Expression Of Tn Research Articles

Tn (N-acetyl-d-galactosamine-O-serine/threonine) immunization protects against hyperoxia-induced lung injury in adult mice through inhibition of the nuclear factor kappa B activity

Prolonged hyperoxia exposure leads to inflammation and acute lung injury. Since hyperoxia activates nuclear factor kappa B (NF-κB) and proinflammatory mediators in lung fibroblasts and murine lungs, and proinflammatory cytokines upregulate Tn (N-acetyl-d-galactosamine-O-serine/threonine) expression in human gingival fibroblasts. We hypothesized connections exist between Tn expression and inflammation regulation. Thus, we immunized adult mice with Tn antigen to examine whether Tn vaccine can protect against hyperoxia-induced lung injury by inhibiting NF-κB activity and cytokine expression through the action of anti-Tn antibodies. Five-week-old female C57BL/6NCrlBltw mice were subcutaneously immunized with Tn antigen four times at biweekly intervals, and one additional immunization was performed at 1 week after the fourth immunization. Four days after the last immunization, mice were exposed to room air (RA) or hyperoxia (100% O2) for up to 96 h. Four study groups were examined: carrier protein + RA (n = 6), Tn vaccine + RA (n = 6), carrier protein + O2 (n = 6), and Tn vaccine + O2 (n = 5). We observed that hyperoxia exposure reduced body weight, increased alveolar protein and cytokine (interleukin-6 and tumor necrosis factor-α) levels, increased mean linear intercept (MLI) values and lung injury scores, and increased lung NF-κB activity. By contrast, Tn immunization increased serum anti-Tn antibody titers and reduced the cytokine levels, MLI values, and lung injury scores. Furthermore, the alleviation of lung injury was accompanied by a reduction in NF-κB activity. Therefore, we proposed that Tn immunization attenuates hyperoxia-induced lung injury in adult mice by inhibiting the NF-κB activity.

Ca2+ metabolic disorder and abnormal expression of cardiac troponin involved in fluoride-induced cardiomyocyte damage

Our previous study indicated that excessive fluoride (F) induces ATP5J and ATP5H proactive expression by interfering cardiomyocyte mitochondrial dysfunction in mice. This study aimed to investigate underlying mechanisms of F¯ induced damage to cardiomyocytes. A total of 100 mg/L F¯ was added to distilled water to treat Kunming mice for 70 days. Pathological and morphological changes in myocardial tissues were observed under transmission electron microscope and light microscope. Content of ATP and ATP enzyme distributed in cardiomyocytes were determined by fluorescence and ATP enzyme staining. Expression levels of troponin (Tn) C, TnI, TnT and tropomyosin (TPM) were measured by immunofluorescence, western blot, and real-time polymerase chain reaction. Contents of Ca2+ in blood, myocardial cells and faeces were also detected by confocal microscopy and ethylenediaminetetraacetic acid. Using 100 mg/L F¯ resulted in nuclaer enrichment, the myocardial fibre breakage and mitochondrial lysis. Following mitochondrial structure damage, contents of ATP and ATP enzyme significantly decreased in the fluoride group. Expression levels of TnT and TnI were significantly down-regulated, whereas that of TPM was up-regulated. Content of Ca2+ in cardiomyocytes of fluoride group visibly increased. Interestingly, contents of Ca2+ in blood and faeces decreased. These findings reveal that excessive F ingestion induces Ca2+ metabolic disorder, and an abnormal expression of cardiac Tn are involved in F-induced cardiomyocyte damage.

Abstract P3-05-14: Expression of the tumor associated carbohydrate antigen Tn and immune effectors in invasive breast cancer

