Role of genes differentially expressed in thyroid carcinogenesis

Thyroid cancer represents the most common endocrine malignancy, and its incidence has increased significantly over the last few decades. Papillary thyroid carcinoma (PTC), the most frequent neoplasia originating from the thyroid epithelium, accounts for about 80% of all thyroid cancers. PTC is characterized by rearrangements of RET and NTRK1 receptor tyrosine kinases, or by activating point mutations in the BRAF serine/threonine kinase or in the RAS genes. Even though the identification of PTC-associated oncogenes has provided a great contribution to the understanding of PTC pathogenesis, the molecular mechanisms underlying the development of this neoplasia, including the role of tumour suppressor genes, are still far from being completely elucidated. Recently, global gene expression analyses have provided new findings contributing to the dissection of thyroid tumour pathogenesis, through the identification of genes discriminating among different histotypes, candidates as new therapeutic targets and possible tumour suppressor genes. Despite the numerous gene expression studies, there are few data addressing the role of differentially expressed genes in the pathogenesis of thyroid tumours. A microarray gene expression profile previously determined in our laboratory identified a list of genes differentially expressed in PTC versus normal thyroid; among those, we selected TIMP3, S100A11 and CITED1 genes for which a role in the pathogenesis of PTC was also suggested by recently published gene and protein expression data. In this PhD project, we performed functional studies in order to assess the role of TIMP3, S100A11 and CITED1 genes in thyroid carcinogenesis, with the aim of unveiling novel mechanisms and identifying novel therapeutic targets in thyroid tumours. TIMP3 (Tissue Inhibitor of Metalloproteinases-3) is a secreted protein able to inhibit extracellular matrix metalloproteinases. TIMP3 gene promoter has been found hypermethylated in thyroid cancer and its downregulation was associated with several aggressive tumour features. To investigate the role of TIMP3 in the pathogenesis of PTC we used an integrated approach including analysis of several gene expression data sets and functional studies. TIMP3 was found to be downregulated in a consistent fraction in PTCs, with respect to normal thyroid. Restoration of TIMP3 in the PTC-derived NIM1 cell line had no effect on growth rate; however, it reduced migration, invasion and anchorage independent growth. The striking effect was observed in vivo, as TIMP3 reduced the tumourigenicity of NIM1 cells by repressing angiogenesis and macrophage infiltration. All these observations suggest a tumour suppressor role in thyroid carcinogenesis. S100A11 (calgizzarin) is a member of the S100 Ca2+-binding protein family, which includes at least 20 proteins. Its role in tumour is not well known, and it appears to have distinct functions in different tumour types. Our microarray analysis showed that S100A11 is overexpressed in PTC compared to normal thyroid. In order to study the role of S100A11 in thyroid carcinogenesis, we performed functional in vitro and in vivo analysis. Analysis of cellular localization in PTC-derived K 1 cell line, revealed that S100A11 was mainly cytoplasmic and was able to trans locate into the nucleus after Ca2+ and TGF-β stimulation. We also found that this translocation did not increase p21 level, a negative regulator of cell growth. Moreover, we found that S100A11 did not alter the activation level of the EGF/EGFR pathway. We then investigated the effect of S100A11 silencing on tumourigenic properties of K 1 cells. We found that S100A11 silencing did not affect cell proliferation, but it exerted a role on the anchorage-independent growth. Analysis of mouse tumour xenografts showed that S100A11 was not involved in the in vivo tumourigenicity of K1 cells. Concomitantly, we assessed the effect of S100A11 gene modulation on the transforming potential of PTC-associated oncogenes. Cotransfection experiments in NIH3T3 cells, which represent a useful model for studying in vitro oncogene activity, showed that S100A11 gene was able to enhance the transforming capability of the PTC-associated TRK-T3 oncogene. Stable NIH3T3 foci expressing TRK-T3 and S100A11 concomitantly to TRK -T3 were produced. Functional studies showed that S I OOA 11 was not involved in the regulation of cell proliferation, whereas it was able to promote invasion, anchorage independent growth and in vivo tumourigenicity of these transformed foci. CITED1 (CBP/p300-Interacting Transactivators with glutamic acid (E) and aspartic acid (D)-rich C-terminal domain 1) is part of a family of transcriptional cofactors that regulates diverse CBP/p300 transcriptional responses. In different microarray studies· CITED 1 was found overexpressed in PTC. It was also proposed as a highly sensitive and specific diagnostic marker, useful in differentiating PTC from benign and malignant thyroid tumours. For this reason, we are interested in the functional role of CITED1 in this histotype. Preliminary experiments involved the analysis of CITED 1 expression in a collection of PTC-derived cell lines and the construction of CITED1 cDNA expression vectors. Further analysis will involve the investigation of its effect on transforming activity of PTC-associated oncogenes and the effect of its gene silencing on the phenotype of a PTC-derived cellular model.