A Novel UBE2T Inhibitor Suppresses Wnt/?-Catenin Signaling Hyperactivation And Gastric Cancer Progression By Blocking RACK1 Ubiquitination

PART1: The role of Wnt / ?-catenin signaling pathway and its inhibitors in gastric cancerObjective: To investigate the roles of aberrant Wnt/?-catenin signaling pathway in GC and examine whether previously developed Wnt pathway inhibitors in preclinical or clinical trials possess inhibitory effects on GC cell growth.Methods: we performed data mining analysis to explore the m RNA levels of?-catenin in GC tissues by the Oncomine platform and Gene Expression Profiling Interactive Analysis(GEPIA).Immunohistochemistry(IHC)were performed to examine the expression difference of ?-catenin between GC and normal tissues.MTT assay were performed to assess the effects of nine inhibitors on GC and GES cell viability.Results: Five independent datasets from Oncomine platform consistently showed that the m RNA levels of ?-catenin in GC tissues were significantly higher than those in normal counterparts.This was further confirmed by Gene Expression Profiling Interactive Analysis(GEPIA).Immunohistochemistry(IHC)assay showed that ?-catenin protein level was significantly upregulated.MTT assays showed that three kinds of the inhibitors(NCB-0846,nicosamide,and PRI-724)have obvious cytotoxicity to GES-1,whereas the others(IWP-2,LF3,ethacrynic acid,ETC-159,LGK-974,and Wnt-C59)cannot effectively suppress HGC27,AGS,and MKN45 cells growth as well as have cytotoxicity to GES-1.Conclusion: Current Wnt pathway inhibitors being studied in preclinical or clinical settings for other cancers are either too cytotoxic or insufficiently efficacious for GC.PART2: Screening and validation of new targets based on the components of ?-catenin destruction complex in Wnt/?-catenin signaling for GCObjective: To investigate the mechanism of down-regulation of ?-catenin destruction complex key component RACK1 in gastric cancer.Methods: Immunohistochemistry(IHC)assays were performed to screen key component from the key components of ?-catenin destruction complex.Immunohistochemistry(IHC),q RT-PCR and West blotting assay were performed to examine the expression levels of RACK1 in GC tissues.The interacting proteins of RACK1 were identified by liquid chromatography–tandem mass spectrometry analysis.The interaction of UBE2 T with RACK1 was confirmed by Co-IP and microscale thermophoresis assays.UBE2T-mediated ubiquitination of RACK1 was performed by ubiquitination assay.Results: IHC and West blotting assays confirmed that the protein level of RACK1 is decreased in GC tissues and low RACK1 protein level correlates with reduced overall survival in GC patients,but q RT-PCR assays showed that RACK1 expression level was similar between GC tissues and paired paracarcinoma tissues.We identified a ubiquitin-conjugating enzyme UBE2 T,which interacts RACK1,by liquid chromatography–tandem mass spectrometry analysis.Co-IP and Microscale thermophoresis assays showed that RACK1 directly interacts with UBE2 T.Our data showed that UBE2 T promotes the ubiquitination of RACK1 by ubiquitination assay.Conclusion: RACK1 as a key component of ?-catenin destruction complex is modified by UPS in GC progressionPART3: The Ubiquitination of RACK1 by UBE2 T positively Regulates Wnt/?-catenin signaling pathwayObjective: To further explore the mechanism that UBE2 T ubiquitinated RACK1 to activate Wnt/?-catenin signaling pathway.Methods: Western blotting was adopted to detect the protein levels of RACK1 after transient transfection of RACK1 plasmids and different concentrations of UBE2 T plasmid.Western blotting assays were performed to detect the effects of UBE2 T knockout on the protein level of RACK1.The specific ubiquitin chain of RACK1 was detected by Immunoprecipitation assay.IHC assay was performed to investigate the expression correlation between UBE2 T and RACK1 and ?-catenin in GC tissues.The effects of UBE2 T C86 on the ubiquitin level of RACK1 were analyzed by Immunoprecipitation and Western bloting assay.In the absence of E3,the effects of UBE2 T on the ubiquitin level of RACK1 were performed in vitro ubiquitination assay.The truncated RACK1 plasmids were constructed by molecular cloning method.Specific lysine sites for ubiquitination of RACK1 catalyzed by UBE2 T were dentified by Co-Immunoprecipitation and Western blotting Assays.Western blotting assay was performed to examine the amount of cytoplasmic and nuclear ?