Growth-Promoting Role of the miR-106a∼363 Cluster in Ewing Sarcoma

Abstract

MicroRNAs (miRs) have been identified as potent regulators of both normal development and the hallmarks of cancer. Targeting of microRNAs has been shown to have preclinical promise, and select miR-based therapies are now in clinical trials. Ewing Sarcoma is a biologically aggressive pediatric cancer with little change in clinical outcomes despite improved chemotherapeutic regimens. There is a substantial need for new therapies to improve Ewing Sarcoma outcomes and to prevent chemotherapy-related secondary sequelae. Most Ewing Sarcoma tumors are driven by the EWS/Fli-1 fusion oncoprotein, acting as a gain-of-function transcription factor causing dysregulation of a variety of targets, including microRNAs. Our previous studies, and those of others, have identified upregulation of miRs belonging to the related miR-17∼92a, miR-106b∼25, and miR-106a∼363 clusters in Ewing Sarcoma. However, the functional consequences of this have not been characterized, nor has miR blockade been explored as an anti-cancer strategy in Ewing Sarcoma. To simulate a potential therapeutic approach, we examined the effects of blockade of these clusters, and their component miRs. Using colony formation as a read-out, we find that blockade of selected individual cluster component miRs, using specific inhibitors, has little or no effect. Combinatorial inhibition using miR “sponge” methodology, on the other hand, is inhibitory to colony formation, with blockade of whole clusters generally more effective than blockade of miR families. We show that a miR-blocking sponge directed against the poorly characterized miR-106a∼363 cluster is a particularly potent inhibitor of clonogenic growth in a subset of Ewing Sarcoma cell lines. We further identify upregulation of miR-15a as a downstream mechanism contributing to the miR-106a∼363 sponge growth-inhibitory effect. Taken together, our studies provide support for a pro-oncogenic role of the miR-106a∼363 cluster in Ewing Sarcoma, and identify miR-106a∼363 blockade, as well as miR-15a replacement, as possible strategies for inhibition of Ewing Sarcoma growth.

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“…Given that miRNAs lie in the central of ceRNA interplay, it is reasonable for us to envision the potentials of cancer-related ceRNAs as therapeutic targets. For example, artificial miRNA sponges containing multiple copies of MREs in tandem have been used to inhibit oncogenic miRNAs and showed tumour suppressive effects in multiple cancer cell lines or xenograft animal models such as Ewing sarcoma,92 renal cancer,93 liver cancer94 and lung cancer 95 96…”

Inhibition of microRNA 128 promotes excitability of cultured cortical neuronal networks

“…1; Supplemental Table S1). To control for effects of lentiviral transduction, we tested two controls that expressed either a nontargeting shRNA or a sponge containing sequences complementary to an artificial miRNA (Ebert et al 2007;Elcheva et al 2009;Dylla and Jedlicka 2013). Each control behaved similarly in comparison with the miR-128 sponge (Supplemental Figs S3,S6).…”