Survivin blocking drug tested on dog lymphoma and bone cancer cells

Abstract

Abstract Canine lymphoma (LSA) and osteosarcoma (OS) have high mortality rates and remain in need of more effective therapeutic approaches. Due to the strong similarities between canine and human LSA and OS, canine LSA and OS are excellent models for the human disease. Survivin, an IAP family member protein that inhibits apoptosis and drives cell proliferation, is commonly elevated in human and canine cancer. Survivin expression is a negative prognostic factor in dogs and humans with LSA and OS. The objective of our research was to determine the effects of survivin inhibition using a locked nucleic acid antisense oligonucleotide (EZN-3042) on canine LSA and canine OS cell lines, with respect to cell proliferation, apoptosis, and chemosensitivity in vitro. Furthermore, we sought to determine the efficacy of EZN-3042 on inhibition of survivin transcription, survivin protein production, and tumor growth in subcutaneous and orthotopic canine OS xenografts. We inhibited survivin using EZN-3042 in two canine LSA and two canine OS cell lines. Survivin inhibition was confirmed by qRT-PCR and western blot. Percent dead and total cell number was assessed by manual cell counting with trypan blue. Growth inhibition was confirmed with a bioreductive fluorometric assay. A caspase-3/7 assay was used to determine levels of apoptosis in EZN-3042 treated cells compared to controls. Cells were then treated with antineoplastic drug doxorubicin (DOX) +/- EZN-3042 and assays repeated. In vivo, nude mice with subcutaneous and orthotopic OS xenografts were given 100 mg/kg EZN3042 intraperitoneally. Survivin inhibition was confirmed with Immunohistochemistry and qRT-PCR analysis. Survivin inhibition in canine LSA and OS cells via EZN-3042 resulted in 34-72% decrease in survivin protein expression and a 1.3-3.4 fold decrease in endogenous survivin mRNA expression. When EZN-3042 treated cells were compared to controls, total and viable cell numbers were decreased, and apoptosis was increased. Survivin inhibition via EZN-3042 enhanced canine LSA and OS cell lines sensitivity to DOX. IHC and qRT-PCR analysis of subcutaneous and orthotopic canine OS xenografts confirmed decreased tumor survivin expression in EZN-3042 treated mice. Mice treated with EZN-3042 in combination with DOX had significantly decreased tumor growth when compared to single agent treatment and control groups. These results demonstrate that survivin inhibition via EZN-3042 decreased LSA and OS cell proliferation, and increased cellular apoptosis and chemosensitivity to DOX, and that EZN-3042 treatment inhibited tumor survivin expression in vivo and significantly decreased tumor growth when combined with DOX. Survivin-directed therapies may be highly effective in treatment of both canine and human LSA and OS, and spontaneous canine cancer may be a valuable model for the evaluation of survivin-targeted treatment. Citation Format: Jenette K. Shoeneman, Eugene J. Ehrhart, III, Joseph B. Charles, Douglas H. Thamm. Survivin inhibition via EZN-3042 in canine models of lymphoma and osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B35.