High Molecular Weight Glycoprotein Research Articles

Immunohistochemical Study of CA 125 in Surface Epithelial Tumors of Ovary in Correlation with Serum Levels

Background:
 Ovarian carcinoma is the 4th leading cancer among women in India. Primary ovarian neoplasms exhibit a wide range of histopathological patterns and tumors with epithelial differentiation are most frequent. Among malignant tumors, most common histological type is serous adenocarcinoma whose diagnosis is established in advanced stages of disease in approximately 75% of patients.
 The most widely used tumor marker in ovarian cancer, often considered “gold standard” is Cancer Antigen125. Cancer Antigen 125 is a high molecular weight glycoprotein which is raised in approximately 90% of patients with advanced epithelial ovarian cancer.
 
 Methods:
 A 2 year prospective study included 81 cases of ovarian neoplasms with surface epithelial differentiation. The specimens were fixed in 10% formalin, routinely processed. Sections of 4-5 microns thickness were obtained from the paraffin block and stained with Hematoxylin & Eosin. The tumors were categorised according to WHO classification. Immunohistochemical analysis of Cancer Antigen 125 was done in all malignant & borderline tumors.
 
 Result:
 A total of 81 cases were studied. There were 15 cases with elevated serum Cancer Antigen 125 levels. Of these 8 showed positive tissue expression. The sensitivity of serum Cancer Antigen 125 was 68.75% & its specificity was 93.8%.
 
 Conclusion: 
 Serum Cancer Antigen 125 is elevated in ovarian tumors especially in malignant surface epithelial tumors & more commonly in serous cystadenocarcinoma. There was a good correlation between serum levels & tissue expression of Cancer Antigen 125.

RvE1 uses the LTB4 receptor BLT1 to increase [Ca2+]i and stimulate mucin secretion in cultured rat and human conjunctival goblet cells

PurposeSpecialized pro-resolving lipid mediator resolvin (Rv) E1 stimulates secretion including mucins from conjunctival goblet cells. RvE1 can use both its ChemR23 receptor and the LTB4 receptor BLT1 to increase [Ca2+]i. The purpose of this study was to determine the expression of ChemR23 and BLT1 and receptors on conjunctival goblet cells and the respective roles these two receptors play in goblet cell responses to RvE1. MethodsGoblet cells were cultured from male rat or human conjunctiva from both sexes. Western blotting analysis, reverse transcription PCR and immunofluorescence microscopy were used to demonstrate the expression of ChemR23 and BLT1 in conjunctival goblet cells. High molecular weight glycoprotein secretion was determined using an enzyme-linked lectin assay. Signaling pathways were studied by measuring the increase in [Ca2+]i using fura 2/AM. ResultsChemR23 and BLT1 and receptors were present on both rat and human conjunctival goblet cells. The BLT1 inhibitors LY293111 and U75302 significantly blocked RvE1-and LTB4-stimulated [Ca2+]i increase. RvE1-and LTB4-stimulated [Ca2+]i and secretion increases were blocked by BLT1-targeted siRNA. RvE1-stimulated [Ca2+]i and secretion increases were also blocked by ChemR23-targeted siRNA. Addition of RvE1 2 min before or simultaneously with LTB4 desensitized the LTB4 [Ca2+]i response. Addition of RvE1 and LTB4 simultaneously caused secretion that was decreased compared to either response alone. ConclusionRvE1, in addition to the ChemR23 receptor, uses the BLT1 receptor to increase [Ca2+]i and stimulate secretion in both rat and human cultured conjunctival goblet cells.

Biosensing of prostate specific antigen (PSA) in human plasma samples using biomacromolecule encapsulation into KCC-1-npr-NH2: A new platform for prostate cancer detection

Prostate-specific antigen (PSA) is a high molecular weight glycoprotein that is used as a marker for the diagnosis of prostate cancer and is therefore important in the medical field. In this study, a novel sandwich type immunoassay was designed based on encapsulation of biotinylated antibody into KCC-1-npr-NH2. KCC-1-npr-NH2 stabilized the stability of the primary antibody. So, encapsulated Ab1 was immobilized on the surface of glassy carbon electrode. Field emission scanning electron microscope (FE-SEM) was employed to monitor the sensor fabrication. The engineered immunosensor was used for the detection of PSA using differential pulse voltammetry (DPVs) and square wave voltammetry (SWVs) techniques. The proposed interface lead to enhancement of accessible surface area for immobilizing a high amount of anti-PSA antibody, increasing electrical conductivity, boosting stability, catalytic properties and biocompatibility. The intensity of electrochemical signals is also increased by the use of AuNPs functionalized with CysA used in secondary antibody (HRP conjugated PSA) structure. Under optimal conditions, the designed immuno-assay provide a good analytical performance for quantifying the PSA marker in the linear range of 1 to 60 μg/l.

Sclerotium rolfsii lectin induces opposite effects on normal PBMCs and leukemic Molt-4 cells by recognising TF antigen and its variants as receptors.

Sclerotium rolfsii lectin (SRL) exerts apoptotic effect against various cancer cells and an antitumor activity on mice with colon and breast cancer xenografts. The current study aimed to explore its exquisite carbohydrate specificity on human peripheral blood mononuclear cells (PBMCs) and leukemic T-cells. SRL, showed strong binding (>98%) to resting/activated PBMCs, leukemic Molt-4 and Jurkat cell lines. The glycans mediated binding to these cells was effectively blocked by mucin and fetuin, exhibiting 97% and 94% inhibition respectively. SRL showed mitogenic stimulation of PBMCs at 10μg/ml as determined by thymidine incorporation assay. In contrast, lectin induced a dose dependent growth inhibition of Molt-4 cells with 58% inhibition at 25μg/ml. Many common membrane receptors in activated PBMCs, Molt 4 and Jurkat cells were identified by lectin blotting. However, membrane receptors that are recognized by SRL in normal resting PBMCs were totally different and are high molecular weight glycoproteins. Treatment of membrane receptors with glycosidases prior to lectin probing, revealed that fucosylated Thomsen-Friedenreich(TF) antigen glycans are increasingly expressed on transformed Molt-4 leukemic cells compared to other cells. The findings highlight the opposite effects of SRL on transformed and normal hematopoietic cells by recognizing different glycan-receptors. SRL has promising potential for diagnostics and therapeutic applications in leukaemia.

Clinical Challenge and Call for Research on Keloid Disorder: Meeting Report from The 3rd International Keloid Research Foundation Symposium, Beijing 2019

Clinical Challenge and Call for Research on Keloid Disorder: Meeting Report from The 3rd International Keloid Research Foundation Symposium, Beijing 2019

Amphiphilic polyanhydride-based recombinant MUC4β-nanovaccine activates dendritic cells.

Mucin 4 (MUC4) is a high molecular weight glycoprotein that is differentially overexpressed in pancreatic cancer (PC), functionally contributes to disease progression, and correlates with poor survival. Further, due to its aberrant glycosylation and extensive splicing, MUC4 is a potential target for cancer immunotherapy. Our previous studies have demonstrated the utility of amphiphilic polyanhydride nanoparticles as a useful platform for the development of protein-based prophylactic and therapeutic vaccines. In the present study, we encapsulated purified recombinant human MUC4-beta (MUC4β) protein in polyanhydride (20:80 CPTEG:CPH) nanoparticles (MUC4β-nanovaccine) and evaluated its ability to activate dendritic cells and induce adaptive immunity. Immature dendritic cells when pulsed with MUC4β-nanovaccine exhibited significant increase in the surface expressions of MHC I and MHC II and costimulatory molecules (CD80 and CD86), as well as, secretion of pro-inflammatory cytokines (IFN-γ, IL-6, and IL-12) as compared to cells exposed to MUC4β alone or MUC4β mixed with blank nanoparticles (MUC4β+NP). Following immunization, as compared to the other formulations, MUC4β-nanovaccine elicited higher IgG2b to IgG1 ratio of anti-MUC4β-antibodies suggesting a predominantly Th1-like class switching. Thus, our findings demonstrate MUC4β-nanovaccine as a novel platform for PC immunotherapy.

