Dottorato in MEDICINA MOLECOLARE

Titolo della tesi: MicroRNAs as diagnostic biomarkers and identification of molecular escape mechanism to tyrosine kinase inhibitors (TKI) in Medullary Thyroid Carcinoma

Medullary thyroid cancer (MTC) is a rare neuroendocrine tumor arising from thyroid C cells, with frequent mutations in the rearranged during transfection (RET) or RAS genes. MTC can be hereditary in 20-25% or sporadic in 75-80% of cases. MTC diagnosis is performed by thyroid nodule biopsy with immunohistochemical positivity for calcitonin (Ctn) and Ctn deregulated serum levels, but sporadic MTC is usually diagnosed as an advanced tumour because it arises as solitary thyroid nodule without symptoms. The determination of tumour biomarkers is of primary importance to personalized medicine, mostly for rare tumours as MTC, whose diagnosis is still challenging due to the lack of non-invasive diagnostic biomarkers. Treatment for localized MTC consists of total thyroidectomy but MTC patients that present metastatic or progressive disease receive treatment with tyrosine kinase inhibitors (TKI) that include Cabozantinib and the recent FDA approved selective RET inhibitor, Pralsetinib (Blu-667). A limitation to the efficacy of TKI therapy is the possibility to develop escape mechanisms. As an example, hypoxia may promote the selection of resistant clones. Activation of hypoxia-inducible pathways as resistant mechanism in MTC tumours has been observed after administration of Cabozantinib, however no studies analysed this event after Blu-667 administration. To address these medical needs, I analysed microRNAs (miRNAs) expression in primary samples derived from MTC patients, both plasma and tissue, with the aim to discover non-invasive MTC diagnostic biomarkers. Moreover, I investigated mechanisms involved in the biological activity of Pralsetinib in a MTC cell line (TT cells), specifically in the context of a hypoxic microenvironment. These experiments allowed the identification and validation of a signature of two circulating microRNAs, miR-26b-5p and miR-451a, in two independent cohorts reporting a significant diagnostic performance for MTC. In the second part of my thesis, I investigate the molecular mechanism underlying response and resistance to the selective RET inhibitor Pralsetinib. Indeed, after in vitro administration of selective RET inhibitor, I have demonstrated the existence of a subpopulation of cells that escape Pralsetinib activity, in a RET-independent manner, specifically through the nuclear translocation and subsequent stabilization of the Gli1 protein, the principal transcriptional activator of the Hedgehog (HH)-pathway. In conclusion, I was able to identify circulating microRNAs as potential diagnostic tool and a possible mechanism of TKI treatment resistance in MTC cell line.