The Role of Canonical Hedgehog Signaling in Canine
Fellow: Vincent Baldanza
Mentor: Angela McCleary-Wheeler
DESCRIPTION (provided by applicant):
Canine osteosarcoma (cOSA), the most common primary bone malignancy in dogs, is a highly aggressive tumor with an estimated micro-metastatic rate of approximately 90%. Even with surgery and chemotherapy, the median survival time is only 10-12 months. The pathogenesis, disease course, and treatment response of cOSA closely parallels that of human pediatric OSA (hOSA), and it has thus been proposed as a spontaneous animal model of the disease. In hOSA, the Hedgehog (HH) developmental signaling pathway has been documented to contribute to the pathogenesis of the disease. Canonical HH signaling occurs through a two receptor mechanism in which the receptor Patched (PTCH) normally maintains an inhibitory function over the receptor Smoothened (SMO) in the absence of its ligands. Binding of its ligands, such as Sonic Hedgehog (SHH) or Indian Hedgehog (IHH), releases the inhibitory effect PTCH has on SMO. This event leads to activation of the downstream cascade, concluding with the activation of the glioma-associated oncogene (GLI) transcription factors that regulate genes responsible for tumorigenesis and metastasis.
In normal bone, the HH pathway tightly regulates growth and differentiation. In hOSA, though, activation of HH or its pathway components appears to play a role in the disease process. While inhibition of GLI and SMO has been shown to suppress hOSA cellular proliferation and growth in vivo, little work has been completed in the canine model. Building off of the data in hOSA, we hypothesize that HH signaling is also active in cOSA, and targeted inhibition of the canonical pathway will negatively impact OSA cell growth and survival. We propose to address our hypothesis with two major aims: 1) determine endogenous protein localization and expression in cOSA tissue samples and cell lines, and 2) examine the downstream effects of targeted HH inhibition in vitro.
In order to characterize the expression patterns of HH receptors, ligands, and transcription factors in cOSA, we will use immunohistochemistry (IHC) and quantitative RT-PCR (qRT-PCR). Twenty cOSA samples have been identified from the CUHA Anatomic Pathology Archives and IHC will be performed using antibodies against SHH, IHH, SMO, PTCH1, GLI1, and GLI2. We have to date optimized and performed IHC for SMO and GLI1. Protein and mRNA expression will be further examined in two established cOSA cell lines, D17 and Abrams, using Western blotting and qRT-PCR. We have begun antibody validation for Western blot using specific siRNA and/or expression plasmids containing the canine genes of interest we have cloned in our lab.
Simultaneously, we will evaluate the effect targeted HH inhibition has on cell proliferation and survival in vitro. We are using a SMO specific inhibitor, vismodegib, which is FDA-approved for human basal cell carcinoma. Canine OSA cell lines will be treated with a range of concentrations of vismodegib over time. Alterations in cell biology will be examined such as proliferation via MTT assay, cell cycle kinetics using flow cytometry, and apoptosis through Annexin-V staining and a colorimetric Caspase-3 activation assay. In addition HH target gene expression will be evaluated using Western blotting and qRT-PCR for Cyclin D1, Bcl-2, PTCH1, and GLI1. We have begun treating these cell lines with vismodegib and have identified promising, early results via MTT assay.
The results provided by these experiments will further explore the role of HH signaling in cOSA and act as a foundation for future experiments and clinical trials exploring more efficacious, targeted therapeutics for the treatment of this disease in dogs. Furthermore, it may provide more evidence for the use of dogs as a relevant, comparative model of this cancer. This research project will also allow me to develop as a veterinary oncologist by exposing me to new research techniques and more critically think about clinical problems.