Positive Feedback Loop Of FAM83A/PI3K/AKT/c-Jun Induces Migration,Invasion And Metastasis In Hepatocellular Carcinoma

BackgroundLiver cancer is currently the second leading cause of cancer-related death globally.Hepatocellular carcinoma(HCC)accounts for 90%of primary liver cancers.The global burden of HCC is increasing and may soon surpass an annual incidence of 1 million cases.HCC has become the major cause of cancer-related deaths in Asia,especially in China.Many factors can induce HCC,such as chronic infection with hepatitis B or hepatitis C viruses,alcohol abuse,and metabolic syndromes related to diabetes and obesity.To date,however,the specific pathogenesis of HCC remains uncertain,and increasing evidence has shown that the factors that induce HCC are related to abnormal expression of tumor-associated genes.However,a more comprehensive understanding of these HCC-related genes is needed.Continuing to discover novel HCC-related genes and studying their molecular mechanisms may promote new breakthroughs for diagnosing,treating and dealing with HCC and HCC-related drug resistanceFAM83A is one of an 8-member family of proteins that share a highly conserved N-terminal domain of unknown function,known as the DUF1669 domain.The UniProt database(https://www.uniprot.orng)describes FAM83A as a probable proto-oncogene that independently functions in activating the PI3K/AKT signaling cascade pathway.FAM83A has been shown to promote cancer and increase proliferation,invasion,stem cell-like traits and drug resistance in tumors,such as lung,breast and pancreatic cancers,and was predicted to be generally involved in many human cancers.However,the role of FAM83A in HCC remains unknown.ObjectionThis study aims to elucidate the role of FAM83A gene in the occurrence and development of HCC from both in vivo and in vitro through the establishment of cell experiments and animal models,and further explore the ways in which FAM83A causes those effects.Based on the above research conclusions,we attempted to provide direction for the significance of FAM83A gene in the treatment and diagnosis of HCC.Methods1.The Cancer Genome Atlas(TCGA)datasets were used to analyze FAM83A expression in HCC tissues and normal liver tissues.10 HCC cell lines and normal human liver cell LO2 were tested to compare the difference of FAM83A expression.Using a tissue array containing 90 paraffin-embedded HCC and peritumoral liver samples to analyze the expression difference of FAM83A in HCC patients and normal population,and its clinical significance.2.Creation of FAM83A overexpressing cell lines and FAM83A expression interference models in Bel-7404 and SMMC-7721 cell lines using lentivirus and small interfering RNA.Cell wound-healing assays,transwell assays and Boyden assays were enforced to investigate the regulation of FAM83A in invasion and migration of HCC cells in vitro.3.Using MTT cytotoxicity to investigate the effects of FMA83A in sorafenib sensitivity.4.Creating a pulmonary metastasis model with lentivirus-transfected HCC cells(Bel-7404 and SMMC-7721)in nude mice to investigate the effects of FAM83A in HCC cell metastasis in vivo.5.Using western blot assay,RT-qPCR assay,and immunohistochemical analysis to explore the possible cancer-promoting mechanisms of FAM83A.6.Using chromatin immunoprecipitation(ChIP)analysis and electrophoretic mobility shift assay(EMSA)further explore the mechanisms of FAM83A gene transcriptional regulationResults1.The Cancer Genome Atlas(TCGA)datasets observed that FAM83A mRNA expression was elevated in HCC tissues.Western blot assays suggested FAM83A protein expression was also elevated in HCC cells.Clinical data showed that high FAM83A expression might be related to poorer progression-free survival times in HCC2.Cell wound-healing assays,transwell assays and Boyden assays showed FAM83A overexpression could promote the invasion and migration capability of HCC cells.When FAM83A expression was interfered the above conclusions can be reversed.3.Overexpression of FAM83A reduced the drug sensitivity of sorafenib in HCC cells.When FAM83A expression was interfered the above conclusions can be reversed.4.Overexpression of FAM83A could promote the metastasis capability of HCC cells in vivo5.FAM83A could activate PI3K/AKT signaling and further induce epithelial-to-mesenchymal transition(EMT)and c-JUN signals.6.c-JUN promoted FAM83A expression by binding to its promoter region and formed a FAM83A/PI3K/AKT/c-Jun positive feedback loop ConclusionHere,we investigated the biological functions of FAM83A in HCC and its possible signaling mechanisms.FAM83A,as a prognostic factor of poor progression-free survival,improves migration,invasion and metastasis and further induces sorafinib resistance in HCC.We further verified an atypical FAM83A/PI3K/AKT/c-JUN positive-feedback loop in HCC cells,which leads to a vicious cycle of cancer-promoting signals.Our data provide evidence that FAM83 A is a cancer promoter in HCC and suggest it as a potential novel therapeutic target.