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.     

Molecular genetics of prostate cancer

The molecular mechanisms underlying the development and progression of prostate cancer are poorly understood. Epidemiological studies have suggested that 5-10% of all prostate cancers are familial, and numerous chromosomal loci have been associated with prostate cancer in multicentre linkage studies. However, no putative susceptibility genes harboured in these chromosomal regions have thus far been identified. Several recurrent chromosomal alterations in prostate cancer have been detected in comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) analysis. The target genes for many of these aberrations are still not known. It seems that the androgen receptor (AR) signalling pathway plays a crucial role in both early development as well as in late progression of the disease. Both germ-line and somatic genetic alterations in the AR gene have been demonstrated in prostate cancer patients. The intention of this review is to summarize the current knowledge of molecular mechanisms in the development of prostate cancer.     

Synergistic effect of a novel antiandrogen, VN/124-1, and signal transduction inhibitors in prostate cancer progression to hormone independence in vitro

This study was carried out to determine the mechanisms associated with loss of androgen dependency and disease progression in prostate cancer. We investigated the role of the androgen receptor and its relationship to other signal transduction proteins. A hormone-refractory prostate cancer cell line [high-passage LNCaP (HP-LNCaP)] was established in vitro. Cells were treated with inhibitors of mammalian target of rapamycin and tyrosine kinase receptors. Expression of these proteins and the androgen receptor were measured by Western immunoblotting. Analysis of the model and various treatments was also assessed through proliferation assays, luciferase activation assays, binding assays, and ELISA. Our novel antiandrogen, VN/124-1, effectively inhibited proliferation of hormone-resistant prostate cancer cell lines (HP-LNCaP), which were no longer sensitive to bicalutamide and had increased expression of the androgen receptor. Treatment with everolimus or gefitinib resulted in an increase in protein expression and activation of the androgen receptor. Conversely, inhibition of the androgen receptor resulted in increased expression of IGFR1beta, pHER2, pmTOR, and pAkt. The addition of bicalutamide to everolimus or gefitinib inhibited cell proliferation in HP-LNCaP cells. However, the addition of VN/124-1 has proven to be superior to bicalutamide, and the combination was synergistic (P<0.05) compared with either agent alone. This study suggests that compensatory cross-talk between the androgen receptor and various signaling pathways may account for decreased sensitivity to androgen receptor antagonists and the progression to hormone-resistant prostate cancer. Furthermore, these findings suggest that inhibition of both pathways may provide effective control in hormone-resistant prostate cancer and restore sensitivity to androgen antagonists in hormone-refractory patients.     

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.     

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.     

Small-interfering RNA-induced androgen receptor silencing leads to apoptotic cell death in prostate cancer

Prostate cancer is the second leading cause of cancer death in the United States and, thus far, there has been no effective therapy for the treatment of hormone-refractory disease. Recently, the androgen receptor (AR) has been shown to play a critical role in the development and progression of the disease. In this report, we showed that knocking down the AR protein level by a small interfering RNA (siRNA) approach resulted in a significant apoptotic cell death as evidenced by an increased annexin V binding, reduced mitochondrial potential, caspase-3/6 activation, and DFF45 and poly(ADP-ribose) polymerase cleavage. The apoptotic response was specifically observed in those siRNA-transfected cells that harbor a native AR gene. No cell death was found in the AR-null prostate cancer cell PC-3 or its subline that has been reconstituted with an exogenous AR gene, as well as two breast cancer cell lines that are AR positive. Moreover, in parallel with the siRNA-induced AR silencing, the antiapoptotic protein Bcl-xL was significantly reduced, which might account for the apoptotic cell death because ectopic enforced expression of Bcl-xL protein partially inhibited apoptosis after AR silencing. Taken together, our data showed that knocking down the AR protein level in prostate cancer cells leads to apoptosis by disrupting the Bcl-xL-mediated survival signal downstream of AR-dependent survival pathway.     

Antisense approaches in prostate cancer

Patients with hormone refractory prostate cancer have limited treatment options and new therapies are urgently needed. Advances in the understanding of the molecular mechanisms implicated in prostate cancer progression have identified many potential therapeutic gene targets that are involved in apoptosis, growth factors, cell signalling and the androgen receptor (AR). Antisense oligonucleotides are short sequences of synthetic modified DNA that are designed to be complimentary to a selected gene's mRNA and thereby specifically inhibit expression of that gene. The antisense approach continues to hold promise as a therapeutic modality to target genes involved in cancer progression, especially those in which the gene products are not amenable to small molecule inhibition or antibodies. The current status and future direction of a number of antisense oligonucleotides targeting several genes, including BCL-2, BCL-XL, clusterin, the inhibitors of apoptosis (IAP) family, MDM2, protein kinase C-alpha, c-raf, insulin-like growth factor binding proteins and the AR, that have potential clinical use in prostate cancer are reviewed.     