Abstract Background: Glycosylation is a post-translational modification generating complex carbohydrate O-glycans. Tn antigen, a N-acetyl-galactosamine-α-O-Ser/Thr residue that is the core glycosylated component of complex mucin-type O-glycans, is expressed in breast cancers. Tn has been associated with invasion, survival and decreased T-cell mediated immune response in several types of cancer including breast cancer. There is therefore a potential rationale to combine Tn vaccine with checkpoint blockers such as PD-1/PDL-1 inhibitors. Little is known regarding the link between the respective expression of Tn antigen, PDL-1, and TILs, in breast cancer. Our study aimed at characterizing Tn expression in a series of invasive breast cancers, and its correlation with immune response. Methods Tn expression was analyzed by immunohistochemistry performed on a tissue microarray of 1864 invasive breast cancer samples (1850 patients for statistical analyses), consisting of 189 (10%) TNBC, 1601 (86%) ER and/or PR positive à checker tumors and 191 (10%) HER-2 positive tumors treated at our institution between 2005 and 2013. Among these cases, 50 luminal B tumors and 50 TNBC were selected for full section and immune analysis, i.e. to study the pattern of Tn expression across whole tumor section and the link between Tn expression and the level of CD3+ and CD8+ infiltrates, and PDL-1 expression. The level of Tn expression was assessed using H-score, combining the percentage of stained cells with staining intensity. Results: In the 100 initial samples, Tn expression was observed in 95%, with a H-Score>10 in 85% of the cases, and with homogeneous staining across the whole tumor section in 86% of the cases. Tn expression was increased in Luminal B subtype as compared to TNBC, with a mean H-Score of 100.9 vs 55, respectively (p <0,0001). The CD3+ infiltrate was more important in TNBC as compared to luminal B tumors (mean stromal CD3+: 34.3% vs 21.2%, p=0,0052). No significant difference was found for the CD8+ infiltrate. PDL-1 expression in stromal cells (≥1%) was observed in 43% of the cases, and increased in TBNC as compared to Luminal B (mean of 7.3% vs 2.2% of cells respectively, p=0.0252). In both tumor subtypes, we observed a positive correlation between the CD3/CD8 infiltrates and PDL-1 expression in stroma cells (p<0,0001), but not with Tn expression. In the TMA cohort, Tn expression was observed in 1723 (92%) tumors with a mean H-Score of 82.75. Similarly, to the results obtained in full section, Tn expression was less intense in TNBC as compared to other subtypes (mean H-Score of 62 vs 85, p<0.001). Tn expression was lower in histological grade I as compared to grade II/III tumors (64,69 for grade I, 88,84 for grade II and 88.99 for grade III, p<0.0001), and significantly higher in HER2 positive tumors versus others (mean H score 62,06 for HER2-/RE-, 119,05 for HER2+ and 80,25 for HER2-/RE+, p<0.0001). Conclusion: Our study shows a lower level of expression of Tn antigen in TNBC as compared to luminal tumors. We could not identify any correlation between Tn antigen expression and immune status of the tumor as defined by CD3+, CD8+ and PDL-1+ infiltrate in tumor stroma. The role of Tn antigen in the HER2-positive subgroup requires further investigation. Citation Format: Verret B, Rossoni C, Lebeherec D, Michiels S, Castanon-Alvarez E, Leclerc C, Delaloge S, Artaud C, Lacroix-Tikri M. Expression of the tumor associated carbohydrate antigen Tn and immune effectors in invasive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-14.

O‐Glycan recognition and function in mice and humans

Glycans function both as direct recognition molecules, where they are bound by a wide variety of glycan‐binding proteins (GBPs), including animal cell receptors, microbial adhesins lectins and toxins, and indirectly glycans function in glycoproteins to facilitate intra‐ and intermolecular interactions that maintain homeostasis and biological integrity. To explore both direct and indirect functions of glycans we have engineered mutations in biosynthetic pathways, most notably in regard to O‐glycan biosynthesis (O‐GalNAc glycans). Normal synthesis of such O‐glycans in animals requires the action of the T‐synthase, a b3‐galactosyltransferase that generates the core 1 O‐glycan Galb1‐3GalNAca1‐Ser/Thr from the Tn antigen precursor GalNAca1‐Ser/Thr. Such normal extended O‐glycans, including those with the core 2 O‐glycan structure and sialyl Lewis x antigen, are recognized by key GBPs, including P‐selectin and L‐selectin. Such specific recognition of O‐glycans by P‐selectin, CLEC2, and other GBPs, directly regulated many cellular interactions and signaling pathways important in development, morphogenesis and organ development. In molecular and biochemical studies we have explored such recognition by generating glycopeptides with specific modifications, including O‐glycans and sulfated tyrosine residues. In terms of additional functions of O‐glycans, we have exploited our discovery that the T‐synthase requires a specific protein chaperone, termed Cosmc, encoded on the X‐chromosome. Cosmc, a resident protein in the endoplasmic reticulum, directly interacts with the newly synthesized T‐synthase to facilitate folding to the active form of the enzyme, that subsequently moves to its functional location in the Golgi apparatus. Thus, Cosmc is the key regulator of Tn antigen expression, and loss of Cosmc, through epigenetic silencing, gene mutations and other pathways, and acquired changes in Cosmc function cause Tn expression in many human tumors and tumor cell lines and in Tn syndrome patients. Targeted deletion of Cosmc leads to loss of normal O‐glycans, expression of the Tn and sialyl Tn antigens, and multiple pathological consequences. In this presentation the loss of Cosmc has been explored in the gastrointestinal tract, where it leads to pathology, chronic inflammation, and changes in the gut microbiome, and in other cell types, such as T cells and B cells, where loss of Cosmc alters cellular maturation and functionalities. Our studies are revealing the multiple ways in which O‐glycans in humans, animals, and other organisms interact within the larger protein‐glycan interactome to regulate biological processes.Support or Funding InformationThis work was supported by NIH grants U01CA168930 and P41GM103694 to R.D.C.

