Investigation Of The Functions Of SGEF Expression In Prostate Cancer Progression

Prostate cancer is a common malignant tumor in males and is the second most frequently diagnosed and mortal cancer among men in the US. In China, the incidence of prostate cancer has been increasing in recent years. Circulating androgens are essential for the onset of prostate cancer through their interactions with the androgen receptor, so removal of testicular androgens by surgical or chemical castration is the most effective therapeutic approach for prostate cancer. Unfortunately, despite being effective in the initial period, after 1 to 3 years, prostate cancer eventually relapses to androgen-independent stage, and androgen-independent prostate cancer has been essentially untreatable. Although the focus of rigorous study, the molecular mechanisms underlying prostate cancer progression from androgen dependence to androgen independence remain poorly understood, so it is urgent to elucidate the pathways of androgen independence, which could lead to the identification of novel therapeutic targets. Our study is aimed to investigate whether SGEF, a human Rho GEF, can play a role in prostate cancer progression and whether it associate the prostate cancer transition from androgen dependence to androgen independence.SGEF, a gene which localized on chromosome 3q25.2, a portion of chromosome 3 (3q25-q26.2) that has been identified as an amplification unit in prostate tumors, was firstly isolated in the identify of androgen-responsive genes. SGEF contains a DH domain followed with a PH domain, which are characteristic of RhoGEFs. SGEF also contains a N-terminal proline-rich domain,two putative nuclear localization signals and a C-terminal SH3 binding domain. Until now, little is known about the function of SGEF protein other than some investigation about its role on regulating reorganization of the cytoskeleton, and its biological function during prostate cancer progression has not been reported.Western blots were performed to determine the expression pattern of SGEF in various cancer cell lines, and SGEF was found expressed in all prostate cancer cell lines we detected. Next we we measured the levels of SGEF mRNA by rRT–PCR and protein by western blot analysis in RWPE-1,BPH,LNCaP and C4-2 cells, and we found that the expression of SGEF in normal prostate cell RWPE-1 and benign prostate hyperplasia cell BPH was very low or almost absent, and the expression of SGEF in androgen-sensitive and non-metastasis LNCaP cells was higher than RWPE-1 and BPH cells, but is lower than the androgen-insensitive and osseous metastatic C4-2 cells. To examine the potential relevance of these findings to clinical prostate cancer, Immunohistochemistry was used to detect the expression of SGEF in 71 specimens of 10 normal prostate tissues,19 benign prostate hyperplasia tissues and 42 prostate cancer tissues. The results revealed that SGEF positive rate and staining in prostate cancer tissues are higher than in normal prostate and BPH tissues. The expression of SGEF in cell lines and clinical tissues indicate that SGEF possesses some characteristics of oncogene.To examine the function of SGEF during prostate cancer progression, we modulated the expression of SGEF by infecting C4-2 cells with SGEF-RNAi-LV(and NC-GFP-LV as a control) . The SGEF-RNAi-LV infected C4-2 cells demonstrated decreased SGEF expression compared with control cells. Under normal culture conditions (containing 8% FBS), MTT assay showed that the growth of SGEF-RNAi-LV infected C4-2 cells was significantly inhibited in contrast with control cells. Colony formation assay and soft agar assay showed the lower clonogenicity and anchorage-independent growth ability of SGEF-RNAi-LV infected C4-2 cells than control cells, respectively. Similar results were obtained in C4-2B cells. These facts suggest that SGEF expression is essential for androgen-dependent growth and survival and may function in the early stage of prostate cancer development. The results from the tissue microarray also revealed a significant correlation between SGEF positive expression and prostate cancer metastasis. To examine whether SGEF expression is associated with cancer cell migration, we investigated the effect of knockdown of SGEF on the migration of C4-2B cells in Transwell migration assays, and found that decreased SGEF expression dramatically blocked the migration of C4-2B cells.To test whether SGEF can also contribute to androgen independent growth and survival of C4-2B cells, growth assay, Colony formation assay and soft agar assay were performed in the absence androgen, and the results reveal that knockdown of SGEF could suppress the androgen independent survival ability and malignant proliferation. We next performed these assay in the presence of bicalutamide, an androgen receptor antagonist, and found that knockdown of SGEF could potentiates the antitumor effects of bicalutamide. These results suggest that SGEF expression may also play a potential role in the development and sustenance of androgen-independent prostate cancer.The Akt signaling pathway plays an important role in prostate cancer development and progression. The abnormal activation of Akt contribute to prostate cancer progression through the regulation of cell survival, cell growth, tumor invasion and metastasis and angiogenesis. Our experimental results showed that knockdown of SGEF reduced phosphorylation of Akt on Thr473, which could be molecular basis mediating prostate cancer malignant progression.Androgen receptor (AR) plays considerable roles in prostate cancer progression. It is an important target of endocrine therapy and prognostic factor. Prostate cancer recurrence after surgery, castration resistance and metastasize to bone suggest the complexity of prostate cancer. AR may need to interact with some selected coregulators for maximal or proper androgen function. It is significant to discover the new AR co-regulators, which is helpful for the elucidation of the molecular mechanisms and cross-talk of signal pathways associated with prostate cancer development and progression and identification of new targets of the therapy.In prostate cancer cells, the transcriptional activity of AR was repressed by SGEF over-expression in androgen-dependent and dose-dependent manner. We also found that SGEF-mediated inhibition of AR transcriptional activity required the GEF activity and depended on structural integrity of itself. The transcriptional activity of AR was enhanced by knockdown of SGEF. The expression level of endogenous PSA reduced when over-expressing SGEF and increased when decreasing the expression of SGEF. The LXXL motif within DH domain of SGEF suggests that it is possible that SGEF interact with AR. To confirm the interaction between SGEF and AR, the GST pull-down assay was performed. The result indicated that SGEF interacted with AR in vitro. We next performed confocal microscope experiment and found SGEF and AR were co-localized in the nucleus. All the results suggest that SGEF may act as a new co-repressor to promote the prostate cancer progression.In conclusion, we found the expression of SGEF was higher in the prostate cancer cells and tissues than normal prostate and benign prostate hyperplasia cells and tissues for the first time and its expression promotes prostate cancer malignant growth and androgen-independent progression. Furthermore, we preliminarily elucidate the molecular mechanisms to promote prostate cancer progression. In addition, we found that SGEF could negatively regulate transcriptional activity of AR and confirmed the interaction between SGEF and AR, which means SGEF may be a novel AR co-repressor. All the date provides novel insights into molecular mechanism mediating prostate cancer malignant progression and identification of new targets of the therapy.