SRC-like Adaptor Protein Can Increase The Sensibility Of Radiation-therapy In Non-small-cell Lung Cancer By Inhibiting ADAM12

Objective: In the worldwide,lung cancer is one of the major cancers causing cancerrelated deaths and almost 80% of the total number of lung cancers are non-small cell lung cancer(NSCLC).Because of delayed diagnosis,most patients lose the opportunities of surgical treatment in clinical treatment.Patients receive traditional radiotherapy,chemotherapy and targeted therapy to treat cancer.C-SRC is an important member of the tyrosine kinase family.It is rarely mutated and highly expressed in 50-80% of lung cancer patients.It is a part of the FAK complex which regulates cell adhesion and migration and plays an important role in the progression of lung cancer and C-SRC protein mediates the development of radiotherapy-resistance in lung cancer.ADAM12,an anti-integrin-metalloproteinase 12,is involved in the progression of cancer progression by regulating epithelial cell mesenchymal transition(EMT)levels of tumor cells in lung cancer and EMT will further promote chemotherapy and radiotherapy resistance in NSCLC.SRC receptor protein(SLAP)belongs to the hematopoietic factor receptor subpopulations and the biological function is to block intracellular signal transduction.The SLAP has a unique C-terminal connected with the SRC tyrosine kinase family with a highly homologous SH3 and SH2 which end of the 18 alkylation of N-term and closely connected with the CBL.The evidence suggests that slap negatively regulates the expression and activation of tyrosine kinases in cancer cells.Because the SH2 domains of SLAP and C-SRC have high homology,SLAP can compete with C-SRCrelated growth factor receptors and mitotic signaling pathway receptors.The purpose of this study is to investigate the role of SRC-like receptor protein(SLAP)in reversing radiotherapy-resistance of non-small cell lung cancer(NSCLC)during radiation therapy by inhibiting phosphorylation of C-SRC.Methods: A549,H1650,and H460 lung cancer cell lines were treated with the C-SRC inhibitor dasatinib and 2Gy or 2Gyx4(2Gy radiotherapy twice a day for four consecutive times)radiotherapy were performed on A549,H1650,H460 cell lines and dasatinib pretreated A549,H1650,and H460 cell lines using a 6mv linear accelerator.The proliferation and apoptosis abilities of the cell lines in each group were observed.A549,H1650,and H460 cell lines were transfected by lentiviral vectors and puromycin was used to select A549,H1650,and H460 cell lines that were stably over-expressing or under-expressing ADAM12 after 72 hours.Western blotting experiments were used to test the level of ADAM12 protein after 5 passages of the cell line.A549,H1650 and H460 cell lines and A549,H1650 and H460 cell lines which are over-expression or under-expression of ADAM12 were subjected to 2Gy or 2Gyx4 radiotherapy by a 6mv linear accelerator.CCK-8 was used to detect the proliferation of cells in each group,the caspase3 assay kit was used to detect the apoptosis in each group,Western blotting was used to detect the expression of ADAM12 and C-SRC in each group.Elisa assay was used to detect the expression of p-AKT in each group.A549,H1650 and H460 cell lines which overexpression of SLAP were constructed by lentiviral vectors and these cell lines were treated with 2Gy and 2Gyx4 radiotherapy respectively.CCK-8 was used to detect the proliferation of cells in each group.The caspase3 kit was used to detect the apoptosis in each group.Western blotting was to detected the expression of ADAM12 and C-SRC proteins in each group and the expression of p-AKT in each group was detected by Elisa.In addition,SLAP was knocked down for rescue experiments in A549,H1650 and H460 cell lines which overexpressed SLAP.SLAP-overexpressing cell lines and negative control cell lines were treated with 2Gy and 2Gyx4 radiotherapy.CCK-8 was used to detect the proliferation of cells in each group.The caspase3 kit was used to detect the apoptosis in each group.Western blotting was used to detect the expression of ADAM12 and C-SRC in each group.Elisa was used to detect the expression of p-AKT,p-MTOR,Cleaved PARP1,CDK1,and CD44 in each group.Wound healing test was used to test the migration ability of cells and transwell assay was used to test cell invasion ability.Finally,C-SRC and AKT were over-expressed