Chiung-Kuei Huang, Ph.D.

Project 1: TET1 in alcoholic liver disease progression.

Chronic alcohol abuse has been linked to abnormal epigenetic modifications that affect the progression of alcoholic liver disease (ALD) by influencing factors in controlling cell death in hepatocytes. One such factor is 5-hydroxymethylcytosine (5hmC), but there is currently little information as to how chronic alcohol consumption affects 5hmC's regulation of cell death. Understanding how alcohol impacts 5hmC formation and consequently cell death pathways will potentially yield therapeutic approaches towards ALD. In our preliminary studies, we found that 5hmC expression is down-regulated in the livers of rats and mice fed with an ethanol diet as well as in human ALD tissues. We further examined the expression levels of enzymes involved in the generation of 5hmC, which include methylcytosine dioxygenase TET1, TET2, and TET3 in ALD samples. It was found that TET1 is significantly down-regulated in human and rodent ALD samples. We determined that using shRNA-TET1 to knockdown TET1 in human hepatocytes significantly suppressed 5hmC formation and promoted cell death as well as the pro-apoptotic gene, HRK. Intriguingly, the treatment of the DNA methylation inhibitor, 5-Azacytidine, could replace the impact of TET1 knockdown on hepatocyte cell death, further suggesting the importance of DNA methylation in TET1-mediated hepatocyte cell death. We then analyzed how TET1 down-regulation is involved in ALD progression by using TET1 knockout mice. It was found that knockout of TET1 substantially elicited liver fibrosis, which is consequently the outcome of wound-healing in chronic liver damage. Thus, our central hypothesis is that ethanol exposure increases hepatocyte cell death to promote ALD progression by suppressing TET1-mediated 5hmC epigenetic changes. Our long-term objective is to clarify the underlying mechanisms by which TET1 modulates ALD progression, and determine if TET1 is a potential therapeutic target in ALD. In this project, we propose two specific aims to evaluate our hypothesis. In aim 1, we will examine how TET1 modulates cell death pathways in ALD progression. We will investigate the enzymatic function of TET1 in regulating cell death pathways by using TET1 catalytic domain, full length TET1, TET1 catalytic domain dead mutation, and TET1 specific inhibitor. In aim 2, we will examine the role of TET1 in ALD progression. We will evaluate the impact of TET1 down-regulation on hepatocyte cell death in wild-type (WT) and TET1 knockout (KO) mice challenged with an alcoholic liquid diet. To further determine the hepatic specific TET1 role in ALD progression, we will generate liver specific TET1 knockout mice by using albumin promoter driven Cre mouse and floxed TET1 mouse. The results will significantly advance our knowledge of the mechanisms by which TET1 modulates hepatocyte cell death and improve our understanding of pathophysiological mechanisms underlying ALD progression. We also anticipate that it will have a broad impact on the understanding of TET1 expression and its relationship to hepatocyte function in general.

Project 2: Aspartate beta-hydroxylase in cholangiocarcinoma tumorigenesis. 

The central focus of this proposal is to understand the role of aspartate beta-hydroxylase (ASPH) in tumorigenesis of cholangiocarcinoma (CCA) and investigate the therapeutic potential of targeting ASPH in preclinical CCA models. The long-term goal of this proposal is to investigate potential targeting approaches for CCA patients. The overall survival of CCA patients with distant metastasis is about 7% and most patients with CCA tumors have no effective therapies. Thus, there is a need to develop an effective therapy for those patients with this deadly disease. We have found that ASPH, which is a 2-oxoglutarate dependent enzyme, is highly expressed in CCA tumors but barely detectable in normal bile ducts. Our pilot studies demonstrated its oncogenic characteristics in CCA progression. Furthermore, we revealed a novel mechanistic link YAP1/ASPH/Notch1/Pin1/RB1 signaling cascade in cholangiocarcinogenesis. More importantly, we show that the YAP1 inhibitor-Verteporfin (VER) substantially suppressed ASPH expression and CCA malignancy. Based on these findings, we hypothesize that the ASPH/RB1 signaling cascade mediated by YAP1 contributes to the early tumorigenesis and late malignant tumor progression of CCA. Three specific aims are proposed to determine the role of ASPH in CCA tumorigenesis. In aim 1, we will determine the effects of ASPH manipulation on CCA tumorigenesis by using different CCA models in vitro and in vivo. Aim 2 will investigate the mechanisms by which ASPH modulates CCA progression via multiple molecular approaches including RNA sequencing. In aim 3, we will repurpose the therapeutic potential of VER in CCA malignancy by using multiple CCA models, including patient-derived xenograft models.