A dynamic microRNA profile that tracks a chemotherapy resistance phenotype in osteosarcoma. Implications for novel therapeutics.

Abstract

Osteosarcoma is a rare primary bone tumor for which no significant therapeutic advancement has been made since the late 1980s despite ongoing efforts. Overall, the five-year survival rate remains about 65%, and is much lower in patients with tumors unresponsive to methotrexate, doxorubicin, and cisplatin therapy. Genetic studies have not revealed actionable drug targets, but our group, and others, have reported that epigenomic biomarkers, including regulatory RNAs, may be useful prognostic tools for osteosarcoma. We tested if microRNA (miRNA) transcriptional patterns mark the transition from a chemotherapy sensitive to resistant tumor phenotype. Small RNA sequencing was performed using 14 patient matched pre-chemotherapy biopsy and post-chemotherapy resection high-grade osteosarcoma frozen tumor samples. Independently, small RNA sequencing was performed using 14 patient matched biopsy and resection samples from untreated tumors. Separately, miRNA specific Illumina DASL arrays were used to assay an independent cohort of 65 pre-chemotherapy biopsy and 26 patient matched post-chemotherapy resection formalin fixed paraffin embedded (FFPE) tumor samples. mRNA specific Illumina DASL arrays were used to profile 37 pre-chemotherapy biopsy and five post-chemotherapy resection FFPE samples, all of which were also used for Illumina DASL miRNA profiling. The National Cancer Institute Therapeutically Applicable Research to Generate Effective Treatments dataset, including PCR based miRNA profiling and RNA-seq data for 86 and 93 pre-chemotherapy tumor samples, respectively, was also used. Paired differential expression testing revealed a profile of 17 miRNAs with significantly different transcriptional levels following chemotherapy. Genes targeted by the miRNAs were differentially expressed following chemotherapy, suggesting the miRNAs may regulate transcriptional networks. Finally, an in vitro pharmacogenomic screen using miRNAs and their target transcripts predicted response to a set of candidate small molecule therapeutics which potentially reverse the chemotherapy resistance phenotype and synergize with chemotherapy in otherwise treatment resistant tumors. Importantly, these novel therapeutic targets are distinct from targets identified by a similar pharmacogenomic analysis of previously published prognostic miRNA profiles from pre chemotherapy biopsy specimens.