SETD3 Promotes The Progression Of Lung Adenocarcinoma By Activating The TNF/NFκB Pathway Through CHD1

Globally,there will be an estimated 19.3 million new cases of cancer in 2020.Breast cancer(11.7%),lung(11.4%),colorectal cancer(10.0%),prostate cancer(7.3%)and gastric cancer(5.6%)was the most common malignant tumor.Lung cancer is the leading cause of cancerrelated death.And the global cancer burden will continue to rise,with an estimated 47% increase in the number of cancer patients to 28.4 million in2040 compared to 2020.Therefore,it is urgent to continue to improve the level of cancer prevention and diagnosis.Studying the etiology of tumor development and elucidating the molecular mechanisms is crucial for improving the prevention,diagnosis and treatment of tumors.Protein methylation is one of the most common forms of protein posttranslational modification.A large number of studies have found that aberrant protein methylation changes are associated with a variety of diseases,including tumors.Protein methylation usually refers to the transfer of the activated methyl group on the donor S-adenosylmethionine(SAM)to an amino acid residue of the acceptor substrate protein,usually lysine and arginine,but also histidine,cysteine and aspartic acid,under the catalysis of different methyltransferases.The SET(Su(var)3-9,enhancer of zeste,trithorax)domain family proteins are a common protein family with protein methyltransferase activity.Most of these proteins were observed to have clear methyltransferase activity and to affect tumor progression.However,the substrate and function of some SET family members are still unclear.For instance,the substrate and biological function of SETD3 in tumors are still unclear.Methods: To investigated the expression level of the protein methyltransferase SETD3 in lung adenocarcinoma and normal lung tissues,we searched the protein expression data at the Clinical Proteomic Tumor Analysis Consortium,a public database.The expression of SETD3 protein in lung adenocarcinoma and normal lung tissues was further verified by Western Blot and immunohistochemistry(IHC).Lentivirus transfection and CRISPR-Cas9 technology were used to construct SETD3 overexpression and knockout lung adenocarcinoma cell lines.Based on these cells,CCK-8 proliferation assay,colony formation assay and subcutaneous xenograft assay in nude mice were performed to investigate the biological effects of SETD3 in LUAD.In order to identify the substrate of SETD3,we performed coimmunoprecipitation(Co IP)and mass spectrometry(LC-MS)analysis in PC9 lung adenocarcinoma cells,and verified the substrate interacting with SETD3 by immunoprecipitation and co-localization immunofluorescence assays.Subsequently,post-translational modification mass spectrometry was used to identify the possible methylation modification sites of the proteins interacting with SETD3,and the site-specific methylation modification antibody was constructed.Co IP and Western Blot experiments were used to verify the regulation of SETD3 on the methylation of its substrate.The effects of SETD3-mediated methylation on the functions of its substrate proteins were investigated by Co IP,Western Blot,in vivo ubiquitination and protein stability assays.To investigate the molecular mechanism by which SETD3 affects the malignant behavior of lung adenocarcinoma,we performed m RNA sequencing on PC9 lung adenocarcinoma cell lines overexpressing empty vector and SETD3 to investigate the molecular pathways that SETD3 may affect.Then RT-q PCR,Western Blot and chromatin immunoprecipitation(Ch IP-q PCR)were used to verify the molecular pathways regulated by SETD3.Results: Using the above methods,we found that the protein level of SETD3 was significantly higher in lung adenocarcinoma tissues than in normal lung tissues.Overexpression of SETD3 promoted the malignant proliferation of lung adenocarcinoma,while knockdown of SETD3 significantly inhibited the proliferation of lung adenocarcinoma.In lung adenocarcinoma,SETD3 interacts with CHD1 and catalyzes the dimethylation of CHD1 at lysine 209.SETD3-mediated dimethylation of CHD1 at lysine 209 reduces the level of ubiquitination and inhibits the ubiquitin-proteasome degradation pathway of CHD1.Mechanistically,SETD3 increases the histone H3K4me3 mark of TNF-NFκB pathway genes through CHD1,activating the transcription of these genes,leading to the activation of TNF-NFκB pathway and promoting the malignant behavior of lung adenocarcinoma.Taken together,this study identified CHD1 as a new substrate for SETD3,which catalyzes the dimethylation of CHD1 at lysine 209.Dependent on its base transferase activity,SETD3 promotes tumor progression through the SETD3\CHD1\TNF-NFκB pathway.