Abstract
Downregulation of microRNAs (miRNAs) at the 14q32 locus stabilizes the expression of cMYC, thus significantly contributing to osteosarcoma (OS) pathobiology. Here, we show that downregulation of 14q32 miRNAs is epigenetically regulated. The predicted promoter regions of miRNA clusters at 14q32 locus showed no recurrent patterns of differential methylation, but Saos2 cells showed elevated histone deacetylase (HDAC) activity. Treatment with 4-phenylbutyrate increased acetylation of histones associated with 14q32 miRNAs, but interestingly, robust restoration of 14q32 miRNA expression, attenuation of cMYC expression, and induction of apoptosis required concomitant treatment with 5-Azacytidine, an inhibitor of DNA methylation. These events were associated with genome-wide gene expression changes including induction of pro-apoptotic genes and downregulation of cell cycle genes. Comparable effects were achieved in human and canine OS cells using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) and the DNA methylation inhibitor Zebularine (Zeb), with significantly more pronounced cytotoxicity in cells whose molecular phenotypes were indicative of aggressive biological behavior. These results suggested that the combination of these chromatin-modifying drugs may be a useful adjuvant in the treatment of rapidly progressive OS.
Highlights
Osteosarcoma (OS), the most common primary tumor of bone, principally affects children and adolescents
We further showed that downregulation of 14q32 miRNAs was not due to recurrent deletion of this locus [7]; we investigated the possibility that downregulation of 14q32 miRNAs was controlled epigenetically through DNA methylation and histone acetylation
DNA methylation pattern observed at these 14q32 miRNA regions did not support the differential expression of these miRNAs in normal bone and OS tumor tissues, suggesting that DNA methylation may not be a primary factor attributing to the downregulation of these miRNAs in human OS
Summary
Osteosarcoma (OS), the most common primary tumor of bone, principally affects children and adolescents. Chromosome 14q32 locus represents one of the most miRNAenriched regions in the human genome, containing .40 distinct miRNAs. Chromosome 14q32 locus represents one of the most miRNAenriched regions in the human genome, containing .40 distinct miRNAs These miRNAs are grouped into at least four clusters; and the locus appears to be involved in several diseases [11], including lymphoblastic leukemias [12], high-grade ovarian tumors [13], gastrointestinal stromal tumors [14], and colon cancer [15]. Homologous regions of these miRNA clusters in mouse are highly conserved [16] and imprinted maternally and paternally. The 14q32 miRNA locus is not recurrently deleted in human OS [7] but its regulation remains incompletely understood
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