BONE REGENERATION AND SOCKET PRESERVATION USING BONE AUGMENTING MATERIALS (HISTOMORPHOMETERIC STUDY IN RABBITS)

Background: The ultimate goal of periodontal therapy is the regeneration of tissues destroyed by periodontal disease Fibronectin belongs to a group of high molecular weight glycoproteins that exist on cell surfaces. It is known to play a role in cell-to-cell and cell-to-substrate adhesion, as well as an essential role in osseointegration due to its capacity to make osteoblasts attach to the extracellular matrix(ECM) components. Combination of Fibronectin and commercially available bone grafts may hold a promising potential of enhanced bone regeneration.Objective: Comparison of the effect of fibronectin, Bovine-Derived Xenograft (BDX)and their combination for socket preservation through histologic and histomorphometric analysis.Material and Method: Thirty six clinically healthy adult New Zealand white male rabbits, were used in this study. The animals were equally divided into three groups; I-control group (defect was left empty as control), II- bio group (bio- oss was applied on the socket ) and III-( Bio Fibro group) bone and fibronectin group( socket was filled with Bovine-Derived Xenograft Bio-Oss® with fibronectin). At 4 and eight weeks the animals were sacrificed. Bone regeneration among the three groups was evaluated through histologic and histomorphometric analysis.Results: The most significant value of area percent of newly formed bone was observed in the (bone and fibronectin) group, then the fibronectin group, with the least amount recorded in the control group. The difference was extremely statistically significant (p<0.0001). The socket is almost filled with newly formed bone trabeculae with organized fibrous tissue in the fibronectin and bone group ( Bio Fibro group ) at eight weeks.Conclusion: Fibronectin is a useful biomaterial for the enhancement of bone formation. Combination of fibronectin and bovine graft seems to lead significantly to favorable bone regeneration and could be efficaciously used in the socket preservation.

Inhibitory activity of salivary glycoproteins on phytohemagglutins (PHA): Possible molecules to enhance nutritional quality of red kidney beans

Food allergy caused by red kidney bean (Phaseolus vulgaris L.) is of serious health concern and is mainly due to its phytohemagglutinins (PHA) content. PHA can enter the circulation after oral uptake and cause IgE mediated allergy. However, studies describing enhancement of nutritional quality of red kidney beans by targeting PHA are not reported. This study was carried out to identify, PHA-inhibitory molecules present in saliva secretions. Results describe that PHA can be effectively inhibited by salivary glycoproteins. Fractionation of salivary proteins by ammonium sulphate precipitation revealed that, PHA-inhibitory proteins can be specifically precipitated at 30-60% of ammonium sulphate saturation. Gel filtration chromatography and lectin-blot analysis of 30-60% ammonium sulphate fraction suggest that only high molecular weight glycoproteins can act as potent inhibitors of PHA. In conclusion, human saliva secretions contain inhibitory glycoproteins which can be used to inhibit PHA effectively. If these glycoproteins are purified to homogeneity, can be used as potent food supplements in order to neutralize allergic potential of PHA, thus increasing the nutritional value of red kidney beans.

Abstract 3829: Generation and characterization of novel antibodies against the β subunit of MUC4

Abstract Mucins are high molecular weight glycoproteins that are primarily expressed by the apical surfaces of respiratory, gastrointestinal and reproductive tracts. They are involved in protection, growth and repair of epithelial surfaces. Apart from its protective function, mucins also regulate diverse functions in normal cells and have a crucial role in oncogenesis. MUC4 is a type-1 transmembrane mucin, which is overexpressed in several cancers and has been implicated in oncogenic transformation, proliferation, invasion, inhibition of apoptosis, and chemoresistance properties associated with these cancer cells. It is a 930 kD glycoprotein, consisting of a large high molecular weight subunit, MUC4α, containing the identical 16 amino acid tandem repeat region and MUC4β, a membrane-bound subunit, containing three EGF (Epidermal Growth Factor)-like domains. The latter subunit is responsible for the oncogenic functions of this molecule and can potentially be targeted. The monoclonal antibody 8G7 has been used extensively to study MUC4 expression. Since 8G7 binds an epitope in the tandem repeat (TR) region on the MUC4α subunit it cannot detect the cleaved cell surface associated form of MUC4 or isoforms that lack the TR region or heavily glycosylated TR regions. The MUC4β subunit however, is attached to the surface of the cell making it an ideal candidate for studying and targeting MUC4. The aim of this study is to generate and characterize monoclonal antibodies (mAbs) against the β subunit of MUC4. Thirty-two mAbs against MUC4β subunit were initially isolated using ELISA screening. Flow cytometry was used to test high affinity binders in MUC4 transfected cells. It was then tested whether these mAbs were able to immunoprecipitate transfected MUC4 from detergent extracted lysates. Finally, four high affinity mAbs (E9 (IgG2b), 6E8 (IgG2b), 1C7 (IgG1), and 3B4 (IgA)) were selected from these analyses. All the four mAbs immunoprecipitated full length MUC4 from native cancer cell lines. All the four mAbs bound to native human MUC4 expressed on cancer cell surface of different cell lines albeit with varying affinities. 3B4 recognized a conformational epitope and exhibited cell surface binding. The mAb 3B4 was internalized into MUC4 expressing cells in a concentration time-dependent manner. 6E8 showed partial internalization while E9 mAb did not show any internalization. Both 3B4 and 8G7 were able to cap MUC4 at the cell surface of cancer cell lines. Our results suggested that these antibodies can potentially block MUC4 function. The mAbs, 3B4, 1C7 and 6E8 inhibited the growth of MUC4 expressing cells in MTT and colony forming assays. Studies are currently underway to evaluate the utility of these novel MUC4β-domain specific mAbs to target MUC4-expressing tumors in vivo. Overall these mAbs can be used as reagents to study MUC4 cleavage and can also be developed into MUC4 specific diagnostic and therapeutic agents. Citation Format: Catherine Orzechowski, Abhijit Aithal, Wade Junker, Prakash Kshirsagar, Surinder Batra, Maneesh Jain. Generation and characterization of novel antibodies against the β subunit of MUC4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3829.

