Mechanism Study Of DUB1 In Regulating Gastric Cancer Progression By Hippo Signaling

According to the 2020 global epidemiological report,more than 1 million new confirmed cases of gastric cancer were reported,while more than half of the cases occurred in China.The carcinogenic process of gastric cancer is a pathological process including gastritis,intestinal metaplasia,and epithelial neoplasia.Due to insidious onset and mild symptoms,most cancer patients are already advanced patients.Its 5-year survival rate is only 35%.Several biomedical studies have shown that H.pylori infection and chronic inflammation participate in gastric cancer development,but the mechanisms of gastric cancer progression remain unclear.Therefore,it is urgent and necessary to develop novel biomarkers and therapeutic targets for gastric cancer.Several studies have identified potential links between the Hippo signaling pathway and gastric cancer.The Hippo signaling pathway plays an important role in several biological processes,including organ size control,tissue homeostasis,carcinogenesis,and the immune response.Activation of Hippo signaling is affected by a phosphorylation cascade,where the mammalian Hippo kinase MST1/2 phosphorylates the LATS1/2 kinase,while the phosphorylated LATS1/2 promotes the phosphorylation of the YAP/TAZ proteins,leading to cytoplasmic localization and protein degradation.If the Hippo signaling pathway is inactivated,the YAP/TAZ becomes unphosphorylated,translocated into the nucleus,and interacts with multiple transcription factors to promote the expression of its target genes.In a H.pylori-induced mouse gastric cancer model,YAP/TAZ overexpression inhibited the progression of gastric cancer.However,despite substantial efforts to develop drugs targeting Hippo signaling,their clinical application is still in an immature stage.TAZ(transcriptional coactivator with PDZ-binding motifs)is a downstream effector protein of Hippo signaling,and is a transcription co-factor of multiple transcription factors,including TEAD.Several studies have suggested that TAZ may be a key factor in carcinogenesis in human cancer.In human cancer samples,TAZ protein levels were increased in breast cancer and gastric cancer,and their expression levels were correlated with a poor prognosis.Thus,targeting TAZ proteins can serve as candidate components of the ubiquitin-proteasome system and can be used to rescue the tumor suppressor function of the Hippo signaling pathway and the progression of gastric cancer progression.Regulation of protein stability is influenced by the balance between the E3 ubiquitin ligase and the deubiquitinase,which is critical for maintaining a normal physiological homeostasis.Disruption of the regulation of protein ubiquitination may lead to multiple human diseases,including cancer.We and other groups have identified several E3 ubiquitin ligase s acting on Hippo pathway effectors,regulating Hippo signaling activity and cancer progression.Although several studies have reported that several deubiquitinases are involved in regulating Hippo signaling,it remains unclear which deubiquitinase produces important effects.Using Deubiquitinating enzymes(DUBs)siRNA screening libraries,we found that DUB1 is a key regulator of Hippo pathway activity in GC patients,while DUB1 was elevated in gastric cancer samples,and the poor prognosis was associated with TAZ expression.However,the function of DUB1 in gastric cancer is not clear,so further exploration of DUB1 function of gastric cancer has an important role and clinical significance,and can provide a new direction for seeking possible therapeutic targets.ObjectiveTo screen the deubiquitinating enzymes that regulate Hippo pathway in gastric cancer;To prove the regulation mechanism of DUB1 protein in Hippo pathway in gastric cancer;It provides a new potential target and strategy for clinical treatment of gastric cancer.Methods1.Screening the interfering RNA library of deubiquitination enzymes to explore the deubiquitination enzymes that can regulate the Hippo pathway.2.The expression of DUB1 and TAZ in gastric cancer tissues and their prognostic relationship were verified through bioinformatics and IH.3.RNA