Glycoprotein screening in colorectal cancer based on differentially expressed Tn antigen.

Colorectal cancer(CRC) is one of the most common cancers worldwide, and the identification of new biomarkers for CRC is valuable for its diagnosis and treatment. We aimed to screen differentially expressed glycoproteins (especially O-glycoproteins) and to identify diagnostic or therapeutic candidates for colorectal cancer(CRC) based on different Tn antigen expression levels. Fresh cancer tissues and adjacent healthy tissues were obtained from CRC patients and classified into three groups based on their Tn antigen expression: CRC with negative Tn expression(CRC Tn‑), CRC with positive Tn expression(CRC Tn+) and normal control without Tn expression(NC). Protein extractions were separated and identified by iTRAQ technology. Glycoproteins and O-glycoproteins were selected using UniProt and DAVID. Deep bioinformatic analysis, including Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes (KO), was used to annotate this O-glycoprotein interaction network. Subsequently, two O‑glycoproteins were verified by western blotting and immunohistochemistry in either LS174T cells or CRC tissues. We found that 330 differentially expressed proteins were identified by iTRAQ between CRC Tn‑ and NC tissues, 317 between CRC Tn+ and NC tissues, and 316 between CRC Tn‑ and Tn+ tissues. Of the 316 proteins, 55 glycoproteins and 19 O‑glycoproteins were identified and analyzed via deep informatics. Namely, different Tn antigen expression levels in CRC led to differential protein expression patterns, especially for glycoproteins and O‑glycoproteins. Decorin and SORBS1, two representative functional O-glycoproteins, were significantly downregulated in the CRC Tn+ tissues compared with the level in the CRC Tn‑ or NC tissues. Based on this deep bioinformatic analysis, Decorin and SORBS1 are hypothesized to be involved in the TGF‑β and PPAR‑γ signaling pathways, respectively.

Effective antitumor therapy based on a novel antibody-drug conjugate targeting the Tn carbohydrate antigen

ABSTRACTAntibody-drug conjugates (ADC), combining the specificity of tumor recognition by monoclonal antibodies (mAb) and the powerful cytotoxicity of anticancer drugs, are currently under growing interest and development. Here, we studied the potential of Chi-Tn, a mAb directed to a glyco-peptidic tumor-associated antigen, to be used as an ADC for cancer treatment. First, we demonstrated that Chi-Tn specifically targeted tumor cells in vivo. Also, using flow cytometry and deconvolution microscopy, we showed that the Chi-Tn mAb is rapidly internalized – condition necessary to ensure the delivery of conjugated cytotoxic drugs in an active form, and targeted to early and recycling endosomes. When conjugated to saporin (SAP) or to auristatin F, the Chi-Tn ADC exhibited effective cytotoxicity to Tn-positive tumor cells in vitro, which correlated with the level of tumoral Tn expression. Furthermore, the Chi-Tn mAb conjugated to auristatin F also exhibited efficient antitumor activity in vivo, validating for the first time the use of an anti-Tn antibody as an effective ADC.

Molecular basis of antibody binding to mucin glycopeptides in lung cancer.

