Fig 2: A schematic diagram showing potential HER2-related signaling pathways and action mechanisms of various inhibitors in HER2-amplified GC. Heterodimerization of HER2 with other HER family members (such as EGFR, HER3, HER4) result in tyrosine kinase activation with the subsequent signaling cascade, including members of MAPK and PI3K/AKT/mTOR pathways. As a result of these signaling pathways activation, different nuclear factors are recruited and modulate the transcription of different genes involved in cell-cycle progression, proliferation, and survival. Trastuzumab inhibits HER2 by targeting its extracellular domain, whereas lapatinib inhibits both HER2 and EGFR by inhibiting the intracellular tyrosine kinases. HER2 targeted therapy could be interrupted by re-activation MAPK and PI3K/AKT/mTOR pathways by compensatory activation of MET, IGF-RI, HER3 or loss of function mutations of tumor suppressor genes such as CSK, PTEN, NF1. In addition, drug resistance could be conferred by loss of function mutations of downstream genes such as KEAP1 and BAX by dysregulation of cellular antioxidants, xenobiotic detoxification enzymes and apoptosis, respectively. Lapatinib combining with lapatinib, SRC inhibitor AZD0530, PI3K inhibitor copanlisib, mTOR inhibitor rapamycin or MEK inhibitor trametinib could counteract the resistance at different level, respectively. A combinational treatment strategy with lapatinib, copanlisib and trametinib is demonstrated more effective for HER2 amplified GC with CSK, PTEN, NF1and KEAP1 mutations in this study

Fig 3: Functional validation of CSK or PTEN null N87 and OE19 cells. a Cell viability of CSK or PTEN knockout OE19 cells treated with indicated doses of lapatinib. OE19 cells were transduced with lentiviruses carrying sgRNAs targeting CSK, PTEN, or non-targeting control sgRNA. The drug resistance of the cells from each group was measured by calculating the relative percentage of cell viability. CSK or PTEN protein expressions were evaluated by western blotting. b Cell viability of CSK or PTEN knockout N87 cells treated with different doses of lapatinib. N87 cells transduced with non-targeting sgRNA as control. Shift between control cells and gene knockout cells in the dose response curve displays the reduced sensitivity to lapatinib in the GC cell lines. CSK or PTEN protein expressions were evaluated by western blotting. c Caspase-Glo 3/7 assay analysis to examine lapatinib induced caspase-3/7 activity after 48 h treatment in CSK or PTEN null cells OE19 and N87 cells. OE19 or N87 cells transduced with virus carrying non-targeting sgRNA as control. Unpaired Student’s t test indicated the following: *significant difference from the control, p < 0.05; **significant difference from the control, p < 0.01

Fig 4: Protein interaction network prediction, gene expression profile and pathway analysis of CSK or PTEN knockout cell lines and Up-regulation of PI3K/AKT and MAPK pathways in the CSK or PTEN knockout GC cells. Protein interaction network (a) and the predicted partners (b) were analyzed by STRING. CSK, PTEN and HER2 (ERBB2) were mapped by searching the STRING database version 10.5. In the resulting protein association network, proteins are presented as nodes which are connected by lines whose thickness represents the confidence level. (c) The bar plot depicts the enriched pathway among the DEGs between CSK null cells (N87-CSK-sgRNA) and parental N87 cells (N87) by KEGG pathway analysis. d The bar plot depicts the enriched pathway among the DEGs between PTEN null cells (OE19-PTEN-sgRNA) and parental OE19 cells (OE19) by KEGG pathway analysis. e The levels of phosphorylated and total proteins of AKT and MAPK were assessed by western blotting in CSK or PTEN knockout OE19 cells after treating with 0.05 uM lapatinib, respectively. OE19 cells transduced with non-targeting sgRNA as control. f The levels of phosphorylated and total proteins of AKT and MAPK were assessed by western blots in CSK or PTEN knockout N87 cells after treating with 0.01 um lapatinib, respectively. N87 cells transduced with non-targeting sgRNA as control. g PTEN and CSK protein expression was examined by western blotting in CSK knockout OE19 cell lines and PTEN knockout OE19 cell lines, respectively. OE19 cells transduced with non-targeting sgRNA as control. h PTEN protein and CSK protein expression was examined by western blotting in CSK knockout N87 cell lines and PTEN knockout N87 cell lines, respectively. N87 cells transduced with non-targeting sgRNA as control

Fig 5: The Cx43 region involved in the recruitment of PTEN and CskC6 glioma cells were incubated with several cell-penetrating peptides containing the indicated sequences of Cx43 fused to biotin for 30 min. (A) The SH3 domain binding motif is shown in grey and the tyrosines phosphorylated by c-Src in red. (B) After 30 min, the cells were lysed, and pull-down assays were carried out with avidin-conjugated agarose beads. Western blots before (lysates) and after avidin pull-down for c-Src, Csk and PTEN showing the enrichment of PTEN and Csk in the complex obtained with TAT-Cx43-266-283-Biotin and, to a lesser extent, with TAT-Cx43-245-283-Biotin. Str-HRP, HRP-conjugated streptavidin. (C) After 30 min, the cells were fixed, and the uptake of peptides bound to biotin was analyzed by fluorescence microscopy. Scale bars: 15 μm.