Glycosylation profiles of breast cancer cells may represent clonal variations of multiple organ metastases.

Glycosylation changes of cancer cells are known to be associated with malignant progression and metastases and potentially determine the organ-selective nature of metastasis as theorized by Paget (Lancet 1:571-573, 1889). Cellular glycans play a variety of roles in the processes of metastasis and may be unique to the cells that metastasize to different organs. We analyzed the glycosylation profiles of the primary tumor and tumors metastasized to lymph node, liver, lung, brain, bone, thyroid, kidney, adrenal, small intestine and pancreas in an autopsy case of breast cancer employing a lectin microarray with 45 lectins. Clustering analysis of the data revealed that metastatic breast cancer cells were categorized into several clusters according to their glycosylation profiles. Our results provide a biological basis to understand differential phenotypes of metastatic breast cancer cells potentially reflecting clonal origin, which does not directly reflect genomic or genetic changes or microenvironmental effects but connects to glycosylation profiles.

Unique Glycoform-Dependent Monoclonal Antibodies for Mouse Mucin 21.

Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.

Mucin 21 confers resistance to apoptosis in an O-glycosylation-dependent manner.

Highly glycosylated mucins protect epithelial surfaces from external insults and are related to malignant behaviors of carcinoma cells. However, the importance of carbohydrate chains on mucins in the process of cellular protection is not fully understood. Here, we investigated the effect of human mucin-21 (MUC21) expression on the susceptibility to apoptosis. MUC21 transfection into HEK293 cells decreased the number of apoptotic cells in culture media containing etoposide or after ultraviolet light irradiation. We used Chinese hamster ovary (CHO) cell variants to investigate the importance of MUC21 glycosylation in the resistance to apoptosis. When MUC21 was expressed in CHO-K1 cells, it was glycosylated with sialyl T-antigen and the cells showed resistance to etoposide-induced apoptosis. MUC21 transfection into Lec2 cells, a variant of CHO cells lacking sialylation of glycans, revealed that the presence of nonsialylated T-antigen also renders cells resistant to etoposide-induced apoptosis. MUC21 was transfected into ldlD cells and the glycosylation was manipulated by supplementation to the medium. Nonsupplemented cells and cells supplemented with N-acetylgalactosamine showed no resistance to etoposide-induced apoptosis. In contrast, these cells supplemented with N-acetylgalactosamine plus galactose expressed sialyl T-antigen and exhibited resistance to etoposide-induced apoptosis. Finally, galectin-3 knockdown in MUC21 transfectants of HEK293 cells did not significantly affect MUC21-dependent induction of apoptosis resistance. The results suggest that T-antigen with or without sialic acid is essential to the antiapoptotic effect of MUC21.

Glycans unique to the relapse-prone subset within triple-negative breast cancer as revealed by lectin array-based analysis of surgical specimens.

INTRODUCTION: Molecular and cellular characteristics of the relapse-prone subset within triple-negative breast cancer (TNBC) remain unclear. Aberrant glycosylation is involved in the malignant behavior of cancer cells. In the present study, we aimed to reveal glycan profiles unique to relapsed TNBC patients. METHODS: Thirty TNBC patients who did not undergo neoadjuvant chemotherapy but postoperative standard adjuvant therapy from 2009 through 2016 at Juntendo Hospital were investigated. TNBC cells were resected from primary breast cancer sections of formalin-fixed surgical specimens using laser-assisted microdissection. The binding intensities of the extracted glycoproteins to 45 lectins were quantified using lectin microarray and compared between relapsed and non-relapsed patients. Immunohistochemical staining with TJA-II lectin in specimen sections was performed. RESULTS: Five patients relapsed during the follow-up (range 37-123 months). Lectin microarray analysis revealed that 7 out of 45 lectins showed significant differences in binding intensity between the relapsed and the non-relapsed group. TJA-II, ACA, WFA, and BPL showed stronger binding in the relapsed group. PNGase F treatment of TNBC cell lysates suggested that TJA-II and ACA bind O-glycans. TJA-II staining of tissue sections revealed strong binding to cell surface membranes and to the cytoplasm of TNBC cells, but not to other types of cells. Significantly more TNBC cells were stained in tissue sections from relapsed than non-relapsed patients. CONCLUSIONS: TNBC cells from relapsed patients showed a unique lectin reactivity, with higher levels of TJA-II (also WFA and BPL) binding than in non-relapsed patients. The results are potentially useful to develop new prognostic and therapeutic tools.

Biological and Clinicopathological Implications of Beta-3-N-acetylglucosaminyltransferase 8 in Triple-negative Breast Cancer.

