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

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.

Molecular Mechanisms Driving Cholangiocarcinoma Invasiveness: An Overview

“…ASPH is a type 2 transmembrane protein that catalyzes the hydroxylation of EGF-like domains in several target proteins, including Notch1 and Jag-1, thereby modulating their function (e.g., by increasing protein stability) 96 . In CCA cells, shRNA-mediated down-regulation of ASPH substantially blunted Notch signaling, as well as cell migration, both in vitro and in vivo 83 . Importantly, increased ASPH expression was noticed at the invasive front of CCA specimens, where high levels of ASPH expression correlated with vascular invasion and poor prognosis 97 .…”

“…Among them are transcription factors belonging to morphogenetic signaling pathways that are active during biliary development, and become reactivated during liver repair and carcinogenesis, including Hedgehog 77 , Wnt 68,78 , and notably, Notch 79–83 signaling. YAP/TAZ are less characterized transcriptional co-activators critically involved in organ development that are drawing much attention in cancer research and thus, will be discussed in detail.…”

“…Inflammatory mediators such as inducible nitric oxide synthase may be partially responsible for Notch1 up-regulation, via activation of JNK1/2 signaling 95 . It is also important to underline that in CCA cells, Notch signaling can be persistently activated even in lack of exogenous stimuli, as demonstrated by Hes1 and Hey1 expression in untreated cells, likely implying an autocrine stimulation of Notch receptors, which might be mediated by Jag-1 79,83 . At the functional level, it was shown that forced overexpression of Notch1 significantly promoted CCA cell migration, along with prototypical EMT-like changes (i.e., down-regulation of E-cadherin, up-regulation of vimentin and α-SMA) 80 .…”

“…In CCA cells, forced Sox9 up-regulation boosted cell migration and invasion, decreased E-cadherin expression, and increased vimentin and α-SMA expression, whereas Sox9 silencing dampened the migratory/invasive capabilities, similarly to what observed with Notch1 manipulation 82 . Recently, aspartate β-hydroxylase (ASPH) has been identified as a critical upstream regulator of Notch1 transcriptional activity in CCA 83 . ASPH is a type 2 transmembrane protein that catalyzes the hydroxylation of EGF-like domains in several target proteins, including Notch1 and Jag-1, thereby modulating their function (e.g., by increasing protein stability) 96 .…”

Aspartate-β-hydroxylase (ASPH): A potential therapeutic target in human malignant gliomas

“…Finally, we conducted in vitro experiments to determine the degree to which treatment of astrocytoma cells with small molecule inhibitors of ASPH’s catalytic activity would be sufficient to decrease cell motility and invasion. The research design was focused on ASPH rather than Humbug because the Type 2 transmembrane structure of ASPH renders its critical catalytic domain accessible to small molecule inhibitor [15, 16] and immune [17, 18] targeting, as demonstrated in other malignancies.…”