| BackgroundGastric cancer(GC)is a multifunctional disease,which is the fourth leading cause of cancer deaths worldwide,and is characterized by asymptomatic early stage.The incidence of GC has decreased since the 1970 s due to medical advances,but it remains a major health concern,especially in East Asian countries,with more than 1million cases reported in 2020.GC is an aggressive malignancy characterized by uncontrolled cell proliferation and metastasis.Yes-associated protein(YAP)and transcription coactivator with PDZ-binding motif(TAZ)are similar proteins that act as transcription coactivators and regulate a variety of cellular processes critical for the progression and metastasis of solid tumors.Increased YAP/TAZ expression and activation have been detected in a variety of solid tumors,including glioblastoma,metastatic breast cancer,and lung cancer cell subtypes.The mechanisms for regulating YAP/TAZ activity are diverse,including inhibition of Hippo pathway upstream mammalian sterility 20-like kinase 1 and 2(MST1/2)and large tumor suppressor homolog 1 and 2(LATS1/2)protein kinases,and non-Hippo-dependent signaling pathways activated by oncogenic protein tyrosine kinases,G-protein coupled receptors(GPCRs),adhesion receptors,a variety of cellular stress signals,and mechanical forces.YAP activity is regulated by its subcellular localization,where cytoplasmic YAP is recruited into destructive complexes for ubiquitination and degradation,while nuclear YAP interacts with TEAD DNA-binding protein to induce gene transcription.Previous studies have shown that the nuclear-cytoplasmic translocation of YAP depends on its phosphorylation status.Specifically,YAP S127 and S397 sites act as negative regulators of YAP nuclear localization,where phosphorylation of S127 residue(p-YAP S127)induces YAP degradation and inhibits its nuclear translocation.Therefore,YAP/TAZ expression is an important downstream target in gastric cancer and other gastrointestinal tumors.Several studies have shown that FAK facilitates YAP transport into the nucleus and triggers its complete activation.However,the underlying molecular mechanisms of FAK and YAP in gastric cancer remain unclear.Focal adhesion kinase(FAK),also known as protein tyrosine kinase 2(PTK2),is a non-receptor intracellular tyrosine kinase and an important member of integrin-mediated signaling pathways.FAK promotes tumor progression,metastasis,and activation of phosphatidylinositol 3-kinase(PI3K)/Akt,p53,and extracellular regulated kinase(ERK)by acting on cancer cells and stromal cells in the tumor microenvironment.In addition,a variety of FAK inhibitors have been tested in clinical trials,and preliminary results show that their respective drugs have cytotoxic effects.Previous studies have confirmed the involvement of FAK in the occurrence of gastric cancer.However,the mechanism of FAK in gastric cancer is currently lack of research.High mobility group protein B1(HMGB1)is highly expressed in gastric cancer tissues and cells,and its overexpression can promote the proliferation of gastric cancer cells.As a cancer cell growth factor,HMGB1 can activate MAPK or PI3K/AKT signaling pathway through RAGE receptor to promote cell proliferation.In addition,HMGB1 can regulate FAK-activated NF-κB signaling pathway to inhibit cancer occurrence.However,the potential molecules of HMGB1 and FAK in gastric cancer have not been reported.The focus of this study was to investigate the role of YAP in regulating the proliferation and migration of gastric cancer cells.The expression level of YAP in clinical tumor specimens was determined by TCGA database.Through in vitro experiments,it was observed that YAP significantly enhanced the proliferation,migration and FAK activation of gastric cancer cells.Furthermore,the importance of YAP-TEAD interaction for these biological effects was confirmed.Subsequently,this study further identified high mobility group protein 1(HMGB1),a previously reported FAK stimulating factor,as a direct transcriptional target of YAP signaling.Furthermore,the results of this study showed that YAP/TEAD up-regulated HMGB1 expression induced FAK phosphorylation and activation of tumor cell proliferation and migration.In summary,the results of this study reveal the function of YAP-HMGB1-FAK axis in regulating the proliferation and migration of gastric cancer cells.Methods1.The pan-cancer level of YAP was detected by TIMER database.The mRNA level of YAP in normal gastric tissues and gastric cancer tissues was detected by GEPIA and UCLAN databases.2.The protein level of YAP in gastric mucosal epithelial cells(GES-1)and