Antagonistic Roles of the Tumor Suppressor miR-210-3p and Oncomucin MUC4 Forming a Negative Feedback Loop in Pancreatic Adenocarcinoma

Background: Pancreatic adenocarcinoma (PDAC) is a deadly cancer with an extremely poor prognosis. MUC4 membrane-bound mucin is neoexpressed in early pancreatic neoplastic lesions and is associated with PDAC progression and chemoresistance. In cancers, microRNAs (miRNAs, small noncoding RNAs) are crucial regulators of carcinogenesis, chemotherapy response and even metastatic processes. In this study, we aimed at identifying and characterizing miRNAs activated downstream of MUC4-associated signaling in pancreatic adenocarcinoma. MiRnome analysis comparing MUC4-KD versus Mock cancer cells showed that MUC4 inhibition impaired miR-210-3p expression. Therefore, we aimed to better understand the miR-210-3p biological roles. Methods: miR-210-3p expression level was analyzed by RT-qPCR in PDAC-derived cell lines (PANC89 Mock and MUC4-KD, PANC-1 and MiaPACA-2), as well as in mice and patients tissues. The MUC4-miR-210-3p regulation was investigated using luciferase reporter construct and chromatin immunoprecipitation experiments. Stable cell lines expressing miR-210-3p or anti-miR-210-3p were established using CRISPR/Cas9 technology or lentiviral transduction. We evaluated the biological activity of miR-210-3p in vitro by measuring cell proliferation and migration and in vivo using a model of subcutaneous xenograft. Results: miR-210-3p expression is correlated with MUC4 expression in PDAC-derived cells and human samples, and in pancreatic PanIN lesions of Pdx1-Cre; LstopL-KrasG12D mice. MUC4 enhances miR-210-3p expression levels via alteration of the NF-κB signaling pathway. Chromatin immunoprecipitation experiments showed p50 NF-κB subunit binding on miR-210-3p promoter regions. We established a reciprocal regulation since miR-210-3p repressed MUC4 expression via its 3′-UTR. MiR-210-3p transient transfection of PANC89, PANC-1 and MiaPACA-2 cells led to a decrease in cell proliferation and migration. These biological effects were validated in cells overexpressing or knocked-down for miR-210-3p. Finally, we showed that miR-210-3p inhibits pancreatic tumor growth and proliferation in vivo. Conclusion: We identified a MUC4-miR-210-3p negative feedback loop in early-onset PDAC, but also revealed new functions of miR-210-3p in both in vitro and in vivo proliferation and migration of pancreatic cancer cells, suggesting a complex balance between MUC4 pro-oncogenic roles and miR-210-3p anti-tumoral effects.

Immunohistochemical Expression of MUC4 in Different Meningioma Subtypes in Comparison to Some Mesenchymal Non-Meningothelial Tumors

BACKGROUND: Meningiomas are the most common primary tumors of the central nervous system worldwide. Routinely used immunohistochemical markers for diagnosis of confusing meningioma cases as epithelial membrane antigen lack specificity and sensitivity. MUC4 is glycosylated membrane-associated mucin expressed by normal epithelia and many cancers. However, it is recently noticed to be expressed in meningiomas. AIM: Intensity of MUC4 expression is needed to be verified whether it is the same among different subtypes or not. MATERIALS AND METHODS: Fifty cases of different intracranial meningioma subtypes and thirty cases of mesenchymal nonmeningothelial tumors were immunohistochemically stained with MUC4 antibody. The results of MUC4 expression intensity were associated with some clinical and pathological parameters. RESULTS: Most studied meningioma cases (84%) showed positive MUC4 expression. Meningothelial meningioma subtype showed characteristic pattern of diffuse and moderate to strong MUC4 staining. While fibroblastic meningioma showed mostly negative staining pattern and focal weak staining pattern if positive. A statistically significant relationship was detected between tumor subtype and intensity of MUC4 expression. On contrary, most included mesenchymal cases were MUC4 negative with statistical significance. Hence, the sensitivity of MUC4 as diagnostic marker for meningioma was 84%, while the specificity was 93.3%. Furthermore, meningioma histologic subtype showed significant relationship with age. CONCLUSION: The current study results suggest that MUC4 could be used as meningioma diagnostic marker with some limitations. Moreover, meningioma should be included in the differential diagnosis of MUC4 positive tumors.

