Shao-Yong Chen

p53 represses androgen-induced transactivation of prostate-specific antigen by disrupting hAR amino- to carboxyl-terminal interaction.

Prostate-specific antigen (PSA) is highly overexpressed in prostate cancer. One important regulator ofPSA expression is the androgen receptor (AR), the nuclear receptor that mediates the biological actions of androgens. AR is able to up-regulate PSA expression by directly binding and activating the promoter of this gene. We provide evidence here that that this AR activity is repressed by the tumor suppressor protein p53. p53 appears to exert its inhibition of human AR (hAR) by disrupting its amino- to carboxyl-terminal (N-to-C) interaction, which is thought to be responsible for the homodimerization of this receptor. Consistent with this, p53 is also able to block hAR DNA binding in vitro. Our previous data have shown that c-Jun can mediate hAR transactivation, and this appears to result from a positive effect on hAR N-to-C interaction and DNA binding. Interestingly, c-Jun is able to relieve the negative effects of p53 on hAR transactivation, N-to-C interaction, and DNA binding, demonstrating antagonistic activities of these two proteins. Importantly, a p53 mutation found in metastatic prostate cancer severely disrupts the p53 negative activity on hAR, suggesting that the inability of p53 mutants to down-regulate hAR is, in part, responsible for the metastatic phenotype.

c-Jun enhancement of androgen receptor transactivation is associated with prostate cancer cell proliferation

Androgens and the androgen receptor (AR) are involved in the growth and progression of prostate cancer. Our previous studies suggest that the proto-oncoprotein c-Jun is an AR coactivator that stimulates AR transactivation by mediating receptor dimerization and subsequent DNA binding. To study the physiological relevance of this c-Jun activity on AR, we have generated stable LNCaP cell lines expressing different levels of c-Jun. These cell lines exhibit a direct correlation between endogenous c-Jun levels and AR transcriptional activity and expression of endogenous androgen-regulated genes. Disruption by antisense RNA of endogenous c-Jun expression in LNCaP cells strongly compromises the androgen-dependent proliferation of these cells. In contrast, expression of a c-Jun mutant, which is fully active in coactivation of AR but deficient in AP-1 transactivation, significantly enhances androgen-dependent proliferation. This finding indicates that the coactivation function of c-Jun is sufficient for regulating androgen-induced growth of LNCaP cells. c-Jun also enhances AR transactivtion in androgen-independent LNCaP cells, which closely mimic hormone-refractory prostate cancer cells in gene expression and growth behavior. Importantly, siRNA-mediated repression of endogenous c-Jun expression results in markedly reduced growth of these cells, strongly suggesting an important biological role for c-Jun in hormone-refractory prostate cancer.

SUMO-3 Enhances Androgen Receptor Transcriptional Activity through a Sumoylation-independent Mechanism in Prostate Cancer Cells

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 androgen-dependent 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.

Makorin RING finger protein 1 (MKRN1) has negative and positive effects on RNA polymerase II-dependent transcription

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 DNA-binding 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 MKRN1s 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.

Expression of a hyperactive androgen receptor leads to androgen-independent growth of prostate cancer cells

Cellular changes that affect the androgen receptor (AR) can cause prostate cancer to transition from androgen dependent to androgen independent, which is usually lethal. One common change in prostate tumors is overexpression of the AR, which has been shown to lead to androgen-independent growth of prostate cancer cells. This led us to hypothesize that expression of a hyperactive AR would be sufficient for androgen-independent growth of prostate cancer cells. To test this hypothesis, stable lune cancer prostate (LNCaP) cell lines were generated, which express a virion phosphoprotein (VP)16-AR hybrid protein that contains full-length AR fused to the strong viral transcriptional activation domain VP16. This fusion protein elicited as much as a 20-fold stronger transcriptional activity than the natural AR. Stable expression of VP16-AR in LNCaP cells yielded androgen-independent cell proliferation, while under the same growth conditions the parental LNCaP cells exhibited only androgen-dependent growth. These results show that expression of a hyperactive AR is sufficient for androgen-independent growth of prostate cancer cells. To study the molecular basis of this enhanced growth, we measured the expression of soluble guanylyl cyclase-alpha1 (sGCalpha1), a subunit of the sGC, an androgen-regulated gene that has been shown to be involved in prostate cancer cell growth. Interestingly, the expression of sGCalpha1 is androgen independent in VP16-AR-expressing cells, in contrast to its androgen-induced expression in control LNCaP cells. RNA(I)-dependent inhibition of sGCalpha1 expression resulted in significantly reduced proliferation of VP16-AR cells, implicating an important role for sGCalpha1 in the androgen-independent growth of these cells.