The Role Of Pyrotinib Combined With Apatinib In HER2-positive Gastric Cancer Treatment And Its Related Mechanisms

Objective: Molecular targeted therapy of gastric cancer has attracted much attention in recent years.Progress has also been made in targeted therapy of human epidermal growth factor receptor 2(HER2)-positive gastric cancer.However,drug resistance has severely limited the efficacy of anti-HER2 therapies.Pyrotinib is a novel,irreversible,pan-HER tyrosine kinase inhibitor.Apatinib is an anti-vascular small molecule inhibitor.Although these two drugs have been used alone for the treatment of gastric cancer,the combined use of pyrotinib and apatinib in HER2-positive gastric cancer has not been reported.This study will evaluate the role of pyrotinib combined with apatinib in HER2-positive gastric cancer in vitro and in vivo.Methods: Western blot and q RT-PCR were used to detect the expression of HER2 in five gastric cancer cell lines,namely NCI-N87,SNU216,SGC7901,MGC-803,and MKN45,and the inhibitory effect of pyrotinib and apatinib on the above cell lines was detected using the CCK-8 method and the half maximal inhibitory concentration(IC50)of the corresponding drugs was calculated.NCI-N87 and SNU216 cells with high HER2 expression were selected for subsequent experiments.NCI-N87 and SNU216 cells were pretreated with pyrotinib,apatinib,or the combination of drugs,respectively.CCK-8 method and colony formation assay were used to examine the inhibitory effect of the drugs on cell proliferation.Transwell assay was used to detect the effects of the drugs on cell invasion.Flow cytometry was used to detect the effects of the drugs on apoptosis.Western blot was used to detect changes in EGFR/HER2 downstream pathways and the expression of apoptosis-related proteins after different treatments.NCI-N87 and SNU216 xenograft models were used to verify the effect of the combination of the two drugs,and the interaction between pyrotinib and apatinib was explored through animal models in vivo.Results: Compared to pyrotinib or apatinib alone,the combination of the two drugs significantly enhanced survival,proliferation,and invasion of HER2-positive gastric cancer cells,as well as promoted cell apoptosis.Pyrotinib and apatinib exhibited synergistic effect in NCI-N87 xenograft models in vivo,with a combination index of less than 1,and the growth inhibition effect of HER2-positive NCI-N87 and SNU216 xenograft tumors of the combination treatment was more obvious than that of monotherapies(P < 0.05 and P < 0.01).The combined application of pyrotinib and apatinib enhanced the inhibition of PI3K/AKT and MAPK signaling pathways and up-regulated the expression of apoptosis-related proteins.Conclusion: The combination of pyrotinib and apatinib has a significantly enhanced antitumor effect in HER2-positive gastric cancer.Objective: Drug resistance of molecular targeted therapies is one of the common limitations in its clinical application.At present,studies have reported the drug resistance and related mechanisms of the classic anti-HER2 drug trastuzumab and other small molecule inhibitors targeting HER2 in HER2-positive gastric cancer.Pyrotinib has been used in a variety of solid tumors including HER2-positive gastric cancer,but the mechanisms of acquired pyrotinib resistance remains unclear.This part of study aims to investigate the occurrence of acquired pyrotinib resistance and explore its underlying mechanisms.Methods: A pyrotinib-resistant gastric cancer cell line NCI-N87-AR was established based on pyrotinib-sensitive NCI-N87 cells.The inhibitory effect of pyrotinib on NCI-N87-AR cells was detected using CCK-8 method and colony formation assay,and the IC50 was calculated.RNA sequencing was used to analyze the differences between parental NCI-N87 cells and pyrotinib-resistant NCI-N87-AR cells.Genes and pathways related to acquired pyrotinib resistance were then screened and verified by western blot and q RT-PCR.NCI-N87 and NCI-N87-AR cells were incubated with exogenous