A flexible summary-based colocalization method with application to the mucin Cystic Fibrosis lung disease modifier locus

Mucus obstruction is a central feature in the Cystic Fibrosis (CF) airways. A genome-wide association study (GWAS) of lung disease by the CF Gene Modifier Consortium (CFGMC) identified a significant locus containing two mucin genes, MUC20 and MUC4. Expression quantitative trait locus (eQTL) analysis using human nasal epithelial (HNE) from 94 CF Canadians in the CFGMC demonstrated MUC4 eQTLs that mirrored the lung association pattern in the region, suggesting that MUC4 expression may mediate CF lung disease. Complications arose, however, with colocalization testing using existing methods: the locus is complex and the associated SNPs span a 0.2Mb region with high linkage disequilibrium and evidence of eQTLs for multiple genes and tissues (heterogeneity). We previously developed the Simple Sum (SS), a powerful colocalization test in regions with heterogeneity, but SS assumed eQTLs to be present to achieve type I error control. Here we propose a two-stage SS (SS2) colocalization test that avoids a prior eQTL assumptions, accounts for multiple hypothesis testing and the composite null hypothesis and enables meta-analysis. We compare SS2 to published approaches through simulation and demonstrate type I error control for all settings with the greatest power in the presence of high LD and heterogeneity. Applying SS2 to the MUC20/MUC4 CF lung disease locus with eQTLs from CF HNE revealed significant colocalization with MUC4 (p = 1.71×10-5) rather than MUC20. The SS2 is a powerful method to inform the responsible gene(s) at a locus and guide future functional studies. SS2 has been implemented in the application LocusFocus (locusfocus.research.sickkids.ca).

Mucin 4: A Sensitive Diagnostic Marker for Pediatric Mucoepidermoid CarcinomaNick Shillingford

Background: Pediatric salivary gland-type neoplasms (SGTNs) pose a significant diagnostic problem due to histo-morphological heterogeneity. Previous reports have shown that Mucin 4 (MUC4) expression is associated with adult mucoepidermoid carcinoma (MEC). We hypothesize that MUC4 is also a sensitive marker for distinguishing MEC from other SGTNs in the pediatric population. Objective: To evaluate MUC4 expression in pediatric SGTNs. Methods: A retrospective review of 74 SGTNs diagnosed between 1993–2015 at Children’s Hospital Los Angeles, Boston Children’s Hospital, and Rhode Island Hospital was performed. H&E sections of 31 MECs were compared to 3 adenoid cystic carcinomas (AdCCs), 6 acinic cell carcinomas (AcCCs), 30 pleomorphic adenomas (PAs), 3 mammary analogue secretory carcinomas (MASCs), and one sialoblastoma (SB). Samples underwent immunohistochemical staining for MUC4, with expression score criteria: 0% positivity = 0, 1-10% = +, 11-50% = ++, 51-90% = +++, >90% = ++++. Results: All MECs were MUC4-positive, with 25 (80.65%) having an expression score ≥ +++. AdCCs and PAs demonstrated no to minimal MUC4-positivity. Subsets of AcCCs and MASCs were unexpectedly MUC4-positive. As a novel marker for pediatric MEC, MUC4’s sensitivity = 100%, specificity = 79.41%, positive predictive value = 75.86%, and negative predictive value = 100%. Conclusion: MUC4 is a sensitive marker for pediatric MEC

Bile accelerates carcinogenic processes in pancreatic ductal adenocarcinoma cells through the overexpression of MUC4

