ASAP2 Regulates The Epithelial-Mesenchymal Transition And Mitochondrial Functions Of Gastric Cancer Cells To Promote The Migration/Invasion And Xenograft Tumor Growth,Respectively

Background&ObjectiveGastric cancer(GC)is the 5th most prevalent cancer and ranks 4th in case fatality rates among all cancers.Metastasis is an essential feature of gastric cancer.However,its underlying mechanism remains unknown.Therefore,the need of the hour is to identify new molecular markers of gastric cancer,investigate its different underlying molecular mechanisms,and develop therapies to suppress advanced disease progression.ARFGAP with SH3 domain,Ankyrin repeat,and PH domain 2(ASAP2),also known as DDEF2,is a member of the ARF GTPase-activating protein(ARFGAP)family.ASAP2 comprises BAR domain,PH domain,ARFGAP domain,Ankyrin repeats,and SH3 domain.It is an ADP-ribosylation factor GTPase-activating protein that activates the downstream proteins ADP-ribosylation factor 6(ARF6)and Rac family small GTPase 1(RAC1).ASAP2 co-localizes with ARF6 to regulate actin aggregation and the remodeling of the plasma membrane.Besides,ASAP2 co-localizes with PYK2 in the Golgi apparatus and plasma membrane and can be tyrosine phosphorylated by the activated PYK2,which further controls ARF-mediated vesicle budding.In addition,ASAP2 involves in phagocytosis in mouse macrophage cell line P388D1,and enhances macrophage efferocytosis in THP-1-derived M2 macrophage.Recent studies have revealed that ASAP2 is also involved in cancer development.ASAP2 has been reported to promote cell migration in pancreatic cancer cell lines Pancl and MiaPaCa2,and to promote cell cycle and cell proliferation in pancreatic cancer by phosphorylating epidermal growth factor receptors(EGFR).However,the role and mechanism of ASAP2 in GC have not been reported.The aim of this study is to explore the role of ASAP2 in GC using bioinformatics,clinical tissue samples and molecular biology experiments to expand the understanding of GC occurrence,development and treatment strategies.Methods1)The expression of ASAP2 in gastric cancer was clarified by analyzing the Cancer Genome Atlas database and detecting 16 cases of gastric and normal tissues by immunohistochemistry and western blotting.2)The metastasis of gastric cancer cells was examined by Transwell assay and wound healing assay after knockdown of ASAP2.3)Mitochondrial function of gastric cancer cells was assessed by measuring oxygen consumption rate and reactive oxygen species after knockdown of ASAP2.4)Immunoprecipitation assays were performed to obtain proteins that bind to ASAP2,which were then identified by mass spectrometry,combined with the GEPIA online tool to find ASAP2-interacting proteins.5)The effect of knockdown of ASAP2 on the growth of gastric cancer cells was assessed in xenograft mouse models in vivo.Results1)ASAP2 is upregulated in human GC.2)ASAP2 promotes migration and invasion of GC cells potentially by promoting EMT.3)ASAP2 attenuates mitochondrial OXPHOS and ROS production of GC cells.4)ASAP2 interacts with ARHGAP21 in mitochondria to regulate its function.5)Knockdown of ASAP2 reduces GC growth in a xenograft mouse model.ConclusionsASAP2 promotes migration and invasion of GC cells by facilitating EMT and potentially promoting xenograft tumor growth of GC cells by inhibiting the mitochondrial functions,including OXPHOS and ROS production,which may serve as a druggable therapeutic target for GC.