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

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 (\u3e95%) 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

Real-World Experience with AGA Guidelines in the Management of Crohn’s Disease following Ileocolonic Resection: A Retrospective Cohort Study

Background. Postoperative endoscopic recurrence (PER) is common in patients with Crohn’s disease (CD) after surgery. The impact of the American Gastroenterological Association (AGA) guideline adherence on PER in real life remains unclear. Methods. The postoperative management of CD patients undergoing ileocolonic resection with anastomosis from 2017 to 2018 was conducted based on the AGA guidelines. Colonoscopies were performed within one year after surgery. Clinical data and risk factors for endoscopic recurrence were analyzed focusing on postoperative pharmacological prophylaxis. Results. All patients were at a high risk of postoperative recurrence according to the AGA guidelines. PER occurred in 29 (28.7%) of these patients. The overall PER rate was 39.2% at one year. The PER rate in patients treated with nitroimidazole, thiopurines, infliximab, or a combination of thiopurines and infliximab for postoperative prophylaxis was 88.1%, 34.1%, 20.5%, and 0%, respectively. Cox regression showed that smoking at the time of surgery and AGA guideline adherence were independent factors associated with PER (HR: 3.75, 95% CI: 1.36-10.33, P=0.01; HR: 0.36, 95% CI: 0.15-0.86, P=0.02). In addition, further investigation revealed that educational background was the main factor related to patients’ nonadherence to AGA guidelines. Conclusions. The majority of CD patients who undergo surgery in clinical practice may be at a high risk of disease recurrence. Thiopurines and infliximab are effective in preventing endoscopic recurrence. Guideline nonadherence is associated with PER at one year, thus indicating that there is room for improvement in adherence to the AGA guidelines

ASPH expression promoted malignant phenotypes in CCA cell lines.

<p><b>(A)</b> MTT assay showing absorbance values measured at 0, 1, 3, and 5 days in H1 cells transfected with empty vector (EV) or ASPH. Right panel demonstrated colony formation in soft agar in H1-EV and H1-ASPH transfected cells. <b>(B)</b> A 5-day MTT and <b>(C)</b> 48 hr transwell migration assay were performed to determine cell proliferation and migration in ETK1 and SSP25 CCA cells infected with lentivirus containing shRNA-luciferase (shLuc), or shRNAs against ASPH (shASPH#1 and shASPH#2). Western blot analysis demonstrated decreased ASPH protein expression. <b>(D)</b> The number of colonies were measured in shLuc, shASPH, EV, ASPH, and ASPH^H675Q transfected H1 cells. <b>(E)</b> MTT absorbance and <b>(F)</b> migrated cell numbers were determined in ETK1 and RBE transfected with EV or ASPH^H675Q. ***, <i>p</i> <0.001; **, <i>p</i> <0.01; *, <i>p</i> <0.05.</p

Functional characterization of a SMI, MO-I-1151 on CCA phenotype.

<p>Relative cell proliferation rates were measured using MTT assay at day 0, 1, 3, and 5 in RBE and SSP25 cells <b>(A). (B)</b> Migrated cell numbers were determined in RBE and SSP25 in the presence or absence of 5 μM MO-I-1151. <b>(C)</b> Colony formation in soft agar was determined in H1 cells treated with or without MO-I-1151 at 5 μM. <b>(D)</b> Formation of CSC spheres were determined in H1 cells treated with DMSO or 5 μM MO-I-1151. (<b>E</b>) MO-I-1151 also increased caspase 3 cleavage in ETK1 and RBE cells. ***, <i>p</i> <0.001; *, <i>p</i> <0.05.</p

ASPH modulated Notch signaling in CCA cells.

<p><b>(A)</b> Protein expression levels of ASPH, activated Notch1, JAG1, HEY1, and HES1 in 5 CCA cell lines. <b>(B)</b> ASPH, cyclin D1, EpCAM, HES1, HEY1, and cleaved caspase-3 in HEK293 cells transfected with WT-ASPH plasmid at concentrations of 0, 0.5, 1, 1.5, 2, and 2.5 μg. <b>(C)</b> Semi-quantitation of immunoblotting results depicted in (<b>B</b>). <b>(D)</b> Expression levels of ASPH, cyclin D1, EpCAM, CD44, HES1, HEY1, and cleaved caspase-3 in H1, RBE, and ETK1 cells infected with lentivirus containing either shLuc or shASPH. <b>(E)</b> pCS2-Notch1-full-length-6MT (pCS2-Notch1-F.L.-6MT), EV, ASPH, ASPH^H675Q, and 12XCSL-DsRedExpressDL (Notch reporter) were co-transfected into HEK293 cells and image of red fluorescence was quantified under a fluorescence microscope. (F) Quantitation of red fluorescence signals are presented. Constitutive active Notch1 (pCS2-Notch1-ΔEMV-6MT) was used as a positive control. Transfection of Notch reporter construct alone was used as a negative control.</p

Anti-tumor effects of targeting ASPH in a rat intrahepatic CCA model of intrahepatic growth as well as subcutaneous tumor growth in nude mice xenograft with a human H1 CCA cell line.

<p><b>(A)</b> Gross morphology and histology (H&E staining) of rat livers inoculated with BDE-Neu-CL#24-shRNA-luciferase (shLuc) or BDE-Neu-CL#24-shRNA-ASPH (shASPH). <b>(B)</b> Tumor volume in rat livers of BDE-Neu cell clone (#24) generated intrahepatic CCA tumors. <b>(C)</b> (Upper) Expression of ASPH, activated Notch1, HES1, and HEY1 in shLuc and shASPH treated in BDE-Neu-CL#24 generated tumors. (Lower) Relative expression abundance was shown by the density measurements. (<b>D</b>) (Upper) Representative IHC images of activated Notch1 in shLuc and shASPH treated rat intrahepatic CCA. (Lower) Number of positive nuclei containing an activated Notch1 signal was calculated. <b>(E)</b> H1 xenograft tumor growth rate and progression was determined in nude mice receiving DMSO or MO-I-1151 treatment every other day at 25 mg/kg. **, <i>p</i> <0.01; *, <i>p</i> <0.05.</p

ASPH upregulated the expression of cyclin D1 expression and accelerated cell proliferation.

<p><b>(A)</b> Relative CCND1 mRNA expression in H1, RBE, SSP25, and ETK1 cells infected with lentivirus containing shLuc or shASPH. <b>(B)</b> Immunoblotting of ASPH, cyclin D1, and intracellular domain of Notch1 (ICN) in RBE-shLuc, RBE-shASPH, ETK1-shLuc, and ETK1-shASPH transfected cells with or without overexpression of ICN. <b>(C)</b> Relative cell proliferation in RBE-shLuc, RBE-shASPH, ETK1-shLuc, and ETK1-shASPH in the presence or absence of 10 μM of the γ-secretase inhibitor DAPT using the MTT assay. <b>(D)</b> Relative cell proliferation rate in RBE-shLuc, RBE-shASPH, RBE-shASPH transfected cells overexpressing ICN. ***, <i>p</i> <0.001; **, <i>p</i> <0.01; *, <i>p</i> <0.05.</p