Analysis of PAX2 gene alterations in renal cell tumors

Dissertation for applying to a Master’s Degree in Molecular Genetics and Biomedicine submitted to the Sciences and Technology Faculty of New University of Lisbon

Background: Renal cell tumors (RCT) represent a heterogeneous group of malignancies arising from the epithelium of the renal tubules. PAX2 (Paired-box 2) is a transcription factor belonging to the evolutionarily conserved paired-box family that is required during development of the central nervous system and genitourinary tract. PAX2, which is considered a proto-oncogene, is normally suppressed at the later stages of embryonic development and reactivated during the carcinogenic process in some cancer models. Previously published data indicate that significant expression of PAX2 protein occurs in three of the most prevalent renal cell tumor subtypes - clear cell RCC (ccRCC), papillary RCC (pRCC) and oncocytoma - but not in chromophobe RCC (chrRCC). Moreover, it has been reported that PAX2 expression could be repressed by aberrant methylation at the end of nephrogenesis. Finally, FISH and cytogenetic analysis, demonstrated loss of chromosome 10 (to which PAX2 is mapped) in 40 to 60% of chrRCC cases. Aims: The main goal of this study was to identify epigenetic and/or genetic alterations affecting the PAX2 gene in a series of renal cell tumors, representing the four major subtypes. Specifically, our aims were to: (1) analyze the expression of PAX2 in different renal cell tumor subtypes, both at mRNA and at protein level; (2) determine whether the regulation of PAX2 expression occurs by epigenetic mechanisms, by assessing its promoter methylation status; (3) analyze the association between PAX2 copy number and gene expression; (4) evaluate the potential use of PAX2 epigenetic/genetic alterations as a biomarker for discrimination between chrRCC and oncocytoma. Methodology: In this study, 120 samples of renal cell tumors (30 of each subtype: ccRCC, pRCC, chrRCC, and oncocytoma) and 4 normal kidney tissues were tested. First, PAX2 protein expression was assessed by immunohistochemistry and the PAX2 mRNA expression levels were determined by quantitative reverse transcription PCR (qRT-PCR), using HPRT1 as an endogenous control. The methylation status of PAX2 promoter was assessed by methylation-specific PCR using two different sets of primers that annealed to adjacent regions in the promoter. The relationship between the number of PAX2 copies and its expression in chrRCC was analyzed by FISH. The chi-square test or Fisher’s exact test were used to seek for differences in the frequency of immunoreactivity for PAX2 protein among the four RCT subtypes....

CI-IPOP-4 and the Calouste Gulbenkian Foundation (Project # 96474)