Role Of TGF?1 Signaling Pathway In Regulating NSCLC Microenvironment And Durg Resistance

Objective: Lung cancer is one of the world's leading cause of mortality and morbidity.Nonsmall cell lung cancer accounts for about 80% of all lung cancers,and above to 75% of patients are in the advanced stage with a 5-year survival rate of 19%.It is known that the growth and development of tumors is closely related to the internal environment in which tumor cells are located.The TGF?1 signaling pathway plays an important role in the occurrence and development of tumors.It mainly promotes the transformation of fibroblasts to myofibroblasts and the secretion of extracellular matrix in the formation of tumor microenvironment.TGF ?1 also activates cancer-associated fibroblasts(CAF)in inducing tumor growth and invasion.Targeting myofibroblasts or CAF to inhibit their induced pulmonary fibrosis and tumorigenesis and metastasis is current research trends.Meanwhile,TGF?1-mediated tumor cell epithelial-mesenchymal transition(EMT)is the cause of congenital and acquired cytotoxic drug resistance.It is in urgent need to establish effective therapy in reversing EMT-related drug resistance.This project focused on study the role and mechanism of TGF?1 and its downstream signaling pathways in regulating CAF tumor nesting and drug resistance in non-small cell lung cancer cells.Methods: It is divided into two parts.1)Induction of fibroblast differentiation by adding TGF?1 in vitro and extract human primary CAF.Using PI3 K and HDAC dual pathway inhibitor CUDC-907 to study the role of TGF?1 in regulating CAFs proliferation,migration,invasion and anti-apoptosis in vitro and in vivo.2)EML4-ALK mutant cell line H3122 was treated with the first and second generation of ALK tyrosine kinase inhibitor consistently in order to aquire drug resistace.TGF?1 expression was evaluated in both H3122 cells and ALK TKIs resistance H3122 respectively.The expression levels of EMT-related signaling regulators(E-cadherin,Vimentin,etc.)in H3122 cells and ALK TKIs resistance H3122 cells were also detected respectively.NKX2-1 and its anti sense Lnc RNA NKX2-1 were also evaluated to figure out the connection between NKX2-1 and TGF ?1.Results: PI3K/HDAC dual pathway inhibitor CUDC-907 inhibited the proliferation,migration and anti-apoptosis ability of CAFs induced by TGF?1,and caused G1-S arrest.In addition,CUDC-907 inhibits the expression of myofibroblast markers,and also significantly inhibits the phosphorylation levels of AKT,mTOR,and Smad2/ 3 as well as promotes the acetylation of histones.It was also verified in bleomycin-induced mouse lung fibrosis and nude mice tumor transplantation models.On the other hand,EML4-ALK cell line undergoes EMT after aquired drug resistance.The expression of TGF?1 is significantly increased whereas the expression of NKX2-1 is significantly reduced.The downregulation of antisense gene Lnc RNA NKX2-1-AS1 in NKX2-1 promoter region may be related to the change of TGF?1 expression.Conclusion: TGF?1 gradually regulates the cell activity of CAF and the expression of PI3K-AKT-mTOR pathway and multiple proteases of the HDAC family.PI3K/HDAC dual pathway inhibitor CUDC-907 has a significant effect on inhibits TGF?1-induced lung and tumor fibrosis which provides an experimental basis for targeting activated myofibroblasts in lung and tumor fibrosis treatment.In addition,TGF?1 plays a vital role in ALK TKI aquired resistance of EML4-ALK mutant non-small cell lung cancer through EMT which may be related to the regulation of NKX2-1 and Lnc RNA NKX2-1-AS1.It provides a new target for patients with ALK TKI aquired resistance.