The Role And Mechanism Of FAM83D Promoting Non-Small Cell Lung Cancer Cell Epithelial-Mesenchymal Transition And Metastasis

BackgroundLung cancer is one of the most common cancers,with morbidity and mortality at the top of malignant tumors and non-small cell lung cancer(NSCLC)accounts for 80-85%of all lung cancer diagnoses.Currently,surgery,chemotherapy and radiotherapy are the main treatments for NSCLC patients,yet 40%of them are first diagnosed as locally advanced or already metastasized to the lymph nodes,brain and liver tissues.These treatments are not ideal for advanced lung cancer patients leading to low overall survival(OS)rate in NSCLC patients.Presently,the molecular mechanism of NSCLC metastasis has not been fully elucidated.Therefore,it is of great significance to study the pathogenesis of NSCLC metastasis in depth in order to find potential therapeutic targets so as to finally improve the survival rate and quality of life for advanced NSCLC patients.Epithelial mesenchymal transition(EMT)is a biological process in which epithelial cells transformed into mesenchymal cells through specific procedures,and plays a pivotal role in cancer metastasis and polyfibrosis diseases.In the EMT process,the intercellular adhesion is weakened,and conversely,the ability of invasion and migration of cells is enhanced,providing a cellular rationale for distant metastasis of malignant tumor.Although many studies have confirmed that EMT has a crucial function in cancer metastasis,the mechanism of EMT development in NSCLC metastasis is not fully understood.FAM83D(family with sequence similarity 83,member D,also named CHICA)is a mitosis related gene locating in the long arm(20q)of the human chromosome 20.FAM83D is first recognized as a spindle protein.Current researches show that FAM83D is amplified and overexpressed in different types of human cancers,including liver cancer,ovarian cancer and lung adenocarcinoma.It is reported that FAM83D promotes cell proliferation,invasion,and EMT by regulating FBXW7/mTOR signaling pathway in breast cancer.Besides,it promotes the occurrence and metastasis of rectal cancer by regulating the FBXW7/Notchl signaling pathway in rectal cancer.It also plays a carcinogenic role by regulating cell cycle in lung adenocarcinoma,and promotes cell proliferation and invasion through regulating MEK/ERK pathway in hepatocellular carcinoma.The above results indicate that FAM83D is closely related to the development of tumors,but it is still unknown whether FAM83D promotes NSCLC cell EMT,invasion and metastasis.The purpose of this study is to explore the role of FAM83D in regulating EMT related cell metastasis in NSCLC cells,and to further study its possible molecular mechanism.Not only did this study supplement the biological function of FAM83D in NSCLC cells,but it further unravel its molecular mechanism providing a new therapeutic target for advanced NSCLC patients.ObjectivesTo investigate the effect and mechanism of FAM83D on proliferation,EMT and metastasis of NSCLC cells.Methods1.Compare FAM83D expression in NSCLC cells and normal lung epithelial cells,as well as NSCLC tissues and its adjacent non-cancerous tissues.(1)qRT-PCR and Western blot detected the expression of FAM83D of 7 NSCLC cell lines and 2 normal lung epithelial cell lines.(2)Collect 40 cases of NSCLC and its adjacent non-cancerous tissues.Immunohistochemical(IHC)method was used to detect the expression level of FAM83D to analyze the difference of FAM83D expression in normal lung tissues,local NSCLC tissues and metastatic NSCLC tissues.2.Explore the effect of FAM83D n proliferation,EMT,invasion and migration of NSCLC cells.(1)BEAS2B cell line that stably expressed high level of FAM83D(BEAS2B-FAM83D)and its control group(BEAS2B-control)were constructed by retrovirus infection.We constructed stably silenced FAM83D cell lines and its control cell lines in A549 and H1299 cells by lentivirus infection,including A549-shcontrol,A549-shFAM83D-1,A549-shFAM83D-2 and H1299-shcontrol,H1299-shFAM83D-1,H1299-shFAM83D-2.The expression of FAM83D in these cell lines was detected by qRT-PCR and Western blot.(2)The effects of FAM83D on the proliferation of NSCLC cells were detected by MTT and colony formation.The effects of FAM83D on NSCLC cell invasion and migration were detected by wound healing,Transwell and Matrigel assays.Finally,the effect of FAM83D on cell EMT was detected by Western blot and immunofluorescence(IF).3.Explore the mechanism of FAM83D regulating EMT,invasion and metastasis of NSCLC cell'(1)Western blot was used to detect the expression levels of AKT/p-AKT,mTOR/p-mTOR,p70S6k/p-p70S6k in the above established cell lines.(2)KM2206(AKT inhibitor)or Rapamycin(mTOR inhibitor)were used in FAM83D overexpressed cell lines and the control groups.Then,Western blot and Transwell assay were conducted to test alteration of EMT markers expression and cell migration ability to confirm that FAM83D enhanced cell EMT and migration ability through regulating AKT/mTOR pathway.Results1.FAM83D expression is elevated in NSCLC cells and tissues.(1)qRT-PCR and Western blot showed that FAM83D expression was highly expressed in NSCLC cells compared to the normal cell lines.(2)Immunohistochemistry showed that the expression of FAM83D was the lowest in the paracancerous tissues,and was relatively high in the NSCLC tissues,and the most significant expression in the NSCLC tissues that had been transferred far away.2.FAM83D promotes NSCLC cell proliferation,EMT,migration and invasion.(1)MTT and colony formation assay confirmed that overexpression of FAM83D could promote BEAS2B cell proliferation,and inversely,silencing FAM83D inhibited the proliferation of A549 compared to their control cells.(2)Western blot and immunofluorescence showed that overexpression of FAM83D significantly increased the expression of the mesenchymal cell markers N-cadherin and Vimentin and inhibited the expression of the epithelial cell markers E-cadherin and ?-catenin in BEAS2B cells.In contrast,silencing FAM83D significantly inhibited the expression of N-cadherin and Vimentin and increased the expression of E-cadherin and ?-catenin in A549 and H1299 cells.Wound healing and Transwell assay showed that overexpression of FAM83D led to enhanced wound healing ability and more cells passing through the Boyden(BD)chamber,while silencing FAM83D inhibited cell spreading ability and less cells passing through the BD chamber.Matrigel assay indicated that the number of cells passing through the matrigel and the BD chamber membrane was increased after FAM83D overexpression.On the contrary,the number of cells passing through the matrigel and the BD chamber membrane was significantly decreased with FAM83D knockdown.3.FAM83D promoted NSCLC cell EMT and invasion through activating AKT/mTORpathway.(1)Western blot results showed that silencing or overexpression of FAM83D did not change protein expression levels of AKT,mTOR and p70S6k.However,FAM83D overexpression significantly increased the levels of p-AKT,p-mTOR and p-p70S6k.On the contrary,the levels of p-AKT,p-mTOR and p-p70S6kwere decreased significantly after FAM83D silencing.(2)In order to further verify that FAM83D regulated the tumor characteristics of NSCLC cells by regulating the AKT/mTOR pathway,we detected the cell migratory ability and the expression of EMT markers using Transwell assay and Western blot separately.The results showed that the effect of FAM83D promoting BEAS2B cell migration was weakened with treatment of MK2206 or Rapamycin.The expression change of EMT markers in BEAS2B cells induced by FAM83D was also inhibited.ConclusionFAM83D can promote NSCLC cell EMT,migration and invasion by activating AKT/mTOR signaling pathway.This study may provide a potential targets for advanced NSCLC treatment.