Deubiquitinating Enzyme USP46 Suppresses The Progression Of Hepatocellular Carcinoma By Stabilizing MST1

Background and Objective:Hepatocellular carcinoma(hereinafter referred to as HCC)is a primary liver tumor with a high incidence and great harm.Liver cancer has the eighth morbidity rate and the second highest mortality rate in the world.The number of deaths due to liver cancer each year has reached 780,000 and is increasing year by year.Due to the high rate of hepatitis B virus infection and other reasons,our country is the country with the highest incidence of liver cancer.At least 400,000 people die from liver cancer each year,accounting for more than half of the world's liver cancer deaths.Most patients with liver cancer have lost the opportunity of radical surgical resection at the time of diagnosis.This is mainly because HCC is prone to invasive growth and early metastasis.Therefore,unraveling the molecular mechanism of the rapid proliferation and metastasis of HCC has become an urgent problem which need to be solved.Ubiquitin-specific protease 46(USP46)is a newly discovered deubiquitinating enzyme in the deubiquitinase family,and its role in cancers has attracted more and more attention.Studies have confirmed that the expression of USP46 in tumor tissues of kidney cancer,lung cancer,and colon cancer is lower than that of normal adjacent tissues.USP46 inhibits tumor growth by inhibiting the proliferation of cancer cells,but in HPV-transformed cancers,USP46 promotes cell proliferation and tumor growth.However,the expression of USP46 in liver cancer and its influence on the progression of liver cancer are still unclear.The transcriptional co-activator YAP is a transcriptional regulator commonly activated in malignant tumors and is closely related to the occurrence and development of liver malignant tumors.YAP is the core transcription factor in the Hippo signaling pathway,and its protein stability and function are controlled by the upstream core kinases MST1/2 and LATS1/2.The extracellular Hippo regulatory signal activates MST1/2 in the cell through transmembrane transduction,and then,MST1/2 activates LATS1/2 to promote the phosphorylation modification of YAP.Phosphorylated YAP remains in the cytoplasm and is degraded by the ubiquitin-proteasome system(UPS).In liver cancer,the Hippo signaling pathway is inhibited,and its core kinase MST1 is low expressed in tumor tissues and inhibits the proliferation and metastasis of liver cancer cells.Based on this,we speculate that USP46 may affect the proliferation and metastasis of liver cancer cells by regulating the activation of MST1 mediated Hippo signaling pathway.In order to verify our hypothesis,first of all,we detected the expression level of USP46 in the tumor tissues and corresponding adjacent tissues of liver cancer patients,and analyzed the relationship between the expression level of USP46 in HCC tissues and the patient's clinical pathological parameters and prognosis.Secondly,using in vitro cell experiments and in vivo animal experiments to clarify the role of USP46 in the proliferation and metastasis of HCC cells.Third,explore the impact of USP46 on the Hippo signaling pathway,and prove whether USP46 affects the proliferation and metastasis of HCC cells by regulating YAP1.Finally,clarify whether USP46 affects the Hippo signaling pathway by regulating the expression of MST1,and clarify the specific molecular mechanism of USP46 regulating MST1.Methods:1.Western blotting and immunohistochemistry(IHC)were used to detect the expression of USP46 in 102 cases of surgically resected liver cancer tissues and their adjacent tissues.Medical records of clinical HCC patients were collected,clinical outcomes were followed up,and the correlation between the expression level of USP46 in HCC tissues and pathological characteristics and outcomes of clinical HCC patients was retrospectively analyzed.2.Sh RNA lentiviral plasmids targeting USP46 was used to construct HCC cell lines that stably reduce USP46,or using overexpression lentiviral plasmids of USP46 to construct HCC cell lines that stably upregulate USP46.CCK-8 assay,cell proliferation assay(Ed U),plate cloning assay and subcutaneous tumor formation model in nude mice were used to detect the effect of up-regulation/down-regulation of USP46 expression on HCC cell proliferation.Transwell invasion assay and orthotopic liver transplantation model of nude mice were used to analyze the effect of changing the expression of USP46 on the invasion and metastasis of HCC cells.3.Using Isobaric Tags for Relative and Absolute Quantitation(i TRAQ)methods to screen whether USP46 affects Hippo signaling pathway.Western blotting was used to detect the expression changes of YAP1 in the down-regulated or up-regulated USP46 cells.Restoring YAP1's expression in cells that up-regulate the expression of USP46,using cell