Carcinogenetic Roles And Underlying Molecular Mechanisms Of BCAR1 In Lung Adenocarcinoma

BackgroundCurrently,lung cancer is the top cancer killer worldwide,leading to a serious threat to human health.Moreover,it is occult and asymptomatic at early stage,resulting in final progression and metastasis.Therefore,most cases were found to be in middle or late stage during diagnosis,with bad treatment efficacy and poor prognosis.The oncogenes of lung adenocarcinoma(LUAD)is very complicated with heterogeneity.Study of targeted molecular is critical for precise treatment of LUAD.Therefore,the explorations of LUAD biomarkers have become timely and hot.Breast cancer anti-estrogen resistance-1(BCAR1)is widely expressed in a variety of tissues and organs,which belongs to Cas protein family.It is not only a cytoskeletal molecule but also an adaptor protein that adheres to the extracellular matrix,which plays a pivotal role in signal transduction,cell growth and development.Studies have demonstrated that BCAR1 plays the carcinogenetic effects in progression and malignant biological behaviors in a variety of cancers,such as cell proliferation,invasion and migration,epithelial-mesenchymal transition(EMT)and chemotherapy resistance.Furthermore,it has an important prognostic value for cancer patients.Previous studies have shown that BCAR1 is highly expressed in LUAD and promotes the EMT of LUAD cells,leading to cell invasion and metastasis as well as poor prognosis.However,the underlying molecular mechanisms of carcinogenesis of BCAR1,e.g.,cell proliferation and anoikis remain unclear and warrant further studies.ObjectiveWe aimed to explore the possible molecular mechanisms of carcinogenetic roles of BCAR1 in LUAD.Methods1.By Protein atlas database,we analyzed the expression of BCAR1 across different cell lines,and BCAR1 expressions in H1975,H1299,and A549 lung adenocarcinoma cells were verified by Western blot(WB);2.BCAR1 was knocked out(KO)by using CRISPR-Cas9 in H1975 and H1299 cells and BCAR1 was overexpression(OE)by c DNA library in A549 cells;3.Cell proliferation,invasion and migration,anoikis,and EMT were evaluated by MTT,Transwell,Anoikis and WB assays;4.BCAR1-OE and BCAR1-NC was constructed in 293 T cells,respectively.And immunoprecipitation mass spectrometry(IP-MS)was conducted for exploration of BCAR1 interacting proteins.The molecular functions of interacting proteins of BCAR1 were analyzed by Gene Ontology database;5.The genes that positively related to BCAR1 were screened in early LUAD patients at The Cancer Genome Atlas(TCGA).IP-MS was used combining with TCGA bioinformatics analysis to screen potential networks of BCAR1 interacting proteins;6.BCAR1 interaction networks and hub genes were analyzed by String,and differential expression and survival analysis of BCAR1,POLR2 A and RAC1 were performed via Gene Expression Profiling Interactive Analysis(GEPIA)and K-M plotter;7.Immunohistochemistry(IHC)assay was carried out to reveal the expression of BCAR1,POLR2 A and RAC1 in 54 patients with early LUAD,and to analyze their correlation or survival analysis;Moreover,WB assay was conducted to verify their expression.8.POLR2 A or RAC1 expression in LUAD cell lines after BCAR1-KO and BCAR1-OE were detected by WB experiment;9.Inhibitor effects of NSC23766 was detected by active RAC1 assay;And antagonizing of the carcinogenetic roles of BCAR1 including cell proliferation,clone formation,invasion,migration,anoikis,and EMT by the RAC1 inhibitor were evaluated by related experiments;10.The interaction of between BCAR1 and POLR2 A or RAC1 were detected by Co-immunoprecipitation(CO-IP)assay;11.Metastasis abilities were detected in tumor-bearing nude mice with BCAR1-KO cells.Results1.BCAR1 is expressed in H1975,H1299,and A549 cells;BCAR1-KO and BCAR1-OE were constructed successfully in these cells.2.Cell proliferation,invasion and migration,anoikis resistance,EMT of cells were inhibited or promoted after BCAR1-KO or BCAR1-OE.Moreover,high expression of BCAR1 predicted poor prognosis in LUAD tissues.The animal experiments demonstrated that metastasis abilities were inhibited in tumor-bearing nude mice with BCAR1-KO cells.3.419 proteins that may interact with BCAR1 were identified by IP-MS assay.Among them,RAC1 is a key regulator of anoikis,and POLR2 A is involved in both catalytic activity and transferase activity.Finally,68 genes were identified after matching with TCGA data.4.POLR2 A expression was significantly positively correlated to BCAR1 expression in LUAD and it can be significantly decreased following BCAR1-KO in cells.Furthermore,RAC1 expression was significantly decreased or increased following BCAR1-KO or BCAR1-OE in cells.The carcinogenetic effects and EMT induced by BCAR1 in LUAD cells can be antagonized by RAC1 inhibitor;5.CO-IP assay demonstrated no direct protein interaction between BCAR1 and POLR2 A or RAC1.ConclusionsOverexpression of BCAR1 promotes cell proliferation,invasion,migration,anoikis resistance,and EMT in LUAD,probably due to up-regulation of POLR2 A and RAC1.