UBE2E2 Promotes Tumor Progression And Metastasis By Modulating Snail And NRF2 In Ovarian Cancer

Background and objective: Ovarian cancer is the most aggressive and lethal gynecologic malignancy and is characterized by the propensity for ovarian cancer cells to “slough off” from the primary tumor site and establish metastases.Dissemination of ovarian cancer cells can lead to inoperable metastatic lesions in the bowel and omentum,which have a poor prognosis despite surgical and chemotherapeutical options.Epithelial-mesenchymal transition(EMT),a major mechanism involved in the initiation of tumor metastasis,involves a wide range of phenotypic and molecular changes leading to cancer cells losing their epithelial characteristics and dedifferentiating into mesenchymal-like cancer stem cells(CSCs).The zinc finger protein SNAI1(also known as Snail,gene name SNAI1),represses the expression of a broad repertoire of epithelial genes,including E-cadherin/CDH1,and is a master regulator of EMT in most cancers.Snail is a highly unstable protein that is rapidly degraded by the proteasome.The scaffold protein p62(sequestosome 1 or SQSTM1),an adaptor that connects ubiquitination with autophagy,was recently reported to be associated with the ubiquitination and proteasomal degradation of the Snail protein in various disease models,such as glioblastoma,bladder tumor,and cardiac fibrosis.However,whether p62 influences the ubiquitination and expression level of Snail in ovarian cancer remains unknown.Ubiquitination,a posttranslational modification,plays a fundamental role in degrading proteins and regulating most cellular processes.Ubiquitination is executed through an enzymatic cascade consisting of Ub-activating enzymes(E1),Ubconjugating enzymes(E2),and Ub protein ligases(E3).In addition to interacting specifically with and discriminating between Ub and ubiquitin-like proteins(Ubls),E2 s are scaffolding proteins/noncovalent regulators in processes that are independent of their enzymatic activity.They discern the type of Ub/UBL chain linkages,contribute to substrate site specificity,and participate in the regulation of a variety of life activities in cells.Through gene expression and functional annotation analyses,we identified the Ub-conjugating enzyme E2E2(UBE2E2)with upregulated expression in ovarian cancer and linked it with detrimental outcomes in patients.UBE2E2(also called Ubc H8)was first reported in 1997 and has been proposed to play a pathological role in various human diseases,including type 2 diabetes(T2D),rheumatic autoimmune disease,Parkinson’s disease,non-small cell lung cancer,hepatocellular adenoma and carcinoma.In addition,UBE2E2 has been identified in relation to adipogenesis and RR intervals.Nevertheless,most of the evidence is based on genome-wide association studies(GWAS)and meta-analyses,and therefore,the molecular function of UBE2E2 remains poorly characterized.Here,we further explored the role and related mechanism of UBE2E2 in the malignant progression and metastasis of ovarian cancer.This study helps to provide novel therapeutic targets for the prevention of ovarian cancer metastasis,and also affords new idea for the development of tumor markers and prognostic indicators.Methods:(1)Bioinformatics analyses: The gene expression analyses and survival analyses were performed by using the Oncomine,Gene Expression Profiling Interactive Analysis(GEPIA),The Human Protein Atlas,KEGG pathway,and KaplanMeier plotter online databases.The Schr?dinger program software was used to simulate the structure of UBE2E2 protein.(2)Cell biology experiments: We knocked out UBE2E2 expression in A2780 cells using the CRISPR-Cas9 system and knocked down UBE2E2 with si RNA transfection.The coding sequence of target genes were subcloned into expression vectors to generate overexpression.Cell proliferation and colony formation assays,wound healing assays,Transwell assays,western blot analysis and coimmunoprecipitation(co-IP),RNA extraction and quantitative RT-PCR(q RTPCR),flow cytometry and immuno-fluorescence staining were performed.