Abstract A14: MiR-34a regulates the invasive capacity of canine osteosarcoma cell lines: A comparative oncology study

Background: Osteosarcoma (OS) is the most common bone tumor in children and dogs; however, no substantial improvement in clinical outcome has occurred in either species over the past 30 years. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and play a fundamental role in cancer. Given that canine OS is a well-validated spontaneous large animal model of the human disease, the purpose of this study was to characterize the impact of miR-34a expression in canine OS tumor lines to better understand its potential role in the biology of this disease. Methods: Real-time PCR was used to assess miR-34a expression in primary canine OS tumors, canine OS cell lines, and normal canine osteoblasts. Canine OSA8 and Abrams cell lines were stably transduced with empty vector or pre-miR-34a lentiviral constructs to determine the consequences of miR-34a on cell proliferation, cell cycle distribution, invasion, and migration. Gene expression profiling of canine OSA8 cells with enforced miR-34a expression was performed using RNA sequencing and changes in mRNA expression were validated using real-time PCR. Results: Real-time PCR demonstrated that miR-34a expression levels were significantly decreased in primary canine OS tumors and canine OS cell lines as compared to normal canine osteoblasts. In canine OS cell lines stably transduced with empty vector or pre-miR-34a lentiviral constructs, overexpression of miR-34a inhibited cellular invasion and migration but had no effect on cell proliferation or cell cycle distribution. Transcriptional profiling of canine OSA8 cells possessing enforced miR-34a expression demonstrated dysregulation of numerous genes, including significant downregulation of multiple putative targets of miR-34a. Moreover, gene ontology analysis of downregulated miR-34a target genes showed enrichment of several biologic processes related to cell invasion and motility. Lastly, we validated changes in miR-34a target gene expression, including decreased expression of KLF4, SEM3A, and VEGFA transcripts in canine OS cells overexpressing miR-34a. Concordant with these data, primary canine OS tumor tissues demonstrated increased expression levels of putative miR-34a target genes. Conclusions: Our data demonstrate that miR-34a expression is significantly decreased in primary canine OS tumor tissues and canine OS cell lines compared to normal canine osteoblasts. These results are concordant with data generated in human OS tumors, suggesting that loss of miR-34a may be fundamental to the disease process in both species. Our data demonstrate that miR-34a contributes to invasion and migration in canine OS cells and suggest that loss of miR-34a may promote a pattern of gene expression contributing to the metastatic phenotype in canine OS. As such, miR-34a may represent a novel target for therapeutic intervention in OS. Citation Format: Cecilia M. Lopez, Peter Y. Yu, Xiaoli Zhang, Ayse Selen Yilmaz, Cheryl A. London, Joelle M. Fenger. MiR-34a regulates the invasive capacity of canine osteosarcoma cell lines: A comparative oncology study [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A14.