| Lung cancer is the most common malignant tumor in the world and the leading cause of death.Non-small cell lung cancer(NSCLC)accounts for about 85% of all lung cancer.Radiotherapy(RT)is widely used in the clinical treatment of NSCLC.However,due to the existence of radioresistance,radiotherapy plays a limited role in the treatment of NSCLC.Some studies have shown that NSCLC radioresistance related pathways mainly include ECM receptor interaction and Focal Adhesion,and the important factor in these pathways,FAK,can affect the nuclear translocation and activation of YAP in response to mechanical activation,but how FAK affects YAP is still unknown.As a part of focal adhesion,Breast Cancer Anti-Estrogen Resistance Protein 1(BCAR1)has been found in many kinds of cancers,which is closely related to the occurrence,development and metastasis of cancer.Some studies have shown that phosphorylated FAK and BCAR1 increase after ionizing radiation(IR)treatment,suggesting that BCAR1 may participate in the process of radiation resistance,but the direct causal relationship between BCAR1 and radiation resistance has not been reported.Our study confirmed that BCAR1 can promote the proliferation of NSCLC cells,and the protein levels of BCAR1 and phosphorylated BCAR1 in the cells treated with IR increased,while the over-expressed BCAR1 protein has the ability to induce the cells to produce radiation resistance.Our results show that the mechanism of radiation resistance induced by BCAR1 is that: BCAR1 can be used as a scaffold to connect YAP and FAK,and YAP can form a ternary complex with FAK protein through BCAR1.The formation of the complex can maintain the stability of YAP,inhibit the degradation of YAP,promote the translocation of YAP into the nucleus,activate the expression of YAP downstream target gene,and thus mediate the generation of radiation resistance of cells.Objective:To explore the relationship between BCAR1 and radiotherapy resistance in NSCLC,and the specific mechanism of BCAR1 affecting radiotherapy resistance.Methods:1.Establish stable overexpression and knockdown BCAR1 cell lines in human and mice NSCLC cell lines respectively,and detect the effect of BCAR1 on the proliferation of NSCLC through colony formation test,MTT test,flow cytometry,Western blot detection of cell cycle-related protein.2.Western blot was used to observe the changes of protein levels of BCAR1 and p-BCAR1 under different time gradients of IR treatment,and the effect of overexpression/knockdown of BCAR1 under IR treatment on the expression of apoptosis protein.Cell survival analysis was used to detect the effect of BCAR1overexpression/knockdown on the radioresistance of NSCLC cells.Western blot and immunofluorescence assay were used to observe the expression of γ-H2 AX.After subcutaneous tumorigenesis,mice were treated with IR to further explore the effect of BCAR1 overexpression on radiosensitivity in vivo.3.KEGG,GO,GSEA database analysis,enrichment of BCAR1 related pathways.The effect of BCAR1 overexpression/knockout on YAP nuclear translocation was detected by nuclear plasma separation test.The GEPIA database analyzed the correlation between BCAR1 and Hippo pathway members.Western blot was used to observe the effect of BCAR1 on HIippo pathway protein.RT-q PCR assay was used to detect the changes of YAP downstream target genes.The luciferase activity of TEAD4 luciferase reporter gene was detected.The effect of BCAR1 overexpression on the stability of YAP was detected by blocking the de novo synthesis of protein with cycloheximide(CHX).The binding between BCAR1 and YAP was detected by immunoprecipitation and immunofluorescence co-localization.The effect of inhibition of YAP activity on radiation resistance induced by BCAR1 overexpression was observed in the recovery experiment.4.Overexpression of FAK interferes with BCAR1 to observe the localization of YAP nucleus.Overexpression or knockdown of BCAR1 was detected by Western blot to observe the expression of phosphorylated FAK.While overexpressing BCAR1,knock down FAK or use FAK inhibitor to observe the changes of cell proliferation,YAP nuclear localization,YAP downstream target gene expression,TEAD4 luciferase reporter gene activity,and γ-H2 AX expression.5.Immunoco-precipitation(Co-IP)was used to detect