Targeting Aspartate Beta-Hydroxylase with the Small Molecule Inhibitor MO-I-1182 Suppresses Cholangiocarcinoma Metastasis.

BACKGROUND: Cholangiocarcinoma is a devastating disease with a 2% 5-year survival if the disease has spread outside the liver. The enzyme aspartate beta-hydroxylase (ASPH) has been demonstrated to be highly expressed in cholangiocarcinoma but not in normal bile ducts and found to stimulate tumor cell migration. In addition, it was found that targeting ASPH inhibits cholangiocarcinoma malignant progression. However, it is not clear whether targeting ASPH with the small molecule inhibitor MO-I-1182 suppresses cholangiocarcinoma metastasis. The current study aims to study the efficacy of MO-I-1182 in suppressing cholangiocarcinoma metastasis. METHODS: The analysis was performed in vitro and in vivo with a preclinical animal model by using molecular and biochemical strategies to regulate ASPH expression and function. RESULTS: Knockdown of ASPH substantially inhibited cell migration and invasion in two human cholangiocarcinoma cell lines. Targeting ASPH with a small molecule inhibitor suppressed cholangiocarcinoma progression. Molecular mechanism studies demonstrated that knockdown of ASPH subsequently suppressed protein levels of the matrix metalloproteinases. The ASPH knockdown experiments suggest that this enzyme may modulate cholangiocarcinoma metastasis by regulating matrix metalloproteinases expression. Furthermore, using an ASPH inhibitor in a rat cholangiocarcinoma intrahepatic model established with BED-Neu-CL#24 cholangiocarcinoma cells, it was found that targeting ASPH inhibited intrahepatic cholangiocarcinoma metastasis and downstream expression of the matrix metalloproteinases. CONCLUSION: ASPH may modulate cholangiocarcinoma metastasis via matrix metalloproteinases expression. Taken together, targeting ASPH function may inhibit intrahepatic cholangiocarcinoma metastasis and improve survival.

Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.

The 5-hydroxymethylcytosine (5hmc) is a newly identified epigenetic modification thought to be regulated by the TET family of proteins. Little information is available about how ethanol consumption may modulate 5hmC formation and alcoholic liver disease (ALD) progression. A rat ALD model was used to study 5hmC in relationship to hepatocyte apoptosis. Human ALD liver samples were also used to validate these findings. It was found that chronic ethanol feeding significantly reduced 5hmC formation in a rat ALD model. There were no significant changes in TET2 and TET3 between the control- and ethanol-fed animals. In contrast, methylcytosine dioxygenase TET1 (TET1) expression was substantially reduced in the ethanol-fed rats and was accompanied by increased hepatocyte apoptosis. Similarly, knockdown of TET1 in human hepatocyte-like cells also significantly promoted apoptosis. Down-regulation of TET1 resulted in elevated expression of the DNA damage marker, suggesting a role for 5hmc in hepatocyte DNA damage as well. Mechanistic studies revealed that inhibition of TET1 promoted apoptotic gene expression. Similarly, targeting TET1 activity by removing cosubstrate promoted apoptosis and DNA damage. Furthermore, treatment with 5-azacitidine significantly mimics these effects, suggesting that chronic ethanol consumption promotes hepatocyte apoptosis and DNA damage by diminishing TET1-mediated 5hmC formation and DNA methylation. In summary, the current study provides a novel molecular insight that TET1-mediated 5hmC is involved in hepatocyte apoptosis in ALD progression.-Ji, C., Nagaoka, K., Zou, J., Casulli, S., Lu, S., Cao, K. Y., Zhang, H., Iwagami, Y., Carlson, R. I., Brooks, K., Lawrence, J., Mueller, W., Wands, J. R., Huang, C.-K. Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.

Aspartate beta-hydroxylase promotes cholangiocarcinoma progression by modulating RB1 phosphorylation.

Cholangiocarcinoma (CCA) is a highly lethal and aggressive disease. Recently, IDH1/2 mutations have been identified in approximately 20% of CCAs which suggests an involvement of 2-oxoglutarate (2-OG) -dependent dioxygenases in oncogenesis. We investigated if the 2-OG dependent dioxygenase, aspartate beta-hydroxylase (ASPH) was important in tumor development and growth. Immunoassays were used to clarify how ASPH modulates CCA progression by promoting phosphorylation of the retinoblastoma protein (RB1). A xenograft model was employed to determine the role of ASPH on CCA growth. Knockdown of ASPH expression inhibited CCA development and growth by reducing RB1 phosphorylation. Expression of ASPH promoted direct protein interaction between RB1, cyclin-dependent kinases, and cyclins. Treatment with 2-OG-dependent dioxygenase and ASPH inhibitors suppressed the interaction between RB1 and CDK4 as well as RB1 phosphorylation. Knockdown of ASPH expression inhibited CCA progression and RB1 phosphorylation in vivo and they were found to be highly expressed in human CCAs. Knockdown of ASPH expression altered CCA development by modulating RB1 phosphorylation, as one of the major factors regulating the growth of these tumors.