Glycopeptides bearing Tn epitopes are emerging targets for cancer diagnosis and immunotherapy. In this study, we analyzed membrane proteins containing O-glycosylated tandem repeat (TR) sequences in lung cancer patients of different types and stages, using gene microarray data in public domain. The expression of Tn and glycopeptide epitopes on the surface of lung cancer cell lines were studied by monoclonal IgG antibodies 14A, 16A, and B72.3. The binding of mAbs to synthetic glycopeptides were studied by surface plasmon resonance. Nine mucin mRNAs were found to be expressed in lung cancer patients but at similar level to healthy individuals. At protein level, a glycopeptide epitope on cancer cell surface is preferably recognized by mAb 16A, as compared to peptide-alone (14A) or sugar-alone epitopes (B72.3). 14A and 16A favor clustered TR containing more than three TR sequences, with 10-fold lower Kd than two consecutive TR. B72.3 preferrably recognized clustered sialyl-Tn displayed on MUC1 but not other O-glycoproteins, with 100-fold stronger binding when MUC1 is transfected as a sugar carrier, while the total sugar epitopes remain unchanged. These findings indicate that clusters of both TR backbones and sugars are essential for mAb binding to mucin glycopeptides. Three rules of antibody binding to mucin glycopeptides at molecular level are presented here: first, the peptide backbone of a glycopeptide is preferentially recognized by B cells through mutations in complementarity determining regions (CDRs) of B cell receptor, and the sugar-binding specificity is acquired through mutations in frame work of heavy chain; secondly, consecutive tandem repeats (TR) of peptides and glycopeptides are preferentially recognized by B cells, which favor clustered TR containing more than three TR sequences; thirdly, certain sugar-specific B cells recognize and accommodate clustered Tn and sialyl-Tn displayed on the surface of a mucin but not other membrane proteins.

Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics

Muscle invasive bladder cancer (MIBC, stage ≥T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin). This decreased the number of invasive lesions and, concomitantly, the expression of STn and also pS6, the downstream effector of the PI3K/Akt/mTOR pathway. In conclusion, STn was found to be marker of poor prognosis in bladder cancer and, in combination with PI3K/Akt/mTOR pathway evaluation, holds potential to improve the stratification of stage disease. Animal experiments suggest that mTOR pathway inhibition could be a potential therapeutic approach for this specific subtype of MIBC.

2160 GIV as a novel marker of recurrence risk in MMR proficient stage II colon cancer

s S387 Material and Methods: Formalin-fixed, paraffin-embedded tissue sections from 186 primary colorectal carcinomas and four normal colorectal tissues were immunostained using specific monoclonal antibodies against Tn and sialyl-Tn antigens. Baseline clinicopathological characteristics and overall 5-year survival were correlated with antigen expression. Results: Of the 186 colorectal carcinomas, Tn antigen is expressed in 70 (37.6%) and sialyl-Tn antigen is expressed in 141 (75.8%). There is no staining for these antigens in normal colorectal tissues. Immunostaining measurements for Tn antigen were significantly associated with the differentiation degree (p = 0.017) of the tumors, but independent of age, gender, tumor location, depth of penetration, status of lymph nodes, lymphovascular invasion, perineural invasion and TNM stage. The expression results of sialyl-Tn antigen revealed more significant associations with the degree of differentiation (p = 0.006) and perineural invasion (p = 0.041). The poor overall survival rate of patients was significantly associated with the positive expression of sialyl-Tn (p = 0.013) but not with the expression of Tn (p = 0.650). Survival at 5 years for patients with sialyl-Tn-negative versus sialyl-Tn-positive tumors was 72.1% versus 49.2%. Cox regression multivariate analysis confirmed that TNM stage (p = 0.001) and sialyl-Tn expression (p = 0.038) were the two most important variables of for predicting overall survival. Conclusions: These results indicate that Tn and sialyl-Tn antigens appear to be useful biomarkers of poorly differentiated adenocarcinomas and mucinous carcinomas. Moreover, the positive expression of sialyl-Tn antigen is an independent predictor of poor prognosis in human colorectal carcinoma. No conflict of interest.

