Role and regulation mechanisms of glycosyltransferases in cancer: focus on B4GALNT2.

This thesis deals with the significance of glycosyltransferases in cancer. It is comprised of three parts. In the first one was established the correlation between the expression level of 114 glycosyltransferases and prognosis using transcriptomic and clinical data of 21 TCGA cancer cohorts, identifying glycosyltransferases generally associated to good or poor prognosis. The second part aimed to investigate the regulation mechanisms of the glycosyltransferase β1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) in colorectal cancer (CRC). B4GALNT2 expression is high in normal colon but is strongly inhibited in CRC. However, high B4GALNT2 correlates with good prognosis in Colorectal Adenocarcinoma TCGA cohort. Beside the certain but not exclusive role of the promoter methylation, other mechanisms responsible for B4GALNT2 downregulation in CRC were investigated. Several transcription factors and miRNAs potentially regulating B4GALNT2 were screened in normal and cancer TCGA samples and in CRC cell lines (CCLE), thus identifying FOXD1 and miR-204-5p as promising B4GALNT2 inhibitory factors in CRC. Their transient transfection in the high-B4GALNT2 expresser CRC cell line GP2d significantly reduced B4GALNT2 mRNA and enzyme activity. Promoter activity was assessed by luciferase assay using a genomic region upstream B4GALNT2 translational start codon, putatively containing FOXD1 binding sites. Transfected GP2d cells with or without FOXD1 cotransfection were assayed for luciferase, showing that in presence of FOXD1 cotransfection the progressive deletion of FOXD1 sites altered luciferase transcription. FOXD1 knockdown in SW948 CRC line lacking B4GALNT2 partially restored B4GALNT2. Hence, FOXD1 and miR-204-5p play a major role in B4GALNT2 downregulation in CRC. The third part of the thesis evaluated the impact of forced B4GALNT2 expression on the neoplastic phenotype of two non-CRC gastrointestinal cancer cell lines. Despite previous studies proved that B4GALNT2 stable transfection in CRC cell lines reduced malignancy, the tendency in non-CRC cell lines was the opposite, indicating that its role of malignancy modulator is CRC specific.