Shiga Toxin Receptor Gb3Cer/CD77: Tumor-Association and Promising Therapeutic Target in Pancreas and Colon Cancer

Abstract

BackgroundDespite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas.Methodology/Principal FindingsTissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors.Conclusions/SignificanceEnhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.

Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains

“…The specifi cities of polyclonal chicken IgY anti-Gb3Cer and anti-Gb4Cer antibodies have been reported in previous publications (57)(58)(59)(60) …”

“…The TLC immunodetection procedure using polyclonal chicken anti-Gb3Cer and anti-Gb4Cer antibodies (1:2000 dilution) was employed as previously described ( 57,59,62,64 ). Alkaline phosphatase-labeled secondary antibodies were used in 1:2000 dilutions.…”

Association of Shiga toxin glycosphingolipid receptors with membrane microdomains of toxin-sensitive lymphoid and myeloid cells

“…The A 1 fragment of the A subunit, nicked by the cell protease furin, irreversibly inactivates eukaryotic protein biosynthesis ( 36 ) and damages DNA, causing the formation of apurinic sites in the nucleus ( 37,38 ), whereas the B subunits are capable of binding to the high-affi nity GSL receptor globotriaosylceramide (Gb3Cer) ( 39,40 ). Food-or waterborne infections of enterohemorrhagic Escherichia coli (EHEC), a certain human pathogenic subtype of Stxproducing E. coli (STEC), can cause severe illnesses such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS) in humans ( 41 ).…”

“…Stx2 combined with anti-Stx2 antibody and polyclonal chicken antibodies against Lc2Cer, Gb3Cer, and Gb4Cer were used in solid-phase binding assays (see "TLC overlay assay" below) for the detection of Lc2Cer and Stx-receptors Gb3Cer and Gb4Cer in the GSL preparations of lymphoid and myeloid cell lines following protocols established previously (40,44,64,(72)(73)(74). The monoclonal mouse IgG-antibody against Stx2 (clone cells/ml in serum-free medium as described previously.…”

Neutral glycosphingolipids in human blood: a precise mass spectrometry analysis with special reference to lipoprotein-associated Shiga toxin receptors

“…The TLC immunodetection procedure using anti-GSL antibodies and Gb3Cer-binding Stx1 and Stx2 in conjunction with anti-Stx1 and anti-Stx2 antibodies, respectively, was employed as previously described ( 50,(55)(56)(57)(58)(59). All primary anti-GSL and alkaline phosphatase-labeled secondary antibodies (Dianova, Hamburg, Germany) were overlayed to the TLC plate in 1:2000 dilutions.…”