Sitagliptin Affects Gastric Cancer Cells Proliferation By Suppressing Melanoma-associated Antigen-A3(MAGE-A3) Expression Through Yes-associated Protein(YAP) Inactivation

Objective: Diabetes and tumors are complex and prevalent health problems worldwide,and are associated with severe acute and chronic complications.In 2030,the prevalence of diabetes among adults worldwide will increase to 7.7%,and will continue to increase in the next few decades.Cancer is the second leading cause of death worldwide,with approximately 8–18% of cancer patients suffering from diabetes.Since some scholars in the 1930 s suggested that diabetes patients may have a significantly increased risk of developing tumors due to glucose metabolism disorders and insulin resistance,this field has attracted the attention of researchers.In the study of gastric cancer,it was found that diabetes not only has common risk factors with gastric cancer,but also can be used as an independent risk factor for gastric cancer,affecting the prognosis of gastric cancer.Therefore,in-depth discussion of the anti-tumor mechanism of hypoglycemic drugs is of great significance for patients with diabetes complicated with tumors.Sitagliptin,as a new oral hypoglycemic agent,has been widely used in many countries and regions.The drug mainly inhibits the inactivation of glucagon-like peptide-1 by selectively inhibiting the activity of dipeptidyl peptidase-4(DPP-4),and improves the endogenous glucagon-like peptide-1 level to lower blood sugar.At present,the interaction between sitagliptin and tumors is still under study.Epidemiological evidence has shown that sitagliptin can reduce the risk of colorectal cancer,breast cancer,oral cancer,prostate cancer,etc.However,the specific mechanism is that sitagliptin is used as a DPP-4 inhibitor,and inhibition of DPP-4 inhibits angiogenesis and epithelial-mesenchymal transition.Other related mechanisms are rarely reported.Moreover,little is known about the effect of sitagliptin on gastric cancer.Therefore,we hope to explore the effect of sitagliptin on gastric cancer and its molecular mechanism.Gene sequencing results show that there are many pathway mutations in the development of gastric cancer,and the Hippo pathway is one of these mutations.The Hippo pathway is a highly conserved growth control signaling pathway in mammals,and its abnormal activation is closely related to the malignant transformation of gastric cancer.The Hippo pathway consists of a series of tandem kinases.The core of this pathway is its lowest downstream co-transcription factor,Yes-associated protein(YAP).The activation of YAP protein is of great significance for cell proliferation and invasion.Genetic studies have confirmed that if YAP is phosphorylated,it stays in the cytoplasm and cannot enter the nucleus,while YAP that is not phosphorylated can enter the nucleus as a co-transcription and therefore exert its cancer-promoting function.Current research on the Hippo pathway has shown that many pathways can affect and alter YAP activity.In the case of energy stress,AMP-activated protein kinase(AMPK)can directly or indirectly phosphorylate YAP,and this regulation is related to cell proliferation and tumor resistance.More importantly,sitagliptin has been reported to affect inflammation,autophagy,and metabolism by regulating AMPK.These mechanisms suggest that AMPK / YAP may be a new target for sitagliptin to inhibit tumor growth.In the study of downstream effectors of YAP,we focused on a family of testis-specific antigens that are specifically overexpressed in tumors and hardly expressed in normal tissues.And in this family,melanoma-associated antigen-A3(MAGE-A3)was found.MAGE-A3 is scarcely expressed in normal tissues,but specifically and highly expressed in gastric cancer tissues.It is expected to become a new tumor marker for early diagnosis of gastric cancer.If sitagliptin can act on MAGE-A3,it will be new options for the treatment of gastric cancer Therefore,we hope that this study will determine whether sitagliptin can inhibit gastric cancer proliferation.If it is effective,then explore the mechanism of sitagliptin inhibiting gastric cancer cell proliferation through in vitro experiments,and provide new therapeutic targets for gastric cancer treatment.Research methods: Human gastric cancer cells(AGS,HGC-27 and MKN45)were cultured in vitro,and treated with different concentrations of sitagliptin.CCK8 and colony formation assay were used to determine whether sitagliptin could inhibit