Bisphenol A facilitates bypass of androgen ablation therapy in prostate cancer

Prostatic adenocarcinomas depend on androgen for growth and survival. First line treatment of disseminated disease exploits this dependence by specifically targeting androgen receptor function. Clinical evidence has shown that androgen receptor is reactivated in recurrent tumors despite the continuance of androgen deprivation therapy. Several factors have been shown to restore androgen receptor activity under these conditions, including somatic mutation of the androgen receptor ligand-binding domain. We have shown previously that select tumor-derived mutants of the androgen receptor are receptive to activation by bisphenol A (BPA), an endocrine-disrupting compound that is leached from polycarbonate plastics and epoxy resins into the human food supply. Moreover, we have shown that BPA can promote cell cycle progression in cultured prostate cancer cells under conditions of androgen deprivation. Here, we challenged the effect of BPA on the therapeutic response in a xenograft model system of prostate cancer containing the endogenous BPA-responsive AR-T877A mutant protein. We show that after androgen deprivation, BPA enhanced both cellular proliferation rates and tumor growth. These effects were mediated, at least in part, through androgen receptor activity, as prostate-specific antigen levels rose with accelerated kinetics in BPA-exposed animals. Thus, at levels relevant to human exposure, BPA can modulate tumor cell growth and advance biochemical recurrence in tumors expressing the AR-T877A mutation.     

B4 androgen ablation: attacking the prostate cancer stem cell

There is increasing evidence that prostate cancers in rodent models and in men contain a cellular subpopulation that displays stem cell properties. These prostate cancer stem cells (PCSCs) lack androgen receptor expression and are increased in castration-resistant disease. In this issue of the JCI, a study from Yoshioka et al. demonstrates that PCSCs are regulated by a pathway in which α6β4 integrin amplifies signaling through ErbB2 and c-Met receptors. Targeting this pathway provides a novel therapeutic strategy for hormone refractory prostate cancer. The prostate gland is thought to contain two distinct stem cell populations, one located in the basal layer and the other located within the luminal compartment. In mouse models, there is evidence that either of these primitive stem cells is capable of giving rise to prostate cancers (1). Development of the normal prostate gland and prostate cancers is dependent on androgenic hormones, the actions of which are mediated by the androgen receptor (AR). In the mouse, although orchiectomy results in marked prostate involution, the gland is completely regenerated following androgen replacement, suggesting the existence of castration-resistant stem cells. Similarly, although androgen ablation remains a mainstay for the treatment of advanced prostate cancer, hormone resistance inevitably develops and, as a result, hormone-refractory metastatic prostate cancer remains incurable. There is increasing evidence that many cancers, including those of the prostate, are driven and maintained by a cellular subpopulation that displays stem cell properties. These properties include self-renewal, which drives tumorigenesis, as well as differentiation, which generates the tumor bulk. Furthermore, studies suggest that unlike AR-driven bulk tumor cell populations, prostate cancer stem cells (PCSCs) lack AR and do not express differentiation markers, such as PSA (2). PCSCs are increased in hormone-refractory prostate cancer in patients as well as in mouse models. In addition to their role in hormone resistance, evidence suggests that these PCSCs may also mediate metastasis to sites such as the bone (3). Given their potential clinical importance, an understanding of the pathways that drive hormone-resistant PCSCs is of considerable importance. In this issue of the JCI, Yoshioka et al. use human prostate cancer specimens and mouse transgenic models to demonstrate an important role for β4 integrin as well as the coexpressed receptor tyrosine kinases ErbB2 and c-Met in the regulation of PCSCs (4).     

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.     

Androgen Receptor Content of the Normal and Hyperplastic Canine Prostate

A procedure was developed for measurement of androgen receptors in cytoplasmic extracts of prostates from intact dogs. The protocol utilized exchange saturation analysis at 15 degrees C employing the synthetic androgen R1881 (17beta-hydroxy-17alpha-methylestra-4,9,11-trien-3-one) as the ligand probe and quantitatively detected total cytoplasmic androgen receptor (R(c), androgen-free receptor, and R(c)A, androgen-occupied receptor) present at the initiation of the assay. This protocol was employed in conjunction with a tissue mince saturation analysis procedure (for quantitation of nuclear androgen receptor) to quantitate total androgen receptor content of normal and hyperplastic prostates obtained from young (2.5- or 4.6-yr old) and aged (12.5-yr old) purebred dogs of known birth date.The total cytoplasmic androgen receptor content (picomoles per prostate) of hyperplastic prostates was 4.6-fold greater than that of normal prostates. The total nuclear androgen receptor content of hyperplastic prostates (picomoles per prostate measured in crude nuclear preparations) was either 5.0- (4.6-yr-old dogs) or 7.8-fold (2.5-yr-old dogs) greater than that of normal prostates. However, androgen receptor content per cell was identical for hyperplastic and normal canine prostates, with the exception that nuclear androgen receptor was diminished in prostates from 2.5-yr-old dogs. The cell content per gram dry weight was identical for hyperplastic and normal canine prostates. We conclude that canine prostate hyperplasia is characterized by coordinate proliferation of androgen receptor-positive and androgen receptor-negative cells and is not a consequence of increased accumulation of 5alpha-dihydrotestosterone due to proliferation of androgen receptors per prostate cell.     

Androgen receptor as a therapeutic target for androgen independent prostate cancer

Prostate cancer is the leading cause of nonskin malignancy and the second leading cause of cancer death in men. Androgen deprivation therapy is the first-line of systemic therapy against advanced prostate cancer. All advanced prostate cancers eventually grow despite castrate levels of testosterone. We review the evidence that androgen independent prostate cancer continues to require androgen receptor activity for growth, the mechanisms of androgen receptor activation in the castrate setting, and possible points of intervention for novel therapies targeting the androgen receptor and 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.