or knock-down in the A549,H1650,and H460 cell lines by lentiviral vectors which had been over-expressed or knocked down SLAP by lentiviral vectors and AKT,p-AKT,and E-cadherin were measured in each cell lines after receiving 2Gyx4 radiotherapy.Wound healing test were used to test cell migration ability and transwell assay were used to test cell invasion ability.Results: After treatment of combination of dasatinib and 2Gy radiotherapy,proliferation of A549,H1650 and H460 cells was significantly reduced and the level of apoptosis was significantly increased.However,after 2Gyx4 radiotherapy in these cell lines,treatment of dasatinib lost these effects.After WB detection,the expression of ADAM12 in A549,H1650 and H460 cell lines was increased after 2Gyx4 radiotherapy.After knockdown of ADAM12 in A549,H1650 and H460 cell lines,these cell lines restored drug sensitivity to dasatinib which combined with radiotherapy of 2Gyx4.Overexpression of SLAP in A549,H1650 and H460 cell lines were established by lentiviral vectors.After 2Gyx4 radiotherapy treatment,the ADAM12 protein of these cell lines which SLAP is overexpressed were remained a low level and its proliferative capacity was in a lower level compared with that of the control group and the expression of p-C-SRC,p-AKT,CDK1,and CD44 was decreased.The expression of p-MTOR and Cleaved PARP1 was increased,and the invasive ability and migration ability were significantly reduced.After knockdown the SLAP in SLAP-overexpressed cell lines,the biological behaviors were returned to previous levels.Overexpression of C-SRC and AKT was transfected into A549,H1650 and H460 cell lines which stably over-expressed SLAP resulted upregulation of p-C-SRC and p-AKT which occurred enhanced migration and invasion.Down-regulation of C-SRC and AKT in A549,H1650 and H460 cell lines which stably low-expressed SLAP decreased cell migration and invasion.Conclusion: C-SRC is an important target gene for the treatment of non-small cell lung cancer.C-SRC inhibitor dasatinib combined with single-dose radiotherapy can increase cell apoptosis and reduce cell proliferation.Continuous four times 2Gy radiotherapy activated the overexpression of ADAM12 protein in lung cancer cell lines,which ADAM12 protein has the ability to promote epithelial-mesenchymal transition(EMT)and EMT can lead to radiotherapy-resistance and chemotherapy-resistance of tumor cells and promote tumor cell migration and invasion.Reduced the expression of ADAM12 in lung cancer cell lines can increase the sensitivity of tumor cells to dasatinib after treatment with 2Gyx4 radiotherapy.Overexpression of ADAM12 protein in lung cancer cell lines can reverse the above-mentioned phenomenon which can demonstrate that ADAM12 can regulate the sensitivity of dasatinib in lung cancer cells.Overexpression of SLAP protein can negatively regulate the expression of ADAM12 protein and downregulate expression of p-C-SRC and increase the apoptosis of lung cancer cells and inhibit cell proliferation.SLAP was knocked down in lung cancer cell lines which were over-expressed SLAP can make the SLAP protein level return to normal level.And then these new cell lines can reverse the above-mentioned biological phenomenon which performed by over-expressed SLAP cell lines after radiotherapy and demonstrating that SLAP can regulate the biological function of lung cancer cell lines by negatively regulates the expression of ADAM12 and p-C-SRC.Lung cancer cell lines which overexpressed SLAP were transfected C-SRC and AKT protein after 2Gyx4 radiotherapy resulted in radiotherapy resistance and higher levels of proliferation,migration,and invasion compared to SLAP overexpressing cell lines.Lung cancer cell lines which have low level of expression of SLAP were knockdown C-SRC and AKT protein after 2Gyx4 radiotherapy resulted in radiotherapy sensitivity and lower levels of proliferation,migration,and invasion compared to SLAP knockdown cell lines.It was proved that SLAP can down-regulate the proliferation ability,invasion ability,migratory ability of tumor cells by negatively regulating phosphorylated C-SRC protein.On the other hand,SLAP can reverse radiotherapy resistance and chemotherapeutic resistance by negatively regulate ADAM12 protein and it can provide new ideas for the combination therapy of chemotherapy and radiotherapy in the clinic.