COP9 Subunits 4 and 5 Target Soluble Guanylyl Cyclase α1 and p53 in Prostate Cancer Cells

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

Abstract 215: Soluble guanylyl cyclase α1 and p53 cytoplasmic sequestration: A novel mechanism for p53 down-regulation in prostate cancer

Abstract Our lab has identified soluble guanylyl cyclase α1 (sGCα1) as a novel androgen- regulated gene involved in prostate cancer cell proliferation. A well-known biological function of sGCα1 is to heterodimerize with sGCβ1 forming the enzyme sGC, which mediates nitric oxide(NO) signaling. Our published data demonstrated that the sGCα1 effect on prostate cancer cell growth is independent of the classical mediators of NO signaling, suggesting that sGCα1 acts via a novel signaling pathway. Furthermore, sGCα1 expression is highly elevated in prostate tumors and this expression is directly correlated to the progression stage of prostate cancer. Interestingly, we have recently discovered that sGCα1 can inhibit the transcriptional activity of p53 via a mechanism that does not depend on classical mediators of NO signaling. Immunoprecipitation and immunocytochemistry assays have shown an association between sGCα1 and p53 and these data suggest that sGCa1 inhibits p53 by mediating the cytoplasmic sequestration of this tumor suppressor protein, which would represent a previously unknown mechanism for p53 regulation. sGCa1 can also regulate in a gene-specific manner p53-regulated gene expression, targeting genes involved in apoptosis. In addition, sGCa1 makes prostate cancer cells resistant to the chemotherapeutic and apoptosis-inducing drug Etoposide. Finally, analysis of prostate tumors has shown a direct correlation in expression between sGCα1 and p53. Collectively, these data suggest that sGCa1 regulation of p53 activity is important in prostate cancer biology and may represent an import mechanism of p53 down-regulation in those prostate cancers that express significant levels of p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 215. doi:10.1158/1538-7445.AM2011-215</jats:p

Ligand-Activated Retinoic Acid Receptor Inhibits AP-1 Transactivation by Disrupting c-Jun/c-Fos Dimerization

AbstractIn the presence of retinoic acid (RA), the retinoid receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), are able to up-regulate transcription directly by binding to RA-responsive elements on the promoters of responsive genes. Liganded RARs and RXRs are also capable of down-regulating transcription, but, by contrast, this is an indirect effect, mediated by the interaction of these nuclear receptors not with DNA but the transcription factor activating protein-1 (AP-1). AP-1 is a dimeric complex of the protooncoproteins c-Jun and c-Fos and directly regulates transcription of genes important for cellular growth. Previous in vitro results have suggested that RARs can block AP-1 DNA binding. Using a mammalian two-hybrid system, we report here that human RARα (hRARα) can disrupt in a RA-dependent manner the homo- and heterodimerization properties of c-Jun and c-Fos. This inhibition of dimerization is cell specific, occurring only in those cells that exhibit RA-induced repression of AP-1 transcriptional activity. Furthermore, this mechanism appears to be specific for the RARs, since another potent inhibitor of AP-1 activity, the glucocorticoid receptor, does not affect AP-1 dimerization. Our data argue for a novel mechanism by which RARs can repress AP-1 DNA binding, in which liganded RARs are able to interfere with c-Jun/c-Jun homodimerization and c-Jun/c-Fos heterodimerization and, in this way, may prevent the formation of AP-1 complexes capable of DNA binding.</jats:p

Androgen up-regulation of Twist1 gene expression is mediated by ETV1

Twist1, a basic helix-loop-helix transcription factor that regulates a number of genes involved in epithelial-to-mesenchymal transition (EMT), is upregulated in prostate cancer. Androgen regulation of Twist1 has been reported in a previous study. However, the mechanism of androgen regulation of the Twist1 gene is not understood because the Twist1 promoter lacks androgen receptor (AR)-responsive elements. Previous studies have shown that the Twist1 promoter has putative binding sites for PEA3 subfamily of ETS transcription factors. Our lab has previously identified Ets Variant 1 (ETV1), a member of the PEA3 subfamily, as a novel androgen-regulated gene that is involved in prostate cancer cell invasion through unknown mechanism. In view of these data, we hypothesized that androgen-activated AR upregulates Twist1 gene expression via ETV1. Our data confirmed the published work that androgen positively regulates Twist1 gene expression and further showed that this positive effect was directed at the Twist1 promoter. The positive effect of androgen on Twist1 gene expression was abrogated upon disruption of AR expression by siRNA or of AR activity by Casodex. More importantly, our data show that disruption of ETV1 leads to significant decrease in both androgen-mediated upregulation as well as basal level of Twist1, which we are able to rescue upon re-expression of ETV1. Indeed, we are able to show that ETV1 mediates the androgen upregulation of Twist1 by acting on the proximal region of Twist1 promoter. Additionally, our data show that Twist1 regulates prostate cancer cell invasion and EMT, providing a possible mechanism by which ETV1 mediates prostate cancer cell invasion. In conclusion, in this study we report Twist1 as an indirect target of AR and androgen regulation through ETV1

Zinc Finger 280B regulates sGCα1 and p53 in prostate cancer cells.