gastric cancer cells(MKN28,AGS,MKN45,HGC27,BGC-823,MGC-803)was detected by Western blot.The transcription level of YAP in gastric mucosal epithelial cells and gastric cancer cells was detected by qRT-PCR.3.The plasmids of YAP knockdown(sh YAP-2,3,4)and overexpression(pc DNA3.1-Flag-YAP)were constructed.The plasmid knockdown and overexpression efficiency were detected by Western blot and qRT-PCR.4.The effects of YAP on the viability,proliferation ability and migration level of gastric cancer cells were detected.The plasmids of YAP knockdown(sh YAP-2,3,4)and overexpression(pc DNA3.1-Flag-YAP)were transfected into AGS and MKN28cells;CCK8,colony formation and Transwell experiments were used to detect the viability,proliferation ability and migration level of AGS and MKN28 cells,respectively.5.The effects of YAP on the phosphorylation level of FAK in gastric cancer cells were detected.The plasmids of YAP knockdown(sh YAP-2,3,4)and overexpression(pc DNA3.1-Flag-YAP)were transfected into AGS and MKN28 cells.The protein levels of FAK and phosphorylated FAK were detected by Western blot.6.The effects of YAP-TEAD interaction on the phosphorylation level of FAK in gastric cancer cells were detected.(1)Verteporfin,a YAP inhibitor,was used to block the YAP-TEAD interaction in gastric cancer cells.The protein levels of YAP,FAK and p-FAK in gastric cancer cells were detected by Western blot.(2)Verteporfin was used to treat gastric cancer cells overexpressing pc DNA3.1-Flag(3)Gastric cancer cells were overexpressed with YAP mutant plasmids(pc DNA3.1-Flag-YAPS127A(a transcriptionally active mutation form of YAP)and pc DNA3.1-Flag-YAPS94A(TEAD binding site mutation)),and the protein levels of Flag-tag,FAK and p-FAK in gastric cancer cells were detected by Western blotting.7.The effects of FAK and FAK inhibitors on the proliferation and migration of gastric cancer cells were detected.Gastric cancer cells were transfected with pc DNA3.1-Flag-FAK,si-FAK-1,2 or pretreated with FAK inhibitors(VS-6063 and PF573228).CCK8,colony formation and Transwell experiments were used to detect the activity,proliferation ability and migration level of AGS and MKN28 cells,respectively.8.The effects of FAK on the subcellular localization of YAP protein in gastric cancer cells were detected.AGS and MKN28 cells were transfected with si-FAK-1,2 and pc DNA3.1-Flag-FAK,respectively;the localization of YAP protein in AGS cells was detected by immunofluorescence assay;the protein expression of YAP in nuclear components and the protein level of p-YAP in cytoplasmic protein were detected by Western blotting;the mRNA expression of YAP target genes CTGF and CYR61 were detected by qRT-PCR.9.The effects of FAK inhibitors on the subcellular localization of YAP protein in gastric cancer cells were detected.FAK inhibitors VS-6063 and PF573228 were used to treat AGS and MKN28 cells.The localization of YAP protein in AGS cells was detected by immunofluorescence assay;the protein expression of YAP in nuclear components and the protein level of p-YAP in cytoplasmic protein were detected by Western blotting;the mRNA expression of YAP target genes CTGF and CYR61 were detected by qRT-PCR.10.The phosphorylation of FAK was detected by regulating HMGB1.(1)After transfection of sh YAP-2,3,4 and pc DNA3.1-Flag-YAP into AGS and MKN28 cells,the expression of HMGB1 protein and mRNA was detected by Western blot and qRT-PCR,respectively.(2)After treatment of Verteporfin into AGS and MKN28 cells,the expression of HMGB1 protein was detected by Western blot.(3)After transfection of YAPS127 A and YAPS94 A into AGS and MKN28 cells,the expression of HMGB1 protein and mRNA was detected by Western blot and qRT-PCR,respectively.(4)After AGS and MKN28 cells were transfected with sh YAP and Flag-HMGB1,the protein levels of Flag-tag,FAK and p-FAK in gastric cancer cells were detected by Western blotting.11.Luciferase report experiments to detect the effects of YAP and its mutants on the activity of HMGB1 promoter.