Functional characterization of BRAFV600E and RET/PTC double mutation and methylation profiling in in vitro model and thyroid tumors

Abstract

Genetic alterations play a crucial role in pathogenesis, diagnosis and treatment of thyroid tumors. The Papillary Thyroid Carcinoma is the most prevalent subtype, and mutations in BRAF and RAS oncogenes and rearrangements involving the RET oncogene are the most frequent. Our group investigated the prevalence of RET/PTC rearrangements and BRAF V600E mutation in a series of CPT (n = 118) and correlated with clinic-pathological features and gene expression profile. In this analysis we identified the co-occurrence of BRAF V600E and RET/PTC in nearly 13% of cases, and the majority occurred in RET/PTC3 isoform. This finding has important clinical implications with respect to prognosis and treatment of patients. Therefore, we will investigate the effect of the presence of both BRAF V600E and RET/PTC3 in Nthy-ori 3-1 cells (immortalized normal thyroid follicular cell) compared the presence of these mutations alone. For that cells, Nthy-ori 3-1 will be transfected with the expression vector pBudCE4.1 containing cDNA of BRAF V600E and RET/PTC3, BRAF V600E, RET/PTC3 or empty. The selected clones will be assessed for effect on activation of MAPK and PIK3/AKT/mTOR pathways and expression of genes associated with iodine metabolism and uptake (NIS, TSHR, TPO, TG and PDS). Moreover, we will evaluate the effect on growth, viability, cell cycle, apoptosis, anchorage independent growth, genetic instability and formation of double strand breaks, and epigenetic profile. Finally, we will evaluate the differential methylation in benign lesions (goiter and follicular adenoma), malignant (papillary carcinoma, follicular carcinoma and anaplastic carcinoma), normal tissue and thyroid correlate with the mutations previously determined. These analyzes will allow a better understanding of genetic and epigenetic events associated with the pathogenesis and/or progression of thyroid tumors. (AU)