(3)Xenograft ovarian cancer mouse models: Human ovarian cancer cells were transplanted orthotopically or injected intraperitoneally into BALB/c nude mice to establish orthotopic xenograft model and intraperitoneal xenograft model.Fluorescence imaging and average radiance values were obtained.The tumor tissues were dissected,photographed,and prepared for western blotting or immunohistochemical(IHC)staining.(4)Patient sample collection: A total of 88 ovarian cancer specimens and 26 normal ovarian tissue specimens were obtained from the Department of Obstetrics and Gynecology.Tissue sections were fixed and immuno-stained with antibodies.Survival information was obtained from the patients and overall survival was determined by Kaplan-Meier analysis.Results:(1)The m RNA and protein expression levels of UBE2E2 were elevated in ovarian cancer specimens compared with normal ovary samples,but no significant difference in the DNA level was found.Patients with high levels of UBE2E2 experienced significantly worse clinical outcomes.(2)The silencing of UBE2E2 blocked ovarian cancer cell proliferation and migration in vitro,downregulated the protein expression of EMT markers Snail,Slug,N-cadherin and increased the expression of an epithelial marker E-cadherin;whereas UBE2E2 overexpression exerted the opposite effects.(3)UBE2E2 upregulated the expression of p62 at both the m RNA and protein levels.Immunofluorescence assays revealed that UBE2E2,which is localized in the nucleus,promoted the expression of p62 in the cytoplasm.However,UBE2E2 overexpression or silencing did not significantly affect LC3 B expression or autophagosome formation.P62 overexpression increased the protein level of Snail and the repression of E-cadherin expression.We did not observe any direct interactions between UBE2E2 and p62 or Snail,or physical interaction between p62 and Snail protein.UBE2E2 overexpression reduced the ubiquitination of Snail protein.Furthermore,overexpression of UBE2E2 or p62 or NRF2 in UBE2E2-depleted cells could partially rescue the Snail-induced EMT.(4)Overexpression of UBE2E2 in ovarian cancer cells upregulated the expression of NRF2 and its target genes,whereas inhibition of UBE2E2 expression had the opposite effect.Exposure to t BHQ,a potent NRF2 activator,increased the amount of NRF2 protein translocated to the nucleus in control cells,but this protective effect was abrogated in UBE2E2-depleted cells.UBE2E2 exerted no significant effect on the m RNA expression level of NRF2.(5)The UBC domain(residues 53-201)of UBE2E2 is sufficient to induce protein translocation into the nucleus.The integrity of the active site Cys139 residue is critical for the cellular location of UBE2E2 and influences its functions in EMT induction.(6)Both C139 A mutant and the N-terminal of UBE2E2(C-terminal-truncated UBE2E2 mutant Δ53-201)were found to cause NRF2 accumulation,which was similar to that of WT-UBE2E2.The N-terminal of UBE2E2(residues 1-52)is necessary and sufficient to interact with NRF2.Over-expression of WT-UBE2E2 and C139AUBE2E2 in UBE2E2-depleted cells mainly caused cytoplasmic NRF2 accumulation.(7)UBE2E2-depleted ovarian cancer cells formed fewer tumor nodules than the control cells in orthotopic xenograft models and intraperitoneal xenograft models.No significant difference in average body weight was found between groups.Western blot and IHC analyses showed that UBE2E2 deletion decreased the levels of p62 and Snail and increased the E-cadherin level in xenograft tumors.Conclusions:(1)UBE2E2 promotes the proliferation,EMT and metastasis of ovarian cancer in vitro and in vivo.(2)UBE2E2 enhances the EMT of ovarian cancer cells by upregulating the expression of p62,stabilizing the expression of Snail and inhibiting its ubiquitin-mediated degradation,but exerts no significant effect on autophagy.(3)The N-terminal of UBE2E2(residues 1-52)is necessary and sufficient to interact with NRF2,and the full-length UBE2E2 protein activates the NRF2-antioxidant response element(ARE)system in ovarian cancer cells.(4)Mutations in the active site cysteine(Cys139)impair both the function and cellular distribution of UBE2E2.