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.     

Modulation of protein phosphatase 2A activity alters androgen-independent growth of prostate cancer cells: therapeutic implications

Earlier we identified PPP2CA, which encodes for the α-isoform of protein phosphatase 2A (PP2A) catalytic subunit, as one of the downregulated genes in androgen-independent prostate cancer. PP2A is a serine/threonine phosphatase and a potent tumor suppressor involved in broad cellular functions; however, its role in prostate cancer has not yet been determined. Here, we have investigated the effect of PP2A activity modulation on the androgen-independent growth of prostate cancer cells. Our data show that the PPP2CA expression and PP2A activity is downregulated in androgen-independent (C4-2) prostate cancer cells as compared with androgen-dependent (LNCaP) cells. Downregulation of PP2A activity by pharmacologic inhibition or short interfering RNA-mediated PPP2CA silencing sustains the growth of LNCaP cells under an androgen-deprived condition by relieving the androgen deprivation-induced cell-cycle arrest and preventing apoptosis. Immunoblot analyses reveal enhanced phosphorylation of Akt, extracellular signal-regulated kinase (ERK), BAD, increased expression of cyclins (A1/D1), and decreased expression of cyclin inhibitor (p27) on PP2A downregulation. Furthermore, our data show that androgen receptor (AR) signaling is partially maintained in PP2A-inhibited cells through increased AR expression and ligand-independent phosphorylation. Pharmacologic inhibition of Akt, ERK, and AR suggest a role of these signaling pathways in facilitating the androgen-independent growth of LNCaP cells. These observations are supported by the effect of ceramide, a PP2A activator, on androgen-independent C4-2 cells. Ceramide inhibited the growth of C4-2 cells on androgen deprivation, an effect that could be abrogated by PP2A downregulation. Altogether, our findings suggest that modulation of PP2A activity may represent an alternative therapeutic approach for the treatment of advanced androgen-independent 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.     

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.     

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.     

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.     

Targeting prostate cancer with conditionally replicative adenovirus using PSMA enhancer.

Prostate cancer is the second most commonly diagnosed cancer in men and accounts for significant mortality and morbidity in the United States. Initially androgen-dependent, prostate cancer ultimately becomes androgen-independent, which makes the disease extremely difficult to cure. In this study, we examined the use of conditionally replication-competent adenovirus for the treatment of hormone-independent prostate cancer. We utilized PSME, an enhancer element for prostate-specific PSMA expression, to control viral E1A protein expression and achieve exclusive virus replication in prostate. Western blotting confirmed that PSME mediated high E1A protein expression in PSMA-positive, androgen-independent prostate cancer cells (C4-2 and CWR22rv), but was much less active in PSMA-negative cancer cells (PC-3 and A549). Consistent with E1A protein expression, the recombinant adenovirus Ad5-PSME-E1a replicated in C4-2 and CWR22rv almost as efficiently as wild type with low levels of androgen, but its replication was significantly attenuated in PSMA-negative cells. In the in vitro killing assay, Ad5-PSME-E1a lysed all C4-2 and CWR22rv cells 5 days after infection, with minimal effect on PSMA-negative cells. In addition, injections of 1.7 x 10(8) plaque-forming units in a CWR22rv xenograft model in nude mice induced significant tumor growth delay, with a substantial necrotic area. These studies suggest that PSME-driven replication-competent adenovirus may be a new therapeutic modality for prostate cancer patients after hormone ablation therapy.     

Prostate cancer: status of current treatments and emerging antisense-based therapies

Prostate cancer, like most other solid tumors, represents a heterogeneous entity consisting of a mixture of androgen-dependent and androgen-independent cells. Although proliferation of prostate tumor cells is often initially androgen-dependent, the inevitable development of androgen-insensitivity in late-stage prostate cancer renders androgen-suppressing treatments ultimately ineffective. Non-hormonal chemotherapy induces apoptosis in actively proliferating cells and is typically of little value, since prostate cancer demonstrates very slow growth kinetics. Objective response rates of < 10% and no improved survival rates have been observed in several hundred clinical studies using both experimental and approved chemotherapeutic agents. An improved understanding of the molecular mechanisms responsible for the onset of the disease, as well as the factors that control the proliferation of prostate cancer cells, have identified key changes in gene expression during cancer progression, especially from androgen-dependent to androgen-independent status. Manipulation of the genes implicated in disease progression represent an important approach for therapeutic intervention. This review summarizes recent progress that has been made with the use of antisense technology with various chemistries to modify gene expression, a strategy that seems to hold great promise for prostate cancer therapy.     

