Requirement Of G?i1/3 Protein On Androgen Induced Proliferation Of Prostate Cancer

Background & Objective:G protein is a heterotrimer with GTP hydrolase activity that binds to guanine nucleotides and has signal transduction.In animals and plants,G protein involved in signal transduction through a very conservative mechanism,thus regulating the growth and development of organisms.Our previous study has demonstrate that G?i1 / 3 protein plays a key role in activating AKT-mTORC1 signaling pathway by tyrosine kinase,such as epidermal growth factor(EGF)and keratinocyte growth factor(KGF).Both G?i1 and G?i3 formed complexes with growth factor receptor binding 2(Grb2)–associated binding protein 1(Gab1)and the EGF receptor(EGFR)and were required for the phosphorylation of Gab1 and its subsequent interaction with the p85 subunit of phosphatidylinositol 3-kinase in response to EGF.Androgen is a steroid hormone that plays an important role in the development and progression of prostate cancer.However,its molecular mechanism has not been fully elucidated.In this study,we investigated whether G?i1/3 plays an important role in androgen-induced cell survival signaling pathway.MethodsTo explore roles of G?i proteins in the mechanism of androgen-mediated development and progression of prostate cancer,we initially used mouse embryo fibroblasts(MEFs)derived from wild type(WT)mice or from mouse embryos deficient in G?i1,G?i3 or Gab1 or transfected with G?i1/G?i3 RNA interference(RNAi),as well as those from embryos doubly deficient in G?i1 and G?i3 or reconstitution of either G?i1 or G?i3 in DKO MEFs.Then we used androgen treated the human prostate cancer cell or human prostate cancer cell knockout of G?i1 or G?i3 gene.After treated with androgen,scratch assay was applied to detect the proliferation of PC3 or DU145 cells silenced G?i1 or G?i3 gene,western blot was used to detect the expression of adapter protein Gab1 and protein involved in MAPK/ERK signal pathway(Erk)and AKT-mTORC1 signal pathway(Akt,S6 K,S6,mTOR,GSK3?/3?).ResultsThe results showed that compared with WT,MEFs lacking G?i1 and G?i3 [termed double-knockout(DKO)cells] or Gab1 were severely impaired in androgen-induced phosphorylation of Akt308 T,Akt473S,GSK-3?,and GSK-3?,as well as the mTORC1 downstream targets 4E-BP1,S6K(389T),and S6.In contrast,loss of either G?i1 or G?i3 led to a small but considerable decrease in the phosphorylation of Akt473 S,4E-BP1(65S),and S6 in response to androgen.Moreover,reconstitution of either G?i1 or G?i3 in DKO MEFs restored phosphorylation of Akt308 T,Akt473S,S6K(389T),and S6 in response to androgen.Immunohistochemical staining showed that the expression of G?i1 / G?i3 protein in prostate cancer tissues was significantly higher than that in adjacent normal tissues and normal tissues.The results on prostate cancer cells or prostate cancer cells knockout G?i1 / G?i3 gene were found to be consistent with the above results when treated with androgen.ConclusionThese results demonstrate that G?i1,G?i3 protein and adapter protein Gab1 play pivotal roles in androgen-induced cell proliferation signaling pathways in prostate cancer,further clarifying the molecular mechanism of androgen-induced proliferation of prostate cancer cell,but also further shows that G?i1 / 3 is expected to become a new target in the treatment of androgen-resistant prostate cancer.