Date of Award
2026
Degree Type
Dissertation
Degree Name
Doctor of Philosophy in Pharmaceutical Sciences
Department
Biomedical and Pharmaceutical Sciences
First Advisor
Ruitang Deng
Abstract
Hepatocellular carcinoma (HCC) is one of the most common forms of liver cancer worldwide, with incidence rates steadily increasing. In 2020, there were approximately 906,000 new cases and 830,000 deaths, with the highest incidence rate observed in Eastern Asia, South-Eastern Asia, and Northern and Western Africa. It is estimated that by 2040, the disease burden will continue to rise to 1.4 million new cases. The U.S. Food and Drug Administration (FDA) has approved tyrosine kinase inhibitors such as Lenvatinib and Sorafenib, as well as combination regimens incorporating immunotherapy and anti-angiogenic agents (e.g., Bevacizumab plus Atezolizumab), as first-line treatments for advanced HCC. Despite advances in cancer therapy, effective treatment options for HCC remain limited. The challenge remains largely due to the complexity of HCC pathogenesis and an incomplete understanding of the underlying molecular mechanisms.
In recent years, the ubiquitin-proteasome system (UPS) and deubiquitinating enzymes (DUBs) have gained significant attention for their roles in regulating protein stability and signaling pathways in cancer. Ubiquitination is a reversible process that can be counteracted by DUBs, which remove ubiquitin from target proteins. As a member of the DUB family, ubiquitin-specific protease 2 (USP2) regulates protein stability, localization, and activity by deubiquitinating its substrates. The human genome encodes more than 100 DUBs, with ubiquitin-specific proteases (USPs) representing the largest family. USP2 exists as three isoforms, USP2a, USP2b, and USP2c, and has been reported to be dysregulated in multiple diseases and cancers.
In Manuscript 1, we demonstrated that USP2 protein and mRNA levels are significantly dysregulated in HCC tumor tissues compared with adjacent non-tumor and normal liver tissues in both human and mouse models. Among the three isoforms, USP2b was identified as the predominant isoform in the liver and was significantly downregulated in HCC tumor tissues. Overexpression of USP2b promoted cell proliferation, colony formation, and wound healing in both HepG2 and Huh7 cells. Interestingly, USP2b overexpression also increased bile acid-induced apoptosis and necrosis in these cells. Unbiased proteomic analysis of USP2-knockout (KO) and parental HepG2 cells further identified USP2-regulated downstream proteins involved in cell proliferation, apoptosis, and tumorigenesis, including serine/threonine kinase 4 (STK4), epidermal growth factor receptor (EGFR), dipeptidyl peptidase 4 (DPP4), and fatty acid binding protein 1 (FABP1). Collectively, these findings provide a molecular basis for developing therapeutic strategies targeting USP2b expression or activity in HCC.
In Manuscript 2, we investigated the mechanisms by which USP2b exerts tumorigenic activity, including promoting cell proliferation, colony formation, and wound healing in HCC cells. Unbiased co-immunoprecipitation (Co-IP)-coupled proteomic analysis, followed by Western blot validation, was used to identify novel USP2b-interacting proteins involved in cell proliferation. Our findings indicate that USP2b promotes cell growth through multiple pathways, including regulation of ribosome biogenesis via eukaryotic translation initiation factor 6 (EIF6), endoplasmic reticulum homeostasis through DDRGK domain-containing protein 1 (DDRGK1), nucleotide biosynthesis through inosine-5’-monophosphate dehydrogenase 2 (IMPDH2), and DNA replication through proliferating cell nuclear antigen (PCNA). These target proteins were dysregulated in HCC tumor tissues and were found to enhance cell growth. Additional proteomic and network analyses identified potential downstream effectors associated with these targets, including ubiquitin-fold modifier-conjugating enzyme 1 (UFC1), ribosomal protein L11 (RPL11), and thymidylate synthase (TYMS). Functional studies showed that these downstream effectors also modulate cell growth, with context-dependent effects. Collectively, these findings establish USP2b as a multifaceted regulator of cell proliferative signaling pathways and provide mechanistic insight into its role in driving cell proliferation and tumor progression in HCC.
In Manuscript 3, we sought to elucidate the underlying mechanisms by which USP2b exerts its effects on bile acid-induced cell death, including apoptosis. Using unbiased co-immunoprecipitation (Co-IP)-coupled proteomic analysis, apoptosis-inducing factor mitochondrion-associated 1 (AIFM1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (YWHAE) were identified as USP2b downstream target proteins. Both proteins were dysregulated in HCC tumor tissues, and functional studies demonstrated that AIFM1 promotes apoptosis, whereas YWHAE exhibits anti-apoptotic activity. STRING network analysis further identified peptidylprolyl isomerase A (PPIA) and Yes-associated protein 1 (YAP1) as potential downstream effectors of AIFM1 and YWHAE, respectively. Overexpression of PPIA and YAP1 suppresses apoptosis, supporting their roles in promoting cell survival. Collectively, these findings suggest that USP2b regulates apoptosis through dual mechanisms, including stabilization of the pro-apoptotic protein AIFM1 and destabilization of the anti-apoptotic regulator YWHAE, providing new insight into the molecular regulation of apoptotic signaling in HCC.
In Manuscript 4, we examined the role of USP2a in HCC and found that it is significantly upregulated in tumor tissues from both human patients and mouse models. USP2a overexpression enhanced cell proliferation, whereas its knockout increased sensitivity to bile acid-induced apoptosis and necrosis. In vivo, USP2a promoted de novo tumor development, as indicated by increased tumor incidence, tumor size, and liver-to-body weight ratio. Proteomic analysis identified novel USP2a target proteins involved in cell proliferation, apoptosis, and tumorigenesis. These include regulators of protein folding (HSPA1A, DNAJA1, TCP1), DNA replication and transcription (RUVBL1, PCNA, TARDBP), and mitochondrial apoptosis (VDAC2). Collectively, these findings demonstrate that USP2a functions as an oncogene, driving HCC progression through multiple downstream pathways.
Recommended Citation
Chen, Qiwen, "THE DYSREGULATION AND MECHANISTIC ROLES OF UBIQUITIN SPECIFIC PEPTIDASE 2 IN THE PATHOGENESIS OF HEPATOCELLULAR CARCINOMA" (2026). Open Access Dissertations. Paper 4578.
https://digitalcommons.uri.edu/oa_diss/4578