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.

Fig 1. ASPH expression promoted malignant phenotypes in CCA cell lines.

(A) 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) A 5-day MTT and (C) 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. (D) The number of colonies were measured in shLuc, shASPH, EV, ASPH, and ASPH^H675Q transfected H1 cells. (E) MTT absorbance and (F) migrated cell numbers were determined in ETK1 and RBE transfected with EV or ASPH^H675Q. ***, p <0.001; **, p <0.01; *, p <0.05.

Fig 2. ASPH modulated Notch signaling in CCA cells.

(A) Protein expression levels of ASPH, activated Notch1, JAG1, HEY1, and HES1 in 5 CCA cell lines. (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. (C) Semi-quantitation of immunoblotting results depicted in (B). (D) 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. (E) 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.

Fig 3. ASPH upregulated the expression of cyclin D1 expression and accelerated cell proliferation.

(A) Relative CCND1 mRNA expression in H1, RBE, SSP25, and ETK1 cells infected with lentivirus containing shLuc or shASPH. (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. (C) 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. (D) Relative cell proliferation rate in RBE-shLuc, RBE-shASPH, RBE-shASPH transfected cells overexpressing ICN. ***, p <0.001; **, p <0.01; *, p <0.05.

Fig 4. Functional characterization of a SMI, MO-I-1151 on CCA phenotype.

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

Fig 5. 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.

(A) 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) Tumor volume in rat livers of BDE-Neu cell clone (#24) generated intrahepatic CCA tumors. (C) (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. (D) (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. (E) 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. **, p <0.01; *, p <0.05.