Avilla, Elvira (2009) Functional role of the CXCR4 chemokine receptor in thyroid cancer. [Tesi di dottorato] (Unpublished)

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Thyroid cancer is the most common endocrine malignancy and its incidence is increasing worldwide. We previously found that normal rat thyroid cells, transduced with papillary thyroid cancer (PTC)-related oncogenes, display an inflammatory signature, that includes cytokines, chemokines and their receptors (Melillo at al., 2005). One of the chemokine receptors we identified, CXCR4, is frequently up-regulated in thyroid cancer and the chemokine SDF-1, a CXCR4 ligand, induces proliferation, survival and invasive ability of PTC cells; moreover, CXCR4 blocking compounds inhibit thyroid cancer growth (Castellone et al., 2004; De Falco et al., 2007). In order to better understand the molecular mechanisms of the biological effects of CXCR4/SDF-1α in thyroid carcer, we performed a global genome expression profile, through DNA microarrays, of CXCR4-expressing human papillary thyroid carcinoma cells (TPC-1) treated or not with SDF-1α. We identified, as transcriptional targets of CXCR4/SDF-1α, two tyrosine-kinase receptors: TYRO3 and AXL. These proteins belong to a small Protein Tyrosine Kinase (PTK) subfamily of receptors that includes three members: TYRO3, AXL and MER, from which this family is named TAM; they can be activated by two ligands, GAS6 and Protein S, are involved in the regulation of immune response, and are overexpressed in some epithelial cancers (Linger et al., 2008). We found that TPC-1 cells, derived from a human PTC, constitutively express TYRO3 and AXL receptors, but SDF-1α stimulation increased their protein level and tyrosine phosphorylation. We found that most of the available thyroid cancer cell lines express both the receptors, albeit to different extent, and AXL was always much more abundant than TYRO3. In most cell lines, the two receptors display high levels of tyrosine-phosphorylation, due to constitutive expression of GAS6. An exception to this rule was the TPC-1 cell line, in which AXL was highly phosphorylated despite the fact that GAS6 is absent. AXL and its ligand GAS6 are also overexpressed in human thyroid carcinoma samples with respect to normal thyroid, as assessed by IHC. The inhibition of TYRO3 and AXL by blocking reagents or RNA interference targeting each receptor or the ligand decreased cell proliferation and resistance to apoptotic stimuli in different thryoid cancer cell lines. In cell lines that expressed both receptors and ligand, the simultaneous blockade of these molecules dramatically affected cell viability. Accordingly, we showed that the stimulation of GAS6-negative TPC-1 cells with exogenous GAS6 increased their proliferation and survival. Finally, we found that the blockade of AXL receptor consistently impaired thyroid carcinoma cell line invasiveness through Matrigel and, moreover, it inhibited tumor growth in nude mice. In this thesis project, we provide evidences that targeting CXCR4/SDF-1 or TYRO3/AXL/GAS6 axis might be exploited as novel anticancer therapyes for human thyroid carcinomas.

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