Cell polarity disruption during HPV carcinogenic processes: analysis of cellular and viral contributions

MARINA BUGNON VALDANO; A.L. CAVATORTA; F. MARZIALI; F. FACCIUTO; E. BOCCARDO; D. GARDIOL.

Workshop; First ICGEB Workshop Human Papillomavirus: From Basic Biology to Cervical Cancer Prevention; 2014

Loss of cell polarity is a hallmark in tumors. We studied different mechanisms that regulate polarity proteins expression, particularly considering those carcinogenic processes associated to Human papillomaviruses (HPV) infections. We are focused on the DLG1 and PAR3 PDZ polarity proteins. Their expression is altered in several tumors but the mechanisms beyond these alterations remain unknown. By luciferase assays, we found that the DLG1 promoter activity is increased significantly in the presence of HPV E6 and/or E7 proteins in epithelial cells in culture. Raft organotypic cultures reproduce epithelial differentiation in vitro and closely mimic the HPV-host interaction. Here we show that in raft cultures expressing HPV E6 and E7, DLG1 and PAR3 were found to be up-regulated and misdistributed, as ascertained by Western Blot and Immunohistochemistry (IHC). These data suggest that the viral proteins could contribute to the changes in the cell proteins expression observed in biopsies. Also, to study if epigenetic regulation mechanisms could contribute to DLG1 regulation in malignant processes, we analyzed the methylation pattern of DLG1 promoter in cultured cell lines, by treatment of DNA with sodium bisulfite and subsequent PCR amplification and sequencing. We designed primers suitable for amplification of bisulfite treated DNA and we found a demethylated state of the promoter in transformed cells. Also, we evaluated the methylation status using biopsies, and we found a difference between normal and tumoral samples, which could be related with the DLG1 expression, as we tested by IHC. Though, this mechanism could also be involved in regulating DLG1 abundance. Together, these results highlight the importance of further studies about the regulation of cell polarity proteins during the transformation processes associated to viral infections.