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Title:Establishment of hedgehog pathway overexpression in osteosarcoma cell lines leading to an anti-apoptotic genotype which can be inhibited by the common anti-fungal and hedgehog pathway inhibitor, itraconazole
Author(s):Connell, Dana Rachelle
Advisor(s):Fan, Timothy M
Contributor(s):Foss, Kari; Barger, Anne
Department / Program:Vet Clinical Medicine
Discipline:Veterinary Clinical Medicine
Degree Granting Institution:University of Illinois at Urbana-Champaign
Abstract:Osteosarcoma (OS) is the most common primary bone cancer in dogs, yet therapies for pets with metastatic OS remain clinically ineffective. With the advent of precision medicine, strong scientific and clinical impetus exists to further investigate druggable molecular perturbations that might contribute to canine OS pathology. Dysregulated activity of the hedgehog (HH) pathway has been identified in diverse cancers in people and animals and leads to overexpression of anti-apoptotic proteins including Bcl-2 and c-FLIP. This produces an anti-apoptotic genotype by influencing both the intrinsic and extrinsic arms of the apoptotic pathway. Itraconazole is an antifungal agent that has been found to inhibit Smoothened (SMO), a crucial G protein-coupled receptor located on the cell membrane and becomes activated on the primary cilium, which allows initiation of HH pathway signaling. We hypothesize that components of the HH pathway are overexpressed in canine OS cell lines with concurrent downstream anti-apoptotic factor overexpression relative to non-malignant osteoblasts. Additionally, itraconazole would inhibit HH pathway signaling by promoting SMO relocation off the primary cilium. RNA transcript and protein expressions of SMO, Bcl-2 and c-FLIP were characterized in three canine OS cell lines and normal canine osteoblast cultures. The IC50 concentrations of itraconazole in OS cell lines were determined, and itraconazole’s effects on the HH pathway signaling partners were evaluated at RNA and protein levels. Confocal microscopy was utilized to confirm the modulation of protein levels after treatment with itraconazole. In addition, the effect of the combinations of itraconazole and commonly used cytotoxic agents, doxorubicin and carboplatin, were explored with regard to cell death. This data was used to assess the tolerability of itraconazole in combination with these chemotherapeutics in a murine model which was then used to explore the effects of itraconazole with or without cytotoxic agents on a metastatic osteosarcoma model. OS cell lines overexpress SMO, Bcl-2, and c-FLIP relative to normal osteoblasts. The IC50 of itraconazole in OS cells ranged from 490-790 nM. Itraconazole effectively perturbs protein expressions of the HH signaling pathway and downstream targets. The combination of itraconazole with carboplatin and itraconazole with doxorubicin was found to be synergistic compared to agents alone with regard to cell death. The combination of cytotoxin and itraconazole was found to be tolerable in mice with no cumulative toxicity. SMO and Bcl-2 are overexpressed in OS cells and itraconazole can disrupt the HH signaling pathway at biologically relevant concentrations, warranting further investigation of itraconazole as adjuvant therapy for OS.
Issue Date:2020-11-18
Rights Information:Copyright 2021 Dana Connell
Date Available in IDEALS:2021-09-17
Date Deposited:2021-05

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