(1)HEK293T,AGS and YAP-deficient cell line MKN45 were transfected with YAP overexpression plasmids to detect the effects of YAP on the activity of HMGB1promoter;(2)HEK293T,AGS and MKN45 were transfected with YAP mutant plasmids YAPS127 and YAPS94 A to detect the effects of YAP mutants on the activity of HMGB1 promoter;12.To detect whether YAP regulates the proliferation and migration of gastric cancer cells by regulating HMGB1/FAK axis.After transfection of sh YAP and pc DNA3.1-Flag-HMGB1 into AGS and MKN28 cells,the activity,proliferation ability and migration level of AGS and MKN28 cells were detected by CCK8,colony formation and Transwell experiments,respectively.Results1.The results of TIMER database analysis showed that YAP was abnormally expressed in a variety of cancers.In addition,the results of GEPIA and UCLAN database analysis showed that compared with normal gastric tissues,the mRNA level of YAP was significantly up-regulated in gastric cancer tissues.2.The results of Western blot and qRT-PCR showed that compared with gastric mucosal epithelial cell GES-1,the protein and mRNA levels of YAP were significantly up-regulated in gastric cancer cells.3.The results of Western blot and qRT-PCR showed that YAP knockdown/overexpression significantly down-regulated/up-regulated the protein and mRNA levels of YAP.4.The results of CCK8 experiment showed that compared with the control group,knockdown/overexpression of YAP up-regulated/inhibited the viability of gastric cancer cells.The results of colony formation experiment showed that knockdown/overexpression of YAP promoted/inhibited the proliferation of gastric cancer cells.The results of Transwell experiment showed that knockdown/overexpression of YAP promoted/inhibited the migration ability of gastric cancer cells.5.The results of Western blot showed that knockdown of YAP significantly down-regulated the p-FAK protein level;overexpression of YAP significantly up-regulated the p-FAK protein level;the total FAK protein level had no significant change.6.The results of Western blot showed that Verteporfin treatment significantly reduced the protein levels of YAP and p-FAK in gastric cancer cells,and the total FAK protein level had no significant change.In addition,Verteporfin treatment significantly reduced the promoting effect of overexpression of YAP on the expression of p-FAK protein.Compared with the control group,the YAP mutant S127 A significantly up-regulated the p-FAK protein level,while the mutant S94 A had no significant effect.7.Silencing FAK or FAK inhibitor significantly reduced the viability,proliferation and migration of gastric cancer cells.Overexpression of FAK resulted in the opposite.8.Immunofluorescence results showed that compared with the control group,the cytoplasmic localization of YAP protein in AGS cells was reduced after overexpression of FAK;Western blot results showed that the protein level of YAP in nuclear protein was significantly increased,and the protein level of p-YAP in cytoplasmic protein was significantly decreased;qRT-PCR results showed that the mRNA expression of YAP target gene CTGF and CYR61 was significantly increased.9.Immunofluorescence results showed that compared with the control group,the FAK inhibitors VS-6063 and PF573228 could significantly reduce the nuclear aggregation of YAP protein in AGS cells;qRT-PCR results showed that the mRNA expression of YAP target gene CTGF and CYR61 was significantly decreased.10.(1)Western blot results showed that knockdown/overexpression of YAP decreased/increased HMGB1 protein and mRNA expression respectively;(2)Verteporfin treatment significantly reduced HMGB1 protein expression;(3)YAPS127A translocation upregulated HMGB1 protein and mRNA expression;however,translocation of YAPS94 A did not change significantly;(4)Knockdown of YAP significantly downregulated FAK phosphorylation,whereas overexpression of HMGB1 reversed the inhibitory effect of YAP knockdown on FAK phosphorylation.11.(1)Overexpression of YAP significantly increased the HMGB1 promoter activity.(2)Overexpression of YAP mutant significantly increased the HMGB1 promoter activity.12.The results showed that overexpression of HMGB1 partially reversed the inhibition of YAP on the proliferation and migration of gastric cancer cells.Conclusion1.YAP is abnormally expressed in a variety of cancers;Compared with normal gastric tissues and cells,YAP is significantly upregulated in gastric cancer tissues and cells.In addition,the expression level of YAP is positively correlated with the prognosis of patients.Knockdown of YAP significantly reduces the proliferation and migration ability of gastric cancer cells.2.In gastric cancer cells,YAP-TEAD interaction regulates the phosphorylation level of FAK.3.In gastric cancer cells,YAP regulates the phosphorylation level of FAK through HMGB1,and then regulates the proliferation and migration ability of gastric cancer cells.4.YAP and its mutant YAPS127 A can significantly increase the activity of HMGB1promoter;HMGB1 is a downstream target gene of YAP.5.Silencing FAK or using FAK inhibitors can inhibit the nucleus aggregation of YAP,forming a positive feedback loop of FAK-YAP. |
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