globotriaosylceramide and Colorectal-Neoplasms

Globotriaosylceramide (Gb3 or CD77) is a tumor-associated carbohydrate antigen implicated in several types of cancer that serves as a potential cancer marker for developing target-specific diagnosis and therapy. However, the development of Gb3-targeted therapeutics has been challenging due to its carbohydrate nature. In the present work, taking advantage of its natural pentamer architecture and Gb3-specific targeting of shiga toxin B subunit (StxB), we constructed a pentameric antibody recruiting chimera by site-specifically conjugating StxB with the rhamnose hapten for immunotherapy of colorectal cancer. The Sortase A-catalyzed enzymatic tethering of rhamnose moieties to the C terminus of Stx1B and Stx2B had very moderate effect on their pentamer architectures and thus the resultant conjugates maintained the potent ability to bind to Gb3 antigen both immobilized on an assay plate and expressed on colorectal cancer cells. All StxB-rhamnose constructs were capable of efficiently mediating the binding of rhamnose antibodies onto HT29 colorectal cancer cells, which was further shown to be able to induce cancer cell lysis by eliciting potent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in vitro. Finally, the best StxB-rhamnose conjugate, i.e. 1B-3R, was confirmed to be able to inhibit the colorectal tumor growth using a HT29-derived xenograft murine model. Taken together, our data demonstrated the potential of repurposing StxB as an excellent multivalent scaffold for developing Gb3-targeted biotherapeutics and StxB-rhamnose conjugates might be promising candidates for targeted immunotherapy of Gb3-related colorectal cancer.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Colorectal Neoplasms; Haptens; Humans; Immunotherapy; Mice; Rhamnose; Shiga Toxin; Trihexosylceramides

Infection with Shiga toxin (Stx)-producing, gram-negative bacteria can induce serious conditions such as dysentery and hemolytic uremic syndrome. In target cells, Stx is internalized by endocytosis, and travels through the Golgi apparatus and the endoplasmic reticulum to reach the cytosol, where it inhibits protein synthesis. Toll-like receptor 4 (TLR4) mediates the recognition of gram-negative bacteria. Here, we have investigated whether the cellular uptake and transport of Stx could involve TLR4. We found that upon small interfering RNA (siRNA)-mediated TLR4 depletion in epithelial colon carcinoma cells, Stx transport to the Golgi was strongly reduced, and this was primarily caused by diminished Stx cellular binding rather than by reduction in toxin uptake or endosome-to-Golgi transport. The reduced cellular binding of Stx upon siRNA-transfection was solely due to TLR4 depletion, because reconstitution of TLR4 expression by the introduction of an siRNA-resistant TLR4 gene completely abolished the TLR4-targeting siRNA-mediated effect. Importantly, the effect of TLR4 depletion was not restricted to cancer cells or epithelial cells, because primary human umbilical vein endothelial cells also displayed reduced Stx binding upon TLR4 depletion. These results indicate that although TLR4 is imperative in innate immunity against gram-negative bacteria, it may be exploited by bacterial toxins, for example Stx, to gain access and entry into cells.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Endothelial Cells; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Golgi Apparatus; Humans; Protein Binding; Protein Transport; Receptors, Cell Surface; RNA, Small Interfering; Shiga Toxin; Signal Transduction; Toll-Like Receptor 4; Trihexosylceramides; Umbilical Veins

The targeting of solid tumors requires delivery tools that resist intracellular and extracellular inactivation, and that are taken up specifically by tumor cells. We have shown previously that the recombinant nontoxic B-subunit of Shiga toxin (STxB) can serve as a delivery tool to target digestive tumors in animal models. The aim of this study was to expand these experiments to human colorectal cancer. Tissue samples of normal colon, benign adenomas, colorectal carcinomas, and liver metastases from 111 patients were obtained for the quantification of the expression of the cellular STxB receptor, the glycosphingolipid globotriaosyl ceramide (Gb(3) or CD77). We found that compared with normal tissue, the expression of Gb(3) was strongly increased in colorectal adenocarcinomas and their metastases, but not in benign adenomas. Short-term primary cultures were prepared from samples of 43 patients, and STxB uptake was studied by immunofluorescence microscopy. Of a given tumor sample, on average, 80% of the cells could visibly bind STxB, and upon incubation at 37 degrees C, STxB was transported to the Golgi apparatus, following the retrograde route. This STxB-specific intracellular targeting allows the molecule to avoid recycling and degradation, and STxB could consequently be detected on tumor cells even 5 days after initial uptake. In conclusion, the targeting properties of STxB could be diverted for the delivery of contrast agents to human colorectal tumors and their metastases, whose early detection and specific targeting remains one of the principal challenges in oncology.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Tumor-Associated, Carbohydrate; Chromatography, Thin Layer; Colorectal Neoplasms; Female; Humans; Intestines; Liver Neoplasms; Male; Middle Aged; Shiga Toxins; Trihexosylceramides