Date of Award

2018

Degree Type

Thesis

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Ruitang Deng

Abstract

Liver cancer is in the ten leading cancer types for estimated new cases and deaths in the United States in 2018, and is often detected at late stages when it is rapidly fatal. Of the types of liver cancers in humans, hepatocellular carcinoma (HCC) is the most lethal and prevalent. An important factor in the development of HCC is Farnesoid X Receptor (FXR). FXR acts as master regulator of bile acid homeostasis, and is involved in lipid, cholesterol, and glucose homeostasis. In addition, it plays a protective role against HCC, the precise mechanisms of which has yet to be identified.

Ubiquitin Specific Peptidase 2 (USP2) has emerged as a promising area of research for cancer therapy for multiple organ systems. Recent studies have shown USP2 to be involved in a variety of cellular processes ranging from circadian rhythms to tumorigenesis. In particular, USP2 has been studied in lung, bladder, breast, prostate, and ovarian cancer. Additionally, USP2 has 3 distinct isoforms, USP2a, USP2b, and USP2c, all of which have unique functions. In manuscript 1, the purpose of the study was to investigate the isoform specific effects of USP2 in hepatocellular carcinoma (HCC) through the use of healthy human liver samples compared to tumor samples, paired human liver tumor and surrounding samples, paired mouse liver tumor and surrounding samples, and the immortalized liver cancer cell line HepG2. Of the three USP2 isoforms, USP2a and USP2b are the most abundant in human and murine liver. Both USP2a and USP2b levels are significantly lower in HCC compared to healthy liver samples. In paired tumor and surrounding liver samples from mice and human, USP2b is consistently decreased within the tumor compared to the surrounding tissue. USP2b was decreased in tumors in all human samples and decreased in over 80% of murine samples. This suggests as the state of the liver worsens in HCC pathology, USP2b will also decrease. In HepG2 cells, both USP2a and USP2b isoforms led to increased proliferation, colony formation, and wound healing. Additionally, USP2b and USP2c lead to increased migration, wound healing, and apoptosis. Previous studies have only recognized USP2b as a circadian gene, making this the first study to report involvement of USP2b in cellular processes beyond its circadian characteristics. Overall, USP2a and USP2b have opposing roles in HCC development, with USP2a displaying a tumor-promotion role and USP2b displaying a tumor-protective role.

In manuscript 2, we present a novel potential mechanism for USP2 regulation in the liver and in HCC development through FXR. Between wild type and FXR-/- mice, there was a dysregulation in USP2 and previously identified downstream targets. To determine if the changes in USP2 were due to FXR regulation, we activated FXR in wild type mice, over-expressed FXR in liver cancer cells, and evaluated the effects of FXR activation on the USP2 promoter. Upon treatment with FXR agonist in wild type mice, USP2 mRNA and protein significantly increased compared to vehicle. Additionally, transfection and activation of FXR in huh7 cells lead to an increase in USP2. Of importance, we found FXR directly regulates USP2 gene expression via dual luciferase assay. This study is the first to report FXR in the regulation of gene, mRNA, and protein expression of USP2 in the liver. Specifically, FXR2 is the predominant isoform in this regulation. In addition, this is also the first study to identify the different USP2 isoforms to be a results of two promoters rather than alternative splicing. As previous studies have found FXR to display a protective effect in HCC, this study identifies a novel mechanism of this protection through USP2.

Available for download on Monday, July 27, 2020

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