globotriaosylceramide and Neoplasm-Metastasis

The B-subunit of the bacterial Shiga toxin (STxB), which is nontoxic and has low immunogenicity, can be used for tumor targeting of breast, colon, and pancreatic cancer. Here, we tested whether human gastric cancers, which are among the most aggressive tumor entities, express the cellular receptor of Shiga toxin, the glycosphingolipid globotriaosylceramide (Gb3/CD77). The majority of cases showed an extensive staining for Gb3 (36/50 cases, 72%), as evidenced on tissue sections of surgically resected specimen. Gb3 expression was detected independent of type (diffuse/intestinal), and was negatively correlated to increasing tumor-node-metastasis stages (P = 0.0385), as well as with markers for senescence. Gb3 expression in nondiseased gastric mucosa was restricted to chief and parietal cells at the bottom of the gastric glands, and was not elevated in endoscopic samples of gastritis (n = 10). Gb3 expression in established cell lines of gastric carcinoma was heterogeneous, with 6 of 10 lines being positive, evidenced by flow cytometry. STxB was taken up rapidly by live Gb3-positive gastric cancer cells, following the intracellular retrograde transport route, avoiding lysosomes and rapidly reaching the Golgi apparatus and the endoplasmic reticulum. Treatment of the Gb3-expressing gastric carcinoma cell line St3051 with STxB coupled to SN38, the active metabolite of the topoisomerase type I inhibitor irinotecan, resulted in >100-fold increased cytotoxicity, as compared with irinotecan alone. No cytotoxicity was observed on gastric cancer cell lines lacking Gb3 expression, demonstrating receptor specificity of the STxB-SN38 compound. Thus, STxB is a highly specific transport vehicle for cytotoxic agents in gastric carcinoma. Mol Cancer Ther; 15(5); 1008-17. ©2016 AACR.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Biomarkers; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Dose-Response Relationship, Drug; Gastric Mucosa; Gene Expression; Humans; Immunotoxins; Molecular Targeted Therapy; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Shiga Toxins; Stomach Neoplasms; Trihexosylceramides

The most devastating aspect of cancer is the emergence of metastases. Thus, identification of potentially metastatic cells among a tumor cell population and the underlying molecular changes that switch cells to a metastatic state are among the most important issues in cancer biology. Here we show that, although normal human colonic epithelial cells lack the glycosphingolipid globotriaosylceramide (Gb(3)), this molecule is highly expressed in metastatic colon cancer. In addition, a subpopulation of cells that are greatly enriched in Gb(3) and have an invasive phenotype was identified in human colon cancer cell lines. In epithelial cells in culture, Gb(3) was necessary and sufficient for cell invasiveness. Transfection of Gb(3) synthase, resulting in Gb(3) expression in noncancerous polarized epithelial cells lacking endogenous Gb(3), induced cell invasiveness. Furthermore, Gb(3) knockdown by small inhibitory RNA in colon cancer epithelial cells inhibited cell invasiveness. Gb(3) is the plasma membrane receptor for Shiga toxin 1. The noncatalytic B subunit of Shiga toxin 1 causes apoptosis of human colon cancer cells expressing Gb(3). Injections of the B subunit of Shiga toxin 1 into HT29 human colon cancer cells engrafted into the flanks of nude mice inhibited tumor growth. These data demonstrate the appearance of a subpopulation of Gb(3) containing epithelial cells in the metastatic stage of human colon cancer and suggest their possible role in colon cancer invasiveness.

Topics: Animals; Caco-2 Cells; Cell Line, Tumor; Cell Transformation, Neoplastic; Colonic Neoplasms; Epithelial Cells; Galactosyltransferases; Glycosphingolipids; Humans; Lasers; Mice; Mice, Nude; Microscopy, Confocal; Microscopy, Fluorescence; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms; Phenotype; Pseudopodia; Shiga Toxins; Trihexosylceramides

The presence of cell surface receptor glycolipid, globotriaosylceramide (Gb3), is essential to confer susceptibility to the E. coli-derived verotoxin (VT). Our earlier studies showed that Gb3 is expressed in ovarian carcinoma cell lines. The Gb3 content of normal ovary, benign and malignant primary ovarian tumors, and their metastases have now been compared by verotoxin thin-layer chromatogram (TLC) overlay of the glycolipid tissue extracts. FITC-labeled VT1 B subunit binding to frozen tumor sections was also monitored histochemically. Low to undetectable levels of Gb3 were found in "normal" ovarian tissue. Gb3 was markedly increased in both benign and malignant tumors, suggesting that increased Gb3 may be related to proliferation, rather than malignancy per se. Mucinous tumors showed the least Gb3 elevation; serous tumors were variable, showing higher levels of Gb3 in less differentiated malignant tumors. By far the highest Gb3 content was observed for secondary ovarian metastases and tumors refractory to chemotherapy. Frozen sections of neoplastic ovarian tissue overlaid with fluorescein-conjugated VT1 B subunit show extensive binding to tumor cells, particularly in poorly differentiated samples and blood vessels adjacent to, and within, the tumor mass. Tumor foci were stained but stromal tissue was consistently negative both in primary tumors and metastases. VT staining of well-differentiated primary ovarian tumor sections was weak, corresponding to their low Gb3 content, but strong staining was observed in sections from a highly differentiated primary tumor from a patient who was unexpectedly refractory to clinical chemotherapy. These studies suggest that verotoxin/Gb3 targeting may provide the basis for new treatments for ovarian cancer.

Topics: Bacterial Toxins; Female; Frozen Sections; Histocytochemistry; Humans; Hyperplasia; Neoplasm Metastasis; Ovarian Cysts; Ovary; Receptors, Cell Surface; Reference Values; Shiga Toxin 1; Trihexosylceramides