sequencing and data analysis were used to analyze the changes of gastric cancer signaling pathway after DUB1 silencing.4.The effects of DUB1 on the proliferation,invasion and migration of gastric cancer were verified by CCK8,trans-well assay,clone formation assay,EdU assay and mouse tumor transplantation assay.5.Through rescue experiment,TAZ was overexpressed in silence DUB1 gastric cancer cells to observe the changes of Hippo downstream target genes and the effects of gastric cancer cell proliferation,migration and invasion.6.Fluorescent reporter assay was used to detect the activity of TEAD reaction elements and determine the effect of DUB1 on the activity of Hippo signaling pathway.7.The localization and interaction of DUB 1 and TAZ were verified by IF assay and CO-IP assay.The plasmids with different domains of DUB1 and TAZ proteins were constructed,and the specific regions of mutual binding were detected.8.Western blot was used to analyze the deubiquitination modification of TAZ by DUB1 through MG132 experiment,CHX experiment,ubiquitination experiment and CO-IP experiment.Results:1.DUB1 expression is elevated in human gastric cancer,and is associated with the whole-genome-wide Hippo signaling activity.By screening DUBs SiRNA libraries,we found that DUB1 significantly modulates the downstream target gene CTGF of YAP/TAZ.Bioinformatics analysis and immunohistochemistry of tissue samples suggested that DUB1 expression was elevated in human gastric cancer and correlated with prognosis.RNA-seq sequencing by silencing DUB1 suggested that DUB1 was associated with Hippo signaling activity in the whole genome of gastric cancer.2.DUB1 promotes the Hippo/TAZ axis signaling pathway in gastric cancer cells.Western blot and QPCR showed that the expression of YAP in gastric cancer cells did not change significantly after DUB1 silencing,while TAZ decreased significantly.Luciferase reporter assay indicated that DUB1 could directly affect the transcriptional activity of TEAD downstream of TAZ.3.DUB1 depletion inhibited the progression of gastric cancer in vitro and in vivo.In vitro experiments confirmed that knockdown of DUB1 could significantly inhibit cell proliferation and clone formation ability,and in vivo cell transplantation tumor showed that knockdown of DUB1 could significantly inhibit the growth of transplanted tumor.4.DUB 1 controls the progression of gastric cancer through the Hippo/TAZ axis.By Rscure assay,TAZ was overexpressed in DUB1 silenced gastric cancer cell line,and Hippo downstream target genes were observed to increase,and the proliferation,migration and invasion functions of gastric cancer cells were restored,which confirmed that DUB1 affected the progression of gastric cancer by regulating TAZ.5.DUB1 associates with TAZ and modulates TAZ stability in gastric cancer cells.Immunofluorescence and nucleoplasmic separation showed that DUB1 and TAZ were co-localized in the nucleus,and CO-IP showed that DUB1 and TAZ were bound.CHX and MG 132 experiments confirmed that DUB1 affected TAZ degradation through the ubiquitin-proteasome pathway.6.DUB1 stabilizes TAZ by inhibiting TAZ K48-linked polyubiquitination.In vivo and in vitro deubiquitination experiments showed that DUB1 directly acts on TAZ and then removes the ubiquitin chain on TAZ.CO-IP experiments with the K48 mutation showed that TAZ was stabilized mainly by inhibiting TAZ K48-linked polyubiquitin.Ubiquitination analysis was performed by mutants of TAZ.The data showed that K39 and K157 were the most likely sites for DUB1 deubiquitination in TAZ protein.Conclusion:DUB1 is an important regulator of Hippo signaling pathway in gastric cancer.In clinical sample analysis,the expression of DUB1 was significantly increased and was directly related to the prognosis of gastric cancer.In a gastric cancer cell model,DUB1 directly affects the progression of gastric cancer by directly inhibiting TAZ K48 polyubiquitin and degrading.In conclusion,in this study,a new deubiquitinase DUB1 was found to lead to the progression of gastric cancer by regulating Hippo/TAZ,and its specific regulatory binding site was also found,which provides a new target and new idea for the treatment of gastric cancer.