Intense investigation is currently devoted to understand to which extent abnormalities of molecules regulating cell cycle progression and apoptosis are involved in the pathogenesis of human neoplasia, searching possible molecular targets for therapy or diagnostic and prognostic markers to be applied in clinical practice. It is now widely accepted that thyroid carcinomas are heterogeneous for morphology, immunophenotype, molecular pathogenesis and clinical outcome, but uncertainty still remains regarding their precise histogenesis. A proportion of cases in fact can not be precisely assigned to well defined entities (e.g. papillary, medullary, anaplastic, muco-epidermoid, etc.) and several mixed forms have been described. In addition, the stem cell derivation of thyroid tumours is still matter of speculation, including the possible role of the so called solid cell nests (SCN). The methodological approach proposed in this research project is able to rapidly detect molecular abnormalities by screening large series of cases, focusing on mixed and/or collision tumors), and to obtain precise information by exploiting more sophisticated and expensive molecular studies on selected groups of highly informative samples. The molecules that will be investigated include the growing regulators of cell cycle and apoptosis, focusing in particular on the complex tumour suppressor pathway involving p53 family members p63 and p73 and their isoforms. Despite the extensive evidence of the relevant role of cell cycle and cell death derangement in thyroid cancer cells, a systematic study aimed to assess the expression of cell cycle regulatory proteins is far from complete, and the possible role of the p53-family pathway has not been previously investigated. We think that the results of this study will be relevant in translational cancer research, allowing us to determine whether aberrant protein profiles are associated with unique tumor types and whether these can represent useful prognostic or therapy markers in thyroid carcinomas.
In particular, the study will take into account the evaluation of cell cycle regulators (including cyclins, cdk-inhibitors), the wnt-pathway (in particular beta-catenin and other inducers of cyclin-D1, the product of a gene that is frequently abnormally expressed in thyroid carcinomas). Finally we will focus on the p53 pathway, searching for abnormal interactions occurring in situ between the expression of p53, its downstream effector p21WAF1, its regulators mdm2 and p14arf, as well as the transactivating (TA-p63) and truncated (N-p63) isoforms of p63, a recently described member of the p53 family. Our interest in this latter gene comes from our preliminary demonstration that solid cell nests of human thyroid in fact express nuclear p63 (unpublished observation). The p63 gene is a member of the p53 tumor suppressor gene family playing an important role in the physiological maintenance of different specialized epithelia. Its gene functions are heterogeneous and complex since it undergoes splicing by alternative transcription from two different promoters, producing as many as six distinct isoforms which exert potentially contrasting effects on the same molecular and cellular targets. Transactivating isoforms (the TA-p63 class) maintain a sequence corresponding to the transactivating domain of p53 and have in fact functions similar to p53 in inducing cell-cycle arrest (mediated by p21WAF1) and apoptosis. The second class, on the other hand, includes forms lacking the NH2-terminal domain (N-p63), produced when the p63 gene is transcribed from the cryptic promoter in intron 3. N-p63 forms act as dominant-negative agents toward transactivation by p53 and p63 itself, inhibiting the activity of p53. When over-expressed, these molecular p63 variants can thus behave as oncogenic molecules. Accordingly, p63 gene amplification and overexpression of N-p63 have been demonstrated in primary lung carcinoma and other malignancies. Further complexity is provided by the recent demonstration of mutual influences occurring between p53 and p63, generated by the suppressing activity of p53 on dominant negative functions of N-p63. In addition, recent experimental evidence has been provided suggesting that the combined function of p63 and p73 is required for p53-dependent apoptosis in response to DNA damage. In this research project we will investigate the expression of p63 isoforms in the development of normal thyroid, solid-cell nests and various thyroid epithelail tumors, comparing the results with those obtained in other endodermal malignancies.
The major aim of this study is to serch for reliable new markers, selected on the basis of reliability and cost-effectiveness, useful for improving the diagnostic evaluation of thyroid carcinomas. In this project we will investigate different types of tumours searching for abnormal expression of molecules involved in the regulation of the cell cycle and apoptosis, with special emphasis on the p53-family pathway involving the isoforms of p63. Histopathological samples will be retrieved from the files of the Department of Pathology, University of Verona, and thoroughly analysed for the expression of investigated molecules using molecular morphology (immunohistochemistry and in situ hybridization). The analysis of p63 isoforms will be performed using different antibodies, specifically recognizing transactivating and truncated dominant-negative isoforms, confirming and extending the results in selected samples using molecular analysis. In addition, a thorough analysis of cell cycle regulators including cyclins, inhibitors of the cyclin-dependent kinase complex p27kip1 and p21waf1, regulators of the p53-pathway, molecules involved in the interaction with microenvironmental components (adhesion molecules such as CD56/N-CAM, fascin, beta.catenin, cadherin, etc) will be investigated in order to shape a molecular profile of different thyroid tumors. The proliferation features will be analysed using the recently described S-Phase Kinase-Associated Protein 2, a regulator of p27kip1 expression.