Identification of a low frequency missense mutation in MUC6 contributing to pulmonary artery hypertension by whole-exome sequencing.

Pulmonary arterial hypertension (PAH) represents a progressive disease characterized by abnormally high blood pressure in the pulmonary artery. Although mutations in the bone morphogenetic receptor 2 (BMPR2) are found in 80% of heritable, their low penetrance suggests that other unidentified genetic modifiers are required for this disease. In this report, whole-exome sequencing (WES) and a linkage analysis were performed on genomic DNA isolated from four affected relatives and one non-affected relative in two PAH families. By focusing on meaningful variants which were presented in the four affected family members, but not presented in the non-affected individual, 49 SNP and eight indel variants in 39 genes were identified as candidates. Further high-throughput multiplex genotyping and Sanger sequencing were carried out to confirm the putative causal mutations in 150 individuals (30 idiopathic PAH [IPAH] patients, 30 chronic thromboembolic pulmonary hypertension [CTEPH] patients, and 90 normal controls). A heterozygous and deleterious mutation in the gene MUC6 (p.Pro1716Ser) was confirmed in the IPAH group (20/30, 67%) and CTEPH group (1/30, 3.33%); no variant was detected in the 90 normal controls. MUC6, which is short for mucin 6, encodes high molecular weight glycoprotein produced by many epithelial tissues and forms an insoluble mucous barrier that protects the lumens. We re-confirmed this low frequency mutation with the 1000 Genomes database across all species; no population or frequency data of this allele were acquired. We also found that this mutation site was highly conserved in different species and predicted MUC6 has the protection function of the airway and pneumoangiogram based on genomic sequence data. The compound heterozygous MUC6 gene mutation (p.Pro1716Ser) suggests a novel disease mechanism leading to PAH.

A detection and quantification label-free tool to speed up downstream processing of model mucins.

Mucins are high-molecular weight glycoproteins (0.25–20 MDa) containing one or more domains that are heavily O-glycosylated. Their implications as targets for cancer treatment have increased the interest in these glycoproteins, mainly in the fields of vaccines and antibodies. However, mucins present high heterogeneity, posing challenges that affect purification processes and quality control analysis. In that sense, it is necessary to develop and improve downstream processes and analytical methods to characterize these products.Here a tool based on biolayer interferometry analysis to improve mucin’s detection and quantification in a fast, simple and label free-way is presented. Taking advantage of lectin recognition of mucins’ carbohydrate structures, several lectins were evaluated and immobilized on streptavidin biosensors. Different assay conditions were optimized and the most suitable lectin, Aleuria aurantia lectin (AAL), was selected. Bovine Submaxillary Gland and human MUC5B mucins were used as proof of concept and were successfully detected and quantified at different stages of purification. High sensitivity levels were achieved with LOD and LOQ of 3.8 μg mL-1 and 11.7 μg mL-1 for BSM, and 0.2 μg mL-1 and 0.6 μg mL-1 for MUC5B. AAL binding specificity was also confirmed with fucose competition assays. Our method represents an advance on mucins detection and quantification since the existing methods present several disadvantages for process development. Hereafter, it can be applied to the optimization of new or already established downstream processes for mucins’ purification.