the binding of BCAR1,YAP and FAK,and a truncated mutant plasmid or point mutation plasmid was established to explore the specific domain of the binding of the three,and to observe whether the formation of the complex of the three caused the radiotherapy resistance of NSCLC.Results:1.BCAR1 is stably overexpressed in A549,H460,LLC cell lines,and stably knockdown BCAR1 in H1299,LK2 cell lines.Colony formation test and MTT test show that BCAR1 has the ability to promote cell proliferation.Flow cytometry showed that the proportion of S phase cells increased significantly after overexpression of BCAR1.Western blot showed that BCAR1 mainly promoted the expression of Cyclin D1 in cyclin.The subcutaneous tumorigenesis experiment in mice showed that BCAR1 promoted the proliferation of NSCLC in vivo.2.Western blot showed that the level of BCAR1 and phosphorylated BCAR1 protein in cells treated with IR increased.Cell survival analysis showed that overexpression(or knockdown)of BCAR1 promoted(or suppressed)cell radioresistance.In addition,after IR treatment,compared with the control cells,the level of apoptotic protein cleaved-PARP and cleaved-capase3 decreased(or increased),and the expression of radiation damage marker γ-H2 AX decreased(or increased).In vivo experiments also showed that overexpression of BCAR1 promoted radiation resistance.3.The analysis of KEGG,GO and GSEA databases showed that the ECM receptor interaction and Focal Adhesion pathways or related pathways were significantly enriched in the differential pathways between the high expression group and the low expression group of BCAR1.Nucleo-cytoplasmic separation experiment showed that BCAR1 overexpression(or knockout)could promote(or inhibit)YAP entry into the nucleus,and promote(or inhibit)the expression of YAP downstream target gene and the activity of TEAD4 luciferase reporter gene.GEPIA database analysis showed that BCAR1 was significantly correlated with the main members of Hippo pathway.Further Western blot analysis showed that overexpression(or knockdown)of BCAR1 could inhibit(or promote)the phosphorylation level of LATS1.In the cells treated with CHX,YAP degradation of BCAR1 overexpressed cells was reduced compared with that of control cells.Co-IP experiment and immunofluorescence co-localization experiment show that BCAR1 can combine with YAP.Inhibition of YAP in BCAR1 overexpression cells will weaken the radiation resistance induced by BCAR1 overexpression.In vivo experiments also confirmed that BCAR1 mediated NSCLC radiation resistance through YAP.4.FAK can promote the nuclear translocation of YAP,but can be eliminated by the knockout of BCAR1.Overexpression(or knockout)of BCAR1 can promote(or inhibit)phosphorylation of FAK at Tyr397 and Tyr925 sites.At the same time,the ability of BCAR1 to promote cell proliferation can be eliminated by knocking down or inhibiting FAK.BCAR1 overexpression promotes the increase of YAP nuclear expression,the increase of YAP downstream target gene expression,and the increase of TEAD4 luciferase reporter gene activity can also be eliminated by inhibiting FAK.The knockdown or inhibition of FAK in BCAR1 overexpression cells can also restore the reduced γ-H2 AX level after BCAR1 overexpression under IR treatment.5.Co-IP experiments show that BCAR1,YAP,and FAK combine with each other,and the binding domain of the three is: BCAR1 combines with FAK and YAP prolinerich region through its SH3 domain,while FAK and YAP cannot bind directly,but indirectly through BCAR1.Transfection BCAR1-ΔSH3 or the P712/715 A mutant of FAK destroyed the formation of the complex of the three,the ability of overexpression of the two to induce YAP nuclear translocation,promote the expression of YAP downstream target gene,and promote the resistance to radiotherapy was reversed.Conclusion:1.BCAR1 can promote the proliferation of NSCLC and induce radiation resistance of NSCLC.2.BCAR1 can play an essential role as a scaffold protein,form a triple complex with YAP and FAK,maintain the stability of YAP,inhibit the degradation of YAP,promote the activation of YAP and nuclear translocation,activate the expression of YAP downstream target genes,and thus mediate the generation of radiation resistance in cells. |
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