CONTINUED FRACTION FOR TRIBONACCI RATIO

We study generalized continued fractions for the expression of tribonacci ratios Tn+k Tn when n is large, and find nth convergents of the continued

Abstract 4983: GALNT3 mediated differential MUC4 glycosylation in the progression of pancreatic cancer

Abstract Purpose: MUC4 is one of the membrane-bound mucins that is expressed de novo during pancreatic cancer (PC) and contributes to pathogenesis. Further, MUC4 is aberrantly glycosylated in PC and some of its tumor-promoting effects are, in part, mediated by interactions facilitated by the glycan epitopes, which are generally masked under normal conditions. Mucins predominantly undergo O-glycosylation that is initiated by a family of enzymes known as GALNTs (UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase), catalyzing the transfer of first sugar residue (GalNAc) to ser/thr residues in mucin tandem repeat domains. Not much is known about the specific glycosyltransferases (GTs) involved in MUC4 glycosylation. Understanding the specific enzymes regulating differential glycosylation of MUC4 in cancer may be important for developing therapeutic strategies for modulating MUC4 function in PC. Thus, the goal of this study was to identify specific GALNT(s) catalyzing MUC4 glycosylation and investigate its role in differential MUC4 glycosylation in PC. Materials & Methods: PCR array analysis was performed in a panel of MUC4 expressing and non-expressing PC cell lines, normal pancreatic cancer cells and MUC4 expressing normal colon cells to identify differentially expressed GTs. To determine the altered glycosylation of MUC4 in normal versus cancerous conditions, Co-immunoprecipitation (Co-IP) studies were conducted using several carbohydrate-specific antibodies. Stable knockdown of GALNT3 was performed in PC and normal colon cells to understand its involvement in differential MUC4 glycosylation. To determine the functional implications of GALNT3 mediated aberrant glycosylation, various in vitro assays such as colony formation and migration were carried out. Results: PCR array and immunoblot analysis demonstrated GALNT3 as one of the GTs upregulated in MUC4 expressing cells as compared to MUC4 non-expressing PC cells. Interestingly, Co-IP analysis revealed differential expression of carbohydrates epitopes such as STn, SLex, SLea on MUC4 in PC and normal colon cells. GALNT3 knockdown in cancer cells resulted in decreased expression of Tn but simultaneous increase in STn epitope. Interestingly, we observed significant increase in colony formation and migration in GALNT3 knockdown cells. This was associated with increase in proliferation and EMT markers in GALNT3 knockdown PC cells such as N-cadherin, vimentin and phosphorylated ErbBs. Conclusion: Overall, our results suggested a correlation of MUC4 and GALNT3 expression, and knockdown of GALNT3 resulted in altered MUC4 glycosylation rather than complete abrogation, suggesting a possible compensatory increase in other GALNTs and other GTs. Further investigations along these lines will provide insight into specific GTs regulating MUC4 glycosylation and glycan epitope-mediated MUC4 functions in cancer. Citation Format: Seema Chugh, Vinayaga Srinivasan Gnanapragassam, Maneesh Jain, Moorthy Ponnusamy, Surinder Kumar Batra. GALNT3 mediated differential MUC4 glycosylation in the progression of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4983. doi:10.1158/1538-7445.AM2015-4983

The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn, Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL.

Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galβ1,3GalNAc) the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar deadadhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours.