Macrophage inducible C-type lectin (Mincle) recognizes glycosylated surface (S)-layer of the periodontal pathogen Tannerella forsythia.

The oral pathogen Tannerella forsythia is implicated in the development of periodontitis, a common inflammatory disease that leads to the destruction of the gum and tooth supporting tissues, often leading to tooth loss. T. forsythia is a unique Gram-negative organism endowed with an elaborate protein O-glycosylation system that allows the bacterium to express a glycosylated surface (S)-layer comprising two high molecular weight glycoproteins modified with O-linked oligosaccharides. The T. forsythia S-layer has been implicated in the modulation of cytokine responses of antigen presenting cells, such as macrophages, that play a significant role during inflammation associated with periodontitis. The macrophage-inducible C-type lectin receptor (Mincle) is an FcRγ-coupled pathogen recognition receptor that recognizes a wide variety of sugar containing ligands from fungal and bacterial pathogens. In this study, we aimed to determine if Mincle might be involved in the recognition of T. forsythia S-layer and modulation of cytokine response of macrophages against the bacterium. Binding studies using recombinant Mincle-Fc fusion protein indicated a specific Ca2+-dependent binding of Mincle to T. forsythia S-layer. Subsequent experiments with Mincle-expressing and Mincle-knockdown macrophages revealed a role for Mincle/S-layer interaction in the induction of both pro- and anti-inflammatory cytokine secretion in macrophages stimulated with T. forsythia as well as its S-layer. Together, these studies revealed Mincle as an important macrophage receptor involved in the modulation of cytokine responses of macrophages against T. forsythia, and thus may play a critical role in orchestrating the host immune response against the bacterium.

Mucin as a diagnostic clue in dermatopathology.

Mucins are high-molecular weight glycoproteins typically found in normal skin in small amounts. There are several reports regarding different types of cutaneous mucinosis. In this report, we discuss mucins in dermatopathology as a diagnostic clue of some well-known entities and some less frequent cutaneous diseases. We also emphasize mucin as a sign in the differential diagnosis between conditions that show histopathological overlap. Lastly, we discuss the locations and circumstances in which mucin could be considered almost normal or physiological.

Functional Consequences of Differential O-glycosylation of MUC1, MUC4, and MUC16 (Downstream Effects on Signaling).

Glycosylation is one of the most abundant post-translational modifications that occur within the cell. Under normal physiological conditions, O-linked glycosylation of extracellular proteins is critical for both structure and function. During the progression of cancer, however, the expression of aberrant and truncated glycans is commonly observed. Mucins are high molecular weight glycoproteins that contain numerous sites of O-glycosylation within their extracellular domains. Transmembrane mucins also play a functional role in monitoring the surrounding microenvironment and transducing these signals into the cell. In cancer, these mucins often take on an oncogenic role and promote a number of pro-tumorigenic effects, including pro-survival, migratory, and invasive behaviors. Within this review, we highlight both the processes involved in the expression of aberrant glycan structures on mucins, as well as the potential downstream impacts on cellular signaling.

Abstract 3411: Role of mucin gene variations in microRNA binding sites in modulating colorectal cancer susceptibility and clinical outcomes

