Regulation Of Tumor Cell Proliferation And Tumorigenicity By GADD45G And USP16

Background and Objective:Hepatocellular carcinoma and lung cancer are two common tumors with rapid progress and high degree of malignancy.The hot topic of current cancer research is to find genes involved in development of these cancers.Growth arrest and DNA damage-inducible 45G(GADD45G)protein is related with cell stress and DNA damage,acting as a tumor suppressor in multifarious tumors.Our previous work has shown that loss of GADD45G-induced cellular senescence is an important event that contributes to liver tumor development.The precise mechanism underlying GADD45G-elicited cell senescence and tumor growth inhibition,however,remains obscure.In the first part of the study,we set to study the mechanism underlying GADD45G-induced cell senescence.USP16 is one of the deubiquitinases that can regulate a variety of biological processes,playing significant roles in modulating cell homeostasis,such as embryonic stem cell differentiation and hematogenesis.However,the function for USP16 in tumorigenesis is still unclear.In the sencod part of the study,we have explored whether USP16 can exert an effect on K-Ras-driven lung tumorigenesis in mice.Results:In the first part of the study,the lentinvirus with Tet-on inducible expression cassettes was used for transducing GADD45G to human liver tumor cells.GADD45G expression induced by Doxycyclin treatment was efficient in eliciting liver tumor cell senescence,wherein h TERT m RNA levels were significantly repressed,coincidently with marked increases in the expression of SIP1,a regulator of h TERT.In cultured liver tumor cells,SIP1 knockdown abrogetd GADD45G-induced senescence and the expression of IL-6 and IL-8.Consistently,SIP1 downregulation significantly counteracted GADD45G-mediated h TERT inhibition and cell-cycle G1 arrest.Moreover,SIP1 inhibition significantly alleviated GADD45G-mediated suppression of tumor growth in vivo.In addition,endogenous GADD45G and SIP1 were upregualted and required in the drug MG132-induced cell senescence.We found that both p38 MAPK and JNK pathways were activated during GADD45G-induced cell senescence.Although inhibition of either p38 MAPK or JNK activity suppressed the GADD45G-induced cell senescence,only the treatment with JNK inhibitor resulted in a decrease in SIP1 expression in tumor cells.In line with these finding,Ch IP experiements further confirmed that c-Jun could efficiently bind to SIP1 promoter in cells with GADD45G overexpression.In clinical HCC specimens,GADD45G and SIP1 were coincidently downregulated in tumor tissues relative to non-tumor tissues.In the second part of the study,we found that expression of H-Ras^V12resulted in increased levels of USP16 protein in human IMR90 fibroblasts and mouse embryonic fibroblasts(MEFs).USP16 deletion(USP16^-/-)in K-Ras ^G12D-expressing MEFs caused inhibition of senescence and impaired cell proliferation.In cultured MEF cells wherein K-Ras^G12D-induced senescence was bypassed by SV40-Large T antigen(SV40T),USP16 deletion was efficient in suppressing cell proliferation.Moreover,the tumor formation capability of K-Ras^G12D/USP16^-/-/SV40T-MEFs in nude mice was much lower than that of K-Ras^G12D/USP16^+/+/SV40T-MEFs,as evidenced by delayed tumor onset and decreases in tumor size.Finally,by employing USP16conditional knockout mice and K-Ras^G12D-driven mouse lung tumor model,we addressed the effects of USP16 on the development of lung tumor.The results revealed that USP16 deletion in lung tissue significantly suppressed lung tumor formation,and that the overall survival of K-Ras^G12D/USP16^-/-mice was delayed.Conclusions:In the process of GADD45G-induced liver tumor cell senescence and inhibition of tumor growth,JNK-mediated SIP1 activation is critically involved in GADD45G-induced liver tumor cell senescence.SIP1 induction is responsible for h TERT inhibition by GADD45G.Both GADD45G and SIP1 are coincidently downregulated in HCC tumor tisssues.These results suggest that defunction of GADD45G-SIP1 regulation contributes to cellular senescence evasion and tumor growth in hepatocellular carcinoma.In the study on the role for USP16 in K-Ras activation-induced lung tumorigenesis,USP16 deletion suppresses K-Ras^G12D-induced proliferation in MEFs and inhibits the tumorigenicity of MEFs co-transformed with K-Ras^G12Dand SV40T.USP16 is required for efficient K-Ras^G12D-driven lung tumor growth in mice.