Studies On The Function And Probable Underlying Mechanism Of SGK1 In Human Prostate Cancer

Serum and glucocorticoid-induced protein kinase 1(SGK1),a serine/threonine protein kinase,is a member of the ’AGC’ subfamily of protein kinases.SGK1 is ubiquitously expressed,and it covers a wide variety of physiological functions(e.g.,transport,hormone release,neuroexcitability,cell proliferation,and apoptosis)and contributes to a multitude of pathophysiological conditions(e.g.,tumor growth,neurodegeneration,fibrosing disease,and the sequelae of ischemia).Increased expression of SGK1 has been observed in several tumors.However,the function and probable underlying mechanism of SGK1 in human prostate cancer is not fully understood.Objective:Here we explored the function of SGK1 and its partial molecular mechanisms from both molecular,cellular and animal levels,and simulated gene therapy for cancer to provide it with some reference theory and experimental basis for PCa treatment.Methods:1.Construction and identification of SGK1-silencing and SGK1-overexpressing stable PCa cell lines.To generate SGK1-silencing and SGK1-overexpressing stable PCa cell lines,PCa cells were infected with lentiviral particles.The lentiviral expression vectors LV2-Control,LV2-shSGK1,LV6-Control and LV6-SGK1(SGK1-overexpression)were purchased from Shanghai Gene Pharma Company(China).In brief,the cells were seeded at 2×105 cells/well in a 6-well plate before lentiviral particle infection and incubated with 2 ml of complete medium for 24 h.Then,cells were infected with lentiviral particles,and after 12 h,the virus-containing medium of infected cells was substituted with fresh complete medium,and infected cells were selected with 4 μg/ml puromycin for 96 h.Empty lentiviral vectors were used as a control.2.Studies on the function and probable underlying mechanism of SGK1 in the growth of human prostate cancerWe investigated the cellular responses to GSK650394 treatment and SGK1 silencing(or overexpression)in human prostate cancer(PCa)cell lines and PC3 xenografts by flow cytometry,western blotting,immunofluorescence,transmission electron microscopy and immunohistochemistry.To discern the relationship between GSK650394-induced autophagy and apoptosis,a vitality assay was performed with PC3 and LNCaP cells pretreated with 5 mM 3MA,10 μM CQ,20μM z-VAD-fmk,or vehicle alone before the addition of GSK650394.Cell proliferation,cell cycle,apoptosis and autophagy were further analyzed using CCK8,flow cytometry,western blotting,immunofluorescence.3.Studies on the function and probable underlying mechanism of SGK1 in human prostate cancer metastasisWe first determined whether SGK1 expression is associated with human PCa progression.Immunohistochemistry staining was performed in 24 primary nonmetastatic PCa(NmPCa)and 21 primary metastatic PCa(mPCa)tissues,all of which contain tumor adjacent normal prostate tissues(AT)and PCa tissues.We investigated the cellular responses to GSK650394 treatment and SGK1 silencing(or overexpression)in human prostate cancer(PCa)cell lines and PC3 xenografts by CCK8,migration and invasion assay,western blotting and immunofluorescence.To discern the relationship between GSK650394-induced autophagy and migration,invasion and metastasis.Cell migration,invasion and autophagy were further analyzed.The expression levels of related proteins and mRNA were detected by Western blotting and RT-qPCR.Results:1.Immunofluorescence and WB results showed that we had successfully constructed SGK1-silencing and SGK1-overexpressing stable PCa cell lines model.2.we demonstrated that SGK1 inhibition,mediated by either GSK650394 or SGK1 shRNA,induced G2/M arrest,apoptosis and autophagy.Furthermore,3MA-mediated autophagy inhibition attenuated SGK1 inhibition-induced apoptosis,suggesting that induction of autophagy precedes apoptosis.Moreover,ectopic expression of SGK1 significantly attenuated the GSK650394-induced effects.Suppression of mTOR and Foxo3a phosphorylation is critical for blockade of SGK1-induced autophagy and apoptosis,at least partially via pFoxo3a(S253)-LC3 and pFoxo3a(S253)-p27 interactions.Dual inhibition of mTOR and SGK1 enhances autophagy activation and leads to synergistic cytocidal effects in PCa cells.3.We found that SGK1 expression positively correlates with human prostate cancer(PCa)progression and metastasis.We show that SGK1 inhibition significantly attenuates EMT and metastasis both in vitro and in vivo.Whereas overexpression of SGK1 dramaticlly promoted the invasion and migration of PCa cells.Our further results suggest that SGK1 inhibition induced antimetastatic effects,at least partially via autophagy-mediated repression of EMT through the downregulation of Snail.Moreover,ectopic expression of SGK1 obviously attenuated the GSK650394-induced autophagy and antimetastatic effects.What’s more,dual inhibition of mTOR and SGK1 enhances autophagy and leads to synergistic antimetastatic effects on PCa cells..Conclusion:1.SGK1 inhibition could significantly inhibit cell proliferation migration,invasion and metastasis of human PCa cells.2.SGK1 inhibition induces autophagy-dependent apoptosis via the mTOR-Foxo3a pathway.3.SGK1 inhibition-induced autophagy impairs prostate cancer metastasis by reversing EMT.Overall,our results indicated that SGK1 can be developed as a promising gene therapy target for prostate cancer.