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

RESEARCH ARTICLE Anti-Tumor Effects of Second Generation βHydroxylase Inhibitors on Cholangiocarcinoma Development and Progression Chiung-Kuei Huang1☯, Yoshifumi Iwagami1☯, Arihiro Aihara1☯, Waihong Chung1, Suzanne de la Monte1, John-Michael Thomas2, Mark Olsen2, Rolf Carlson1, Tunan Yu1, Xiaoqun Dong3¤, Jack Wands1* 1 Liver Research Center, Warren Alpert Medical School of Brown University and Rhode Island Hospital, Providence, Rhode Island, United States of America, 2 Department of Medical Chemistry, College of Pharmacy Glendale, Midwestern University, Glendale, Arizona, United States of America, 3 Department of Biomedical and Pharmaceutical Science, College of Pharmacy, The University of Rhode Island, Pharmacy Building, 7 Greenhouse Road, Kingston, Rhode Island, United States of America ☯ These authors contributed equally to this work. ¤ Current address: Department of Internal Medicine, College of Medicine, The University of Oklahoma Health Sciences Center, 975 NE10th Street, Oklahoma City, Oklahoma, United States of America * Abstract OPEN ACCESS Citation: Huang C-K, Iwagami Y, Aihara A, Chung W, de la Monte S, Thomas J-M, et al. (2016) Anti-Tumor Effects of Second Generation β-Hydroxylase Inhibitors on Cholangiocarcinoma Development and Progression. PLoS ONE 11(3): e0150336. doi:10.1371/journal. pone.0150336 Editor: Justin L Mott, University of Nebraska Medical Center, UNITED STATES Received: August 20, 2015 Accepted: February 11, 2016 Published: March 8, 2016 Copyright: © 2016 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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. Data Availability Statement: All relevant data are within the paper and its supporting information files. Funding: Funding for this work was provided by National Institutes of Health R01CA-123544 and 8P20GM103430-12. Competing Interests: The authors have declared that no competing interests exist. 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]. The 5-year survival rate is 15% for PLOS ONE | DOI:10.1371/journal.pone.0150336 March 8, 2016 1 / 18 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]. However, the underlying molecular mechanisms involved in CCA development and growth remain elusive. To clarify the cellular factors responsible for initiation and progression of CCAs, several genetically engineered murine models have been developed, such as a double knockout of SMAD4 and PTEN genes driven by an albumin promoter [3]; specific overexpression of the intracellular domain (ICN) of Notch1 driven by an albumin promoter [4]; a knockout of NF2 [5]; overexpression of mutant K-rasG12D and partial deletion of p53 driven by an albumin promoter [6], as well as a direct knockout of p53 driven by the CK19 promoter [7]. Most of these genetic changes have been previously described in human tumors based on whole-exome sequencing of liver fluke-related and non-infection-related bile duct tumors[8]. Notch signaling has been critically involved in CCA’s pathogenesis since overexpression of the ICN in the liver led to CCA development in animal models [4]. Aspartate β-hydroxylase (ASPH) is a Type II transmembrane protein and belongs to the αketoglutarate-dependent dioxygenase family [9]. ASPH catalyzes the hydroxylation of aspartyl and asparaginyl residues located in the epidermal growth factor (EGF)-like domain of various proteins [10]. ASPH has been described to be overexpressed in placenta, as well as the embryo during different stages of development but has very low or negligible expression in adult tissues [11]. Interestingly, ASPH becomes re-expressed in tumors of liver, pancreas, lung and colon [12–14], suggesting that ASPH may be an oncogene involved in the transformation of normal cells to a malignant phenotype [15]. This hypothesis raises the possibility that targeting ASPH to reduce its level or activity may suppress tumor growth and inhibit cellular migration and invasion [9, 16]. Previous studies have shown that the transcriptional expression of ASPH is regulated through insulin -insulin-like growth factor 1 stimulated MAPK/ERK and PI3K/AKT pathways [17]. Importantly, in hepatocellular carcinoma (HCC), Notch signaling can be activated directly by ASPH upregulation [9] to promote tumor cell migration, invasion and metastases. Since activation of Notch signaling is proposed to play a key role in the pathogenesis of CCA, inhibition of this signaling pathway may produce anti-tumor effects [4]. Therefore, we hypothesized that overexpression of the ASPH protein could be a major factor for CCA development and progression; and targeting this enzyme with a potent second generation small molecule inhibitor (SMI) of β-hydroxylase activity that was developed by rational drug design based on the crystal structure of the catalytic site, would constitute a novel therapeutic approa (...truncated)