Abstract B05: MicroRNA-206 drives rhabdomyosarcoma differentiation through downregulation of PAX7

The goal of this work is to understand the mechanism of microRNA-206 (miR-206) induced differentiation in rhabdomyosarcoma (RMS). RMS is the most common soft tissue sarcoma of childhood with both histology and gene expression suggestive that RMS tumor cells resemble a developmental arrest of skeletal muscle. miR-206 is a skeletal muscle specific miRNA expression increases through normal muscle development and has decreased levels in RMS compared to normal skeletal muscle. Exogneous miR-206 replacement drives differentiation and cell cycle exit of RMS. However, the target genes responsible for the relief of the development arrest are largely unknown. Using a combinatorial approach with mRNA microarrays and tandem mass tag (TMT) proteome profiling we identified 165 mRNAs and 272 proteins downregulated in miR-206 mimic transfected RMS cells. Potential targets were validated with 3'UTR reporter assays as well as real-time PCR and immunoblot from miR-206 mimic transfected cells. Further analysis with genetic rescue identified the key targets responsible for the RMS differentiation block as PAX7, PAX3, CCND2, and NOTCH3. However, siRNA knockdown of these targets alone and in a pooled combination failed to induce differentiation to the same extent as miR-206 despite knockdown levels comparable to miR-206. Thus, target identification collectively is key to understanding the mechanism of miR-206 function in RMS, suggesting the one miRNA-one target model to be insufficient for the complex phenotype of miR-206 induced differentiation. Genetic deletion of miR-206 in a mouse model of RMS accelerated and exacerbated tumor development indicating that both in vitro and in vivo miR-206 possesses tumor suppressive properties. In addition, Notch3 and Pax7 levels were increased in knockout tumors compared to wild type, providing further evidence for these genes as key targets of miR-206 in RMS. These results illustrate that miR-206 relieves the differentiation arrest in RMS and suggests that miR-206 replacement therapy could be a potential therapeutic strategy. Citation Format: Jason A. Hanna, Matthew R. Garcia, Jonathan C. Go, David Finkelstein, Kiran Kodali, Vishwajeeth Pagala, Eric N. Olson, Junmin Peng, Mark E. Hatley. MicroRNA-206 drives rhabdomyosarcoma differentiation through downregulation of PAX7. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer