Androgens and the androgen receptor (AR) act in cells by modulating gene expression. Through gene microarray studies, we have identified Ets Variant Gene 1 (ETV1) as a novel androgen-regulated gene. Our data demonstrate that ETV1 mRNA and protein are up-regulated in response to ligand-activated AR in androgen-dependent LNCaP cells, but there is no detectable ETV1 expression in normal prostate cells. The ETV1 promoter is induced by androgens and recruits the AR in the context of chromatin. ETV1-regulated endogenous matrix metalloproteinase genes can be induced by ligand-activated AR. In contrast to the hormone-induced expression in androgen-dependent LNCaP cells, ETV1 expression in androgen-independent LNCaP cells is high and unresponsive to androgen. This androgen-independent ETV1 expression contrasts with the hormone-dependent expression observed for TMPRSS2 in these androgen-independent prostate cancer cells. ETV1 is overexpressed in prostate cancer independent of the TMPRSS2:ETV1 translocation. Disruption of ETV1 expression in both androgen-dependent and androgen-independent prostate cancer cells significantly compromises the invasion capacity of these cells, suggesting an important role for ETV1 in prostate cancer metastasis. Collectively, these results demonstrate that ETV1 expression transitions from androgen-induced to androgen-independent as prostate cancer cells switch from hormone-dependent to hormone-refractory and suggest that this transition may be in part responsible for the elevated levels of ETV1 observed in prostate tumors. Additionally, our data provide an indirect mechanism of AR regulation of gene expression, via the transactivation of the transcription factor ETV1.

The growth and progression of prostate cancer are dependent on androgens and androgen receptor (AR), which act by modulating gene expression. Utilizing a gene microarray approach, we have identified the a1-subunit gene of soluble guanylyl cyclase (sGC) as a novel androgen-regulated gene. A heterodimeric cytoplasmic protein composed of one a and one b subunit, sGC mediates the widespread cellular effects of nitric oxide (NO). We report here that, in prostate cancer cells, androgens stimulate the expression of sGCa1. A cloned human sGCa1 promoter is activated by androgen in an AR-dependent manner, suggesting that sGCa1 may be a direct AR target gene. Disruption of sGCa1 expression severely compromises the growth of both androgendependent and androgen-independent AR-positive prostate cancer cells. Overexpression of sGCa1 alone is sufficient for stimulating prostate cancer cell proliferation. Interestingly, the major growth effect of sGCa1 is independent of NO and cyclic guanosine monophosphate, a major mediator of the sGC enzyme. These data strongly suggest that sGCa1 acts in prostate cancer via a novel pathway that does not depend on sGCb1. Tissue studies show that sGCa1 expression is significantly elevated in advanced prostate cancer. Thus, sGCa1 may be an important mediator of the procarcinogenic effects of androgens.

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Multidrug resistance-associated proteins (MRPs) may mediate multidrug resistance in tumor cells. Using a gene array analysis, we have identified MRP4 as an androgen receptor (AR)-regulated gene. Dihydrotestosterone induced MRP4 expression in both androgen-dependent and -independent LNCaP cells, whereas there was little detectable expression in PC-3 or normal prostate epithelial cells. Disruption of MRP4 expression renders LNCaP cells more sensitive to the cytotoxic effects of methotrexate but not etoposide. Analysis of human tissues showed detectable MRP4 expression only in metastatic prostate cancer. These results suggest that AR induction of MRP4 mediates resistance of PC cells to nucleotide-based chemotherapeutic drugs.