-catenin after transient transfection of RACK1 mutant plasmids or UBE2TC86 mutant plasmids in HEK-293 T cells.Results: Western blotting assays showed that overexpression of UBE2 T leads to a significant dose-dependent reduction in total protein levels of RACK1 and UBE2T-/-AGS cells had higher RACK1 protein levels than wild AGS cells.UBE2 T catalyses the K48 linked polyubiquitination of RACK1.IHC Assays showed that the level of UBE2 T is negatively correlated with RACK1 but positive correlated with ?-cateninin GC tissues.UBE2 T overexpression promotes polyubiquitination of RACK1,which depended on the catalytic site cysteine 86.UBE2 T promotes the degradation of RACK1 by inducing its polyubiquitination at K172,K225,and K257 without any E3 ligase.UBE2 T promotes ?-catenin translocation into the nucleus and hyperactivates Wnt/?-catenin signaling pathway in GC by inducing RACK1 degradation.Conclusion: UBE2 T induces the ubiquitination and degradation of RACK1 at K172,K225,and K257 residues independent of E3 ligase,which in turn can lead to activate Wnt/?-catenin signaling pathway.PART4: The expression and biological role of ubiquitin conjugating enzyme UBE2 T in gastric cancerObjective: To investigate the effects of UBE2 T on gastric cancer progression.Methods: We performed data mining analysis to explore the m RNA levels of UBE2 T in GC and multiple cancers tissues by the Oncomine platform and GEPIA.Western Blotting and q RT-PCR assays were performed to examine the expression of UBE2 T in GC and adjacent normal tissues.IHC assay was performed to assess the expression of UBE2 T in GC and adjacent normal tissues to analyze the correlation between UBE2 T expression and clinicopathological parameters.The expressions of UBE2 T protein in normal gastric epithelial cells and GC cell lines were determined by Western Blotting assays.The impact of UBE2 T Knockout on cell proliferation was conducted by colony formation and xenograft tumor experiments.Results: Data from Oncomine and GEPIA datasets showed that the m RNA level of UBE2 T was significantly higher in GC tissues.GEPIA database showed that the m RNA level of UBE2 T was also upregulated in 24 additional cancer types.IHC assays showed that the protein levels of UBE2 T are obviously increased and UBE2 T expression is positive correlated with tumor size,depth of invasion,lymph node metastasis,clinical stage,and poor prognosis.Colony-formation assay showed that knockout of UBE2 T decreased the ability of the proliferation in GC cells.UBE2 T knockout significantly suppresses the growth of GC cells xenografts tumor in vivo.Conclusion: UBE2 T knockout suppresses malignant progression and UBE2 T is a promising target for GC therapy.PART5: Discovery of a novel UBE2 T small molecule Inhibitors and its anti-tumor effects on gastric cancer cellsObjective: To investigate the discovery of UBE2 T inhibitors and its anti-tumor effects on gastric cancer cells.Methods: UBE2 T inhibitors in the Chem Div and SPECS small-molecular compounds library were docked and scored by computational virtual screening software Schrodinger,and the small-molecular compounds with ideal ranking scores were selected.MTT assays were performed to examine the effects of these selected inhibitors on the growth of GC cells,and the median inhibition concentrations(IC50)was determined.MST assays were performed to analyze the specificity of the small-molecular compound to UBE2 T protein.Co-Immunoprecipitation and Western blotting Assays was performed to analyze the effects of small-molecular compound on the ubiquitin level of RACK1.Western blotting Assays was used to detect the amount of cytoplasmic,nuclear and total protein after the inhibitor was added to GC cells.The impact of UBE2 T inhibitors on GC cell proliferation and migration was conducted by colony formation,traswell assays and xenograft tumor experiments.Results: We attempted to identify 18 novel small-molecule inhibitors for UBE2 T by performing computational virtual screens.MTT assays showed that only M435-1279 significantly inhibited the growth of HGC27,AGS,and MKN45 cells.The data also showed that the compound didn't block the interaction between RACK1 and UBE2 T but obviously inhibited the ubiquitination of RACK1 and the hyperactivation of the Wnt/ ?-catenin pathway.UBE2 T inhitors M435-1279 significantly suppresses the proliferation and migration of GC cells in vitro,and the growth of xenografts tumor in vivo.Conclusion: The small-molecule inhibitors M435-1279 are considered as potent UBE2 T enzyme inhibitors for gastric cancer therapy.