Abstract A15: MiR-377 targets E2F3 and alters the NF-κB signaling pathway through MAP3K7 in malignant melanoma
Authors: Zehavi Liron , Layani Adi , Jacob-Hirsch Jasmine , Sidi Yechezkel , Leibowitz-Amit Raya , Avni Dror 
Published: May 2016

Background: The incidence of cutaneous malignant melanoma continues to rise, and once the disease metastasizes it is almost inevitably fatal. We were the first to report that a large micro-RNA (miRNA) cluster on human chromosome 14q32, implicated in many types of cancers, is significantly down-regulated in melanoma, and have been studying this ‘tumor-suppressor miRNA cluster’ for several years now. MiR-377, one of the miRNAs located within this cluster, was studied in this work.

Methods: qRT-pCR was used to quantify miR-377 levels in melanoma cell lines and samples. Melanoma cell lines ectopically expressing miR-377 were generated by stable transfection and mRNA expression was assessed using mRNA arrays. Potential targets of miR-377 were identified through luciferase reporter assays. Cellular proliferation, migration and soft-agar colony formation were monitored in control and miR-377-expressing cells using cell biology techniques and protein expression was assessed by western blot.

Results: miR-377 is expressed in normal melanocytes but not in melanoma cell lines or samples. Its ectopic stable expression in melanoma cell lines decreased their proliferative and migratory capacity and their colony-forming capability. mRNA arrays of melanoma cells over-expressing miR-377 pointed to several down-regulated mRNAs that have putative binding sites for miR-377 in their 3'UTR, of which both E2F3 and MAP3K7 were found to be direct targets of miR-377.

E2F3, a potent transcriptional inducer of cell-cycle progression, was found to be elevated in melanoma cell lines, but decreased following ectopic expression of miR-377. Ectopic miR-377 also led to a decrease in the activity of a reporter plasmid containing three E2F DNA-binding sites linked to a luciferase cDNA sequence, demonstrating that miR-377 down-regulates E2F3-induced transcription.

MAP3K7, a serine/threonine kinase along the MAPK signaling pathway, was over-expressed in melanoma but decreased following ectopic expression of miR-377. MAP3K7 is known to be involved in the activation of NF-κB. MiR-377 over-expression led to decreased activity of a reporter plasmid containing two NF-κB DNA-binding sites and to decreased output along the NF-κB signaling pathway.

Conclusion Our results suggest that miR-377 is an important negative regulator of E2F and of the MAP3K7/NF-κB signaling pathway in melanoma cells. The NF-κB signaling has been implicated in the acquisition of resistance to B-Raf inhibition. It is tempting to speculate that silencing of miR-377 in melanoma promotes the tumorigenic and metastatic potential of the cells through activation of these pathways. Indeed we found in preliminary results that overexpression of miR-377 in melanoma cells resistance to PLX-4032 (Zelboraf), re-sensitized the cells to the drug.

Citation Format: Liron Zehavi, Adi Layani, Jasmine Jacob-Hirsch, Yechezkel Sidi, Raya Leibowitz-Amit, Dror Avni. MiR-377 targets E2F3 and alters the NF-κB signaling pathway through MAP3K7 in malignant melanoma. [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 Res 2016;76(6 Suppl):Abstract nr A15.