Effects Of FAM122A On HCC Cell Growth And Doxorubicin-induced Cytotoxicity

Our previous study showed that FAM122A is a newly identified endogenous inhibitor of protein phosphatase 2A(PP2A).FAM122A directly interacts with A?and B55 subunits of PP2A,and promotes the polyubiquitination and degradation of the C?subunit,thus resulting in decreasing its phosphatase activity.FAM122A protein is highly conserved in mammals,but so far the physiological function of FAM122A and its possible role in diseases are still largely unknown.PP2A participates in various cellular biological processes,and its dysfunction has been found in many diseases,especially as an important tumor suppressor in the occurrence and development of cancers.Several lines of evidences show that the abnormal expression of PP2A subunits and decreased phosphatase activity are found in hepatocellular carcinoma(HCC)patients.Therefore,we tried to investigate the potential role of FAM122A in HCC.With the application of CRISPR/cas9-mediated FAM122A knockout HCC cell lines(SMMC-7721 and PLC/PRF/5),stable FAM122A overexpression cells by lentivirus infection,xenograft models,and combined with RNA sequencing,we investigated the potential function of FAM122A in HCC occurrence and development,and related mechanisms.In addition,we also tested the effect of FAM122A modulation on doxorubicin-induced cytotoxity.The results were obtained as following:(1)FAM122A is required for HCC cell growth and proliferation.SRA database show that the expression of FAM122A mRNA is relatively higher in tumor tissues,compared to that of tumor-free tissues by analyzing 66-paired HCC cohorts.FAM122A deletion inhibited the cellular growth,proliferation and malignant transformation of HCC cells in vitro and in vivo,as determined by CCK8,plate and soft agar colony formation assay and xenograft experiments.Consistently,FAM122A overexpression promoted the proliferation potential and transformation of HCC cells,as well as tumor growth in xenografts.Subsequently,the xenograft tumors from FAM122A knockout HCC cells showed less Ki67 staining cells,compared with NC cells.These results suggested that FAM122A has an essential role in HCC cell growth and proliferation.(2)Deletion of FAM122A causes cell cycle arrest,downregulation of AKT phosphorylation and inhibition of proliferation-related genes.In order to investigate the mechanism of FAM122A in modulating HCC cell growth and proliferation,the apoptosis state and cell cycle distribution in HCC cells with or without FAM122A were examined.Although the percentages of Annexin V~+/PI~+cells did not change apparently,FAM122A knock out presents cell cycle arrests,with G2/M phase arrest in SMMC-7721 and G1 phase arrest in PLC/PRF/5 cells.Consequently,RNA sequencing was performed in SMMC-7721 cells with or without FAM122A.Gene Ontology(GO)analysis showed FAM122A deletion reduced the expression of genes related to proliferation.Further,KEGG results showed these decreased proliferation-correlated genes were mainly involved in PI3K/AKT and MAPK signaling pathways.In addition,FAM122A deletion induced the reduction of phosphorylated AKT at the Thr308 and Ser473.(3)FAM122A deletion enhances Doxorubicin-induced cytotoxicity.Accumulating evidence show enhanced PP2A activation and/or decreased AKT activity can increase the sensitivity of chemotherapy and radiotherapy.Doxorubicin is a commonly used chemotherapeutic drug in HCC patients on clinic.We found that FAM122A deletion significantly increase Doxorubicin-induced DNA damage response and apoptosis,as assessed by?H2AX foci formation and protein accumulation,together with activated caspase-3/PARP cleavage.On the contrary,FMA122A overexpression antagonized Doxorubicin-triggered DNA damage and apoptosis.Collectively,FAM122A may be a potential target for HCC treatment.