The Ocular Surface Glycocalyx and its Alteration in Dry Eye Disease: A Review.

Many studies have revealed that transmembrane mucins, large glycoproteins with heavily glycosylated glycans, are essential for maintaining ocular surface epithelium lubrication and wettability. Recent reports indicate that transmembrane mucins and galectin-3, a chimera type of galectin that binds ß-galactoside in the glycan, play a crucial role in maintaining the epithelial glycocalyx barrier. This review summarizes current evidence regarding the role of galectin-3, the role of the three major transmembrane mucins (i.e., MUC1, MUC4, and MUC16), in the maintenance of ocular surface wettability and transcellular barrier. Pathological mechanisms of glycocalyx barrier disruption and epithelial surface wettability decreases in dry eye disease are also summarized. Lastly, new ophthalmic drugs that target transmembrane mucin are described.

TNFα-Induced Mucin 4 Expression Elicits Trastuzumab Resistance in HER2-Positive Breast Cancer.

PURPOSE: Although trastuzumab administration improved the outcome of HER2-positive breast cancer patients, resistance events hamper its clinical benefits. We demonstrated that TNFα stimulation in vitro induces trastuzumab resistance in HER2-positive breast cancer cell lines. Here, we explored the mechanism of TNFα-induced trastuzumab resistance and the therapeutic strategies to overcome it. EXPERIMENTAL DESIGN: Trastuzumab-sensitive breast cancer cells, genetically engineered to stably overexpress TNFα, and de novo trastuzumab-resistant tumors, were used to evaluate trastuzumab response and TNFα-blocking antibodies effectiveness respectively. Immunohistochemistry and antibody-dependent cell cytotoxicity (ADCC), together with siRNA strategy, were used to explore TNFα influence on the expression and function of its downstream target, mucin 4 (MUC4). The clinical relevance of MUC4 expression was studied in a cohort of 78 HER2-positive breast cancer patients treated with adjuvant trastuzumab. RESULTS: TNFα overexpression turned trastuzumab-sensitive cells and tumors into resistant ones. Histopathologic findings revealed mucin foci in TNFα-producing tumors. TNFα induced upregulation of MUC4 that reduced trastuzumab binding to its epitope and impaired ADCC. Silencing MUC4 enhanced trastuzumab binding, increased ADCC, and overcame trastuzumab and trastuzumab-emtansine antiproliferative effects in TNFα-overexpressing cells. Accordingly, administration of TNFα-blocking antibodies downregulated MUC4 and sensitized de novo trastuzumab-resistant breast cancer cells and tumors to trastuzumab. In HER2-positive breast cancer samples, MUC4 expression was found to be an independent predictor of poor disease-free survival (P = 0.008). CONCLUSIONS: We identified TNFα-induced MUC4 expression as a novel trastuzumab resistance mechanism. We propose MUC4 expression as a predictive biomarker of trastuzumab efficacy and a guide to combination therapy of TNFα-blocking antibodies with trastuzumab. Clin Cancer Res; 23(3); 636-48. ©2016 AACR.

MUC4 regulates cellular senescence in head and neck squamous cell carcinoma through p16/Rb pathway.

The limited effectiveness of therapy for patients with advanced stage head and neck squamous cell carcinoma (HNSCC) or recurrent disease is a reflection of an incomplete understanding of the molecular basis of HNSCC pathogenesis. MUC4, a high molecular weight glycoprotein, is differentially overexpressed in many human cancers and implicated in cancer progression and resistance to several chemotherapies. However, its clinical relevance and the molecular mechanisms through which it mediates HNSCC progression are not well understood. This study revealed a significant upregulation of MUC4 in 78% (68/87) of HNSCC tissues compared with 10% positivity (1/10) in benign samples (P=0.006, odds ratio (95% confidence interval)=10.74 (2.0-57.56). MUC4 knockdown (KD) in SCC1 and SCC10B HNSCC cell lines resulted in significant inhibition of growth in vitro and in vivo, increased senescence as indicated by an increase in the number of flat, enlarged and senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells. Decreased cellular proliferation was associated with G0/G1 cell cycle arrest and decrease expression of cell cycle regulatory proteins like cyclin E, cyclin D1 and decrease in BrdU incorporation. Mechanistic studies revealed upregulation of p16, pRb dephosphorylation and its interaction with histone deacetylase 1/2. This resulted in decreased histone acetylation (H3K9) at cyclin E promoter leading to its downregulation. Orthotopic implantation of MUC4 KD SCC1 cells into the floor of the mouth in nude mice resulted in the formation of significantly smaller tumors (170±18.30 mg) compared to those (375±17.29 mg) formed by control cells (P=0.00007). In conclusion, our findings showed that MUC4 overexpression has a critical role by regulating proliferation and cellular senescence of HNSCC cells. Downregulation of MUC4 may be a promising therapeutic approach for treating HNSCC patients.

The MUC4 mucin mediates gemcitabine resistance of human pancreatic cancer cells via the Concentrative Nucleoside Transporter family.

The fluorinated analog of deoxycytidine, Gemcitabine (Gemzar), is the main chemotherapeutic drug in pancreatic cancer, but survival remains weak mainly because of the high resistance of tumors to the drug. Recent works have shown that the mucin MUC4 may confer an advantage to pancreatic tumor cells by modifying their susceptibility to drugs. However, the cellular mechanism(s) responsible for this MUC4-mediated resistance is unknown. The aim of this work was to identify the cellular mechanisms responsible for gemcitabine resistance linked to MUC4 expression. CAPAN-2 and CAPAN-1 adenocarcinomatous pancreatic cancer (PC) cell lines were used to establish stable MUC4-deficient clones (MUC4-KD) by shRNA interference. Measurement of the IC50 index using tetrazolium salt test indicated that MUC4-deficient cells were more sensitive to gemcitabine. This was correlated with increased Bax/BclXL ratio and apoptotic cell number. Expression of Equilibrative/Concentrative Nucleoside Transporter (hENT1, hCNT1/3), deoxycytidine kinase (dCK), ribonucleotide reductase (RRM1/2) and Multidrug-Resistance Protein (MRP3/4/5) was evaluated by quantitative RT-PCR (qRT-PCR) and western blotting. Alteration of MRP3, MRP4, hCNT1 and hCNT3 expression was observed in MUC4-KD cells, but only hCNT1 alteration was correlated to MUC4 expression and sensitivity to gemcitabine. Decreased activation of MAPK, JNK and NF-κB pathways was observed in MUC4-deficient cells, in which the NF-κB pathway was found to have an important role in both sensitivity to gemcitabine and hCNT1 regulation. Finally, and in accordance with our in vitro data, we found that MUC4 expression was conversely correlated to that of hCNT1 in tissues from patients with pancreatic adenocarcinoma. This work describes a new mechanism of PC cell resistance to gemcitabine, in which the MUC4 mucin negatively regulates the hCNT1 transporter expression via the NF-κB pathway. Altogether, these data point out to MUC4 and hCNT1 as potential targets to ameliorate the response of pancreatic tumors to gemcitabine treatment.

The mucin MUC4 and its membrane partner ErbB2 regulate biological properties of human CAPAN-2 pancreatic cancer cells via different signalling pathways.