the proliferation of gastric cancer cells.Then,on the basis of clarifying the effect of sitagliptin on gastric cancer cells,the experimental methods of immunofluorescence and nuclear plasma separation were used to detect the changes of nuclear YAP expression after sitagliptin treatment.After that,the method was tested by Western Blot in the expression of related pathway proteins(AMPK,P-AMPK,LATS,P-LATS,YAP,P-YAP,MAGE-A3),and used inhibitors and transfection methods to improve the recovery experiment,so that the upstream and downstream relationships between the molecules were more precise.When the effect of sitagliptin on various pathway molecules was clarified,the methods of immunohistochemistry,Kaplan-Meier plotter survival curve,and knockdown of MAGE-A3 in vitro were used to determine the relationship between MAGE-A3 and gastric cancer cell proliferation and prognosis.Finally,the molecular mechanism of sitagliptin inhibiting the proliferation of gastric cancer cells was explored.Results: The experimental results of CCK8 and colony formation assay showed that sitagliptin had an inhibitory effect on the proliferation and cloning ability of gastric cancer cells,and this inhibitory effect was concentration-dependent.Explore the molecular mechanism of sitagliptin on the premise of inhibiting the proliferation of gastric cancer cells.First,the results of immunofluorescence and nuclear plasma separation showed that after treatment with sitagliptin,the expression of YAP in the nucleus was reduced.In addition,two known downstream effector genes of YAP were also reduced,which reflected to some extent that sitagliptin could inhibit the expression of nuclear YAP.Afterwards,Western Blot were used to detect the phosphorylation levels of Hippo pathway-related proteins.The results showed that AGS,HGC-27 and MKN45 all had peak phosphorylation levels,and sitagliptin had a concentrationdependent activation of YAP phosphorylation.Since sitagliptin could activate the Hippo pathway,the AMPK pathway was futher teated.The results showed that sitagliptin could activate AMPK,and the phosphorylated AMPK peaks of the three gastric cancer cell lines were all within 15 minutes.At the same time,10 ?m AMPK inhibitor—compound C,were pretreated to the cells for 2 hours to inhibit AMPK activity,and then tested the phosphorylation level of Hippo pathway-related proteins.The results showed that the AMPK inhibitor could block the activation of Hippo pathway by sitagliptin.Finally,the utility gene of YAP after nuclear entry was determined.Bioinformatics methods showed that MAGE-A3 had the best correlation with YAP compared to several other representative cancer-testis specific antigens that were highly expressed in gastric cancer.Therefore,MAGE-A3 was selected for subsequent experiments.First,the treatment of sitagliptin could decrease the mRNA and protein levels of MAGE-A3;secondly,the transfection of YAP 5SA mutant plasmids allowed YAP to continue to enter the nucleus,which not only promoted the proliferation of gastric cancer cells,but also restored the effect of sitagliptin on MAGEA3 expression.YAP knockdown could also reverse the effect,which further confirmed the regulation of YAP on MAGE-A3.In the study of MAGE-A3 and gastric cancer prognosis,immunohistochemistry results showed: MAGE-A3 was closely related to gastric cancer differentiation and lymphatic metastasis,but was not related to age,tumor size,Her2 or Lauren.Kaplan-Meier plotter survival curve also supported that the survival rate of patients with high expression of MAGE-A3 was significantly lower than that of patients with low expression of MAGE-A3.Selected the gastric cancer cell line HGC-27 with relatively high expression of MAGE-A3 for MAGE-A3 knockdown in vitro.The results of CCK8 and clones showed that MAGE-A3 was closely related to the proliferation of gastric cancer.Conclusion: Research shows that sitagliptin can inhibit the proliferation of gastric cancer cells.This effect may be due to AMPK activation.Subsequently,activated AMPK promotes the phosphorylation of YAP,thereby inhibiting YAP entering into the nucleus.This can prevent the expression of gastric cancer prognostic factor MAGE-A3 at the transcription level and improve the prognosis of gastric cancer.Taken together,these results illustrate the potential anticancer mechanism of sitagliptin in the treatment of gastric cancer.However,the existence of other anti-tumor mechanisms of sitagliptin and its safety in human use need further study.