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) remains difficult to treat and new molecular targets are needed. Here, we investigated the impact of glycosyltransferase genes on TNBC patient survival. PATIENTS AND METHODS: mRNA expression levels of 101 glycosyltransferase genes in TNBC patients were compared for correlation with patient survival using The Cancer Genome Atlas data. An antibody to ß-3-N-acetylgluco-saminyltransferase 8 (B3GNT8) was applied to investigate B3GNT8 protein distribution and expression levels in 23 TNBC surgical specimens. RESULTS: B3GNT8 mRNA levels inversely correlated with relapse-free survival (p<0.01) and overall survival (p<0.05) in TNBC patients. Anti-B3GNT8 antibody binding was observed as dots in the cytoplasm of cancer cells. These dots were supposed to correspond to B3GNT8 protein in tumour cells, but their number was smaller in relapsed patients than in non-relapsed patients. CONCLUSION: B3GNT8 mRNA expression levels in TNBC tumour tissues are potentially useful in distinguishing patients with favourable and poor clinical outcomes.

Mucin 21/epiglycanin modulates cell adhesion.

The molecular structure of mouse Mucin 21 (Muc21)/epiglycanin is proposed to have 98 tandem repeats of 15 amino acids and three exceptional repeats with 12 or 13 amino acids each, followed by a stem domain, a transmembrane domain, and a cytoplasmic tail. A cDNA of Muc21 having 84 tandem repeats of 15 amino acids was constructed and transfected using a Venus vector into HEK 293T cells. The fluorescent cells, which were considered to express Muc21, were nonadherent. This antiadhesion effect was lessened when constructs with smaller numbers of tandem repeats were used, suggesting that the tandem repeat domain plays a crucial role. Cells expressing Muc21 were significantly less adherent to each other and to extracellular matrix components than control cells. Antibody binding to the cell surface integrin subunits alpha5, alpha6, and beta1 was reduced in Muc21 transfectants in a tandem repeat-dependent manner, whereas equal amounts of proteins were detected by Western blot analysis. Muc21 was expressed as a large glycoprotein that was highly glycosylated with O-glycans at the cell surface, as detected by flow cytometry, Western blotting, and lectin blotting. Although at least a portion of Muc21 was glycosylated with sialylated glycans, removal of sialic acid did not influence the prevention of adhesion.

Identification and expression of human epiglycanin/MUC21: a novel transmembrane mucin.

The gene for the human orthologue of mouse epiglycanin, a mucin expressed on mammary carcinoma TA3-Ha cells but not TA3-St cells, was identified by homology search to a mouse epiglycanin cDNA fragment identified by representational difference analysis between TA3-Ha and TA3-St cells. The open reading frame of this gene was cloned from human cervical carcinoma ME-180 cells. It consists of a mucin domain with 28 nonidentical tandem repeats of 45 nucleotides each corresponding to a threonine/serine-rich peptide, a stem domain, a transmembrane domain, and a cytoplasmic tail. The cloned cDNA with a FLAG sequence was expressed in K562 cells. A combination of immunoprecipitation with a polyclonal antibody specific for the cytoplasmic tail and Western blotting analysis with an anti-FLAG antibody and lectins revealed a mucin-like component as the gene product. Analysis by the use of tissue cDNA libraries indicated that the gene is expressed in lung, large intestine, thymus, and testis among 16 normal tissues tested. The polyclonal antibody specific for a synthetic peptide from the cytoplasmic tail, when tested for its reactivity with normal lung tissues, reacted with epithelia of bronchi and bronchioli but not with alveoli. All of 24 lung adenocarcinomas specimens tested were reactive with the antibody, whereas reactivity was observed with only 2 out of 24 squamous and none out of 24 small cell lung carcinomas. This is a novel transmembrane mucin and designated as MUC21.

Protective role for nitric oxide during the endoplasmic reticulum stress response in pancreatic beta-cells.

Higher requirements for disulfide bond formation in professional secretory cells may affect intracellular redox homeostasis, particularly during an endoplasmic reticulum (ER) stress response. To assess this hypothesis, we investigated the effects of the ER stress response on the major redox couple (GSH/GSSG), endogenous ROS production, expression of genes involved in ER oxidative protein folding, general antioxidant defense, and thiol metabolism by use of the well-validated MIN6 beta-cell as a model and mouse islets. The data revealed that glucose concentration-dependent decreases in the GSH/GSSG ratio were further decreased significantly by ER-derived oxidative stress induced by inhibiting ER-associated degradation with the specific proteasome inhibitor lactacystin (10 microM) in mouse islets. Notably, minimal cell death was observed during 12-h treatments. This was likely attributed to the upregulation of genes encoding the rate limiting enzyme for glutathione synthesis (gamma-glutamylcysteine ligase), as well as genes involved in antioxidant defense (glutathione peroxidase, peroxiredoxin-1) and ER protein folding (Grp78/BiP, PDI, Ero1). Gene expression and reporter assays with a NO synthase inhibitor (Nomega-nitro-L-arginine methyl ester, 1-10 mM) indicated that endogenous NO production was essential for the upregulation of several ER stress-responsive genes. Specifically, gel shift analyses demonstrate NO-independent binding of the transcription factor NF-E2-related factor to the antioxidant response element Gclc-ARE4 in MIN6 cells. However, endogenous NO production was necessary for activation of Gclc-ARE4-driven reporter gene expression. Together, these data reveal a distinct protective role for NO during the ER stress response, which helps to dissipate ROS and promote beta-cell survival.

