The Efficacy And Drug Resistance Mechanism Study Of Multi-Target Inhibitors In Gastric Cancer

Background and Objective:Gastric cancer(GC)is one of the most common malignant gastrointestinal tumors in China,and remains the third common malignancy and leading cause of death.Due to the occult onset and the lack of specificity of clinical manifestations,more than 80%of gastric cancer has been in the advanced stage when diagnosed,and the overall prognosis of chemotherapy is poor.Molecular targeted therapy has become a new treatment direction and research hotspot for cancer.At present,the targeted therapies that have been proved to benefit GC patients include trastuzumab,apatinib and ramuzumab.Multi-target tyrosine kinase inhibitors have been applied to cancer therapy and have become a new breakthrough.As one of the representatives,anlotinib,which was developed independently by China,is a novel multi-target tyrosine kinase inhibitor that can effectively inhibit VEGFR,PDGFR,FGFR,C-kit and other kinases,and then inhibit tumor angiogenesis and growth.Many studies have confirmed that it has strong anti-tumor activity and is well tolerated by patients.It has been approved by NMPA for the clinical therapy of NSCLC and soft tissue sarcoma.This study focused on anlotinib,a novel multi-target inhibitor,to explore its antitumor efficacy in gastric cancer and the possible mechanism of drug resistance in subsequent treatment,providing theoretical and practical basis for GC therapy.Methods:1.The CCK8 proliferation assay,colony formation assay,apoptosis analysis by flow cytometry were conducted to detect the anti-tumor effect of anlotinib on gastric cancer cells in vitro.2.The subcutaneous xenograft model and the pulmonary metastasis model via caudal vein of nude mice were established to explore the anti-tumor effect of anlotinib on gastric cancer cells in vivo.3.We established anlotinib resistant GC cell lines,AGS-R and MGC803-R,and investigated the toxicologic effects through cell survival and colony formation in vitro.4.The transcriptional activity of TGF-β signaling pathway in anlotinib-resistant GC cell lines was verified by dual luciferase reporter assay.Western blot assay was used to detect changes of important proteins in TGF-β signaling pathway.5.GC cells were exposed to anlotinib,TGF-β inhibitor and combination,respectively.The anti-tumor effect of anlotinib on GC after inhibiting TGF-β signaling pathway was explored through a series of in vitro and in vivo function experiments.6.Anlotinib resistant GC cells were exposed to anlotinib,TGF-β inhibitor and combination,respectively.A series of function assays in vitro and in vivo was used to verify the role of TGF-β signaling pathway in anlotinib resistance of GC.7.RNA-sequencing was then performed to screen anlotinib resistance-associated genes between AGS and AGS-R cells.The role of candidate gene in anlotinib resistance was explored by gain-and loss-of-function analyses.8.Western blot and q RT-PCR assays were used to detect the expression of ENPP2 in anlotinib-resistant GC cells with treatment of TGF-β inhibitor,and the online database was employed to analyze the correlation between TGF-β signaling pathway and ENPP2.Results:1.At the cellular level,anlotinib could significantly inhibit the growth of gastric cancer cells and promote apoptosis in a dose-dependent manner.2.At the animal level,anlotinib inhibited subcutaneous tumorigenesis and metastasis of gastric cancer cells in nude mice.3.Compared with parental gastric cancer cells,AGS-R and MGC803-R showed significant resistance to anlotinib.4.The dual luciferase reporter assay and Western Blot assay indicated that TGF-βsignaling pathway was activated in anlotinib-resistant cells.Transcriptome sequencing showed that more up-regulated genes were enriched in TGF-β signaling pathway in anlotinib-resistant cells.5.The anti-tumor effect of anlotinib combined with TGF-β inhibitor LY364947 in gastric cancer cells in vitro and in vivo was significantly better than that of single drug treatment.6.At the same time,TGF-β inhibitor can significantly reverse anlotinib resistance in gastric cancer.7.In addition to TGF-β signaling pathway playing a role in anlotinib resistance,the transcriptomic profiling indicated that ENPP2 which is negatively correlated with prognosis of gastric cancer was found among significantly up-regulated differential genes in anlotinib-resistant cells.Overexpression of ENPP2 conferred and knockdown of ENPP2 attenuated the anlotinib resistance of GC cells.8.GEPIA database analysis showed that TGF-β signaling pathway and ENPP2 expression were positively correlated in GC.After being exposed to TGF-β inhibitor,WB and q PCR results showed that ENPP2 expression was down-regulated in anlotinibresistant GC cells,which verified the positive regulatory relationship between them.Conclusion:1.In this study,we confirmed the anti-tumor effect of anlotinib on GC and successfully established anlotinib-resistant GC cells to illustrate the importance of TGF-β signaling pathway in the mechanism of anlotinib resistance in GC.Results showed that the TGF-β signaling pathways was activated in the resistant cells.Inhibition of TGF-β signaling pathway can enhance the anti-tumor effect of anlotinib and reverse the GC resistance to anlotinib.Our study provided a theoretical and practical basis for the multi-target inhibitors in the treatment of GC and provided a reference for subsequent resistance mechanisms in the meantime.2.In addition to TGF-β signaling pathway possibly promoting anlotinib resistance in GC,through transcriptome sequencing and experimental demonstration,we still found that ENPP2 plays a role in the resistance of GC to anlotinib,suggesting that ENPP2 may be a potential target of GC combined with anlotinib therapy.