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Title: Functional characterization and clinical relevance of ubiquitin conjugating enzyme E2T in hepatocellular carcinoma
Authors: Ho, Pui Yu
Degree: Ph.D.
Issue Date: 2019
Abstract: Hepatocellular carcinoma (HCC) is one of the common cancers worldwide. Most HCC patients present at an advanced stage when resection or liver transplantation is not feasible. Even after surgery, the long-term prognosis of HCC remains unsatisfactory due to high recurrence rates. Chemotherapy remains the principle alternative for treating unresectable HCC. However, the efficacy is limited due to chemoresistance. Increasing evidence showed the critical role of cancer stem cells (CSCs) on tumor relapse and therapeutic resistance. Since normal stem cells and CSCs share high similarity in genetic profile, we would like to identify the molecules/pathways crucial in liver CSCs by determining what molecules involved in normal liver stem cells during liver regeneration. For this purpose, partial hepatectomy mouse model was employed to analyze the change in genetic profile in regenerating liver. Comparison of expression profiles between early regenerating liver and intact liver revealed the upregulation of DNA Damage Response pathways in self-renewing liver, in which Ubiquitin Conjugating Enzyme E2T (UBE2T) was the most significantly upregulated. This, together with the publicly available dataset (GSE5975) shows upregulation of UBE2T in EpCAM-enriched liver CSC populations, suggest the potential role of UBE2T on regulation of cancer stemness. By qPCR analysis, UBE2T was overexpressed in 91% of the clinical HCC specimens and associated with aggressive phenotype and poorer patients' survival. Significant overexpression of UBE2T in protein level was also confirmed by Western Blot and IHC analyses. By lentiviral based knock-down and ectopic overexpression approaches, we demonstrated the role of UBE2T in regulation of liver CSC properties including self-renewability and expression of CSC markers. Alternation of UBE2T expression level also affected HCC invasiveness and drug resistance. Apart from in vitro functional assays, UBE2T also found to regulate in vivo tumorigenicity and CSC frequency. In orthotopic HCC model, UBE2T was found to play pivotal role in lung metastasis in vivo. In order to identify downstream target of UBE2T for regulation of cancer stemness, tandem affinity purification coupled with mass spectrometry (TAP/MS) was employed. Upon analysis, Mule, a E3 ubiquitin ligase, was identified to be the novel protein binding partner of UBE2T. Being the E2 ubiquitination enzyme, UBE2T was found to physically bind and regulate the protein expression of Mule via ubiquitination. Recently, Mule is suggested to further directly degrade protein of β-catenin. Therefore, we further examined the effect of UBE2T on β-catenin expression. Consistently, we found that overexpression of UBE2T increased β-catenin expression via degradation of Mule. Opposite effects were observed when UBE2T expression was suppressed. Furthermore, the effect of UBE2T on β-catenin activity was further confirmed by Immunofluorescence (IF). These effects were offset when E2 activity of UBE2T was impaired by replacing wild-type form of UBE2T with the E2-dead mutant (C86A). In conclusion, we have uncovered the novel role of UBE2T signaling cascade in regulation liver CSCs. UBE2T regulates liver CSC functions through Mule-mediated β-catenin degradation. Our study not only provides a mechanistic insight for tumor recurrence and drug resistance, but also open a new therapeutic avenue for treatment of HCC.
Subjects: Hong Kong Polytechnic University -- Dissertations
Liver -- Cancer -- Treatment
Cancer cells
Stem cells
Pages: xiv, 149 pages : color illustrations
Appears in Collections:Thesis

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