Abstract Mucins, high molecular weight glycoproteins predominantly expressed at the epithelial surface of tissues, provide protection for colon surface under normal physiological conditions. Mucinous colorectal carcinoma is generally defined as having greater than 50% of the tumor area with a mucinous differentiation by histologic examination. MiRNAs have recently emerged as important regulators for altered mucin expression during malignant development. MiRNAs are short non-coding RNAs that regulate gene expression by binding to the 3’untranslated regions (3’UTR) of target mRNA thereby hampering protein translation or inducing mRNA destabilization. Aberrant miRNA expression and/or function are frequently observed in colorectal cancer (CRC). Polymorphisms in miRNA binding sites may affect miRNA binding to target genes, resulting in differential mRNA and protein expression and susceptibility to common diseases. We hypothesize that variations in mucin genes may modulate signaling response affecting cancer susceptibility, cancer survival and efficacy of chemotherapy. Thirteen polymorphisms in nine mucin genes (MUC6, MUC7, MUC13, MUC14, MUC15, MUC17, MUC20, MUC21 and MUC24) were analyzed in DNA samples of 1111 cases and 1469 controls from the Czech Republic. Investigated variants were also studied in association with clinical outcome in all patients provided with detailed information on follow up. Genetic variations in mucin genes were associated with clinical outcome. In particular, CRC patients carrying the CC genotype for rs886403 in MUC21 gene displayed a shorter survival and higher recurrence risk (HR 1.69; 95% CI 1.13-2.46; p = 0.01 and EFS: HR 1.99; 95% CI 1.38-2.84; p = 0.0002, resp.). The observed association was strikingly pronounced in colon cancer patients while individuals with rectal cancer and carrying variant CC genotype of rs4729655 in MUC17 displayed better overall survival (HR 0.27; 95% CI 0.14-0.54; p = 0.0002). Expanding our knowledge on mucin involvement in CRC may help us better understand the etiopathogenesis of this disease and thereby contribute to the development of new treatment strategies. The present results identified plausible candidate SNPs potentially affecting miRNAs that target mucin genes in relation to CRC patients’ survival. Work supported by IGA MZ: NT 15-26535A Citation Format: Petra Bendova, Veronika Vymetalkova, Barbara Pardini, Fabio Rosa, Cornelia di Gaetano, Ludmila Vodickova, Tomas Buchler, Alessio Naccarati, Pavel Vodicka. Role of mucin gene variations in microRNA binding sites in modulating colorectal cancer susceptibility and clinical outcomes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3411.

Abstract 4592: MUC13 interaction with receptor tyrosine kinase HER2 drives pancreatic ductal adenocarcinoma progression

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States and has a very poor survival rate due to late diagnosis. MUC13 is a recently identified high molecular weight glycoprotein that is upregulated in PDAC and its progression is allowed via alterations of multiple signaling pathways. MUC13 is aberrantly expressed in PDAC and generally correlates with increased expression of HER2, however, the underlying mechanism remains poorly understood. MUC13 consists of three EGF-like domains that may serve as a ligand for EGF receptors, such as HER2, and modulate EGFR signaling pathways. We sought to better characterize the interaction of MUC13 with HER2 in PDAC. Methods: MUC13 and HER2 interaction was studied using reciprocal co-immunoprecipitation, immunofluorescence, proximity ligation, Western blotting, co-capping assays in human PDAC cell lines and immunohistofluorescence techniques in human tissues. Tissue microarrays prepared from formalin-fixed, paraffin-embedded specimens of PDAC were assessed for expression of MUC13 and HER2 using our own laboratory generated anti-MUC13 mouse monoclonal antibody (MAb) through confocal immunofluorescence. The association of MUC13 and HER2 co-localization with nuclear chromatin organization was analyzed to study the stage or degree of aggressiveness of the pancreatic cancer using Dapi stained confocal images of tissues. Results: MUC13 co-localizes and interacts with HER2 in PDAC cell lines. The results from this study demonstrate that MUC13 functionally interacts and activates HER2 at Tyrp1248 in PDAC cells, leading to stimulation of HER2 signaling cascade including, ERK1/2, FAK, AKT and PAK1 as well as regulation of the growth, cytoskeleton remodeling, motility and invasion of PDAC cells - all collectively contributing to PDAC progression. The interaction between MUC13-HER2 binding resulting in their tumorigenic characteristics likely occurs at the 1st and 2nd but not the 3rd domains of MUC13 as the EGF 1 and 2 deletion mutant constructs of MUC13 failed to promote proliferation and invasion of cells. These phenotypic effects of MUC13-HER2 co-localization could be effectively compromised by depleting MUC13. MUC13-HER2 co-localization also held true in PDAC human tissues with a strong functional correlation that contributed to an increased degree of disorder and cancer aggressiveness. Conclusions: 1) MUC13 can be detected in formalin-fixed paraffin-embedded tissues using our anti-MUC13 MAb. 2) MUC13 co-localizes and activates HER2 and its downstream signaling cascade promoting PDAC progression in both cell lines and human tissue. 3) This process is reversed by depletion of MUC13. 4) This MUC13-HER2 interaction may potentially be manipulated for targeted therapeutics in patients harboring PDAC. Citation Format: Sheema Khan, Mara C. Ebeling, Mohammad Sikander, Murali M. Yallapu, Tomoko Ise, Satoshi Nagata, Stephen W. Behrman, Nadeem Zafar, Jim Y. Wan, Hemendra M. Ghimire, Peeyush Sahay, Prabhakar Pradhan, Meena Jaggi, Subhash C. Chauhan. MUC13 interaction with receptor tyrosine kinase HER2 drives pancreatic ductal adenocarcinoma progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4592.

