| BackgroundGastric cancer is one of the most common malignant tumors in our country,and the updated data show that it ranks 3rd in terms of incidence and mortality.With the development of molecular biology,molecular targeted therapy has become a hot topic in the comprehensive treatment of gastric cancer,among which human epidermal growth factor receptor 2(HER2)is the first target that has been proven to be effective.Trastuzumab,a humanized monoclonal antibody against HER2,inhibits tumor growth by antagonizing the transmission of HER2 signaling pathway.In clinical practice,trastuzumab treatment significantly prolonged the overall survival of patients with HER2-positive metastatic gastric cancer.However,most patients developed drug resistance within 1 year of effective treatment.XB130 is a newly discovered connector protein that activates several tumor-associated signaling pathways and is also activated by phosphorylation of various tyrosine kinases including HER2,epidermal growth factor receptor(EGFR)and SRC.Therefore,it is widely involved in the differentiation,proliferation and invasion of tumor cells,and is considered as a potential key target for tumor diagnosis and treatment.In this study,we intend to focus on the role and mechanism of XB130 in the development of trastuzumab resistance in gastric cancer.Contents and methods1.Human gastric cancer trastuzumab-resistant cells were established by doseescalation method,and CCK8 assay was performed to detect the sensitivity to trastuzumab.2.Western blot was used to detect the protein levels of XB130,AKT,p-AKT,SRC,p-SRC(Tyr416,Tyr527)and PTEN in parental and resistant cells,and QRTPCR was used to detect the levels of XB130 and PTEN.3.Co-IP to detect the binding of XB130 to PI3K p58α in parental and resistant cells;treat resistant cells with SRC inhibitor to detect the changes in the binding levels of XB130 to PI3K p58α.4.Transfected with XB130 shRNA,XB130 shRNA combined with Myr-Akt to detect changes of sensitivity to trastuzumab and p-AKT expression in resistant cells.5.Bioinformatics to predict the binding site of XB130 and PTEN,and luciferase reporter gene assay to determine the relationship between XB130 and PTEN gene promoter activity.6.Transfection of resistant cells with PTEN wt/mut,XB130 shRNA combined with PTEN shRNA to detect sensitivity to trastuzumab and changes in p-AKT and pSRC expression;7.Established tumor-bearing mice models with parental/resistant cells and resistant cells XB130 shRNA/NC respectively,ploted the tumor growth curve after trastuzumab treatment;and detected the expression of XB130,PTEN,SRC/p-SRC in tumor by IHC.8.The clinical data of patients with metastatic gastric cancer treated with trastuzumab were collected,and the expression of XB130,PTEN and SRC/p-SRC in tumor tissues before/after progression were detected by IHC.9.Differences were compared by ANOVA or Student’s t-test(two-sided)using SPSS 17.0 and GraphPad Prism.Data were expressed as mean±standard deviation,P<0.05 indicated that the difference is statistically significant.Results1.The constructed resistant cells were less sensitive to trastuzumab.2.The constructed resistant cells highly expressed XB130,p-AKT,p-SRC(Tyr416)and lowly expressed PTEN.3.The binding of XB130 to PI3K p85 was found to be tighter in trastuzumabresistant cells by Co-IP,which was weakened through using the SRC inhibitor Saracatinib.4.Silencing XB130 caused a decrease in p-AKT expression and an increased sensitivity to trastuzumab;while co-transfection with XB130 shRNA and Myr-Akt showed the opposite result.5.XB130 could bind to the PTEN promoter and negatively regulate PTEN gene activity.6.Simultaneous silencing of XB130 and PTEN resulted in unchanged of pAKT and restoration of resistance to trastuzumab in resistant cells.7.Mice tumor model were constructed and found that XB130 knockdown mice treated with trastuzumab showed significant reduction in tumor size;intratumoral XB130 and p-SRC expression were significantly reduced and PTEN was significantly increased.8.The comparison of pre/post trastuzumab treatment in HER2+gastric cancer patients revealed that the levels of XB130 and p-SRC were strikingly increased,while PTEN was remarkably decreased in tumor tissues after treatment.Conclusions1.XB130 is involved in regulating the resistance to trastuzumab in HER2+gastric cancer.2.XB130 regulates the development of trastuzumab resistance by activating AKT through binding PI3K p85α under the mediation of SRC kinase.3.XB130 mediates trastuzumab resistance by forming a positive feedback loop between SRC-XB130-PTEN through regulating PTEN gene transcription.4.Animal experiments and clinical case data confirm that XB130 is involved in regulating trastuzumab resistance in HER2+gastric cancer. |
