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Transmembrane 4 superfamily 3 (TM4SF3) was identified as a novel androgen-regulated gene in prostate cancer (PCa) cells. Our data demonstrate that TM4SF3 exhibits androgen-induced repression of the mRNA but up-regulation of the protein. The androgen positive effect on the TM4SF3 protein is of significant interest in view of the procancer functions of both androgens and tetraspanin proteins. Androgen positively regulates TM4SF3 protein stability by inhibiting its proteasome-dependent degradation. This androgen stabilization of TM4SF3 is involved in promoting PCa cell invasion and migration of both androgen-dependent and androgen-independent PCa cells. Although confirming androgen up-regulation of the TM4SF3 protein, we observed that TM4SF3 is localized not only to the membrane, but also, surprisingly, the nuclei of PCa cells. This novel nuclear localization of TM4SF3 depends on androgen-induced nuclear localization of androgen receptor (AR) in both androgen-dependent and androgen-independent PCa cell lines. TM4SF3 interacts with AR both in PCa cell types and in vitro, strongly suggesting a direct interaction. This direct interaction is required for the stabilization of not only TM4SF3, but also remarkably AR, because down-regulation of TM4SF3 resulted in reduced AR protein levels. As expected of an important AR regulator, TM4SF3 regulates androgen-dependent gene expression in and proliferation of PCa cells. Importantly, a direct correlation between AR and TM4SF3 protein levels and nuclear colocalization were also observed in prostate tumors, strongly suggesting that the mutual stabilization resulting from the AR-TM4SF3 interaction is found in tumors and that this interaction is important in PCa biology.

Our laboratory previously has identified soluble guanylyl cyclase α1 (sGCα1) as a direct target of androgen receptor and essential for prostate cancer cell growth via a pathway independent of nitric oxide (NO) signaling. We identified the COP9 signalosome subunit 4 (CSN4) as a novel interacting partner for sGCα1. Importantly, the CSN4-sGCα1 interaction inhibits sGCα1 proteasomal degradation. Consistent with this, disruption of CSN4 led to a significant decrease in prostate cancer cell proliferation, which was significantly but not completely rescued by sGCα1 overexpression, opening the possibility of an additional target of CSN4. Interestingly, immunoprecipitation experiments showed that p53 is found in the CSN4-sGCα1 cytoplasmic protein complex. However, in contrast to sGCα1, p53 protein stability was compromised by CSN4, leading to prostate cancer cell survival and proliferation. Interestingly, we observed that CSN4 was overexpressed in prostate tumors, and its protein level correlates directly with sGCα1 and inversely with p53 proteins, mimicking what was observed in prostate cancer cells. Our data further showed that CSN4 silencing decreased CSN5 protein levels and suggest that the CSN4 effects on sGCα1 and p53 proteins are mediated by CSN5. Lastly, our study showed that caseine kinase-2 (CK2) was involved in regulating p53 and sGCα1 protein stability as determined by both disruption of CK2 expression and inhibition of its kinase activity. Collectively, our study has identified a novel endogenous CSN4-CSN5-CK2 complex with sGCα1and p53 that oppositely controls the stability of these 2 proteins and provides prostate cancer cells an important mechanism for survival and proliferation.

Our laboratory has previously identified soluble guanylyl cyclase α1 (sGCα1) as a novel androgen-regulated gene essential for prostate cancer cell proliferation. sGCα1 expression is highly elevated in prostate tumors, contrasting with the low expression of sGCβ1, with which sGCα1 dimerizes to mediate nitric oxide (NO) signaling. In studying its mechanism of action, we have discovered that sGCα1 can inhibit the transcriptional activity of p53 in prostate cancer cells independent of either classical mediators of NO signaling or the guanylyl cyclase activity of sGCα1. Interestingly, sGCα1 inhibition of p53-regulated gene expression was gene specific, targeting genes involved in apoptosis/cell survival. Consistent with this, overexpression of sGCα1 makes prostate cancer cells more resistant to etoposide, a chemotherapeutic and apoptosis-inducing drug. Immunoprecipitation and immunocytochemistry assays show a physical and direct interaction between sGCα1 and p53 in prostate cancer cells. Interestingly, sGCα1 induces p53 cytoplasmic sequestration, representing a new mechanism of p53 inactivation in prostate cancer. Analysis of prostate tumors has shown a direct expression correlation between sGCα1 and p53. Collectively, these data suggest that sGCα1 regulation of p53 activity is important in prostate cancer biology and may represent an important mechanism of p53 down-regulation in those prostate cancers that express significant levels of p53.

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.