The Study Of Targeted Inhibition Of HER2-SHCBP1-PLK1 Non-canonical Pathway To Sensitize Trastuzumab In Gastric Cancer

Objective:The monotherapy targeting HER2 has limited efficacy in gastric cancer and the mechanism has not been elucidated nowadays.The study is intended to hunt for aberrant HER2 downstream signaling pathway and develop relative potential therapeutic targets and drugs to further improve the effect of HER2-targeted therapy in gastric cancer.Methods:The study firstly screened out HER2-correlated proteins and evaluated the relationship between SHCBP1 expression and clinical characteristics by bioinformatics,immunoprecipitation and mass spectrometry analysis,and other experiments.In vitro cell experiments and in vivo CDX(cellderived xenograft)animal model experiments were carried out to assess the effect of SHCBP1 expression on trastuzumab sensitivity in gastric cancer.Secondly,immunoprecipitation,cellular immunofluorescence,deletion,mutagenesis and GST pulldown experiments were performed to investigate the mechanism of HER2 receptor activation leading to the SHCBP1 nuclear translocation,promoting mitosis in gastric cancer cell and further involving in the regulation of trastuzumab sensitivity.Then,the model of SHCBP1-PLK1 complex was constructed by the homology modeling of SHCBP1,protein-protein docking and other experiments.Based on the model,small molecule inhibitors were virtually screened.In vitro cell experiments were performed to validate the effect of the inhibitor TFBG targeting the SHCBP1-PLK1 interaction.Finally,the efficacy of TFBG sensitizing trastuzumab in the treatment of gastric cancer was further evaluated by in vitro cell experiments and in vivo CDX animal model.Results:1.A critical HER2 downstream regulator,SHCBP1,was identified.And the treatment effect of trastuzumab was poor for gastric cancer patients with high expression of SHCBP1.The knockdown of SHCBP1 expression could significantly sensitize trastuzumab to inhibit the growth and proliferation of gastric cancer.2.Under the stimulation of EGF,SHCBP1 dissociates from Shc1 and translocates into the nucleus.SHCBP1 could interact directly with PLK1 to enhance MISP phosphorylation,contributing to cell mitosis and promoting cell proliferation in the nucleus.The blockade of SHCBP1-PLK1-MISP signaling could enhance the effect of trastuzumab on inhibiting the proliferation of gastric cancer cells.3.By constructing the SHCBP1-PLK1 complex model,the study identified the four core amino acids(K474,Y485,H489 and L490)on the binding surface of SHCBP1-PLK1 interaction and accordingly,the inhibitor TFBG was screened.Subsequently,MST(Microscale thermophoresis)and immunoprecipitation experiments were performed to further validate the targeting to the complex and efficacy of TFBG.4.The SHCBP1-PLK1 complex inhibitor TFBG could synergistically sensitize trastuzumab to inhibit the growth and proliferation of gastric cancer in vitro and in vivo.The mechanistic study revealed that TFBG functions mainly by inhibiting the activation of SHCBP1-PLK1-MISP signaling.Conclusion:The hyperactivation of SHCBP1-PLK1-MISP non-canonical pathway,a HER2 downstream signaling,could promote trastuzumab resistance in the treatment of gastric cancer and the inhibitor TFBG targeting the pathway could enhance the effect of trastuzumab in gastric cancer.