Osteosarcoma (OSA) represents the most common primary bone tumor in humans and pet dogs. Little progress has been made with regard to viable treatment options in the past three decades and patients presenting with metastatic disease continue to have a poor prognosis. Recent mouse studies have suggested that microRNA-34a (miR-34a) may have anti-tumor activities in human OSA models. Due to the conservation of microRNA across species, we hypothesized that a bioengineered miR-34a prodrug (tRNA/miR-34a) would have similar effects in canine OSA, providing a valuable preclinical model for development of this therapeutic modality. Using a panel of canine OSA cell lines, we found that tRNA/miR-34a reduced viability, clonogenic growth, and migration and invasion while increasing tumor cell apoptosis. Furthermore, canine OSA cells successfully process the tRNA/miR-34a into mature miR-34a which reduces expression of target proteins such as platelet derived growth factor receptor alpha (PDGFRα), Notch1 and vascular endothelial growth factor (VEGF). Additionally, our subcutaneous OSA xenograft model demonstrated in vivo tumor growth delay, increased necrosis and apoptosis by tRNA/miR-34a, and decreased cellular proliferation ability. Taken together, these data support that this novel microRNA-based therapy may possess clinical utility in a spontaneously-occurring large animal model of OSA, which can then serve to inform the clinical development of this therapy for human OSA patients.

This study was undertaken to investigate the therapeutic effect of a genetically engineered, human pre-microRNA-34a prodrug in canine osteosarcoma (OS), validating a robust preclinical model for the development of this proposed agent in humans. Although osteosarcoma is the most prevalent primary bone tumor in children and young adults, the low overall incidence presents an obstacle to timely development and testing of new agents. However, the incidence of canine OS is nearly 12 times that in humans, providing a useful preclinical model with spontaneously occurring OS. Despite multimodal therapeutic protocols, up to 40% of human and 90% of dogs will not survive long-term. Therefore, there is a clear need to identify new therapeutic agents for OS treatment. MicroRNAs (miRs) are small, noncoding RNAs that control relevant cellular pathways in tumorigenesis. MiR-34a, a downstream component of the p53 tumor suppressor, is downregulated in OS tumors and its reduced expression linked to poor therapeutic response. Recent evidence in human OS cell lines has shown that a novel genetically engineered human tRNA/miR-34a prodrug has anti-tumor effects following intracellular processing into mature miR-34a. We transfected a panel of canine OS cell lines (D17, HMPOS, Abrams and Gracie) with the human tRNA/miR-34a prodrug and evaluated the downstream effects to validate canine OS as a preclinical model. We measured miR-34a levels following transfection using quantitative real-time PCR. Proliferation was assessed via bioreductive and clonogenic assays. Western blot and immunofluorescence microscopy were used to investigate miR-34a target protein expression. Caspase 3/7 activity and TUNEL staining were used to measure apoptotic effects. In vitro properties associated with metastatic ability were assessed via transwell migration and invasion assays using Boyden chambers. We found significant increases in mature miR-34a levels following transfection, indicating that canine cells effectively process the prodrug. Increased miR-34a was associated with reduced PDGFRα protein expression and dose- and time-dependent inhibition of proliferation. Compared to control and MSA-transfected cells, miR-34a significantly reduced clonogenic cell growth, increased caspase 3/7 activity, and increased TUNEL positivity. We also found a significant inhibition of canine OS cell migration and invasion following transfection with miR-34a prodrug. Taken together, our findings demonstrate the successful internalization and processing of human miR-34a prodrug by canine OS cells. The increased miR-34a levels produce the same anti-tumor effects seen in human OS cells. These results strongly supports the use of canine OS as a preclinical model for the development of this therapy in human OS. Citation Format: Fernando Alegre Guerra, Kellie Snider, Amanda Ormonde, Ai-Ming Yu, Luke Wittenburg. Canine osteosarcoma as a platform to investigate the therapeutic potential of a genetically engineered pre-microRNA prodrug in human osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4406.