Investigation Of Molecular Mechanisms Of PC-1 In Prostate Cancer Progression And Its Role In Therapies

PC-1, a new member of the Tumor Protein D52 (TPD52) family, is induced by androgen and specific expression in prostate tissues. The expression of PC-1 is low in androgen-dependent LNCaP cells and up-regulated in the androgen-dependent and osseous metastatic C4-2 subline. Immunohistochemical experiments reveal that PC-1 gene expression is associated with prostate cancer malignant progression and is prevalently up-regulated in andvanced prostate cancer tissues. We reproted Previously that PC-1 expression plays an important role in mediating prostate cancer progression, its expression promotes prostate cancer growth and androgen-independent progression. These results indicate that PC-1 possesses some characteristics of oncogene. However, the clinical biological functions of PC-1 gene and the mechanisms and the downstream effectors of PC-l are not clearly understood. In this study, further investigation is performed to elucidate the clinical biological functions of PC-1 gene in prostate cancer progression and the molecular events and the downstream effectors of this gene.1) Here, we used LNCaP and C4-2 prostate cancer cell lines with diferent expression of PC-1 treated with casodex, an AR antagonist, it was found that PC-1 expression continued to promote prostate cancer proliferate compared with untreated condition. Next, we analyzed the expression of growth-related genes following casodex treatment, the Rb, cyclinE, p21 and p27 which implicate in the cell cycle regulation were found regulated by PC-1. This results showed that PC-1 abrogated sensitivity to casodex by regulate growth-related genes and cell cycle progression.2) Moreover, we analysed if PC-1 expression affected the PCa cells sensitivity to mTOR antagonist rapamycin and PI3K inhibitor LY294002. The experimental data demonstrated that the expression of PC-1 failed to alter PCa cells sensitivity to LY294002. In contrast, PC-1 overexpression resulted in marked resistance to rapamycin. In the PCa cell lines, the effect of PC-1 appeared to decrease rapamycin-induced cytostasis and autophagy. After establishing an association between PC-1 activity and rapamycin sensitivity, we sought to investigate how PC-1 may affect the mTOR signaling cascade. We found that the expression of 4E-BP1 protein was specifically increased in cells expressing PC-1 and limited effect of rapamycin on 4E-BP1. Next, direct proteins interaction between PC-1 and 4E-BP1 was demonstrated by co-immunoprecipiation,in vitro binding and GST-pull down assays. PC-1 was also found to decrease polyubiquitination -mediated degradation of 4E-BP1 and subsequently improved its stability. Further, we examined PC-1 and 4E-BP1 expression in prostate specimens, results showed that high expression of 4E-BP1 in human prostate cancers correlated with PC-1 activation.3) Radiotherapy is still a promising, effective, and definitive treatment option for men with localized prostate cancer. To investigate the effect of PC-1 protein on cellular radiosensitivity, the growth curve and survival assays followingγ-ray irradiation demonstrated that depletion of PC-1 significantly increased PCa cells radiosensitivity. Theγ-H2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in PC-1 knock down cells. Furthermore, PC-1 was found to be upregulated by IR, partly colocalize withγ-H2Ax. The silencing of PC-1 strongly inhibited radiation-induced phosphorylation of DNA-PKcs at S2056 as well as repair of DNA DSBs as measured by theγ-H2Ax foci assay. A G2/M arrest was induced in parental C4-2 cell after 4Gy irradiation. However, PC-1 silenced C4-2 cells exhibited a noticeable delay on the initiation and elimination of radiation-induced G2/M arrest and the expression of cyclin B1 was continuously activated as compared to parental cells.4) Affymetrix microarray assay demonstrated that the expression of downstream mTOR components as EIF4A2, EIF4G3 and EIF5A2 were altered in LNCaP-pc-1 cell was consistent with the results described above. Cell cycle-control genes were also regulated by PC-1. In addition, the genes like JAG1, THBS1, DSC3, MAP2, TUBA3 and NEDD9 involved in cell motility, adhesion and cytoskeleton were differentially expressed in the presence or absence of PC-1.5) It was shown that FLNa may be one of the potential PC-1 interaction proteins by yeast two-hybrid experiment, direct proteins interaction between PC-1 and FLNa was confirmed by co-immunoprecipiation,in vitro binding and GST-pull down assays. PC-1 binds to FLNa through its distinctive domain in N-terminal and leuzine zipper motif. In LNCaP cell, immunostaining of PC-1 and FLNa revealed a distinct co-localization of these two proteins in membrane ruffles. RNA-interference-based depletions of PC-1 lead to defective migration in C4-2 cell by wound-healing assay. These results showed that PC-1 localized to membrane ruffles with FLNa and may regulated actin cytoskeleton organization and cell migration.6) In addition, to investigate the function of FLNa in PCa progression, LNCaP and C4-2 cells with expression of endogenous FLNa knocked down by RNAi were established. A series of experimental data demonstrated that FLNa was in negative correlation with prostate cancer growth and anchorage independent colony formation in vitro. These results indicated that FLNa possessed characteristics of anti-oncogene and may play a particular role in prostate cancer progression.In conclusion, our data provide cellular and molecular evidence to show that PC-1 was involved in many molecular events including cell proliferation, differentiation, adhesion, migration, cell cycle progression and DNA damage and repair by regulating various signaling pathways in prostate cancer progression. Our study provides novel insights into molecular mechanism mediating prostate cancer malignant progression and the protentiation of PC-1 as anticancer target.