Date of Award
Doctor of Philosophy in Pharmaceutical Sciences
Biomedical and Pharmaceutical Sciences
Hepatocellular carcinoma (HCC), or liver cancer, is the sixth most common cancer worldwide, and the third leading cause of cancer-related deaths. In 2023, an estimated 41,210 new HCC cases and 29,380 deaths have been reported in the United States with an overall 5-year relative survival rate of 20.8%. It has been predicted that by 2025, there will be more than one million cases of HCC globally. Unfortunately, there are limited effective treatments and current treatment options available are invasive and burdensome. HCC patients are usually diagnosed at the late stages of liver cancer when the only viable treatment options are liver hepatectomies or liver transplantation. In our studies, we discovered two novel mechanisms that can aid in developing targeted therapies for HCC.
Ubiquitin specific peptidase 2 (USP2) is a deubiquitinating enzyme known to modulate cell cycle progression, circadian rhythm, and carcinogenesis in the liver. USP2 and its isoforms USP2a and USP2b are dysregulated in human HCC tissues and farnesoid x receptor knockout (FXR-KO)-HCC mouse models. In HCC tissues, USP2a has shown tumor-promoting properties, while USP2b has tumor-suppressing characteristics. FXR has been defined as a potential target for HCC treatment, as it has helped to significantly reduce pathophysiological severities in diseases that are precursors for HCC development. However, the mechanism by which FXR regulates USP2 isoforms and its contribution to the pathogenesis of HCC is largely unknown.
In our first study, we discovered that FXR activation by obeticholic acid (OCA) treatment significantly upregulated USP2a and USP2b mRNA and protein expression in C57BL/6 wild-type (WT) mice and in human hepatoma Huh7 cells. On the contrary, FXR antagonism by DY268 treatment significantly downregulated USP2a and USP2b expression in WT mice and Huh7 cells. In FXR-KO mice, OCA treatment and DY268 treatment had no significant change in USP2a or USP2b expression compared to the vehicle-treated group. Interestingly, transfection of FXR isoforms, FXRα1 and FXRα2, showed that USP2a and USP2b transactivation was predominantly regulated by FXRα2, while FXRα1 had limited effects in Huh7 cells. In silico analysis of USP2a and USP2b promoters using bioinformatics software revealed novel USP2a and USP2b-FXR response elements (FXREs). Targeted mutagenesis of the FXREs completely abolished inducibility of the USP2a and USP2b promoters in OCA-treated Huh7 cells. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) demonstrated direct binding and abundant recruitment of FXR to the human and mouse USP2a and USP2b promoters. Coregulator co-activator-associated arginine methyltransferase 1 (CARM-1) was significantly recruited to the human USP2a and USP2b promoters upon FXR activation. In mice, FXR was found to specifically bind to the predicted USP2a and USP2b-FXREs. The coactivator CARM-1 was recruited to the mouse USP2a promoter, while coactivators glutamate receptor-interacting protein 1 (GRIP-1) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) were recruited to the mouse USP2b promoter upon FXR activation. Overall, manuscript 1 displays the established correlation between the expression of USP2 isoforms and their direct regulation by FXR, indicating its contribution to the pathogenesis of HCC.
In manuscript 2, we present the mechanism in which estrogen receptor alpha (ERα) downregulates and directly interacts with USP2a and USP2b. Estrogen has been considered to play a protective role in the pathogenesis of HCC as men statistically develop HCC at higher rates than women, and this could be on the account of women having higher estrogen levels. Studies have been conducted proving that estrogen can inhibit the progression of HCC due to its anti-inflammatory effects. However, studies identifying the transcriptional regulation of USP2a and USP2b by ERα in the pathogenesis of HCC have not been established. In our second study, we found that ligand-activated ERα by estradiol (E2) treatment significantly decreases USP2a and USP2b mRNA and protein levels. We found that ERα represses the transactivation of USP2a and USP2b, and directly binds to the USP2a and USP2b promoter. Interestingly, we identified the estrogen response element (ERE) and discovered ERα WT recruitment adjacent to the FXRE in the USP2a and USP2b promoters.
It has been reported that ERα variants, ERα 36, ERα 46, and ERαΔE5, are considered predictors of poor prognosis in HCC. In this second study, we found that ERα predominantly represses the transactivation of USP2b, even in the presence of FXRα2. We also found that ERα 46 and ERαΔE5 significantly repress the FXRα2-mediated transactivation of USP2a and USP2b. Taken together, this discovery displays the direct downregulation of USP2a and USP2b by ERα. This study also demonstrates the possible interference of ERα and FXRα2 in USP2b regulation, as well as ERα 46 and ERαΔE5 interference with FXRα2-mediated transactivation of USP2a and USP2b. Most importantly, these novel mechanisms provide the molecular basis for developing therapies to treat HCC through modulating USP2a and USP2b expression by FXR or ER activation.
Ali, Winifer M., "UNDERLYING MECHANISMS OF UBIQUITIN SPECIFIC PEPTIDASE 2 ISOFORMS REGULATION IN HEPATOCELLULAR CARCINOMA BY THE FARNESOID X RECEPTOR SIGNALING PATHWAY" (2023). Open Access Dissertations. Paper 1561.