Identification and Functional Characterization of a Human GalNAc α2,6-Sialyltransferase with Altered Expression in Breast Cancer

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Abstract
We sought to identify genes with altered expression during human breast cancer progression by applying mRNA comparisons of normal and tumor mammary cell lines with increasingly malignant phenotypes. The gene encoding a new sialyltransferase (STM) was found to be down-regulated in tumor cells. Abnormal expression and enzymatic activities of sialyltransferases in tumor cells result in the formation of tumor-associated carbohydrate antigens that can be used for the better understanding of the disease process and are applied for tumor diagnosis and immunotherapy. Altered glycosylation patterns of the MUC1 mucin, in particular, is a target antigen for immunotherapy of breast and other cancers. Total RNAs from multiple normal mammary epithelial cell strains and tumor cell lines were compared by differential display and the differential expression of selected cDNAs was confirmed by Northern analyses. Recombinant STM was expressed in COS-7 cells. The substrate and linkage specificity of STM was examined using various oligosaccharides and O-glycosylated proteins as acceptor substrates. The chromosomal localization of the SIATL1 gene was assigned by somatic cell hybrid analysis. A human sialyltransferase gene was identified by differential display as being down-regulated in breast tumor cell lines as compared to normal mammary epithelial cell strains, and the corresponding full-length cDNA (stm) was cloned. The encoded protein of 374 amino acid residues contained the L- and S-sialylmotifs, two catalytic regions conserved in all functional sialyltransferases. Recombinant STM is an active GalNAc α2,6-sialyltransferase with Galβ1,3 GalNAc-O-Ser/Thr and (+/−Neu5Acα2,3) Galβ1,3GalNAc-O-Ser/Thr acceptor specificity. The SIATL1 gene, encoding STM, was mapped to the long arm of human chromosome 17 at q23-qter, a region that is non-randomly deleted in human breast cancers. However, Southern analyses indicated that SIATL1 is usually not grossly rearranged in breast tumors. Northern analyses showed that the gene was widely expressed in normal human tissues, as well as in normal breast and prostate epithelial cell lines, but significantly down-regulated or absent in corresponding tumor cell lines. Our findings suggest that aberrant expression of STM sialyltransferase in tumors could be a feature of the malignant phenotype. In breast cancers, the MUC1 mucin is overexpressed and contains shorter O-glycans as compared to the normal mucin. Because STM catalyzes the synthesis of O-glycans, cloning and characterization of its substrate specificity will contribute to the understanding of the molecular mechanisms underlying the aberrant gly-cosylation patterns of O-glycans and the formation of mucin-related antigens in human breast cancers.