proliferation test(Ed U)and nude mouse subcutaneous tumor model to analyze the changes of HCC cells' proliferation in each group,transwell invasion test and nude mouse liver orthotopic xenograft model were used to analyze the changes of HCC cells' invasion and metastasis in each group.Through these experiments,our purpose is to explore whether USP46 affects the proliferation and metastasis of HCC cells by regulating the expression of YAP1.4.The binding of USP46 to YAP1 was detected by immunoprecipitation and Western blotting.In down-regulated or up-regulated USP46 cells,q RT-PCR and Western blotting were used to detect the expression changes of MST1.Transfecting MST1 overexpression plasmid into HCC cells that with stable downregulation of USP46 to increase the expression of MST1,or transfecting sh RNA targeting MST1 into HCC cells that with stable up-regulation of USP46 to reduce the expression of MST1.Western blotting was used to detect the changes in the expression of YAP1.At the same time,cell proliferation test(Ed U)and transwell invasion test were used to detect changes in the proliferation and metastasis ability of HCC cells.5.Co-immunoprecipitation and western blotting were used to detect the binding of USP46 and MST1.In HCC cells that up-regulated the expression of USP46,cycloheximide(CHX)was used to block protein synthesis,and then comparing MST1 protein degradation rate between control group and USP46-overexpressing group.In HCC cells that downregulated the expression of USP46,the proteasome inhibitor MG132 was added to block protein degradation,and then comparing the changes of MST1 protein between control group and USP46-silenced group.The expression of USP46 was up-regulated or down-regulated in HCC cells,and the ubiquitination level of MST1 was detected by in vivo ubiquitination experiments.Results:1.The results of immunohistochemistry and western blotting showed that the expression of USP46 in HCC tissues was lower than that in adjacent tissues.In addition,low expression of USP46 was associated with clinical features of HCC,including larger tumor size,advanced TNM stage,more venous invasion,and tumor microsatellite formation.Survival analysis showed that the overall survival rate of HCC patients with low USP46 expression in tumor tissues was significantly lower than that of HCC patients with high USP46 expression in tumor tissues.2.In vitro cell tests,we found that up-regulation of USP46 expression may repress the proliferation and invasion capacity of HCC cells,while down-regulation of USP46 can promote the proliferation and invasion of HCC cells.In vivo animal experiments also further confirmed that up-regulation of USP46 can inhibit the growth and metastasis of HCC,while down-regulation of USP46 can promote the growth and metastasis of HCC.3.We used i TRAQ to explore the changes in the proteome of HCC cells when USP46 was downregulated.KEGG analysis showed that the Hippo signaling pathway changes most significantly in HCC cells that down-regulated USP46.The results of Western blotting further confirmed that down-regulation of USP46 can increase the expression of YAP1,while over-expression of USP46 can decrease the expression of YAP1.The results of the recuse experiment showed that overexpression of YAP1 can restore the reduction of HCC cells' proliferation and invasion caused by up-regulation of USP46.4.Co-immunoprecipitation results show that USP46 does not directly bind to YAP1.However,the protein expression of MST1,a upstream kinase of YAP1,is regulated by USP46.When MST1 overexpression plasmid was transfected into HCC cells that with stable downregulation of USP46 to increase the expression of MST1,the expression of YAP1 was decreased,which in turn driven to a diminish in the proliferation and invasion capacity of HCC cells.Transfecting MST1 targeting sh RNA into HCC cells that with stable up-regulation of USP46 to reduce the expression of MST1 can cause an increase of YAP1's expression,which in turn to increase HCC cells' proliferation and invasion.5.The result of co-immunoprecipitation confirms that USP46 binds to MST1 directly.Overexpression of USP46 can reduce the degradation rate of MST1,thereby increasing the stability of MST1 protein.The proteasome inhibitor MG132 can block the regulatory effect of USP46 on MST1,suggesting that USP46 affects the degradation of MST1 protein.In vivo ubiquitination experiments further confirmed that USP46 inhibits the ubiquitination level of MST1.Conclusions:1.USP46 is low expressed in HCC and is related to the prognosis of HCC patients.2.USP46 inhibits the proliferation and metastasis of HCC cells.3.USP46 suppresses the progression of HCC by inhibiting YAP1 expression.4.USP46 maintains the activation of Hippo pathway by increasing the expression of MST1 protein in HCC cells.5.USP46 stabilizes MST1 protein by inhibiting its ubiquitination.