SHIP2 Inhibits Migration And Invasion Of Gastric Cancer Cells By Interacting With IQGAP2

Background&Objective Gastric cancer（GC）is one of the most common malignant tumors in the world,ranking the fifth in morbidity and the third in mortality,accounting for the first place in malignant tumors of the digestive tract.SHIP2（Src-homology2-containing inositol 5-phosphatase 2）is a member of the polyphosphate inositol-5-phosphatase family which is implicated in type 2 diabetes and Alzheimer’s disease.However,the role of SHIP2 in tumorigenesis and progression is complicated,and its"pro-tumor"or"anti-tumor"role depends on tissue context.The reason is that SHIP2,as a multidomain protein,can regulate the PI3K/Akt signaling pathway by its intermediate 5-phosphatase catalytic domain,and also interact with various cytokine receptors,adaptor proteins,cytoskeletal proteins,and phosphatases,regulates cellular insulin signaling pathways（case processes involved in diabetes and metabolic diseases）,cytoskeletal organization and function（affects cellular receptors mediate biological behaviors such as endocytosis,adhesion,migration,proliferation,and apoptosis）as well as immune system function through its own N-terminal SH2 domain and C-terminal PRD and SAM domains.Our previous study has been demonstrated that SHIP2 is frequently downregulated in gastric cancer,and reduced SHIP2 expression promotes tumorigenesis and proliferation of gastric cancer via activation of the PI3K/Akt signaling.To further clarify the molecular mechanisms of SHIP2 as a tumor suppressor in gastric cancer,in this study we first screened the SHIP2-binding protein IQGAP2 by immunoprecipitation and mass spectrometry analyses,then verified the endogenous interaction of SHIP2 and IQGAP2 and their role in migration and invasion of gastric cancer cells.Methods The binding proteins of SHIP2 in gastric cancer cells were screened by immunoprecipitation assays and mass spectrometry analyses.The protein and mRNA levels of IQGAP2、SHIP2 in normal gastric mucosal epithelial cells GES-1 and a group of gastric cancer cell lines were detected by western blot and qPCR.The interaction of IQGAP2 and SHIP2 was analysed by co-immunoprecipitation assays,and the endogenous binding of them was confirmed by immunofluorescence colocalization assays.A series of SHIP2 domain deletion mutants were constructed and transfected into human kidney blast cell 293T with wild-type IQGAP2 plasmid,and the domains of SHIP2 interacting with IQGAP2 were determined by co-immunoprecipitation assays.In the further functional experiments,we established stable gastric cancer cell lines SGC-7901.shSHIP2,SGC-7901.shIQGAP2,SGC-7901.shSHIP2/IQGAP2,MGC-803.shSHIP2,MGC-803.shIQGAP2,MGC-803.shSHIP2/IQGAP2 by shRNA knockdown of SHIP2 or IQGAP2 alone or their combination.The expression of SHIP2,IQGAP2,phosphorylated Akt,and downstream adhesion molecules E-cadherin,β-catenin,and N-cadherin in stable cells were detected by western blot.The effects of SHIP2 and IQGAP2 on migration and invasion of gastric cancer cells were detected by wound healing assays and transwell assays.To further clarify the effects of the interaction of SHIP2 and IQGAP2 on gastric cancer cell migration and invasion,we used lentivirus-mediated specific shRNA to knockdown IQGAP2 in the stable gastric cancer cell line transduced by lentiviruses carrying vector overexpressing SHIP2 or control vector（SGC-7901.SHIP2 and SGC-7901.NC）;the expression of SHIP2,IQGAP2,phosphorylated Akt,and downstream adhesion molecules E-cadherin,β-catenin,and N-cadherin of gastric cancer cells were detected by western blot;the effects on migration and invasion of gastric cancer cells were detected by wound healing assays and transwell assays.In addition,the stable knockdown cell line of SGC-7901.shSHIP2 was used to transfect wild-type SHIP2 and the deletion mutant SHIP2^Δ935-1258（deletion the domain interacting with IQGAP2）.The expression of SHIP2,IQGAP2,phosphorylated Akt and downstream adhesion molecules E-cadherin,β-catenin and N-cadherin were detected by western blot.The effects of exogenous overexpression of SHIP2 and SHIP2^Δ935-1258 on migration and invasion of gastric cancer cells were examined by transwell assays.Results1)Although SHIP2 is down-regulated in gastric cancer cells compared with normal gastric mucosal epithelial cell line GES-1,there is no significant difference in the expression level of IQGAP2 between GES-1 and a panel of gastric cancer cell lines.2)In gastric cancer cells,SHIP2 binds to IQGAP2 endogenously through PRD and SAM domains,and they colocalize in the cytoplasm of gastric cancer cells.3)SHIP2 and IQGAP2 inhibit migration and invasion of gastric cancer cells synergistically,which is associated with decreased Akt phosphorylation and the expression of downstream adhesion molecules E-cadherin,β-catenin and N-cadherin.4)SHIP2 inhibits migration and invasion of gastric cancer cells by interacting with IQGAP2 by PRD and SAM domain,which is associated with decreased Akt phosphorylation,and subsequent up-regulation of E-cadherin,β-catenin and the down-regulation of N-cadherin.Conclusion SHIP2 interacts with IQGAP2 through PRD and SAM domains in gastric cancer cells,resulting in decaeased Akt activity,and subsequent up-regulation of E-cadherin,β-catenin and down-regulation of N-cadherin,thereby inhibits migration and invasion of gastric cancer cells.