The promise of glycomics for discovery of new biomarkers

Glycosylation, the attachment of carbohydrates to a protein or lipid, is the most abundant and structurally complex post-translational modification taking place in cells. Approximately two percent of the human genome codes for proteins involved in the glycosylation pathways that modify proteins and lipids. Carbohydrates themselves can be modified in multiple ways, undergoing for example acetylation, sulfation, methylation, phosphorylation, or sialylation. This results in a cellular glycan repertoire composed of vastly diverse and information-rich molecules. Glycans are central to numerous intra-cellular and inter-cellular processes including cell growth, differentiation, adhesion, and cellcell recognition. To date ninety nine distinct human glycosylation disorders, many affecting multiple organ systems, have been identified. The extent of cellular glycosylation is dynamic, changing in response to the phenotype, metabolic state, and developmental stage of the cell. Changes in the expression of one’s cellular glycan profile and the synthetic enzymes involved in their synthesis, as well as the repertoire of one’s serum anti-glycan antibodies, are observed during cellular transformation and metastasis. For example, changes in N-glycan branching, the extent of sialylation or the rate of glycosylation of mucins, the over expression of sialylated gangliosides, and loss of expression of glycosylphosphatidylinositol (GPI), have all been correlated with malignant disease and its progression. This makes glycans promising molecules for the discovery of biomarkers to detect and monitor disease. However, until recently the study of carbohydrates as potential biomarkers has suffered from a lack of tools and technologies capable of carrying out rapid, robust analysis of structural and functional changes in cellular glycans, anti-glycan antibodies, and serum glycoproteins and glycolipids. The development of glycan array technology for determining the binding specificity of glycan binding proteins and serum anti-glycan antibodies, and advances in high-throughput analytical technologies capable of defining glycan structure, are now opening up new avenues for the discovery of glycan biomarkers with specific prognostic and diagnostic value. This special issue of Cancer Biomarkers will review the clinical findings and genetic and glycobiologic basis of glycosylation disorders, and the need for better serum based diagnostic biomarkers, beyond the presently used blood test for N-linked glycan disorders. New glyco-analytical tools for study of glycosylation changes in serum glycoproteins and how such data can improve our understanding of disease pathways and lead to discovery of pre-clinical and clinical glycan based disease biomarkers will be discussed. Likewise, the use of glycan array technology to identify new disease biomarkers, and the power of these arrays to determine carbohydrate binding specificity for use as a diagnostic tool, using flu as a model system will be presented. Finally, we will look at the presence of glycosylphosphatidylinositol anchored proteins in serum and how they may serve as biomarkers for detection of cancer, and how truncation of O-glycans (expression of the tumor-associated carbohydrate antigen Tn and/or Sialyl Tn antigen) through genetic and epigenetic pathways, or metabolic regulation results in malignant disease.

Expression of tumor-associated carbohydrate antigen Tn in cancer cells is stimulated by cytokine secreted from cancer cells

Meeting abstracts Tumour-associated carbohydrate antigen (TACA) Tn is always observed on the cell surface of malignant tumour cells. Tn is presented as mucin-type carbohydrate. It has been reported to be associated with cancer progression, but the regulation on Tn expression in cancer cells is not

Reciprocal relationship of Tn/NF‐κB and sTn as an indicator of the prognosis of oral squamous cell carcinoma

In order to determine whether the expression of tumour-associated carbohydrate antigens (Tn/sTn) and a representative inflammation marker, nuclear factor-κB (NF-κB), is associated with the invasiveness of oral squamous cell carcinoma (OSCC), this study has attempted to investigate the correlation of the aforementioned markers with the well-established invasive pattern grading score (IPGS) and clinicopathological parameters. Specimens from 143 OSCC patients with classified clinicopathological parameters and IPGS were stained immunohistochemically using anti-Tn, sTn and NF-κB antibodies. Our results showed that the expression of both Tn and NF-κB was correlated positively with staging (P = 0.036; P = 0.015), recurrence (P < 0.001; P < 0.001) and distant metastasis (P = 0.005; P = 0.009), as well as with IPGS, while the expression of sTn was correlated inversely. In addition, poor survival was associated with overexpression of Tn and NF-κB but not with expression of sTn. Our results indicate that a reciprocal relationship between Tn and sTn expression may serve as a reliable indicator for OSCC prognostic evaluation. In addition, expression of Tn rather than sTn may play an important role in deeply invasive OSCC via regulation of NF-κB signalling.

AB0128 Expression of aberrantly glycosylated mucins (tn and sialyl tn antigens) and muc1 in labial salivary gland of patients with sjogren’s syndrome.

BackgroundPrimary Sjogren syndrome (SS) is an autoimmune exocrinopathy. Mucin are present on apical surfaces of all healthy wet surface epithelia of human body and on the glands of simple secretory...

Reciprocal expression of tumour-associated carbohydrate antigens (Tn/sTn) and nuclear factor-B serves as an indicator of the prognosis of oral squamous cell carcinoma

To determine whether expression of tumour-associated carbohydrate antigens (Tn/sTn) and a representative inflammation marker, nuclear factor-B (NF-B), is related to the invasiveness of oral squamous cell carcinoma (OSCC) by correlating these markers with the wellestablished invasive pattern grading score (IPGS) and clinicopathological parameters. Specimens from 143 OSCC patients with clinicopathological parameters were classified and scored by the IPGS system, followed by an immunohistochemical study of the expression of Tn, sTn and NF-B. Our results showed that the expression of both Tn and NF-B was significantly correlated with staging, recurrence, and distant metastasis. Both Tn and NF-B expression were positively correlated with IPGS; however, sTn expression was inversely correlated with IPGS. In addition, overexpression of Tn and NF-B was closely correlated with poor survival, whereas expression of sTn was inversely correlated with the patients’ prognosis. Our results indicate there is a reciprocal relationship between Tn and sTn expression and that these may serve as reliable indicators for evaluation of prognosis. They are also worthy of consideration as a therapeutic target for treatment of OSCC. In addition, expression of Tn rather than sTn may play an important role in late invasive OSCC via regulation of NF-B signalling.

