ARTICLE ABSTRACTPurpose: Targeting human epidermal growth factor receptor 2 (HER2) therapy is currently considered as the standard treatment for HER2-positive (HER2+) advanced gastric cancer. However, as seen in recent clinical trials, most of HER2+ gastric cancer are actually unresponsive to HER2-targeted agents, including lapatinib. The aim of this study is to identify the responsible receptor tyrosine kinases (RTK) potentially conferring lapatinib unresponsiveness in HER2+ gastric cancer and elucidate the molecular mechanism underlying this RTKs-induced resistance.Experimental Design: A functional RNAi screen targeting human RTKs and related growth factors was used to identify candidate RTKs conferring lapatinib unresponsiveness in HER2+ gastric cancer cells. Independent siRNAs transfection and corresponding ligands supplement were performed to validate the effects of candidate RTKs on lapatinib sensitivity. Cross-talks of pathways involved were analyzed via Western blot analysis. Cell apoptosis and cell motility were detected using FACS system and Transwell assay. Immunohistochemistry was used to analyze protein expression in clinical samples.Results: MET, HER3, insulin-like growth factor (IGF)-1R, and INSR were identified to mediate lapatinib unresponsiveness in HER2+ gastric cancer cells. Activation of these bypass RTKs attenuated lapatinib-induced apoptosis and suppression of cell motility, mechanistically because of restimulating the shared downstream AKT or ERK signaling, as well as restimulating WNT signaling and epithelial-to-mesenchymal transition (EMT)–like process. Patients' specimens revealed that these unresponsiveness-conferring RTKs were particularly enriched in the majority of patients with HER2+ gastric cancer.Conclusions: MET, HER3, IGF1R, and INSR pathways activation represent novel mechanism underlying lapatinib unresponsiveness in HER2+ gastric cancer. Combination strategy may be recommended in treating patients with HER2+ gastric cancer with these pathways activation. Clin Cancer Res; 20(17); 4559–73. ©2014 AACR.