Abstract 183: BRAF oncogene induces miR-17-92 cluster in thyroid cells and iodine counteracts this effect

Abstract

Abstract Background: High dose iodine intake is used as a prophylactic measure to protect thyroid from deleterious effects of radioactive iodine in case of nuclear accidents. In vitro, high concentration iodine delays oncogenic effects of RET/PTC3 in thyroid follicular cells, although its role during BRAF oncogene activation remains unclear. BRAF mutation (T1799A) is the most prevalent alteration during thyroid follicular cells oncogenesis leading to thyroid cancer. BRAFT1799A is associated to aggressive thyroid tumors and poor prognosis. High levels of microRNAs (miRNAs) miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b and miR-92, components of miR-17-92 cluster, are associated with several aggressive tumors such as lung cancer and thyroid carcinoma. Aberrant miRNA expression may cooperate to tumorigenesis as it regulates protein levels of different target mRNA from several signaling pathways. Therefore, in this study we aim at analyzing the role of iodine in miRNAs activated by BRAFT1799A oncogene. Methods: Cell culture and treatment: BRAF9-6 cells derived from rat thyroid follicular cells express conditionally BRAFT1799A in the presence of doxaciclin (Dox, at 1µg/mL). Early BRAFT1799A activation was analyzed after 48 hours of induction (Dox group). Five-days long iodine treatment was performed using culture media containing 10-5M sodium iodide (NaI group), and dox was added at day 3 (Dox+NaI group). Control group (Ctr) was performed in the absence of Dox and NaI; gene expression: detection of miR-18a, miR-19a, miR-19b and snoRNA was performed by qPCR. protein levels: Smad4 protein expression was investigated by Western-blotting using specific monoclonal antibodies against Smad4. Alfa-tubulin was detected as load control. Results: Early effects of BRAFT1799A in miRNA expression involve activation of miR-17-92 cluster in thyroid follicular cells. Conditional induction of BRAFT1799A (Dox group) results in a 85.8- fold, 634.3 fold and 141.6-fold increase in miR-19a, miR-19b and miR-18a, respectively, compared to control (Ctr group). Moreover, treating cells with iodine before BRAF activation (Dox+NaI group) resulted in partial or complete abrogation of this effect, with expression levels of miR-18a, miR-19a and miR-19b in 2.7-fold, 2.36-fold and 0.66-fold of Ctr group, respectively. Bioinformatic target prediction for miR-19a and miR-19b shows that Smad4 mRNA contain predicted binding sites in the 3′-UTR for these miRNAs. Smad4 is the key transducer of TGFB inhibitory signalling, frequently misbalanced in cancer. BRAF induction reduced Smad4 protein levels, effect abolished by previous iodine treatment, suggesting Smad4 as a possible target of miR-19a and miR-19b. Conclusion: Iodine exerts a protective effect in thyroid follicular cells, abrogating BRAFT1799A induced activation of miR-17-92 cluster key oncogenic miRNAs, miR-19a and miR-19b, and restoring Smad4 protein levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 183. doi:1538-7445.AM2012-183