Search

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.

The Zinc Finger (ZNF) 280B protein was identified as an unexpected target of an shRNA designed for sGCα1. Further analysis showed that these two proteins are connected in another way, with 280B up-regulation of sGCα1 expression. Knock-down and over-expression experiments showed that 280B serves pro-growth and pro-survival functions in prostate cancer. Surprisingly however, these pro-cancer functions of 280B are not mediated by sGCα1, which itself has similar functions in prostate cancer, but by down-regulated p53. The p53 protein is a second target of 280B in prostate cancer, but unlike sGCα1, p53 is down-regulated by 280B. 280B induces p53 nuclear export, leading to subsequent proteasomal degradation. The protein responsible for p53 regulation by 280B is Mdm2, the E3 ubiquitin ligase that promotes p53 degradation by inducing its nuclear export. We show here that 280B up-regulates expression of Mdm2 in prostate cancer cells, and this regulation is via the Mdm2 promoter. To demonstrate an in vivo relevance to this interaction, expression studies show that 280B protein levels are up-regulated in prostate cancer and these levels correspond to reduced levels of p53. Thus, by enhancing the expression of Mdm2, the uncharacterized 280B protein provides a novel mechanism of p53 suppression in prostate cancer.

Among the many identified androgen-regulated genes, sGCα1 (soluble guanylyl cyclase α1) appears to play a pivotal role in mediating the pro-cancer effects of androgens and androgen receptor. The classical role for sGCα1 is to heterodimerize with the sGCβ1 subunit, forming sGC, the enzyme that mediates nitric oxide signaling by catalyzing the synthesis of cyclic guanosine monophosphate. Our published data show that sGCα1 can drive prostate cancer cell proliferation independent of hormone and provide cancer cells a pro-survival function, via a novel mechanism for p53 inhibition, both of which are independent of sGCβ1, NO, and cGMP. All of these properties make sGCα1 an important novel target for prostate cancer therapy. Thus, peptides were designed targeting sGCα1 with the aim of disrupting this protein's pro-cancer activities. One peptide (A-8R) was determined to be strongly cytotoxic to prostate cancer cells, rapidly inducing apoptosis. Cytotoxicity was observed in both hormone-dependent and, significantly, hormone-refractory prostate cancer cells, opening the possibility that this peptide can be used to treat the usually lethal castration-resistant prostate cancer. In mouse xenograft studies, Peptide A-8R was able to stop tumor growth of not only hormone-dependent cells, but most importantly from hormone-independent cells. In addition, the mechanism of Peptide A cytotoxicity is generation of reactive oxygen species, which recently have been recognized as a major mode of action of important cancer drugs. Thus, this paper provides strong evidence that targeting an important AR-regulated gene is a new paradigm for effective prostate cancer therapy.

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.