinical and clinical study outcomes to determine their predictive value of preventive efficacy.

Differential regulation of detoxification enzymes in hepatic and mammary tissue by hops (Humulus lupulus) in vitro and in vivo.

SCOPE: Hops contain the phytoestrogen, 8-prenylnaringenin, and the cytoprotective compound, xanthohumol (XH). XH induces the detoxification enzyme, NAD(P)H-quinone oxidoreductase (NQO1) in vitro; however, the tissue distribution of XH and 8-prenylnaringenin and their tissue-specific activity have not been analyzed. METHODS AND RESULTS: An orally administered hop extract and subcutaneously injected XH were administered to Sprague-Dawley rats over 4 days. LC-MS-MS analysis of plasma, liver, and mammary gland revealed that XH accumulated in liver and mammary glands. Compared with the low level in the original extract, 8-prenylnaringenin was enriched in the tissues. Hops and XH-induced NQO1 in the liver, while only hops reduced NQO1 activity in the mammary gland. Mechanistic studies revealed that hops modulated NQO1 through three mechanisms. In liver cells, (i) XH modified Kelch-like ECH-associated protein leading to nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation and antioxidant response element (ARE) activation; (ii) hop-mediated ARE induction was partially mediated through phosphorylation of Nrf2 by PKC; (iii) in breast cells, 8-prenylnaringenin reduced NQO1 likely through binding to estrogen receptorα, recruiting Nrf2, and downregulating ARE-regulated genes. CONCLUSION: XH and 8-prenylnaringenin in dietary hops are bioavailable to the target tissues. While hops and XH might be cytoprotective in the liver, 8-prenylnaringenin seems responsible for hop-mediated NQO1 reduction in the mammary gland.