R. W. DeVere White

Tumor suppressive miR-124 targets androgen receptor and inhibits proliferation of prostate cancer cells

Although prostate cancer (CaP) is the most frequently diagnosed malignant tumor in American men, the mechanisms underlying the development and progression of CaP remain largely unknown. Recent studies have shown that downregulation of the microRNA miR-124 occurs in several types of human cancer, suggesting a tumor suppressive function of miR-124. Until now, however, it has been unclear whether miR-124 is associated with CaP. In the present study, we completed a series of experiments to understand the functional role of miR-124 in CaP. We detected the expression level of miR-124 in clinical CaP tissues, evaluated the influence of miR-124 on the growth of CaP cells and investigated the mechanism underlying the dysregulation of miR-124. We found that (i) miR-124 directly targets the androgen receptor (AR) and subsequently induces an upregulation of p53; (ii) miR-124 is significantly downregulated in malignant prostatic cells compared to benign cells, and DNA methylation causes the reduced expression of miR-124; and (iii) miR-124 can inhibit the growth of CaP cells in vitro and in vivo. Data from this study revealed that loss of miR-124 expression is a common event in CaP, which may contribute to the pathogenesis of CaP. Our studies also suggest that miR-124 is a potential tumor suppressive gene in CaP, and restoration of miR-124 expression may represent a novel strategy for CaP therapy.

miR-30 as a tumor suppressor connects EGF/Src signal to ERG and EMT

Src tyrosine kinase (Src) is implicated in the development of bone metastasis and castration resistance of prostate cancer. Src inhibitors are currently being tested in clinical trials for such diseases. Understanding the molecular and cellular actions of Src inhibitors holds the key to future improvement of this line of therapy. Here we describe the microRNA expression profiles modulated by two Src inhibitors and demonstrate that the miR-30 family members are the most prominently induced species. Consistent with its tumor suppressor role, miR-30 is downmodulated by oncogenic signals such as epidermal growth factor (EGF) and hepatocyte growth factor, and is generally underexpressed in prostate cancer specimens. A number of epithelial-to-mesenchymal transition (EMT)-associated genes are predicted targets of miR-30. Among these genes the Ets-related gene (ERG) is the most frequently overexpressed oncogene in prostate cancer activated by genomic fusion events between promoter upstream sequences of the TMPRSS2 and coding sequences of ERG. We showed by ERG 3′ untranslated region reporter and mutagenesis assays that ERG is a direct target of miR-30. Overexpression of miR-30 in prostate cancer cells suppresses EMT phenotypes and inhibits cell migration and invasion. It also inhibits the in vitro and in vivo growth of VCaP cells, which depends on TMPRSS2-ERG for proliferation. TMPRSS2-ERG is generally regulated by androgen at the transcriptional level. Our finding reveals a new post-transcriptional mechanism of TMPRSS2-ERG regulation by Src and growth signals via miR-30 providing a rationale for targeting ERG-positive castration-resistant tumors with Src inhibitors.

Targeting autophagy overcomes Enzalutamide resistance in castration-resistant prostate cancer cells and improves therapeutic response in a xenograft model

Macro-autophagy is associated with drug resistance in various cancers and can function as an adaptive response to maintain cell survival under metabolic stresses, including androgen deprivation. Androgen deprivation or treatment with androgen receptor (AR) signaling inhibitor (ARSI), Enzalutamide (MDV-3100, ENZA) or bicalutamide induced autophagy in androgen-dependent and in castration-resistant CaP (castration-resistant prostate cancer (CRPC)) cell lines. The autophagic cascade triggered by AR blockage, correlated with the increased light chain 3-II/I ratio and ATG-5 expression. Autophagy was observed in a subpopulation of C4-2B cells that developed insensitivity to ENZA after sustained exposure in culture. Using flow cytometry and clonogenic assays, we showed that inhibiting autophagy with clomipramine (CMI), chloroquine or metformin increased apoptosis and significantly impaired cell viability. This autophagic process was mediated by AMP-dependent protein kinase (AMPK) activation and the suppression of mammalian target of rapamycin (mTOR) through Raptor phosphorylation (Serine 792). Furthermore, small interfering RNA targeting AMPK significantly inhibited autophagy and promoted cell death in CaP cells acutely or chronically exposed to ENZA or androgen deprivation, suggesting that autophagy is an important survival mechanism in CRPC. Lastly, in vivo studies with mice orthotopically implanted with ENZA-resistant cells demonstrated that the combination of ENZA and autophagy modulators, CMI or metformin significantly reduced tumor growth when compared with control groups (P<0.005). In conclusion, autophagy is as an important mechanism of resistance to ARSI in CRPC. Antiandrogen-induced autophagy is mediated through the activation of AMPK pathway and the suppression of mTOR pathway. Blocking autophagy pharmacologically or genetically significantly impairs prostate cancer cell survival in vitro and in vivo, implying the therapeutics potential of autophagy inhibitors in the antiandrogen-resistance setting.