L-rhamnose and lactulose decrease serum triacylglycerols and their rates of synthesis, but do not affect serum cholesterol concentrations in men.

Colonic short-chain fatty acids (SCFA) may affect hepatic lipid metabolism. Lactulose increases colonic acetate production, whereas L-rhamnose increases propionate. To test the effects of oral L-rhamnose and lactulose for 28 d on fasting concentrations and hepatic synthesis of lipids in humans, 18 men were administered 25 g/d of L-rhamnose, lactulose, or d-glucose for 4 wk in a partially randomized crossover design, with blood collected from fasting subjects on the first and last day of each period. Cholesterol and triacylglycerol (TG) synthesis rates were determined using deuterated water uptake rate over the last 24 h of each period. Postprandial blood lipids, and glucose and insulin were assessed in 11 subjects on d 28. Fasting serum cholesterol was unchanged; however, when expressed as a percentage change, TG were decreased, relative to baseline (P < 0.04), by L-rhamnose (-10%) and lactulose (-10%), compared with D-glucose, which increased serum TG (+11%). Net TG-fatty acid (TGFA) synthesis on d 28 was lower with L-rhamnose (2.42 +/- 0.38 g/d) and lactulose (2.62 +/- 0.35 g/d) than with D-glucose (2.96 +/- 0.31 g/d, P < 0.01). We conclude that these results do not support a primary role for propionate in the cholesterol-lowering effect of soluble fiber. However, both lactulose and L-rhamnose lowered serum TG (expressed as a percentage change) and TGFA synthesis, compared with d-glucose, which increased them. Although these data are consistent with inhibition of TGFA synthesis by SCFA, other aspects of the metabolism of these sugars cannot be ruled out as putative agents of their TG-lowering effects.

Temporal changes of multiple redox couples from proliferation to growth arrest in IEC-6 intestinal epithelial cells.

Changes in intracellular redox couples and redox reactive molecules have been implicated in the regulation of a variety of cellular processes, including cell proliferation and growth arrest by contact inhibition. However, the magnitude, direction, and temporal relationship of redox changes to cellular responses are incompletely defined. The present work sought to characterize redox and metabolic changes associated with proliferative stages to contact inhibition of growth in rat IEC-6 intestinal epithelial cells. From the first day of culture until 1 day before confluence, an increase in GSH concentrations and a significant reduction in the redox potential of the GSSG/2GSH couple were observed. These changes were accompanied by a decrease in relative reactive oxygen species (ROS) and nitric oxide (NO) concentrations and oxidation of the redox potential of the NADP(+)/reduced NADP and NAD(+)/NADH couples. Postconfluent cells exhibited a significant decrease in GSH concentrations and a significant oxidation of the GSSG/2GSH couple. When cell proliferation decreased, relative ROS concentrations increased (P < 0.01), whereas NO concentrations remained unchanged, and the NAD(+)/NADH couple became more reduced. Together, these data indicate that the redox potential of distinct couples varies differentially in both magnitude and direction during successive stages of IEC-6 growth. This finding points out the difficulty of defining intracellular redox status at particular stages of cell growth by examining only one redox species. In addition, the data provide a numerical framework for future research of regulatory mechanisms governed by distinct intracellular redox couples.

L-Rhamnose increases serum propionate after long-term supplementation, but lactulose does not raise serum acetate.

BACKGROUND: Acute ingestion of the unabsorbed sugar l-rhamnose in humans raises serum propionate, whereas acute ingestion of lactulose raises serum acetate. It is not known whether short-chain fatty acid concentrations in urine and feces reflect those in blood. OBJECTIVE: The objective was to test the effects of oral l-rhamnose and lactulose for 28 d on acetate and propionate concentrations in serum, urine, and feces. DESIGN: Eleven subjects ingested 25 g l-rhamnose, lactulose, or d-glucose (control) for 28 d in a partially randomized crossover design. One fecal sample, hourly blood samples, and all urine samples were collected over 12 h on the last day of each phase. RESULTS: The increase in serum propionate was greater after l-rhamnose than after lactulose (P < 0.05). The effect of lactulose on serum acetate was not significant, but lactulose raised the acetate:propionate ratio compared with d-glucose or l-rhamnose in serum (P < 0.005) and urine (P < 0.02). Flatulence was significantly greater after lactulose and l-rhamnose than after d-glucose (P < 0.0001), an effect that lasted 4 wk with lactulose but only 1 wk with l-rhamnose. CONCLUSIONS: This study confirmed that l-rhamnose ingestion over 28 d continues to selectively raise serum propionate in humans. Although serum acetate did not increase significantly after lactulose, the serum acetate:propionate ratio was significantly different after l-rhamnose and lactulose, which suggests that these substrates could be used to examine the role of colonic acetate and propionate production in the effect of dietary fiber on lipid metabolism. Changes in the ratio of urinary acetate to propionate reflected those in serum.