Abstract
BackgroundHairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Notch signaling and HES1 expression have been linked to growth and survival in a variety of human cancer types and have been associated with increased metastasis and invasiveness in human osteosarcoma cell lines. Osteosarcoma (OSA) is an aggressive cancer demonstrating both high metastatic rate and chemotherapeutic resistance. The current study examined expression of Notch signaling mediators in primary canine OSA tumors and canine and human osteosarcoma cell lines to assess their role in OSA development and progression.ResultsReverse transcriptase - quantitative PCR (RT-qPCR) was utilized to quantify HES1, HEY1, NOTCH1 and NOTCH2 gene expression in matched tumor and normal metaphyseal bone samples taken from dogs treated for appendicular OSA at the Colorado State University Veterinary Teaching Hospital. Gene expression was also assessed in tumors from dogs with a disease free interval (DFI) of <100 days compared to those with a DFI > 300 days following treatment with surgical amputation followed by standard chemotherapy. Immunohistochemistry was performed to confirm expression of HES1. Data from RT-qPCR and immunohistochemical (IHC) experiments were analyzed using REST2009 software and survival analysis based on IHC expression employed the Kaplan-Meier method and log rank analysis. Unbiased clustered images were generated from gene array analysis data for Notch/HES1 associated genes.Gene array analysis of Notch/HES1 associated genes suggested alterations in the Notch signaling pathway may contribute to the development of canine OSA. HES1 mRNA expression was elevated in tumor samples relative to normal bone, but decreased in tumor samples from dogs with a DFI < 100 days relative to those with a DFI > 300 days. NOTCH2 and HEY1 mRNA expression was also elevated in tumors relative to normal bone, but was not differentially expressed between the DFI tumor groups. Survival analysis confirmed an association between decreased HES1 immunosignal and shorter DFI.ConclusionsOur findings suggest that activation of Notch signaling occurs and may contribute to the development of canine OSA. However, association of low HES1 expression and shorter DFI suggests that mechanisms that do not alter HES1 expression may drive the most aggressive tumors.
Highlights
Hairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling
In order to explore the hypothesis that Notch signaling would be altered in canine OSA compared to normal bone samples, the current study examines the expression of NOTCH1 and 2 receptors and signaling targets, HES1 and HEY1, in canine OSA samples from patients with known outcome and normal bone tissues
Gene expression analysis of Notch/HES1-associated genes groups normal and OSA bone samples, but does not distinguish disease free interval (DFI) groups To assess the biological relevance of Notch/HES1 signaling in canine osteosarcoma, probesets including Notch receptor ligands, effectors, or targets of either the canonical Notch pathway or HES1 were selected from Canine 2.0 gene array data and analyzed for differential gene expression as described in materials and methods
Summary
Hairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Standard of care therapy for both human and canine OSA patients remains a combination of surgery and chemotherapy, with five-year survival rates reported in humans as high as 70% [1,8] and median survival in canine patients around 200 days [2]. In both human and canine patients approximately 80% are estimated to have micrometastases at presentation, some of whose tumors are refractory to chemotherapy [2,8]. The focus of recent research, has turned toward molecular characterization of primary tumors, especially aberrant gene and/or protein expression that might correlate with prognosis or chemotherapy sensitivity
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