A 90 kDa fragment of filamin A promotes Casodex-induced growth inhibition in Casodex-resistant androgen receptor positive C4-2 prostate cancer cells

Prostate tumors are initially dependent on androgens for growth, but the majority of patients treated with anti-androgen therapy progress to androgen-independence characterized by resistance to such treatment. This study investigates a novel role for filamin A (FlnA), a 280 kDa cytoskeletal protein (consisting of an actin-binding domain (ABD) followed by 24 sequential repeats), in androgen-independent (AI) growth. Full-length FlnA is cleaved to 170 kDa (ABD+FlnA1–15) and 110 kDa fragments (FlnA16–24); the latter is further cleaved to a 90 kDa fragment (repeats 16–23) capable of nuclear translocation and androgen receptor (AR) binding. Here, we demonstrate that in androgen-dependent LNCaP prostate cancer cells, the cleaved 90 kDa fragment is localized to the nucleus, whereas in its AI subline C4–2, FlnA failed to cleave and remained cytoplasmic. Transfection of FlnA16–24 cDNA in C4–2 cells restored expression and nuclear localization of 90 kDa FlnA. Unlike LNCaP, C4–2 cells proliferate in androgen-reduced medium and in the presence of the AR-antagonist Casodex. They also exhibit increased Akt phosphorylation compared to LNCaP, which may contribute to their AI phenotype. Nuclear expression of 90 kDa FlnA in C4–2 cells decreased Akt phosphorylation, prevented proliferation in androgen-reduced medium and restored Casodex sensitivity. This effect was inhibited by constitutive activation of Akt indicating that FlnA restored Casodex sensitivity in C4–2 cells by decreasing Akt phosphorylation. In addition, FlnA-specific siRNA which depleted FlnA levels, but not control siRNA, induced resistance to Casodex in LNCaP cells. Our results demonstrate that expression of nuclear FlnA is necessary for androgen dependence in these cells.

Inappropriate activation of androgen receptor by relaxin via Β-catenin pathway

We have previously demonstrated that human H2-relaxin can mediate androgen-independent growth of LNCaP through a mechanism that involves the activation of the androgen receptor (AR) signaling pathway. The goal of the current study is to elucidate the mechanism(s) by which H2-relaxin causes activation of the AR pathway. Our data indicate that there is cross-talk between AR and components of the Wnt signaling pathway. Addition of H2-relaxin to LNCaP cells resulted in increased phosphorylation of protein kinase B (Akt) and inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β) with subsequent cytoplasmic accumulation of β-catenin. Immunoprecipitation and immunocytochemical studies demonstrated that the stabilized β-catenin formed a complex with AR, which was then translocated into the nucleus. Chromatin immunoprecipitation analysis determined that the AR/β-catenin complex binds to the proximal region of the prostate-specific antigen promoter. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, using LY294002, prevented both H2-relaxin-mediated phosphorylation of Akt and GSK-3β and translocation of β-catenin/AR into the nucleus. Knockdown of β-catenin levels using a β-catenin-specific small interfering RNA inhibited H2-relaxin-induced AR activity. The combined data demonstrate that PI3K/Akt and components of the Wnt pathway can facilitate H2-relaxin-mediated activation of the AR pathway.

Molecular biology of prostate cancer

SummaryCarcinoma of the prostate (CaP) is a very prevalent tumor among men. However thus far, relatively little information is known about the molecular mechanisms involved in the development, progression and metastasis of this disease. This article reviews the current state of knowledge of five selected molecular aspects of human CaP: tumor suppressor genes, metastasis suppressor genes and related biological events (allelic loss and DNA methylation), oncogenes (including growth factors and their receptors), the anti-apoptosis gene BCL2, and the human androgen receptor gene (hAR). Alterations of these genes in structure and expression as well as the frequencies of these molecular events are discussed to synthesize an understanding of documented genetic alterations that occur in CaP and their possible relation to the biology of the disease.