The mucin MUC4 and its membrane partner the ErbB2 oncogenic receptor are potential interacting partners in human pancreatic tumour development. However, the way they function is still largely unknown. In this work, we aimed to identify the cellular mechanisms and the intracellular signalling pathways under the control of both ErbB2 and MUC4 in a human pancreatic adenocarcinomatous cell line. Using co-immunoprecipitation and GST pull-down, we show that MUC4 and ErbB2 interact in the human pancreatic adenocarcinomatous cell line CAPAN-2 via the EGF domains of MUC4. Stable cell clones were generated in which either MUC4 or ErbB2 were knocked down (KD) by a shRNA approach. Biological properties of these cells were then studied in vitro and in vivo. Our results show that ErbB2-KD cells are more apoptotic and less proliferative (decreased cyclin D1 and increased p27kip1 expression) while migration and invasive properties were not altered. MUC4-KD clones were less proliferative with decreased cyclin D1 expression, G1 cell cycle arrest and altered ErbB2/ErbB3 expression. Their migration properties were reduced whereas invasive properties were increased. Importantly, inhibition of ErbB2 and MUC4 expression did not impair the same signalling pathways (inhibition of MUC4 expression affected the JNK pathway whereas that of ErbB2 altered the MAPK pathway). Finally, ErbB2-KD and MUC4-KD cells showed impaired tumour growth in vivo. Our results show that ErbB2 and MUC4, which interact physically, activate different intracellular signalling pathways to regulate biological properties of CAPAN-2 pancreatic cancer cells.

The MUC4 membrane-bound mucin regulates esophageal cancer cell proliferation and migration properties: Implication for S100A4 protein.

MUC4 is a membrane-bound mucin known to participate in tumor progression. It has been shown that MUC4 pattern of expression is modified during esophageal carcinogenesis, with a progressive increase from metaplastic lesions to adenocarcinoma. The principal cause of development of esophageal adenocarcinoma is the gastro-esophageal reflux, and MUC4 was previously shown to be upregulated by several bile acids present in reflux. In this report, our aim was thus to determine whether MUC4 plays a role in biological properties of human esophageal cancer cells. For that stable MUC4-deficient cancer cell lines (shMUC4 cells) were established using a shRNA approach. In vitro (proliferation, migration and invasion) and in vivo (tumor growth following subcutaneous xenografts in SCID mice) biological properties of shMUC4 cells were analyzed. Our results show that shMUC4 cells were less proliferative, had decreased migration properties and did not express S100A4 protein when compared with MUC4 expressing cells. Absence of MUC4 did not impair shMUC4 invasiveness. Subcutaneous xenografts showed a significant decrease in tumor size when cells did not express MUC4. Altogether, these data indicate that MUC4 plays a key role in proliferative and migrating properties of esophageal cancer cells as well as is a tumor growth promoter. MUC4 mucin appears thus as a good therapeutic target to slow-down esophageal tumor progression.

Membrane-tethered mucins have multiple functions on the ocular surface.

Membrane-tethered mucins are large glycoproteins present in the glycocalyx along the apical surface of all wet-surfaced epithelia of the body, including that of the ocular surface. Originally thought to function only in epithelial surface lubrication and hydration, data now indicate that the mucins are multifunctional molecules, each having unique as well as common functions. This review summarizes current knowledge regarding the three major membrane mucins of the ocular surface, MUC1, MUC4, and MUC16. The mucins vary in their ocular surface distribution, size, structural motifs, and functions. The ectodomains of each are released into the tear film and are, thus, a component of the soluble mucins of the tear film. Both animal and in vitro models for their study are herein described, as are alterations of the mucins in ocular surface disease.

Pancreatic cancer cells resistance to gemcitabine: the role of MUC4 mucin.

BACKGROUND: A major obstacle to the successful management of pancreatic cancer is to acquire resistance to the existing chemotherapeutic agents. Resistance to gemcitabine, the standard first-line chemotherapeutic agent for advanced and metastatic pancreatic cancer, is mainly attributed to an altered apoptotic threshold in the pancreatic cancer. The MUC4 transmembrane glycoprotein is aberrantly overexpressed in the pancreatic cancer and recently, has been shown to increase pancreatic tumour cell growth by the inhibition of apoptosis. METHODS: Effect of MUC4 on pancreatic cancer cells resistance to gemcitabine was studied in MUC4-expressing and MUC4-knocked down pancreatic cancer cell lines after treatment with gemcitabine by Annexin-V staining, DNA fragmentation assay, assessment of mitochondrial cytochrome c release, immunoblotting and co-immunoprecipitation techniques. RESULTS: Annexin-V staining and DNA fragmentation experiment demonstrated that MUC4 protects CD18/HPAF pancreatic cancer cells from gemcitabine-induced apoptosis. In concert with these results, MUC4 also attenuated mitochondrial cytochrome c release and the activation of caspase-9. Further, our results showed that MUC4 exerts anti-apoptotic function through HER2/extracellular signal-regulated kinase-dependent phosphorylation and inactivation of the pro-apoptotic protein Bad. CONCLUSION: Our results elucidate the function of MUC4 in imparting resistance to pancreatic cancer cells against gemcitabine through the activation of anti-apoptotic pathways and, thereby, promoting cell survival.

