Abstract 540: Characterization of miR-9 expression and activation in canine osteosarcoma
Authors: Fenger Joelle M. , Couto Jason I. , Bear Misty D. , Volinia Stefano , Modiano Jaime F. , Breen Matthew , London Cheryl A. , Kisseberth William C. 
Published: May 2015

Abstract
Introduction: Osteosarcoma (OSA) is the most common malignant bone tumor in children and dogs. Despite aggressive treatment, no improvement in survival times has been achieved in the past 15 years with 40% of children and 90% of dogs still dying from disease. MicroRNAs (miRs) are non-protein coding RNAs that have been implicated as having a fundamental role in cancer. We analyzed primary canine OSA tumors and canine osteoblasts for miR expression and found miR-9 to be highly expressed in OSA tumors and cell lines as compared to normal osteoblasts. We hypothesize that miR-9 overexpression generates a pattern of gene dysregulation that contributes to the pathogenesis of OSA.
Methodology: Canine bone marrow derived stem cells were differentiated in vitro into osteoblasts and expression of bone-specific markers (ALP, OP, BMP-2) was detected by immunocytochemistry or RT-PCR. MiR expression profiling was performed on 48 primary canine OSA tumors and 3 osteoblast cultures using the NanoString nCounter human microRNA Expression Assay. Taqman miRNA assays were used to measure miR-9 expression in primary canine OSA tumors, OSA cell lines, primary osteoblast cultures, and commercially available osteoblast cell lines. Canine OSA16 and osteoblast cell lines were transduced with lentiviral pre-miR-9 or empty control vector constructs and cells were evaluated for differences in proliferative capacity, apoptosis, and the ability to migrate through Matrigel.
Results and Conclusions: We identified 84 miRs (p ≤ 0.01) differentially expressed in primary canine OSA tumors compared to canine osteoblasts. MiR-9 was highly expressed in primary OSA tumors and cell lines as compared to normal osteoblast cell lines or primary cultures. Canine OSA16 and normal osteoblast cell lines, which express low levels of miR-9 were transduced with miR-9 lentiviral constructs resulting in high levels of miR-9 expression. Overexpression of miR-9 did not effect cell proliferation or apoptosis, but enhanced invasion through matrigel in both malignant OSA cells and normal osteoblasts. Our findings suggest that miR-9 may play an important role in regulating OSA cell invasion and suggest that overexpression of miR-9 in vivo may promote metastasis in canine OSA. Transcriptional and proteomic profiling of osteoblasts overexpressing miR-9 is underway to define key differences in mRNA and protein expression and phosphorylation that may contribute to the metastatic phenotype in canine OSA. This work will provide significant new data regarding the impact of miR-9 on molecular pathways in OSA, thereby laying the foundation for the development and testing of novel therapeutics in both children and dogs affected by this disease.
Citation Format: Joelle M. Fenger, Jason I. Couto, Misty D. Bear, Stefano Volinia, Jaime F. Modiano, Matthew Breen, Cheryl A. London, William C. Kisseberth. Characterization of miR-9 expression and activation in canine osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 540. doi:10.1158/1538-7445.AM2014-540