Dysregulation and Oncogenic Activities of Ubiquitin Specific Peptidase 2a in Hepatocellular Carcinoma

Qiwen Chen, Xinmu Zhang, Christina Nadolny, Winifer Ali1, Syed Hashmi1, Ruitang Deng

Hepatocellular carcinoma (HCC) is one of the most common forms of liver cancer worldwide and accounts for 90% of liver cancer cases. By 2025, it is estimated to have more than one million cases globally. Hepatitis B virus and hepatitis C virus are the main risk factors for HCC development. Early-stage HCC patients may profit from multiple treatment options including surgical resection, liver transplantation, arterial embolization, radioembolization, systemic targeted agent, or liquid biopsy. For patients with advanced HCC, sorafenib is the only approved therapy. The treatment of HCC depends on the tumor stage, patient performance status, and liver function reserved. However, only a small percentage of patients respond well to these treatments, and recurrent diseases remain high. Currently, there is a limited effective option in treating HCC patients. There is an urge to develop more effective therapies for HCC. However, the challenge remains due to the complexity of HCC pathogenesis and the lack of understanding of the complex mechanism. In recent years, the ubiquitin-proteasome system (UPS) and deubiquitinating enzymes (DUBs) have emerged as an important topic for understanding the mechanism of HCC and other cancers. The ubiquitin-proteasome system is one of the most important post-translational modification pathways in eukaryotic cells that regulates cell functions including cell cycle progression, DNA repair, and signal transduction. Ubiquitination can be reversed by deubiquitinating enzymes (DUBs) such as ubiquitin specific peptidase 2 (USP2) that can cleave ubiquitin or ubiquitinated proteins from targeted proteins. As a DUB, USP2a play a critical role in protein degradation and its mRNA and protein level was found to be unregulated in HCC tumor. In addition, USP2a exhibited oncogenic activities by promoting cell proliferation, colony formation and wound healing. Through an unbiased coimmunoprecipitation (Co-IP)-couple proteomic analysis and Western blot of USP2a overexpression pull down results in novel USP2a target proteins involved in cell proliferation, apoptosis and tumorigenesis including HSPA1A, DNAJA1, TCP1, RUVBL1, PCNA. TARDBP and VDAC2. Modulating USP2a expression or its downstream activities can be the molecular basis for developing therapies for HCC.

Peroxisome proliferator-activated receptor alpha (PPARɑ) activates multidrug resistanceassociated protein 3/ATP binding cassette subfamily C member 3 (MRP3/ABCC3) in human hepatocytes

Gina M. Gallucci, Colleen M. Hayes, David N. Assis, James L. Boyer, Nisanne S. Ghonem

Multidrug resistance protein 3 (MRP3)/ATP-dependent transporter of the ATP-binding cassette family 3 (ABCC3) is a transporter located along the basolateral membrane of hepatocytes, that transports glucuronide metabolites, including bilirubin and bile acid-glucuronides, out of the liver and into systemic circulation. Bile acid glucuronidation is a phase II pathway of detoxification that conjugates glucuronic acid to parent bile acids which results in compounds that are less cytotoxic and more readily excreted. Bile acid-glucuronides are substrates for MRP3, which transports them out of the liver and into systemic circulation for subsequent renal excretion. As such, and due to its role in bile acid detoxification, MRP3 may be a therapeutic target for the treatment of cholestatic liver diseases, which are characterized by bile acid accumulation and toxicity. The peroxisome proliferator-activated receptor alpha (PPARα) ligand fenofibrate upregulates bile acid-glucuronides in the serum of cholestatic patients and upregulates MRP3 messenger RNA in cultured primary human hepatocytes. However, it is unknown how MRP3 is transcriptionally regulated. This study investigates the transcriptional regulation of MRP3/ABCC3 by PPARα. The MRP3 mRNA and protein expression in HepG2 cells were both significantly increased after treatment with fenofibrate (50, 125 µM), compared to vehicle control. In silico analysis of the MRP3 promoter identified multiple PPARα response elements (PPREs). Direct binding of human PPARα to the MRP3/ABCC3 promoter was demonstrated by electrophoretic mobility shift (EMSA) assays. Further investigation will include the activation and binding of PPARα to the MRP3 promoter in vivo by ChIP. Thus far, these data demonstrate that fenofibrate directly binds to functional PPREs within the MRP3 promoter, which contributes to the mechanistic understanding of adjunct fenofibrate for the treatment of cholestatic liver diseases.