Diagnostic potential of mucins in pancreatic juice for pancreatic cancer.

222 Background: Pancreatic juice remains an underutilized resource for diagnosing pancreatic cancer. Mucins are high molecular weight glycoproteins differentially upregulated in pancreatic cancer, and we hypothesize that their profile in pancreatic juice may have diagnostic potential. Methods: Pancreatic juice was obtained during endoscopy from non-healthy non-pancreatic control (NHPC, n = 57), chronic pancreatitis (CP, n = 23), and pancreatic cancer (PC, n = 23) patients. Sandwich ELISA was used to detect MUC1, MUC4, MUC5AC, CA125, and CA19-9. Kruskal-Wallis test and Wilcoxon rank sum test for group and pairwise comparison was done with p &lt; 0.05 as significant. Logistic regression with ROC curve modeling of log transformed data was done for each biomarker individually and in combination to determine odds ratio (OR), sensitivity (SN), and specificity (SP) for PC. Results: PC vs NHPC: MUC5AC had the best individual performance for diagnosing PC with an OR = 2.78 (95% CI = 1.51-5.13), AUC = 0.81, and optimal SN/SP of 0.83 and 0.67, respectively. CA125 was increased in PC with an OR = 2.31 (95% CI = 1.4-4.0), AUC = 0.73, and optimal SN/SP of 0.88 and 0.67. CA19-9 was increased in PC with an OR = 1.5 (95% CI = 1.2-1.8), AUC = 0.76, and optimal SN/SP of 0.73 and 0.70. A combination of MUC1, MUC5AC, CA125, and CA19-9 outperformed all individual markers and had the largest AUC (0.89) with optimal SN/SP of 0.84 and 0.79. PC vs CP: MUC1 concentration in PC was significantly less than CP with an OR = 0.21 (95%CI = 0.088-0.49), AUC = 0.82, and optimal SN/SP of 0.87 and 0.78. PC vs NHPC+CP: MUC1 was decreased significantly in PC with an OR = 0.65 (95% CI = 0.44-0.96), AUC = 0.69, and optimal SN/SP of 0.87 and 0.63. CA125 was increased in PC with an OR = 1.64 (95%CI = 1.1-2.4), AUC = 0.66, and optimal SN/SP of 0.67 and 0.64. CA19-9 was increased in PC with an OR = 1.32 (95%CI = 1.1-1.6), AUC = 0.68, and optimal SN/SP of 0.63 and 0.67. A combination of MUC1, MUC5AC, CA125, and CA19-9 had an AUC = 0.86 with optimal SN/SP of 0.87 and 0.77 for PC. Conclusions: MUC1, MUC5AC, CA125, and CA19-9 combination provides a significantly improved diagnostic panel compared to any individual marker in pancreatic juice for detecting malignancy.

Advances in thyroglobulin assays and their impact on the management of differentiated thyroid cancers

Thyroglobulin (Tg) is a high molecular weight glycoprotein located mainly in thyroid follicles, where thyroid hormones are synthesized and stored. In patients with differentiated thyroid cancer of follicular origin, serum Tg levels become undetectable following total thyroidectomy and iodine-131 remnant ablation. It is a key biomarker to follow-up patients with differentiated thyroid cancer, in combination with neck ultrasound monitoring. The measurement of Tg in the wash-out of the needle used for fine needle aspiration biopsy is a valuable aid to the diagnosis of lymph node metastasis. The presence of anti-thyroglobulin antibodies affects reliability of Tg results measured in serum or plasma. Systematic investigation of such antibodies is required to validate any Tg assay. Elevated or rising levels of anti-thyroglobulin antibodies can in turn be used as a surrogate tumor marker of thyroid cancer. The development of second-generation Tg assay (automated, highly sensitive) has enabled significant advances in the management of differentiated thyroid cancer: early detection of persistent or recurrent disease and follow-up care simplified in low-risk patients. Testing of serum Tg can also be useful in evaluating other clinical situations such as congenital hypothyroidism, endemic goiter and thyrotoxicosis factitia.

