MicroRNAs (miRs) are a novel class of cellular bioactive molecules with critical functions in the regulation of gene expression in normal biology and disease. MiRs are frequently misexpressed in cancer, with potent biological consequences. However, relatively little is known about miRs in pediatric cancers, including sarcomas. Moreover, the mechanisms behind aberrant miR expression in cancer are poorly understood. Ewing sarcoma is an aggressive pediatric malignancy driven by EWS/Ets fusion oncoproteins, which are gain-of-function transcriptional regulators. We employed stable silencing of EWS/Fli1, the most common of the oncogenic fusions, and global miR profiling to identify EWS/Fli1-regulated miRs with oncogenesis-modifying roles in Ewing sarcoma. In this report, we characterize a group of miRs (100, 125b, 22, 221/222, 27a and 29a) strongly repressed by EWS/Fli1. Strikingly, all of these miRs have predicted targets in the insulin-like growth factor (IGF) signaling pathway, a pivotal driver of Ewing sarcoma oncogenesis. We demonstrate that miRs in this group negatively regulate the expression of multiple pro-oncogenic components of the IGF pathway, namely IGF-1, IGF-1 receptor, mammalian/mechanistic target of rapamycin and ribosomal protein S6 kinase A1. Consistent with tumor-suppressive functions, these miRs manifest growth inhibitory properties in Ewing sarcoma cells. Our studies thus uncover a novel oncogenic mechanism in Ewing sarcoma, involving post-transcriptional derepression of IGF signaling by the EWS/Fli1 fusion oncoprotein via miRs. This novel pathway may be amenable to innovative therapeutic targeting in Ewing sarcoma and other malignancies with activated IGF signaling.

To identify microRNAs (miRNAs) associated with estrogen receptor (ESR1) status, we profiled luminal A, ESR1+ breast cancer cell lines versus triple negative (TN), which lack ERα, progesterone receptor and Her2/neu. Although two-thirds of the differentially expressed miRNAs are higher in ESR1+ breast cancer cells, some miRNAs, such as miR-222/221 and miR-29a, are dramatically higher in ESR1− cells (~100 and 16 fold higher, respectively). MiR-222/221 (which target ESR1 itself) and miR-29a are predicted to target the 3' UTR of Dicer1. Addition of these miRNAs to ESR1+ cells reduces Dicer protein, whereas antagonizing miR-222 in ESR1− cells increases Dicer protein. We demonstrate via luciferase reporter assays, that these miRNA directly target the Dicer1 3’UTR. In contrast, miR-200c, which promotes an epithelial phenotype, is 58 fold higher in the more well-differentiated ERα+ cells and restoration of miR-200c to ERα− cells causes increased Dicer protein, resulting in increased levels of other mature miRNAs typically low in ESR1− cells. Together our findings explain why Dicer is low in ERα negative breast cancers, since such cells express high miR-221/222 and miR-29a levels (which repress Dicer) and low miR-200c (which positively affect Dicer levels). Furthermore, we find that miR-7, which is more abundant in ERα cells and is estrogen regulated, targets growth factor receptors and signaling intermediates such as EGFR, IGF1R and IRS-2. In summary, miRNAs differentially expressed in ERα+ versus ERα− breast cancers actively control some of the most distinguishing characteristics of the luminal A and TN subtypes, such as ESR1 itself, Dicer and growth factor receptor levels.

Ewing's sarcoma is an aggressive pediatric cancer of the bone and soft tissue, in which patients whose tumors have a poor histological response to initial chemotherapy have a poor overall prognosis. Therefore, it is important to identify molecules involved in resistance to chemotherapy. Herein, we demonstrate that the DNA-repair protein and transcriptional cofactor, EYA3, is highly expressed in Ewing's sarcoma tumor samples and cell lines compared with mesenchymal stem cells, the presumed cell of origin of Ewing's sarcoma, and that it is regulated by the EWS/FLI1 fusion protein transcription factor. We further demonstrate that EWS/FLI1 mediates upregulation of EYA3 via repression of miR-708, a microRNA that targets the EYA3 3′UTR, rather than by binding the EYA3 promoter directly. Importantly, we demonstrate that high levels of EYA3 significantly correlate with low levels of miR-708 in Ewing's sarcoma samples, suggesting that this miR-mediated mechanism of EYA3 regulation holds true in human cancers. Because EYA proteins are important for cell survival during development, we examine, and demonstrate, that loss of EYA3 decreases survival of Ewing's sarcoma cells. Most importantly, knockdown of EYA3 in Ewing's sarcoma cells leads to sensitization to DNA-damaging chemotherapeutics used in the treatment of Ewing's sarcoma, and as expected, after chemotherapeutic treatment, EYA3 knockdown cells repair DNA damage less effectively than their control counterparts. These studies identify EYA3 as a novel mediator of chemoresistance in Ewing's sarcoma and define the molecular mechanisms of both EYA3 overexpression and of EYA3-mediated chemoresistance.

<p>PDF file - 652K, miR-708 increases apoptosis and sensitizes A673 Ewing's sarcoma cells to etoposide likely due to decreased DNA</p>