Anti-Tumor Effects of Second Generation β-Hydroxylase Inhibitors on Cholangiocarcinoma Development and Progression.

Chiung-Kuei Huang,Xiaoqun Dong,Waihong Chung,John-Michael Thomas,Rolf Carlson,Yoshifumi Iwagami,Arihiro Aihara,Tunan Yu,Suzanne De La Monte,Mark Olsen,Jack Wands

doi:10.1371/journal.pone.0150336

Chiung-Kuei Huang, Xiaoqun Dong + Show 9 more

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https://doi.org/10.1371/journal.pone.0150336

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Abstract

Cholangiocarcinoma (CCA) has a poor prognosis due to widespread intrahepatic spread. Aspartate β-hydroxylase (ASPH) is a transmembrane protein and catalyzes the hydroxylation of aspartyl and asparaginyl residues in calcium binding epidermal growth factor (cbEGF)-like domains of various proteins, including Notch receptors and ligands. ASPH is highly overexpressed (>95%) in human CCA tumors. We explored the molecular mechanisms by which ASPH mediated the CCA malignant phenotype and evaluated the potential of ASPH as a therapeutic target for CCA. The importance of expression and enzymatic activity of ASPH for CCA growth and progression was examined using shRNA “knockdown” and a mutant construct that reduced its catalytic activity. Second generation small molecule inhibitors (SMIs) of β-hydroxylase activity were developed and used to target ASPH in vitro and in vivo. Subcutaneous and intrahepatic xenograft rodent models were employed to determine anti-tumor effects on CCA growth and development. It was found that the enzymatic activity of ASPH was critical for mediating CCA progression, as well as inhibiting apoptosis. Mechanistically, ASPH overexpression promoted Notch activation and modulated CCA progression through a Notch1-dependent cyclin D1 pathway. Targeting ASPH with shRNAs or a SMI significantly suppressed CCA growth in vivo.

Highlights

Cholangiocarcinomas (CCAs) are very aggressive tumors with high mortality due to early intrahepatic invasion and subsequent metastatic spread
Aspartate β-hydroxylase (ASPH) has been previously shown to be upregulated in human CCA tumors by immunohistochemistry (IHC) using a monoclonal antibody (FB50); and more importantly, enhanced levels of ASPH were correlated with reduced survival rates as well as intrahepatic and lymph node metastases [13, 28]
Since hepatocytes and bile duct cells develop from a common precursor [29], it was of interest to evaluate the expression of ASPH in CCA and compare it to that in hepatocellular carcinoma (HCC) cell lines

Summary

Introduction

Cholangiocarcinomas (CCAs) are very aggressive tumors with high mortality due to early intrahepatic invasion and subsequent metastatic spread. The CCAs are classified into 3 subtypes as intrahepatic, extrahepatic, or hilar tumors [1]. Targeting ASPH Inhibits Cholangiocarcinoma Growth localized disease and ~2% for advanced CCAs with distant metastases, respectively (American Cancer Society). Over the past several decades the incidence of CCAs has been rising in the United States [2]. Several potential risk factors have been identified to be associated with the development of CCAs, including age over 65, biliary stones, chronic infection with liver flukes, hepatitis B and C viruses, liver cirrhosis, and primary sclerosing cholangitis [1]. The underlying molecular mechanisms involved in CCA development and growth remain elusive

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