cefoxitin and Carcinogenesis

Core 1-derived mucin-type O-glycans (O-glycans) are a major component of gastric mucus with an unclear role. To address this, we generated mice lacking gastric epithelial O-glycans (GEC C1galt1-/-). GEC C1galt1-/- mice exhibited spontaneous gastritis that progressed to adenocarcinoma with ∼80% penetrance by 1 yr. GEC C1galt1-/- gastric epithelium exhibited defective expression of a major mucus forming O-glycoprotein Muc5AC relative to WT controls, which was associated with impaired gastric acid homeostasis. Inflammation and tumorigenesis in GEC C1galt1-/- stomach were concurrent with activation of caspases 1 and 11 (Casp1/11)-dependent inflammasome. GEC C1galt1-/- mice genetically lacking Casp1/11 had reduced gastritis and gastric cancer progression. Notably, expression of Tn antigen, a truncated form of O-glycan, and CASP1 activation was associated with tumor progression in gastric cancer patients. These results reveal a critical role of O-glycosylation in gastric homeostasis and the protection of the gastric mucosa from Casp1-mediated gastric inflammation and cancer.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Carcinogenesis; Caspase 1; Female; Gastric Mucosa; Gastritis; Glycosylation; Homeostasis; Humans; Inflammation; Male; Mice; Mucins; Mucus; Neoplasms; Polysaccharides; Stomach Neoplasms

Aberrant O-glycosylation is frequently observed in colorectal cancer (CRC) patients, but it is unclear if it contributes intrinsically to tumorigenesis. Here, we investigated the biological consequences of aberrant O-glycosylation in CRC. We first detected the expression profile of Tn antigen in a serial of human CRC tissues and then explored the genetic and biosynthetic mechanisms. Moreover, we used a human CRC cell line (LS174T), which express Tn antigen, to assess whether aberrant O-glycosylation can directly promote oncogenic properties. It showed that Tn antigen was detected in around 86% human primary and metastatic CRC tissues. Bio-functional investigations showed that T-synthase and Cosmc were both impaired in cancer tissues. A further analysis detected an occurrence of hypermethylation of Cosmc gene, which possibly caused its loss-of-function and a consequent inactive T-synthase. Transfection of LS174T cells with WT Cosmc restored mature O-glycosylation, which subsequently down-regulated cancer cell proliferation, migration and apoptotic-resistant ability. Significantly, the expression of MUC2, a heavily O-glycosylated glycoprotein that plays an essential role in intestinal function, was uniformly reduced in human CRC tissues as well as in LS174T cells. These data suggest that aberrant O-glycosylation contributes to the development of CRC through direct induction of oncogenic properties in cancer cells.

Topics: Aged; Antigens, Tumor-Associated, Carbohydrate; Apoptosis; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Glycosylation; Humans; Male; Middle Aged; Molecular Chaperones; Mucin-2; Transfection

Immature truncated O-glycans such as Tn antigen are frequently detected in human colorectal cancer (CRC); however, the precise pathological consequences of Tn antigen expression on CRC are unknown. T-synthase is the key enzyme required for biosynthesis of mature O-glycans. Here we investigated the functional roles of Tn antigen expression mediated by T-synthase deficiency in CRC cells.. To knock out T-synthase, we used CRISPR-Cas9 technology to target C1GALT1, the gene encoding T-synthase, in a CRC cell line (HCT116). Deletion of T-synthase was confirmed by western blotting, and expression of Tn antigen was determined by flow cytometry in HCT116 cells. We then assessed the biological effects of T-synthase deficiency on oncogenic behaviors in HCT116 cells. Furthermore, we analyzed the mechanistic role of T-synthase deficiency in cancer cells by determining the epithelial-mesenchymal transition (EMT) pathway.. We showed that forced knockout of T-synthase in HCT116 cells significantly induced Tn antigen expression, which represented the occurrence of aberrant O-glycosylation. Loss of T-synthase significantly enhanced cell proliferation and adhesion, as well as migration and invasiveness in culture. More importantly, we demonstrated that T-synthase deficiency directly induced classical EMT characteristics in cancer cells. E-cadherin, a typical epithelial cell marker, was markedly decreased in T-synthase knockout HCT 116 cells, accompanied by an enhanced expression of mesenchymal markers including snail and fibronectin (FN).. These findings indicate that T-synthase deficiency in CRC cells not only is responsible for aberrant O-glycosylation, but also triggers the molecular process of EMT pathway, which may translate to increased invasiveness and metastasis in cancers.