The Effect of Sirolimus on Prostate-Specific Antigen (PSA) Levels in Male Renal Transplant Recipients Without Prostate Cancer

In kidney recipients, the immunosuppressant sirolimus has been associated with a decreased incidence of de novo posttransplant malignancies (including prostate cancer). But the effect of sirolimus on the prostate‐specific antigen (PSA) blood level, an important prostate cancer screening tool, remains unknown. We studied male kidney recipients >50 years old (transplanted from January 1994 to December 2006) without clinical evidence for prostate cancer. Pre‐ and posttransplant PSA levels were analyzed for 97 recipients (n = 19 on sirolimus, n = 78 on tacrolimus [control group]). Pretransplant PSA was similar for sirolimus versus tacrolimus recipients (mean, 1.8 versus 1.7 ng/mL, p = 0.89), but posttransplant PSA was significantly lower for recipients on sirolimus (mean, 0.9 versus 1.9 ng/mL, respectively, p < 0.001). The mean difference between pretransplant and posttransplant PSA was −0.9 ng/mL (50.0%, p = 0.006) for the sirolimus group versus +0.2 ng/mL (+11.8%, p = 0.24) for the tacrolimus group. By multivariate analysis, only pretransplant PSA and immunosuppression with sirolimus independently impacted posttransplant PSA. Our data strongly suggest that sirolimus is associated with a significant PSA decrease in kidney recipients. Future studies must investigate the clinical implications of our findings for the use of PSA for prostate cancer screening in male kidney recipients on sirolimus.

External vacuum devices: a clinical comparison with pharmacologic erections

SummaryThe purpose of the present study was to compare the satisfaction rates obtained for vacuum constriction devices with those achieved using intracavernous pharmacologic injections in a group of patients afflicted with erectile dysfunction. The subjects were stratified into three groups: group 1 failed to achieve an adequate erection on pharmacologic injection, group 2 achieved satisfactory erection following pharmacologic injection, and group 3 was left untreated. All patients were given a vacuum constriction device. We assessed their satisfaction using a questionnaire. The data suggest high satisfaction rates in all three groups. Of particular interest was that over half of the patients who had successfully been treated with pharmacologic injections switched to the vacuum constriction device at the end of the study. The data indicate high levels of patient satisfaction with the vacuum constriction devices, even among subjects in whom prior alternative impotence therapy had been successful.

Flow cytometric evaluation of bladder cancer : recommendations of the NCI flow cytometry network for bladder cancer

SummaryThe National Cancer Institute-supported Flow Cytometry Network for Bladder Cancer concluded that when properly used, DNA flow cytometry of bladder irrigation specimens can be a clinically useful laboratory procedure to monitor patients with bladder cancer. It recommended the use of this technique in managing patients with low-stage disease, particularly flat carcinoma in situ. The method has limited value in managing patients with high-stage (i.e., muscle-invasive) carcinoma; it is not recommended for screening subjects in the absence of a clinical suspicion of or a history of bladder tumors. DNA histograms alone are not sufficient for diagnosis or clinical action but require correlation with other clinical information. Bladder irrigation specimens collected during cystoscopy or by vigorous barbotage via a number 18 Foley catheter may be processed for flow cytometric analyses. Criteria for sampling adequacy have been established and are presented in this report. Optimal results are obtained with fresh specimens that are stained and examined promptly after collection. Preservation procedures are described for cases in which fresh specimens cannot be evaluated. Used with appropriate quality-control measures, commercially available flow cytometers can provide clinically useful information. Staining protocols using propidium iodide are recommended by the Network. Staining protocols are described for isolated nuclei and whole cells (see Appendix). The presence of bladder cancer is signaled by the identification of cell populations with clearly aneuploid DNA content. Quality-control measures and issues of inter- and intralaboratory differences in histogram configuration and analysis must be considered in the interpretation of results. Although the Network participants recognize the potential value of additional markers, these were not evaluated.

Deoxyribonucleic acid ploidy in the irradiated prostate

SummaryA retrospective study was performed to determine whether the ploidy of localized prostate cancer was correlated with the response to radiation therapy. Prostate tissue was obtained from 69 patients prior to and at least 22 months following definitive radiation therapy. A positive postradiation biopsy of a hypoechoic lesion obtained under transrectal ultrasound guidance was taken as evidence of a failure to respond to therapy. Only 20% of these patients had negative posttherapy biopsies. This poor response rate was similar, regardless of whether the pretreatment tumor was diploid or aneuploid. Treatment failure was correlated with an increased incidence of aneuploid histograms after therapy. Prior to therapy, 80% of the tumors were diploid; after therapy, only 56% were diploid, whereas nontetraploid-aneuploid tumors had increased 3-fold. Among 44 patients who had diploid tumors prior to therapy, the residual prostate cancer was tetraploid in five cases and nontetraploid-aneuploid in ten cases.