stem cell factor(SCF).CCK-8 method and colony formation assay were performed to examine the role of SCF in pyrotinib resistance.Western blot was used to detect the changes of SCF downstream pathways.Results: The sensitivity of NCI-N87 and NCI-N87-AR cells to pyrotinib was significantly different(P < 0.01).The IC50 of pyrotinib of the two cells were 0.04 ± 0.01μM and 4.15 ± 0.28μM,respectively.The proliferation ability of NCI-N87-AR cells was significantly stronger than that of NCI-N87 cells after pyrotinib pretreatment(P < 0.001),indicating that NCI-N87-AR cells were resistant to pyrotinib.A comparative analysis of the RNA sequencing results of the two cells revealed that the up-regulation of KITLG and PIK3R1 and the PI3K/AKT and MAPK pathways were related to the occurrence of acquired pyrotinib resistance,and were verified by western blot and q RT-PCR.SCF promoted the survival and proliferation of NCI-N87 and NCI-N87-AR cells through c-kit-mediated activation of the PI3K/AKT and MAPK pathways.Conclusion: The upregulation of SCF may lead to the occurrence of acquired pyrotinib resistance in HER2-positive gastric cancer through activation of the PI3K/AKT and MAPK pathways mediated by c-kit.Objective: In the previous part of the study,we found that the upregulation of SCF may lead to the occurrence of acquired pyrotinib resistance in HER2-positive gastric cancer through activation of the PI3K/AKT and MAPK pathways mediated by c-kit.Blocking the activation of alternate pathways by inhibiting c-kit receptor may be a potential solution to pyrotinib resistance in HER2-positive gastric cancer.In addition to selectively targeting vascular endothelial growth factor receptor 2(VEGFR2),apatinib can also effectively inhibit c-kit receptor.Studies have shown that patients with HER2-positive gastric cancer may benefit from apatinib treatment after trastuzumab resistance.However,the role of apatinib in pyrotinib resistance remains unclear.This part of the study mainly investigates the role of apatinib in acquired pyrotinib resistance and its related mechanisms.Methods: Western blot was used to detect the effects of apatinib at different concentrations on SCF/c-kit and downstream pathways.NCI-N87-AR cells were pretreated with pyrotinib,apatinib,or the combination of drugs,respectively.CCK-8 method and colony formation assay were used to examine the inhibitory effect of the drugs on cell proliferation.Transwell assay was used to detect the effects of the drugs on cell invasion.Flow cytometry was used to detect the effects of the drugs on apoptosis.Western blot was used to detect changes in SCF/c-kit downstream pathways and the expression of apoptosis-related proteins after different treatments.NCI-N87-AR xenograft models were used to examine the effect of the combination of the two drugs.Results: The inhibitory effect of apatinib on c-kit phosphorylation and downstream PI3K/AKT and MAPK pathways increased with its increasing concentration.SCF promoted the activation of PI3K/AKT and MAPK pathways in NCI-N87 cells,while apatinib inhibited c-kit-mediated activation of PI3K/AKT and MAPK pathways in NCI-N87-AR cells.Compared to pyrotinib or apatinib alone,the combination of the two drugs significantly enhanced survival,proliferation and invasion of NCI-N87-AR cells,as well as promoted cell apoptosis.The growth inhibition effect of NCI-N87-AR xenograft tumors of pyrotinib combined with apatinib was more obvious than that of monotherapies(P < 0.05 and P < 0.01).The combined application of pyrotinib and apatinib enhanced the inhibition of SCF/ckit signaling and downstream PI3K/AKT and MAPK pathways and up-regulated the expression of apoptosis-related proteins.Conclusion: Apatinib can inhibit the activation of SCF/c-kit signaling and its downstream PI3K/AKT and MAPK pathways by targeting c-kit,thereby reversing acquired pyrotinib resistance.The combination of pyrotinib and apatinib has a significantly enhance antitumor effect in pyrotinib-resistant HER2-positive gastric cancer.