Targeting Cell-Bound MUC1 on Myelomonocytic and Monocytic Leukemias and Leukemic Stem Cells: Therapeutic Implications

Monocytic neoplasms comprise a heterogeneous group of hematologic malignancies including chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5), and monocytic sarcoma. Monocytic or granulomonocytic hyperplasia is a finding frequently-if not invariably-shared by these different entities, as is a poor therapeutic outcome in the absence of hematopoietic stem cell transplantation. Cell surface molecules aberrantly expressed or overexpressed by leukemic cells represent potential disease-specific therapeutic targets. MUC1, a polymorphic type I high molecular weight glycoprotein represents such a molecule. MUC1 consists of an extracellular domain containing 20 to 125 tandem repeats of a 20 amino acid-long sequence, followed by a transmembrane domain and a short cytoplasmic tail leading to intracellular signaling. Cleavage of MUC1 yields two unequal chains: a large extracellular alpha subunit containing the tandem repeat array bound in a strong non-covalent interaction to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Essentially all anti-MUC1 antibodies reported to date target the highly immunogenic tandem repeat of the MUC1 alpha chain. Because the alpha chain binds the cell-bound domains of MUC1 only intermittently in an 'on-and-off' manner, agents directed against the alpha chain will not effectively target MUC1+ cells. In contrast, the MUC1 SEA domain represents a stable structure fixed to the cell surface at all times. We therefore generated mAbs that specifically recognize the cell-bound MUC1 SEA domain. One of them, a partially humanized murine mAb termed DMB-5F3 was used to examine the expression of MUC1 on AML cells by flow cytometry. A series of twenty-two AML samples (blood-derived n=12; bone marrow-derived n=10; AML0=2, AML1=2, AML2=10, AML4=1, AML5=5, AML6=2) collected either at the time of diagnosis or at relapse were analysed for MUC1 expression by flow cytometry. A murine mammary tumor cell line stably transfected with human MUC1 DNA served as control. Blasts cells from 5 AML samples highly expressed MUC1, and significantly, all were of monocytic or myelomonocytic lineage (AML4=1, AML5=4). Leukemic stem cells (CD34pos or CD34neg linneg) from the MUC1+ AMLs were examined and likewise found to express MUC1. In addition, AML cell lines MV411, MOLM14, and SHI-1 derived from monocytic leukemic lineage clearly expressed cell surface MUC1, while non- monocytic leukemic cell lines U937, K562, and HL60 had little or no expression. Normal monocytes and monocytes derived from patients with activated monocytosis were also found to express MUC1. Based on these findings we examined MUC1 expression in a series of myelomonocytic leukemia (CMML and JMML). In fifteen CMML samples examined (type 1 n=11, type 2 n=4) (blood n=7, BM n=7) 92%-100% (median 99.7%) of CD14+CD56+ CMML cells bound mAb DMB-5F3 to cell-surface MUC1. CD14+CD16+CD56+ blast cells from 2 pts with JMML were also found to express MUC1 (between 64% and 71 % positive). Based on these findings we conclude that expression of MUC1 is restricted to monocytic and myelomonocytic leukemias and that MUC1 represents an effective target for leukemic immunotherapy. Significantly, anti-MUC1 mAb also targets monocytic leukemic stem cells, reinforcing its therapeutic potential. The fact that the anti-MUC1 antibody DMB-5F3 can enter cells and thereby ferry Ab-bound toxin opens the way for us to demonstrate leukemic cell killing with anti-MUC1 mAb-immunotoxin conjugates. DisclosuresMoreau:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

The mucin MUC4 is a transcriptional and post-transcriptional target of K-ras oncogene in pancreatic cancer. Implication of MAPK/AP-1, NF-κB and RalB signaling pathways

The membrane-bound mucin MUC4 is a high molecular weight glycoprotein frequently deregulated in cancer. In pancreatic cancer, one of the most deadly cancers in occidental countries, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras remains clinically undruggable. Targeting early downstream K-ras signaling in cancer may thus appear as an interesting strategy and MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-rasG12D mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-κB signaling pathways. In vitro, transfection of constitutively activated K-rasG12V in pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was found to be mediated at the transcriptional level by AP-1 and NF-κB transcription factors via MAPK, JNK and NF-κB pathways and at the post-transcriptional level by a mechanism involving the RalB GTPase. Altogether, these results identify MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer.