Expression,prognosis And Functional Significance Of CNN2 Related Mechanotransduction In Lung Cancer

Background:Lung cancer has the highest morbidity and mortality among malignant tumors both worldwide and in China^[1,2],usually diagnosed at advanced stage.Tumor itself greatly disrupts the mechanical environment of the lung,trasforming the primary location from soft tissue that dilates and contracts cyclicly in response to respiratory movement and airflow to rigid,non-aerated solid tissue^[3].Number of studies have shown disregualted mechanotranduction in tumors,including stiff ECM,activation of integrin-FA and its downstream pathways,and the polymerization and depolymerization of actin stress fibers remodelling,lead to survival,invasion and metastasis of tumor cells^[4-7],but few is about lung cancer.Calponin 2（CNN2）is an actin binding protein^[8],colocalized with myosin IIA and tropomyosin^[9]in actin-stress fibres.Our previous studies have shown that CNN2 is regulated by extracellular mechanical signals,stabilizing the cytoskeleton by regulating the polymerization and depolymerization of the actin stress fibres^[10,11].CNN2 was widely expressed in different organs,especially in lung,lymph node and appendix^[12],however,the functional significance in these organs is still unclear.In various cells,the down-regulation of CNN2 is accompanied with increased proliferation,migration and decreased adhesion^[9,13].CNN2 decreasing accelerated the dynamic remodeling of actin microfilament,which is the characteristic of tumor cells.Calponin 1,which is the homology isoform of CNN2,is also downregulated in variety of tumors(（leiomyosarcoma,hepatocellular carcinoma,basal cell carcinoma,and prostate cancer）,and considered as a tumor suppressor gene^[8,14,15].At the same time,overexpression of CNN2 in prostate cancer cell lines inhibited cell proliferation and migration^[13],however the function of CNN2 in lung cancer is still unknown.Aims:We aimed to explore the expression pattern and prognostic value of CNN2 in pan-cancer across multiple databases,then combine the transcriptome and proteomic data from CNN2-KD lung cancer cells,together with cell biological behaviour experiment and3D Spherical cell migration test to preliminarily explore the expression,functional and prognostic significance of CNN2 in lung cancerMethods:We analyzed the m RNA expression and clinical data from TCGA,GTEx and CCLE database,to verify the expression,prognostic,role of CNN2 in pan-cancer,and correlation analysis of its expression in lung adenocarcinoma with microenvironment and immune cell infiltration.We furthter performed CNN2 knockdown in 2D cultured lung cancer cells to observe the phenotype of CNN2-KD lung cancer cells and stress fibre remodelling.We also performed 3D spherical cell migration test to verify the results obtained in 2D experiments.In the next,we co-analysis the data from TCGA and the transcriptomic and proteomic data from CNN2-KD lung cancer cell lines,to found the correlation and mechanism between CNN2 and LUAD.Results:We found CNN2 expression was downregulated in most cancers,including lung cancer compared with primary organ.The m RNA levels of CNN2 alone from TCGA seemed not associated with survival in lung cancer.GO enrichment,KEGG pathway,GSEA and GSVA analysis showed,that the CNN2 expression is significantly associated with pathways correlated with mechanotransduction,including the cell-ECM adhesion and cell-cell junction related pathways.The ESTIMATEScore and immune infiltration analysis showed,CNN2 expression is more related to tumor stromal,especially CAF but not immune cell infiltration.Through cell proliferation,adhesion,wound healing and transwell migration/invasion tests,it was confirmed that down-regulation of CNN2 would increase the migration and reduce the adhesion of lung cancer cells,but had no significant effect on cell proliferation and invasion.Further immunofluorescence staining showed that the stress fibers around CNN2-KD lung cancer cells were significantly decreased,which may be the reason for their reduced adhesion and increased migration.The expression of CNN2 in lung cancer cells was extremely low in 3D culture compared to 2D plates,and overexpression of CNN2 significantly inhibited the dissemination and migration of lung cancer cells.However,we also found lung cancer cells were more likely to diffuse and migrate outward in stiffer ECM than in softer matrix.This indicate that the low expression of CNN2 in lung cancer cells is important to maintain the ability to dissemination in 3D culture.In the next,we extracted m RNA and total protein from CNN2-KD and NC groups of A549 lung cancers cells for transcriptomic and proteomic analysis.The gene clusters most closely related with the expression of CNN2,whether in transcriptome or proteome,enriched in pathways,such as cell-matrix adhesion,integrin-binding and stress fibres,which related to mechanotransduction.We further combined analysis the TCGA transcriptome,lung cancer cell transcriptome and proteome to obtain a gene set,which contained 9 genes commonly related with CNN2 in all 3 datasets.7 of these genes were enriched in focal adhesion,cell-substrate junction,collagen-containing extracellular matix,acting binding and integrin binding.We further set these 7 genes together with CNN2 as a gene cluster,from ss GSEA score and consensus cluster analysis,LUAD can be clustered into 2 subtypes.The CNN2 related gene cluster was significantly associated with the survival of lung adenocarcinoma patients.Conclusion:The expression of CNN2 is low in lung adenocarcinoma compared with primary lung tissue.It regulates the adhesion and migration of lung cancer cells through ECM-integrin-FA-stress fiber related mechanotransduction pathways,and ultimately affects patients’survival.