P130cas Exerts Radiotherapy Resistance In Non-small Cell Lung Cancer Through FAK-YAP Signaling Pathway

Purpose:p130cas is a member of the cas family.p130 cas is involved in the occurrence and development of a variety of human cancers.It can promote the proliferation,invasion,and metastasis of non-small cell lung cancer.Radiotherapy is an important method for the treatment of non-small cell lung cancer and an important method of combined surgery.However,most patients will have the effect of radiotherapy resistance.but it is unclear whether p130 cas plays a role in the radiotherapy of lung cancer and the mechanism of action.Patients and methods: First,we constructed p130 cas stably overexpressing H460 and A549 lung cancer cell lines.Western blot was used to detect the phosphorylation level of p130 casrelated FAK-SRC pathway and pull-down technology was used to detect the activity of Rac1-GTPrS.We further detected the expression level of YAP nuclear protein by western blot and the level of target mRNA downstream of YAP by qPCR.At the same time,the level of transcription enhancement factor TEAD4 was detected by the luciferase reporter gene and our Co-immunoprecipitation was used to detect the interaction between p130 cas and YAP.We inhibited the phosphorylation level of FAK and forcedly inactivated mutants transfected with RAC1,and then performed the same test.At the same time,Western blot and MTS experiments and colony formation experiments were used to study the biological role of p130 cas in the sensitivity of non-small cell lung cancer cells to radiotherapy.We used immunofluorescence analysis to detect changes in the ?H2AX cell line of p130 cas overexpressed after radiotherapy.Next,in order to explore the specific mechanism of p130 cas resistance to radiotherapy,we added the FAK inhibitor pf562271 to the stably transfected cell lines,detected the changes in radiosensitivity after transient interference with FAK,added the YAP inhibitor verteporfin and transfected the shyap plasmid In the future,the changes of radiosensitivity will be tested to explore the specific mechanism of p130cas' s resistance to radiotherapy.Results:We used western blot to detect increased FAK and SRC phosphorylation levels in p130cas-overexpressed cells.We used pull-down tests to detect increased Rac1 small GTPase activity after P130 cas overexpression;YAP nuclear protein expression increased,while YAP protein downstream target mRNA The mRNA levels of CTGF and CYR61 increased,the level of transcription enhancement factor TEAD4 detected by the luciferase reporter gene increased,and co-immunoprecipitation results showed that p130 cas could bind to YAP and FAK.Transfected and untransfected p130 cas cell lines were irradiated,and the p-ATM and p-CHK2 and r-H2 AX protein levels were detected.It was found that p-CHK2 and p-ATM expression were down-regulated after p130 cas overexpression,and p130 cas could inhibit Activation of DNA damage repair pathways in cell lung cancer after radiotherapy.At the same time,western blot and immunofluorescence were used to detect p130 cas overexpression.After r-H2 AX levels were found,p130 cas overexpressed cells were irradiated with r-H2 AX to increase protein expression.Immunofluorescence showed more damage.Focus,above we have concluded that p130 cas can enhance the radiotherapy resistance of non-small cell lung cancer.We added the specific phosphorylation inhibitor pf562271 of FAK and interfered with FAK protein,and then detected the levels of r-H2 AX by western blot and immunofluorescence.It was found that after FAK was inhibited,the radiotherapy resistance of 130 cas was reversed.At the same time,the radiotherapy resistance of p130 cas was also reversed after adding the veterporfin inhibitor of YAP and transfection of the shyap plasmid.Conclusion: p130 cas can increase the nuclear expression of YAP protein through the activation of FAK and Rac1 small GTPase,the mRNA levels of downstream target mRNAs CTGF and CYR61 increase,and the luciferase reporter gene shows that the fluorescence intensity of TEAD4 increases after p130 cas overexpression.The addition of FAK-specific phosphorylation inhibitor pf562271 increased YAP nuclear protein,increased mRNA levels of CTGF and CYR61,and increased luciferase reporter TEAD4.p130 cas exerts radiotherapy resistance by inhibiting the activation of the damage repair pathway.When FAK phosphorylation is inhibited,FAK protein levels are reduced,YAP and TEAD4 are inhibited from binding,and YAP protein levels are inhibited after radiotherapy resistance is reversed.Therefore,p130 cas can be reversed.Through the expression and activation of FAK and YAP to exert radiotherapy resistance,p130 cas can become a key therapeutic target for radiosensitivity.