Androgens are important for male sexual development, which depend on the cognate receptor, the androgen receptor. The transcriptional activity of the androgen receptor, like other nuclear receptors, is regulated by accessory proteins that can have either positive or negative effects. Through a yeast functional screen, we have identified SUMO-3 as a regulator of androgen receptor activity in prostate cancer cells. SUMO-3 is one of three eukaryotic proteins that become post-translationally conjugated to their target proteins in a manner analogous to the attachment of ubiquitin. In primary prostate epithelial cells, PrEC, and the prostate cancer cells, PC-3, SUMO-3 has a weak negative effect on androgen receptor transcriptional activity. In contrast, SUMO-3 and it close relative SUMO-2 strongly enhance transactivation by endogenous androgen receptor in LNCaP cells. This positive effect is observed in both androgendependent and androgen-independent LNCaP cells. Interestingly, SUMO-1, unlike SUMO-3 and SUMO-2, can inhibit, but not stimulate, androgen receptor activity. Mutational analysis of the androgen receptor and SUMO-3 demonstrates that the SUMO-3-positive activity does not depend on either the sumoylation sites of the androgen receptor or the sumoylation function of SUMO-3. Stable overexpression of SUMO-3 in LNCaP cells significantly enhances the androgen-dependent proliferation of these cells. Additionally, siRNA-mediated repression of SUMO-2 significantly inhibits the growth of both androgen-dependent and -independent LNCaP cells. Collectively, these results suggest (i) a novel mechanism for elevating AR activity through the switch of SUMO-3 from a weak negative regulator in normal prostate cells to a strong positive regulator in prostate cancer cells and (ii) a proliferative role for SUMO-3 and SUMO-2 in the growth of prostate cancer cells that is independent of sumoylation of the androgen receptor.The physiological functions of the androgens testosterone and 5␣-dihydrotestosterone (DHT) 4 are mediated by the androgen receptor (AR) (reviewed in Ref. 1), a member of the nuclear receptor superfamily (reviewed in Refs. 2-6). Through the regulation of target genes, androgens and AR play an essential role in male sexual development and the proper development and function of male reproductive organs, such as prostate and epididymis (7). Patients with 5␣-reductase II deficiency, which results in low levels of DHT, have ambiguous external genitalia and a highly underdeveloped and impalpable prostate (8, 9). Reduction or loss of AR activity in males results in androgen insensitivity syndrome (10). In addition to normal prostate development, AR is essential for the initiation and progression of prostate cancer. The best demonstration of this is the effectiveness of anti-androgen and androgen ablation therapy in inhibiting the development of prostate cancer in the early stage of the disease (11). However, local recurrences and metastases will eventually develop in most, if not all, patients after therapy, and prostat...

Through its transcriptional activities, the proto-oncoprotein c-Jun can regulate cellular proliferation, survival, and differentiation. We have established a novel yeast assay that screens for repressors of c-Jun transcriptional activity. This screen led to the identification of a ubiquitously expressed novel RING zinc finger protein, termed Makorin RING zinc finger protein 1 (MKRN1), recently shown to act as an E3 ubiquitin ligase. Overexpression of MKRN1 in mammalian cells inhibited the transcriptional activities of not only c-Jun, but also the nuclear receptors, the androgen receptor, and the retinoic acid receptors. Truncation analysis indicates that both the amino and carboxy termini are required for this transrepression activity. Surprisingly, when fused to the heterologous DNAbinding domain of GAL4, MKRN1 activates, rather than inhibits, a GAL4-responsive reporter plasmid. In addition, truncation of either the amino- or carboxy-terminal half of MKRN1 disrupts its transactivation activity, the same observation that was made on its transrepression activity. These results demonstrate that MKRN1 has transcriptional activity and suggest that its transrepression and transactivation functions are mediated by the same mechanism. Interestingly, disruption of MKRN1's ubiquitin ligase activity does not affect its inhibitory transcriptional activity. Thus, MKRN1 may represent a nuclear protein with multiple nuclear functions, including regulating RNA polymerase II-catalyzed transcription.

Background Serum androgen concentrations decline with age in male Brown Norway rats and castration induces apoptosis of luminal secretory epithelial cells in the ventral but not in the dorsal and lateral prostate lobes. Clusterin has been described as an androgen-repressed gene and a protein with either anti- or pro-apoptotic actions. Methods We measured clusterin mRNA and protein levels, the effects of aging and castration on clusterin protein levels and clusterin immunolocalization within the prostatic ductal network in the prostate lobes of young and aged rats. Results Whereas levels of clusterin mRNA and protein expression measured by RT-PCR and Western blot, respectively, were higher in the ventral and lateral lobes of aged (24 months) compared to young (4 months) rats, no age-dependent differences were observed in the dorsal lobe. Clusterin expression was localized by immunohistochemistry exclusively to the proximal duct segment of young rats, but extended to the distal segment of the ventral and lateral lobes of aged rats. Despite an age-related decrease in serum testosterone concentration, clusterin gene expression was not altered in the dorsal lobe. After castration, levels of clusterin expression increased significantly in the ventral and lateral lobes despite the absence of epithelial cell apoptosis in the latter. In castrated rats, clusterin expression extended throughout the proximal-distal duct regions of the prostate lobes of young and aged rats. Conclusion Regulation of clusterin expression in the prostate lobes of aging rats appears complex and is neither directly repressed by androgen nor dependent on apoptotic-induced stress.