MiR-182 promotes cancer invasion by linking RET oncogene activated NF-κB to loss of the HES1/Notch1 regulatory circuit

Background: Dominant-activating mutations in the RET proto-oncogene, a receptor tyrosine kinase, are responsible for the development of medullary thyroid carcinoma (MTC) and causative for multiple endocrine neoplasia (MEN) type 2A and 2B. These tumors are highly aggressive with a high propensity for early metastasis and chemoresistance. This attribute makes this neoplasia an excellent model for probing mechanisms underlying cancer progression. Methods: The expression level of miR-182 was measured in MTC tumor specimens and in TT cells by real-time RT-PCR. TT cells and modified NThy-ori 3.1 that stably express RETM918T were used to investigate RET-dependent regulation of miR-182. Identification and validation of miR-182 targets and pathways was accomplished with luciferase assays, qRT-PCR, Western blotting and immunofluorescence. In vitro, overexpression and knockdown experiments were carried out to examine the impact of miR-182 and HES1 on invasion and migration. Results: We found that miR-182 expression is significantly upregulated in MTC patient samples and tumor-derived cell lines harboring mutated RET. Inhibition of RET oncogenic signaling through a dominant-negative RET Delta TK mutant in TT cells reduces miR-182, whereas overexpression of RETM918T in NThy-ori 3.1 cells increases miR-182 levels. We further show that overexpression of this miRNA in NThy. miR-182 cells promotes the invasive and migratory properties without affecting cell proliferation. MiR-182 is upregulated after RET induced NF-kappa B translocation into the nucleus via binding of NF-kappa B to the miR-182 promoter. Database analysis revealed that HES1, a repressor of the Notch pathway, is a target of miR-182, whose upregulation correlates with loss of HES1 transcription in MTC tissue samples and mutant RET cell lines. Moreover, we demonstrated that the 3'UTR of the HES1 mRNA bearing the targeting sequence for miR-182 clearly reduced luciferase reporter activity in cells expressing miR-182. Decreased expression of HES1 promotes migration by upregulating Notch1 inhibitor Deltex1 and consequent repression of Notch1. Conclusion: We demonstrate a novel mechanism for MTC aggressiveness in which mutated RET/NF-kappa B-driven expression of miR-182 impedes HES1 activation in a negative feedback loop. This observation might open new possibilities to treat RET oncogene associated metastatic cancer.