Different Levels of Sialyl-Tn Antigen Expressed on MUC16 in Patients With Endometriosis and Ovarian Cancer

Although CA125 antigen is a useful marker for ovarian cancer, its expression is also elevated in endometriosis. The purpose of this study was to develop an assay method for evaluating differentially glycosylated MUC16 (CA125 core protein) in patients with endometriosis and ovarian cancer. We prepared MUC16-enriched fractions from peritoneal fluid of patients with endometriosis and conditioned medium of ovarian carcinoma-3 cells by gel filtration, and evaluated the expression of sialyl-Le, Tn, and sialyl-Tn antigens by dot blot analysis. A sandwich enzyme-linked immunosorbent assay was developed to measure the level of sialyl-Tn antigen expressed on MUC16 (sTn/MUC16). The level of sTn/MUC16 was compared between patients with endometriosis (n = 21) and ovarian cancer (n = 36) and in ovarian cancers with different clinical diagnostic criteria. Furthermore, distribution of MUC16 and sialyl-Tn antigen in ovarian cancer tissues was observed immunohistochemically. Sialyl-Tn antigen was markedly detectable in the MUC16-enriched fractions from conditioned medium of ovarian carcinoma-3 cells but negligible in those from the peritoneal fluid of the patients with endometriosis. The level of sTn/MUC16 determined by a sandwich enzyme-linked immunosorbent assay was significantly higher in the patients with ovarian cancer than that in the patients with endometriosis (P < 0.001). An elevated level of sTn/MUC16 was detected in 44% of the patients with ovarian cancer but not all the patients with endometriosis. This level increased more prominently in the patients with ovarian cancer than that of MUC16 as both the clinical stage and cytological grade advanced. An elevated level of sTn/MUC16 was frequently found in the patients with serous and endometrioid carcinomas. Consistent with this, sialyl-Tn antigen was colocalized with MUC16 in serous and endometrioid ovarian cancer tissues. Estimation of the sTn/MUC16 level may be useful for discriminating endometriosis from ovarian cancer and for evaluating the clinical stage, cytological grade, and histological type of ovarian cancer.

Cosmc Is Silenced in Human Tn4 B Cells through Hypermethylation of the Gene Promoter

BackgroundMucin‐type O‐glycosylation is regulated through core 1 β3‐galatosyltransferase (T‐synthase) and its specific molecular chaperone Cosmc. Human Cosmc is encoded by its gene on Xq24. Genetic alterations of Cosmc, including point mutations and gene deletions, have been identified in several human diseases. Tn4 B cells from patient with Tn‐syndrome‐like features have no transcript of Cosmc and express the Tn antigen (GalNAcα1‐Ser/Thr).Study ObjectiveTo elucidate the molecular mechanism as to how human Tn4 B cells lack Cosmc transcripts.MethodsTreat Tn4 B cells with 5‐Aza‐2′‐deoxycytidine (5‐Aza‐CdR) to test whether it can induce expression of Cosmc; and perform bisulfite sequencing to examine the methylation status of the Cosmc promoter.ResultsTreatment of Tn4 B cells with 5‐Aza‐CdR results in expression of Cosmc transcripts, restores the activity of T‐synthase, and correct the O‐glycosylation. Bisulfite sequencing showed that CG dinucleotides in the Cosmc promoter in Tn4 B cells are exclusively methylated. Interestingly, other X‐linked genes, OGT and PIG‐A are not silenced in the Tn4 B cells.ConclusionHypermethylation of the Cosmc promoter can completely silence the gene, which is another mechanism that can lead to expression of Tn and STn antigens, which may shed the light on our better understanding of the epigenetic alteration leading to the tumor antigen expression in human tumors.

ChemInform Abstract: The Tn Antigen — Structural Simplicity and Biological Complexity

AbstractReview: 335 refs.