A novel class of pyranocoumarin anti-androgen receptor signaling compounds

Androgen and the androgen receptor (AR)-mediated signaling are crucial for prostate cancer development. Novel agents that can inhibit AR signaling in ligand-dependent and ligand-independent manners are desirable for the chemoprevention of prostate carcinogenesis and for the treatment of advanced prostate cancer. We have shown recently that the pyranocoumarin compound decursin from the herb Angelica gigas possesses potent anti-AR activities distinct from the anti-androgen bicalutamide. Here, we compared the anti-AR activities and the cell cycle arrest and apoptotic effects of decursin and two natural analogues in the androgen-dependent LNCaP human prostate cancer cell culture model to identify structure-activity relationships and mechanisms. Decursin and its isomer decursinol angelate decreased prostate-specific antigen expression with IC(50) of approximately 1 mumol/L. Both inhibited the androgen-stimulated AR nuclear translocation and transactivation, decreased AR protein abundance through proteasomal degradation, and induced G(0/1) arrest and morphologic differentiation. They also induced caspase-mediated apoptosis and reactive oxygen species at higher concentrations. Furthermore, they lacked the agonist activity of bicalutamide in the absence of androgen and were more potent than bicalutamide for suppressing androgen-stimulated cell growth. Decursinol, which does not contain a side chain, lacked the reactive oxygen species induction and apoptotic activities and exerted paradoxically an inhibitory and a stimulatory effect on AR signaling and cell growth. In conclusion, decursin and decursinol angelate are members of a novel class of nonsteroidal compounds that exert a long-lasting inhibition of both ligand-dependent and ligand-independent AR signaling. The side chain is critical for sustaining the anti-AR activities and the growth arrest and apoptotic effects.     

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.     

Monogene and polygene therapy for the treatment of experimental prostate cancers by use of apoptotic genes bax and bad driven by the prostate-specific promoter ARR(2)PB

We have shown that adenovirus-mediated manipulation of apoptotic genes such as bax could be a therapeutic option for prostate cancer. Unfortunately, the response of experimental prostate tumors to a single therapeutic gene of the apoptotic pathway is short-lived, and most of these tumors relapse after a short period of time. In this investigation we present data generated with adenovirus AvARR(2)PB-Bad, in which the apoptotic gene bad was placed under the control of the dihydrotestosterone (DHT)-inducible third-generation probasin-derived promoter ARR(2)PB. This therapeutic virus was given alone or in combination with other therapeutic viruses to a variety of in vitro and in vivo experimental models of prostate cancer. On infection with AvARR(2)PB-Bad, DHT-induced Bad overexpression occurred specifically in androgen receptor-positive (AR(+)) cells of prostatic derivation. The apoptotic effect of AvARR(2)PB-Bad (group 1) was compared with that of AvARR(2)PB-Bax (which overexpresses the apoptotic protein Bax) (group 2), with that of the combination AvARR(2)PB-Bad plus AvARR(2)PB-Bax (group 3), and with that of the control virus AvARR(2)PB-CAT (group 4) in the cell line LNCaP. In addition to identifying the modality of apoptosis induction by overexpressed Bad, the results suggested that group 3 contained more apoptotic cells than any other group. In additional studies, AR(+) androgen-dependent LNCaP cells or AR(+) and androgen-independent C4-2 cells were injected subcutaneously into nude mice. Four groups of six LNCaP or C4-2 tumors were treated with the same combinations of viruses discussed above for groups 1, 2, 3, and 4. Treatment resulted in decreased tumor size in groups 1, 2, and 3 compared with group 4. There was a better response in group 3 compared with group 2, and in group 2 compared with group 1. A better response in group 3 was confirmed during a 8-week follow-up period, in which no treatment was administered. Two LNCaP and C4-2 tumors of group 3 disappeared at the end of treatment and did not recur after an 8-week follow-up period. The data suggest that polygene therapy with apoptotic molecules is more effective in experimental models of androgen-dependent or -independent prostate cancer than monogene therapy.     

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.     

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.     

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.