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

Androgen up-regulation of Twist1 gene expression is mediated by ETV1

Twist1, a basic helix-loop-helix transcription factor that regulates a number of genes involved in epithelial-to-mesenchymal transition (EMT), is upregulated in prostate cancer. Androgen regulation of Twist1 has been reported in a previous study. However, the mechanism of androgen regulation of the Twist1 gene is not understood because the Twist1 promoter lacks androgen receptor (AR)-responsive elements. Previous studies have shown that the Twist1 promoter has putative binding sites for PEA3 subfamily of ETS transcription factors. Our lab has previously identified Ets Variant 1 (ETV1), a member of the PEA3 subfamily, as a novel androgen-regulated gene that is involved in prostate cancer cell invasion through unknown mechanism. In view of these data, we hypothesized that androgen-activated AR upregulates Twist1 gene expression via ETV1. Our data confirmed the published work that androgen positively regulates Twist1 gene expression and further showed that this positive effect was directed at the Twist1 promoter. The positive effect of androgen on Twist1 gene expression was abrogated upon disruption of AR expression by siRNA or of AR activity by Casodex. More importantly, our data show that disruption of ETV1 leads to significant decrease in both androgen-mediated upregulation as well as basal level of Twist1, which we are able to rescue upon re-expression of ETV1. Indeed, we are able to show that ETV1 mediates the androgen upregulation of Twist1 by acting on the proximal region of Twist1 promoter. Additionally, our data show that Twist1 regulates prostate cancer cell invasion and EMT, providing a possible mechanism by which ETV1 mediates prostate cancer cell invasion. In conclusion, in this study we report Twist1 as an indirect target of AR and androgen regulation through ETV1.</jats:p

Peptide B targets soluble guanylyl cyclase α1 and kills prostate cancer cells.

Among androgen-regulated genes, soluble guanylyl cyclase α1 (sGCα1) is significant in promoting the survival and growth of prostate cancer cells and does so independent of nitric oxide (NO) signaling. Peptides were designed targeting sGCα1 to block its pro-cancer functions and one peptide is discussed here. Peptide B-8R killed both androgen-dependent and androgen-independent prostate cancer cells that expressed sGCα1, but not cells that do not express this gene. Peptide B-8R induced apoptosis of prostate cancer cells. Importantly, Peptide B-8R does not affect nor its cytotoxicity depend on NO signaling, despite the fact that it associates with sGCα1, which dimerizes with sGCβ1 to form the sGC enzyme. Just as with a previously studied Peptide A-8R, Peptide B-8R induced elevated levels of reactive oxygen species (ROS) in prostate cancer cells, but using a ROS-sequestering agent showed that ROS was not responsible the cytotoxic activity of Peptide B-8R. Interestingly, Peptide B-8R induced elevated levels of p53 and phosphorylated p38, but neither of these changes is the cause of the peptide's cytotoxicity. Additional drugs were used to alter levels of iron levels in cells and these studies showed that Peptide B-8R activity does not depend on Ferroptosis. Thus, future work will be directed at defining the mechanism of cytotoxic action of Peptide B-8R against prostate cancer cells

A peptide against soluble guanylyl cyclase α1: a new approach to treating 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

An sGCα1 siRNA also targets 280B in prostate cancer cells.

<p>(<b>A, B, C</b>) LNCaP cells were infected with empty lentivirus or lentiviruses expressing one of two different sGCα1 shRNAs (7, 8) and sGCα1 expression was measured by real-time PCR (A) or Western blotting (B), and cell density was measured by MTT assay (C). (<b>D</b>) The diagram shows the nucleotide homology between siRNA 7 and 280B. (<b>E, F</b>) LNCaP, C81, and CWR22-RV1 cells were subjected to the same infections and 280B levels were measured by real-time PCR (E) or Western blotting (F). (<b>G, H</b>) C81 cells were transfected with Control (Ctrl), sGCα1 siRNA (7,8), 280B siRNA, and 280B plus sGCα1 siRNA 8, and sGCα1 levels were measured by Western blotting (G), and cell density was measure by MTT assay (H). All expression levels are relative to the first condition (A, D), and it was set to 1. β-actin served as a loading control in the Western blots. Bar graphs represent averages of three independent experiments plus SD. Asterisks indicate statistical significance (P<0.01).</p