Recent advances in systemic therapy: Advances in systemic therapy for HER2-positive metastatic breast cancer.

Human epidermal growth factor receptor (HER)2 over-expression is associated with a shortened disease-free interval and poor survival. Although the addition of trastuzumab to chemotherapy in the first-line setting has improved response rates, progression-free survival, and overall survival, response rates declined when trastuzumab was used beyond the first-line setting because of multiple mechanisms of resistance. Studies have demonstrated the clinical utility of continuing trastuzumab beyond progression, and further trials to explore this concept are ongoing. New tyrosine kinase inhibitors, monoclonal antibodies, PTEN (phosphatase and tensin homolog) pathway regulators, HER2 antibody-drug conjugates, and inhibitors of heat shock protein-90 are being evaluated to determine whether they may have a role to play in treating trastuzumab-resistant metastatic breast cancer.

MUC4, a multifunctional transmembrane glycoprotein, induces oncogenic transformation of NIH3T3 mouse fibroblast cells.

Numerous studies have established the association of MUC4 with the progression of cancer and metastasis. An aberrant expression of MUC4 is reported in precancerous lesions, indicating its early involvement in the disease process; however, its precise role in cellular transformation has not been explored. MUC4 contains many unique domains and is proposed to affect cell signaling pathways and behavior of the tumor cells. In the present study, to decipher the oncogenic potential of MUC4, we stably expressed the MUC4 mucin in NIH3T3 mouse fibroblast cells. Stable ectopic expression of MUC4 resulted in increased growth, colony formation, and motility of NIH3T3 cells in vitro and tumor formation in nude mice when cells were injected s.c. Microarray analysis showed increased expression of several growth-associated and mitochondrial energy production-associated genes in MUC4-expressing NIH3T3 cells. In addition, expression of MUC4 in NIH3T3 cells resulted in enhanced levels of oncoprotein ErbB2 and its phosphorylated form (pY(1248)-ErbB2). In conclusion, our studies provide the first evidence that MUC4 alone induces cellular transformation and indicates a novel role of MUC4 in cancer biology.

The membrane-bound mucins: how large O-glycoproteins play key roles in epithelial cancers and hold promise as biological tools for gene-based and immunotherapies.

Membrane-bound mucins belong to an ever-increasing family of O-glycoproteins that share a structure conserved throughout evolution. Typically, membrane-bound mucins contain a long extracellular domain, a hydrophobic transmembrane domain, and a short cytoplasmic tail. They are modular proteins and have a structural organization containing Pro/ Thr/Ser-rich O-glycosylated domains and EGF-like domains. The biological roles of mucins arise from their structures. MUC1 and MUC4 modulate biological properties of the cell, alter its behavior and modulate cell signaling pathways associated with tumorigenesis. Altered expression and post-translational modifications confer an important role to MUC1 and MUC4 in tumor progression, metastasis, and cancer cell chimioresistance. Moreover, increasing knowledge about their animal counterparts has made possible a greater understanding of their pathophysiological role in vivo. Most biological functions attributed to MUC4 are based on the structural homology with its rat homologue. From these results, the development of new biological tools targeting mucins has been increasing and the recent attention given to these complex molecules may bring hope for improved cancer treatments in the future. This review discusses the structure/function of MUC1 and MUC4 membrane-bound mucins in relation to cancer cell behavior and cell signaling pathways associated with tumorigenesis, as well as their potential as biological tools for gene therapy and immunotherapy approaches.