EBP1, an ErbB3-binding protein, is decreased in prostate cancer and implicated in hormone resistance

Aberrant activation of the androgen receptor (AR) by the ErbB2/ErbB3 heterodimer contributes to the development of hormone resistance in prostate cancer. EBP1, an ErbB3-binding protein, acts as an AR corepressor. As EBP1 is decreased in preclinical models of hormone-refractory prostate cancer, we studied the expression of EBP1 in human prostate cancer. We found that the expression of the EBP1 gene was significantly decreased in prostate cancer tissues compared with benign prostate at both mRNA and protein levels. Restoration of EBP1 expression in the hormone-refractory LNCaP C81 cell line led to an amelioration of the androgen-independent phenotype based on established biological criteria and a reduction in the expression of a cohort of AR target genes. The ability of the ErbB3 ligand heregulin (HRG) to stimulate growth and AKT phosphorylation of hormone-refractory prostate cancer cells was abolished. Abrogation of EBP1 expression by short hairpin RNA in hormone-dependent LNCaP cells, which undergo apoptosis in response to HRG, resulted in HRG-stimulated cell growth. Restoration of EBP1 expression decreased the tumorigenicity of C81 xenografts in female mice, whereas elimination of EBP1 expression enhanced the ability of LNCaP cells to grow in female mice. Our data support a role for EBP1 in the development of hormone-refractory prostate cancer via inhibition of both AR- and HRG-stimulated growth and present a novel strategy for treating androgen-refractory prostate cancer.     

Chemical ablation of androgen receptor in prostate cancer cells by the histone deacetylase inhibitor LAQ824

Androgen receptor plays a critical role in the development of primary as well as advanced hormone-refractory prostate cancer. Therefore, ablation of androgen receptor from prostate cancer cells is an interesting concept for developing a new therapy not only for androgen-dependent prostate cancer but also for metastatic hormone-refractory prostate cancer, for which there is no effective treatment available. We report here that LAQ824, a cinnamyl hydroxamatic acid histone deacetylase inhibitor currently in human clinical trials, effectively depleted androgen receptor in prostate cancer cells at nanomolar concentrations. LAQ824 seemed capable of depleting both the mutant and wild-type androgen receptors in either androgen-dependent and androgen-independent prostate cancer cells. Although LAQ824 may exert its effect through multiple mechanisms, several lines of evidence suggest that inactivation of the heat shock protein-90 (Hsp90) molecular chaperone is involved in LAQ824-induced androgen receptor depletion. Besides androgen receptor, LAQ824 reduced the level of Hsp90 client proteins HER-2 (ErbB2), Akt/PKB, and Raf-1 in LNCaP cells. Another Hsp90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), also induced androgen receptor diminution. LAQ824 induced Hsp90 acetylation in LNCaP cells, which resulted in inhibition of its ATP-binding activity, dissociation of Hsp90-androgen receptor complex, and proteasome-mediated degradation of androgen receptor. Consequently, LAQ824 blocked androgen-induced prostate-specific antigen production in LNCaP cells. LAQ824 effectively inhibited cell proliferation and induced apoptosis of these prostate cancer cells. These results reveal that LAQ824 is a potent agent for depletion of androgen receptor and a potential new drug for prostate cancer.     

Development of a prostate-specific promoter for gene therapy against androgen-independent prostate cancer

Androgen ablation has been the standard treatment for metastasized prostate cancer. In most cases, however, prostate cancer cells eventually lose androgen dependency and become refractory to the conventional endocrine therapy. Androgen-independent prostate cancer is characterized by a heterogeneous loss of androgen receptor (AR) expression among tumor cells. Prostate-specific promoters such as prostate-specific antigen and rat probasin (rPB) promoters have been examined in the development of gene therapy targeted to prostate cancer. However, those promoters require binding of the androgen–AR complex to the androgen-response element and are active only in the androgen-dependent prostate cancer cell lines and not in the androgen-independent cell lines. To target transgene expression in androgen-independent prostate cancer, we designed a prostate-specific promoter that is activated by the retinoids–retinoid receptor complex instead of the androgen–AR complex. The modified rPB promoters expressed transgenes in response to retinoid in both androgen-dependent and androgen-independent prostate cancer cells and not in other cancer cell lines or in human normal cells, in vitro and in vivo. Furthermore, the combination of retinoid treatment and adenovirus-mediated gene transfer of the modified rPB-driven HSV-tk gene resulted in a significant growth suppression of the androgen-independent prostate cancer cells in the presence of the prodrug ganciclovir. This study suggests that tailoring of the hormone-responsive elements may offer a new therapeutic opportunity against the hormone-refractory stage of prostate cancer.     

Androgen antagonist activity by the antioxidant moiety of vitamin E, 2,2,5,7,8-pentamethyl-6-chromanol in human prostate carcinoma cells

Antioxidants, such as vitamin E, are being investigated for efficacy in prostate cancer prevention. In this study, we show that the antioxidant moiety of vitamin E, 2,2,5,7,8-pentamethyl-6-chromanol (PMCol), has antiandrogen activity in prostate carcinoma cells. In the presence of PMCol, the androgen-stimulated biphasic growth curve of LNCaP human prostate carcinoma cells was shifted to the right. The PMCol-induced growth shift was similar to that produced by treatment with the pure antiandrogen bicalutamide (i.e., Casodex), indicative of androgen receptor (AR) antagonist activity. The concentration of PMCol used was below the concentration required to affect cell growth or viability in the absence of androgen. Using an AR binding competition assay, PMCol was found to be a potent antiandrogen in both LNCaP and LAPC4 cells, with an IC(50) of approximately 10 micro M against 1 nM R1881 (methyltrienolone; a stable, synthetic androgen). Prostate-specific antigen release from LNCaP cells produced by androgen exposure with either 0.05 or 1.0 nM R1881 was inhibited 100% and 80%, respectively, by 30 micro M PMCol. Also, PMCol inhibited androgen-induced promoter activation in both LNCaP and LAPC4 cells. However, PMCol did not affect AR protein levels, suggesting that the inhibitory effects of PMCol on androgenic pathways were not due to decreased expression of the AR. Therefore, growth modulation by the antioxidant moiety of vitamin E in androgen-sensitive prostate carcinoma cells is due, at least in part, to its potent antiandrogenic activity.     

Myc confers androgen-independent prostate cancer cell growth

Prostate cancer is one of the most diagnosed and mortal cancers in western countries. A major clinical problem is the development of androgen-independent prostate cancer (AIPC) during antihormonal treatment. The molecular mechanisms underlying the change from androgen dependence to independence of these tumors are poorly understood and represent a challenge to develop new therapies. Based on genetic data showing amplification of the c-myc gene in AIPC, we studied the ability of c-myc to confer AIPC cell growth. Human androgen-dependent prostate cancer cells overexpressing c-myc grew independently of androgens and presented tumorigenic properties in androgen-depleted conditions. Analysis of signalling pathways by pharmacological inhibitors of the androgen receptor (AR) or by RNA interference directed against AR or c-myc showed that c-myc acted downstream of AR through multiple growth effectors. Thus c-myc is required for androgen-dependent growth and following ectopic expression can induce androgen-independent growth. Moreover, RNA interference directed against c-myc showed that growth of human AIPC cells, AR-positive or -negative, required c-myc expression. Furthermore, we showed that c-myc-overexpressing cells retain a functional p53 pathway and thus respond to etoposide.     

Signal transduction in prostate cancer progression

Prostate cancer is the most frequently diagnosed cancer among men and the second leading cause of male cancer deaths in the United States. When prostate cancer initially presents in the clinic, the tumour is dependent on androgen for growth and, therefore, responsive to the surgical or pharmacological ablation of circulating androgens. However, there is a high rate of treatment failure because the disease often recurs as androgen-independent metastases. Surprisingly, this late-stage androgen-independent prostate cancer almost always retains expression of the AR (androgen receptor), despite the near absence of circulating androgens. Although late-stage prostate cancer is androgen-independent, the AR still seems to play a role in cancer cell growth at this stage of disease. Therefore a key to understanding hormone-independent prostate cancer is to determine the mechanism(s) by which the AR can function even in the absence of physiological levels of circulating androgen. This review will focus on the role of growth factor signalling in prostate cancer progression to androgen independence and thus outline potential molecular areas of intervention to treat prostate cancer progression.     

Calcitrol (1alpha,25-dihydroxyvitamin D3) inhibits androgen glucuronidation in prostate cancer cells

Calcitriol (1alpha,25-dihydroxyvitamin D(3)), the active metabolite of vitamin D, has recently emerged as a promising therapeutic agent in the treatment of prostate cancer, the second most common cause of cancer death in American males. In the present study, we have analyzed the effects of calcitriol treatment on the expression and activity of the UDP-glucuronosyltransferase (UGT) 2B15 and 2B17 in prostate cancer LNCaP and 22Rv1 cells. These two enzymes share a crucial role in the inactivation of androgens in the human prostate. We report that calcitriol treatment results in lower glucuronide conjugation of the active androgen dihydrotestosterone and its reduced metabolites androstane-3alpha-diol and androsterone in LNCaP cells. The same treatment also drastically decreased the mRNA and protein levels of UGT2B15 and UGT2B17 in LNCaP and 22Rv1 cells. Using casodex, an androgen receptor (AR) antagonist, and AR-specific small interfering RNA probes, we show that calcitriol requires a functional AR to inhibit the expression of the UGT2B17 gene in LNCaP cells. By contrast, transient transfection and site-directed mutagenesis experiments revealed that calcitriol down-regulates UGT2B15 promoter activity through a responsive region between positions -171 and -113 bp. In conclusion, the present study identifies the vitamin D receptor activator calcitriol as a negative regulator of the UGT2B15- and UGT2B17-dependent inactivation of androgens in prostate cancer LNCaP cells. Androgens promote prostate cancer cell proliferation; thus, the reduction of their inactivation could have a limiting effect of the calcitriol antiproliferative properties in prostate cancer cells.     

Sodium selenite inhibits interleukin-6-mediated androgen receptor activation in prostate cancer cells via upregulation of c-Jun

BACKGROUND: It has been suggested that interleukin-6 (IL-6) may modulate androgen receptor (AR) action to accelerate prostate cancer (PCa) progression. Selenium compounds are highly recommended as a promising chemopreventive agent for PCa. This study was to determine if selenium can repress IL-6 mediated AR action in PCa progression. METHODS: Cell proliferation, prostate-specific antigen, gene transfer, and Western blot assays were used to study the effects of sodium selenite and methylseleninic acid on IL-6 mediated AR action on an AR expressing human prostate cancer cell line, LNCaP. RESULTS: We found that sodium selenite, but not methylseleninic acid, significantly (p<0.05) inhibited IL-6-induced trans-activating activity of AR and cell proliferation in LNCaP cells. Interestingly, although sodium selenite did not show effect on activation of both STAT3 and ERK1/2 in the presence of IL-6, an increased expression of c-Jun was detected in cells after treatment with sodium selenite. Indeed, we showed overexpression of c-Jun blocked IL-6-induced AR activation. CONCLUSIONS: Taken together, our results suggest that sodium selenite not methylseleninic acid can inhibit IL-6-mediated AR activation by increased c-Jun in LNCaP cells. Sodium selenite may be a proper selenium form for further testing its potency on intervening IL-6-mediated PCa progression.     

Suberoylanilide hydroxamic acid (vorinostat) represses androgen receptor expression and acts synergistically with an androgen receptor antagonist to inhibit prostate cancer cell proliferation

Growth of prostate cancer cells is initially dependent on androgens, and androgen ablation therapy is used to control tumor growth. Unfortunately, resistance to androgen ablation therapy inevitably occurs, and there is an urgent need for better treatments for advanced prostate cancer. Histone deacetylase inhibitors, such as suberoylanilide hydroxamic acid (SAHA; vorinostat), are promising agents for the treatment of a range of malignancies, including prostate cancer. SAHA inhibited growth of the androgen-responsive LNCaP prostate cancer cell line at low micromolar concentrations and induced caspase-dependent apoptosis associated with chromatin condensation, DNA fragmentation, and mitochondrial membrane depolarization at higher concentrations (>/=5 mumol/L). Gene profiling and immunoblot analyses showed a decrease in androgen receptor (AR) mRNA and protein in LNCaP cells cultured with SAHA compared with control cells, with a corresponding decrease in levels of the AR-regulated gene, prostate-specific antigen. Culture of LNCaP cells in steroid-free medium markedly sensitized the cells to SAHA. Moreover, a combination of low, subeffective doses of SAHA and the AR antagonist bicalutamide resulted in a synergistic reduction in cell proliferation and increase in caspase-dependent cell death. Addition of exogenous androgen prevented the induction of cell death, indicating that suppression of androgen signaling was required for synergy. At the subeffective concentrations, these agents had no effect, alone or in combination, on proliferation or death of AR-negative PC-3 prostate cancer cells. Our findings indicate that SAHA is effective in targeting the AR signaling axis and that androgen deprivation sensitizes prostate cancer cells to SAHA. Consequently, combinatorial treatments that target different components of the AR pathway may afford a more effective strategy to control the growth of prostate cancer cells.     

Anticancer efficacy of simvastatin on prostate cancer cells and tumor xenografts is associated with inhibition of Akt and reduced prostate-specific antigen expression

Prostate cancer is the second-leading cause of cancer-associated death among men in the United States. There has been renewed interest in the potential therapeutic benefits of statins for cancer. Simvastatin, a widely used generic drug for preventing cardiovascular events, is well known for its effects on cellular proliferation and inflammation, two key processes that also determine the rate of tumor growth. Although a growing body of evidence suggests that statins have the potential to reduce the risk of many cancers, there are discrepancies over the pro- and anticancer effects of statins. In the current study, we sought to investigate the effects of simvastatin on the Akt pathway in prostate cancer cells with respect to the regulation of various cell functions in vitro and tumor growth in vivo. Time- and dose-dependent effects of simvastatin on LNCaP (androgen-dependent) and PC3 (androgen-independent) cells indicate that treatment with simvastatin at concentrations as low as 25 μM was sufficient to inhibit serum-stimulated Akt activity. Akin to this, treatment with simvastatin significantly inhibited serum-induced cell migration, invasion, colony formation, and proliferation. Simvastatin-mediated effects on colony formation were rescued by adenovirus-mediated expression of constitutively active Akt (myristoylated Akt) in PC3 cell lines. A PC3 xenograft model performed in nude mice exhibited reduced tumor growth with simvastatin treatment associated with decreased Akt activity and reduced prostate-specific antigen (PSA) levels. Our findings demonstrate the therapeutic benefits of simvastatin for prostate cancer and suggest a link between simvastatin, regulation of Akt activity, and PSA expression in prostate tumors.     

Mechanisms of selenium down-regulation of androgen receptor signaling in prostate cancer

Prevention trials showed that selenium reduced prostate cancer incidence by 50%, establishing selenium as a promising chemopreventive agent for prostate cancer. Selenium inhibited human prostate cancer cell growth, blocked cell cycle progression at multiple transition points, and induced apoptotic cell death. Previous studies showed a novel mechanism of selenium anticancer action in which selenium markedly reduces androgen signaling and androgen receptor (AR)-mediated gene expression, including prostate-specific antigen (PSA), in human prostate cancer cells. The molecular mechanisms of selenium-mediated down-regulation of AR signaling are not clear. In this study, a systemic approach was taken to examine the modification of androgen signaling by selenium in human prostate cancer cells. In addition to reduced AR mRNA expression, selenium was found to initially increase the stability of AR mRNA within 6 hours while decreasing the stability of AR mRNA after 8 hours. Selenium increased AR protein degradation and reduced AR nuclear localization. Scatchard analysis indicated that selenium did not affect ligand binding to AR in LNCaP cells. Chromatin immunoprecipitation analyses showed that DHT increased the recruitment of AR and coactivators, such as SRC-1 and TIF-2, to the promoter of the PSA gene, and that recruitment was greatly diminished in the presence of 5 micromol/L selenium. On the other hand, selenium enhanced the recruitment of corepressors, such as SMRT, to the promoter of the PSA gene. Taken together, these results suggest that selenium disrupts AR signaling at multiple stages, including AR mRNA expression, mRNA stability, protein degradation, nuclear translocation, and recruitment of coregulators.     

The xenoestrogen bisphenol A induces inappropriate androgen receptor activation and mitogenesis in prostatic adenocarcinoma cells

Treatment for prostatic adenocarcinoma is reliant on the initial androgen dependence of this tumor type. The goal of therapy is to eliminate androgen receptor activity, either through direct inhibition of the receptor or through inhibition of androgen synthesis. Although this course of therapy is initially effective, androgen-refractory tumors ultimately arise and lead to patient morbidity. Factors contributing to the transition from a state of androgen dependence to the androgen-refractory state are poorly understood, but clinical evidence in androgen-refractory tumors suggests that the androgen receptor is inappropriately activated in these cells. Thus, the mechanisms that contribute to inappropriate (androgen-independent) activation of the androgen receptor (AR) is an area of intensive research. Here we demonstrate that bisphenol A (BPA), a polycarbonate plastic monomer and established xenoestrogen, initiates androgen-independent proliferation in human prostatic adenocarcinoma (LNCaP) cells. The mitogenic capacity of BPA occurred in the nanomolar range, indicating that little BPA is required to stimulate proliferation. We show that BPA stimulated nuclear translocation of the tumor-derived receptor (AR-T877A), albeit with delayed kinetics compared with dihydrotestosterone. This translocation event was followed by specific DNA binding at androgen response elements, as shown by electrophoretic mobility shift assays. Moreover, the ability of BPA to stimulate AR-T877A activity was demonstrated by reporter assays and by analysis of an endogenous AR target gene, prostate-specific antigen. Thus, BPA is able to activate AR-T877A in the absence of androgens. Lastly, full mitogenic function of BPA is dependent on activation of the tumor-derived AR-T877A. These data implicate BPA as an inappropriate mitogen for prostatic adenocarcinoma cells and provide the impetus to study the consequence of BPA exposure on prostate cancer.     

组蛋白去乙酰化酶抑制剂FK228对前列腺癌LNCaP细胞抑制效应的细胞信号通路研究

目的研究组蛋白去乙酰化酶抑制剂FK228对前列腺癌LNCaP细胞抑制作用的细胞信号机制。方法Western blot分析细胞蛋白乙酰化水平、细胞信号通路蛋白及细胞周期相关蛋白的表达。结果FK228能够使细胞内蛋白乙酰化水平增高,乙酰化组蛋白H3积累,多种细胞信号通路的相关蛋白如雄激素受体、HER-2、Raf-1、Akt、CDK4等呈时间依赖性和剂量依赖性被清除。结论FK228能够同时阻断对细胞生长具有重要作用的多条细胞信号通路,从而对前列腺癌LNCaP细胞发挥抑制作用。     

Effect of zinc on regulation of insulin-like growth factor signaling in human androgen-independent prostate cancer cells

BackgroundProstate cancer is one of the most frequently diagnosed cancers in men. Progression of these tumors is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. Among these, insulin-like growth factors (IGFs) signaling pathway contributes a major role. In this study, we examined the effect of zinc on insulin-like growth factors signaling in prostate cancer cells.MethodsHuman androgen-independent prostatic carcinoma (PC-3) cells were treated with different concentrations of zinc (20–100 µmol/l) for 24 and 48 h. Cell viability was performed by 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Insulin-like growth factor binding protein-3 (IGFBP-3), insulin-like growth factor-I receptor (IGF-IR), insulin receptor substrate-1 (IRS-1) and IRS-2, phosphatidylinositol-3 kinase (PI-3 K), protein kinase B or Akt, phosphorylated Akt (p-Akt), extracellular regulated kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), and cyclin D1 protein levels were assessed by Western blot analysis. Apoptosis was confirmed by 4′,6′-diaminido-2-phenylindole dihydrochloride (DAPI) staining, and mitochondrial membrane potential was performed using rhodamine-123 staining method.ResultsZinc significantly reduces the cell viability of PC-3 cells. It decreases the protein levels of IGF-IR, IRS-1, and IRS-2 and increases the level of IGFBP-3. Zinc reduces the levels of PI-3 K, Akt, ERK1/2, and cyclin Dl. Loss of mitochondrial membrane potential and apoptotic cell death were also observed in zinc-treated cells.ConclusionThis study suggests that zinc decreases the survival of androgen-independent prostate cancer cells by modulating the expression of IGF system components and its signaling molecules. Thus, zinc may be qualified as a potential agent for the treatment of prostate cancer.     

Inhibition of androgen receptor signaling by selenite and methylseleninic acid in prostate cancer cells: two distinct mechanisms of action

The development of prostate cancer and its progression to a hormone-refractory state is highly dependent on androgen receptor (AR) expression. Recent studies have shown that the selenium-based compound methylseleninic acid (MSeA) can disrupt AR signaling in prostate cancer cells. We have found that selenite can inhibit AR expression and activity in LAPC-4 and LNCaP prostate cancer cells as well but through a different mechanism. On entering the cell, selenite consumes reduced glutathione (GSH) and generates superoxide radicals. Pretreatment with N-acetylcysteine, a GSH precursor, blocked the down-regulation of AR mRNA and protein expression by selenite and restored AR ligand binding and prostate-specific antigen expression to control levels. MSeA reacts with reduced GSH within the cell; however, N-acetylcysteine did not effect MSeA-induced down-regulation of AR and prostate-specific antigen. The superoxide dismutase mimetic MnTMPyP was also found to prevent the decrease in AR expression caused by selenite but not by MSeA. A Sp1-binding site in the AR promoter is a key regulatory component for its expression. Selenite decreased Sp1 expression and activity, whereas MSeA did not. The inhibition of Sp1 by selenite was reversed in the presence of N-acetylcysteine. In conclusion, we have found that selenite and MSeA disrupt AR signaling by distinct mechanisms. The inhibition of AR expression and activity by selenite occurs via a redox mechanism involving GSH, superoxide, and Sp1.     

Bortezomib sensitizes HCC cells to CS-1008, an antihuman death receptor 5 antibody, through the inhibition of CIP2A

Previously, we have shown that bortezomib overcame TRAIL resistance in hepatocellular carcinoma (HCC) cells via the inhibition of Akt. Here, we report that bortezomib sensitizes these TRAIL-resistant cells, including Huh-7, Hep3B, and Sk-Hep1, to CS-1008, a humanized agonistic antihuman death receptor 5 antibody. Cancerous inhibitor of protein phosphatase 2A (CIP2A) mediated the sensitizing effect of bortezomib to CS-1008 through inhibiting protein phosphatase 2A (PP2A) activity. Combination treatment of bortezomib and CS-1008 downregulated CIP2A in a concentration- and time-dependent manner, and increased PP2A activity in HCC cells. Importantly, ectopic expression of CIP2A decreased Akt-related PP2A activity, indicating that CIP2A negatively regulates Akt-related PP2A activity in HCC cells. Moreover, silencing CIP2A by short interfering RNA enhanced CS-1008-induced apoptosis in HCC cells and ectopic expression of CIP2A in HCC cells abolished CS-1008-induced apoptosis, indicating that CIP2A plays an important role in the sensitizing effect of bortezomib to CS-1008. Finally, our in vivo data showed that CS-1008 and bortezomib combination treatment decreased tumor growth significantly. In conclusion, bortezomib sensitized HCC cells to CS-1008 through the inhibition of CIP2A.