Topics: Antigens, Tumor-Associated, Carbohydrate; Carcinogenesis; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Galactosyltransferases; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Neoplasm Invasiveness

Chronic inflammation and composition of the colon microbiota have been associated with colorectal cancer in humans. The human commensal enterotoxigenic Bacteroides fragilis (ETBF) is linked to both inflammatory bowel disease and colorectal cancer and, in our murine model, causes interleukin 17A (IL-17A)-dependent colon tumors. In these studies, we hypothesized that persistent colonization by ETBF is required for tumorigenesis.. We established a method for clearing ETBF in mice, using the antibiotic cefoxitin. Multiple intestinal neoplasia mice were colonized with ETBF for the experiment duration or were cleared of infection after 5 or 14 days. Gross tumors and/or microadenomas were then evaluated. In parallel, IL-17A expression was evaluated in wild-type littermates.. Cefoxitin treatment resulted in complete and durable clearance of ETBF colonization. We observed a stepwise increase in median colon tumor numbers as the duration of ETBF colonization increased before cefoxitin treatment. ETBF eradication also significantly decreased mucosal IL-17A expression.. The timing of ETBF clearance profoundly influences colon adenoma formation, defining a period during which the colon is susceptible to IL-17A-dependent tumorigenesis in this murine model. This model system can be used to study the microbiota-dependent and molecular mechanisms contributing to IL-17A-dependent colon tumor initiation.

Topics: Animals; Bacteroides fragilis; Carcinogenesis; Cefoxitin; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Enterotoxins; Humans; Mice

Human pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies in the world and despite great efforts in research types of treatment remain limited. A frequently detected alteration in PDACs is a truncated O-linked N-acetylgalactosamine (GalNAc) glycosylation with expression of the Tn antigen. Changes in O-glycosylation affect posttranslationally modified O-GalNAc proteins resulting in profound cellular alterations. Tn antigen is a tumor associated glycan detected in 75-90 % of PDACs and up to 67 % in its precursor lesions. Since the role of Tn antigen expression in PDAC is insufficiently understood we analyzed the impact of COSMC mediated Tn antigen expression in two human PDAC cell lines on cellular oncogenic properties.. Forced expression of Tn antigen on O-glycosylated proteins in pancreatic cancer cells was induced by lentiviral-mediated knockdown of the COSMC chaperone, which prevented O-glycan elongation beyond the initial GalNAcα1- residue on O-linked glycoproteins. Altered O-GalNAc glycosylation was analyzed in human pancreatic cancer cell lines Panc-1 and L3.6pl using Western and Far-Western blot as well as immunocytochemical techniques. To assess the biological implications of COSMC function on oncogenic properties, cell viability assays, scratch assays combined with live cell imaging, migration and apoptosis assays were performed. Lectin based glycoprotein enrichment with subsequent mass spectrometric analysis identified new cancer O-GalNAc modified proteins. Expression of Tn antigen bearing Nucleolin in patient derived PDAC tumor specimens was evaluated and correlated with clinicopathological data.. Tn antigen expression was induced on various O-GalNAc glycoproteins in COSMC deficient cell lines compared to the control. Proliferation was reduced (p < 0.001) in COSMC knockdown cells, whereas migration was increased (p < 0.001) and apoptosis was decreased (p = 0.03), highlighting the importance of Tn antigen expression on metastatic and anti-apoptotic behavior of PDAC derived cells. Nucleolin was identified as O-GalNAc modified protein in COSMC deficient PDAC cell lines. Interestingly, immunohistochemical staining and co-localization studies of patient derived PDACs revealed poor survival for patients with strong co-localization of Tn antigen and Nucleolin (p = 0.037).. This study substantiates the influence of altered O-glycan (Tn/STn) expression on oncogenic properties in pancreatic cancer and identifies O-GalNAc modified Nucleolin as novel prognostic marker.

Topics: Adenocarcinoma; Antigens, Tumor-Associated, Carbohydrate; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glycosylation; Humans; Mass Spectrometry; Molecular Chaperones; N-Acetylgalactosaminyltransferases; Neoplasm Proteins; Nucleolin; Pancreatic Neoplasms; Phosphoproteins; Polysaccharides; RNA-Binding Proteins; RNA, Messenger

A high expression of O-glycosylated proteins is one of the prominent characteristics of ovarian carcinoma cells associated with cell migration, which would be attributed to the upregulated expression of glycosyltransferases. Therefore, elucidating glycosyltransferases and their substrates may improve our understanding of their roles in tumor metastasis. In the present study, we reported that knockdown of polypeptide N-acetylgalactosaminyltransferase 14 (GALNT14) by small interfering RNA significantly suppressed the cell migration and altered cellular morphology. Immunoprecipitation and western blot analyses indicated that GALNT14 contributed to the glycosylation of transmembrane mucin 13 (MUC13), which was significantly higher in ovarian cancer cells compared with the normal/benign ovary tissues. Furthermore, interleukin-8 (IL-8), which could regulate the migration ability of epithelial ovarian cancer (EOC) cells, had no remarkable effect on the expression of GALNT14 and the tumor-associated carbohydrate epitope Tn antigen. In addition, extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor modulated the expression levels of GALNT14. Our findings provide evidence that GALNT14 may contribute to ovarian carcinogenesis through aberrant glycosylation of MUC13, but not through the IL-8 pathway. These data provide novel insights into understanding the function of MUC13 on neoplasm metastasis and may aid in the development of new anticancer drugs for EOC.

Topics: Antigens, Tumor-Associated, Carbohydrate; Carcinogenesis; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Movement; Female; Glycosylation; Humans; Interleukin-8; MAP Kinase Signaling System; Mucins; N-Acetylgalactosaminyltransferases; Neoplasm Metastasis; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Polypeptide N-acetylgalactosaminyltransferase