Pancreatic cancer (PC) is one of the leading causes of mortality rate globally and is usually associated with obstructive jaundice (OJ). Up to date, there is no clear consensus on whether biliary decompression should be performed prior to surgery and how high levels of serum bile affects the outcome of PC. Therefore, our study aims were to characterise the effect of bile acids (BAs) on carcinogenic processes using pancreatic ductal adenocarcinoma (PDAC) cell lines and to investigate the underlying mechanisms. Liquid chromatography-mass spectrometry was used to determine the serum concentrations of BAs. The effects of BAs on tumour progression were investigated using different assays. Mucin expressions were studied in normal and PDAC cell lines and in human samples at gene and protein levels and results were validated with gene silencing. The levels of BAs were significantly higher in the PDAC + OJ group compared to the healthy control. Treating PDAC cells with different BAs or with human serum obtained from PDAC + OJ patients enhanced the rate of proliferation, migration, adhesion, colony forming, and the expression of MUC4. In PDAC + OJ patients, MUC4 expression was higher and the 4-year survival rate was lower compare to PDAC patients. Silencing of MUC4 decreased BAs-induced carcinogenic processes in PDAC cells. Our results show that BAs promote carcinogenic process in PDAC cells, in which the increased expression of MUC4 plays an important role. Based on these results, we assume that in PC patients, where the disease is associated with OJ, the early treatment of biliary obstruction improves life expectancy.

Targeting interleukin-17 receptor B enhances gemcitabine sensitivity through downregulation of mucins in pancreatic cancer

Pancreatic cancer is the fourth leading cause of death worldwide due to its poorest prognoses with a 7% 5-year survival rate. Eighty percent of pancreatic cancer patients relapse after chemotherapy and develop early metastasis and drug resistance. Resistance to nucleoside analog gemcitabine frequently used in first-line therapy is an urgent issue in pancreatic cancer treatment. Expression of mucin (MUC) glycoproteins has been shown to enhance chemoresistance via increased cell stemness. Here we show interlukine-17 receptor B (IL-17RB) expression is positively correlated with MUC1 and MUC4 expression in pancreatic cancer cells and tumor tissue. Moreover, IL-17RB transcriptionally up-regulates expression of MUC1 and MUC4 to enhance cancer stem-like properties and resistance to gemcitabine. These results suggest IL-17RB can be a potential target for pancreatic cancer therapy. Indeed, treatment with IL-17RB-neutralizing antibody has a synergistic effect in combination with gemcitabine for killing pancreatic cancer cells. Altogether, these findings provide feasible applications for IL-17RB-targeting therapy in pancreatic cancer treatment.

Utility of Immunohistochemistry for MUC4 and GATA3 to Aid in the Distinction of Pleural Sarcomatoid Mesothelioma From Pulmonary Sarcomatoid Carcinoma

Context.— Distinguishing pulmonary sarcomatoid carcinoma from pleural sarcomatoid mesothelioma is challenging because of overlapping histology, immunophenotype, and clinical features. Reliable immunohistochemical markers to aid in this distinction would be very valuable. Recent studies have proposed that MUC4 expression is common in sarcomatoid carcinoma but not in sarcomatoid mesothelioma, with the converse pattern reported for GATA3. Objective.— To further explore the utility of MUC4 and GATA3 in distinguishing pulmonary sarcomatoid carcinoma from sarcomatoid mesothelioma. Design.— Well-characterized cases of sarcomatoid carcinoma (n = 32) and sarcomatoid mesothelioma (n = 64) were included. Diagnoses were confirmed by thoracic pathologists with incorporation of immunophenotype, clinical, and radiographic features. Whole-tissue sections were stained for GATA3 and MUC4. Results.— Patients with sarcomatoid carcinoma and sarcomatoid mesothelioma had similar mean age and male predominance. GATA3 was positive in 63 of 64 sarcomatoid mesotheliomas (98%; 42 diffuse, 16 patchy, 5 focal), and 15 of 32 sarcomatoid carcinomas (47%; 3 diffuse, 8 patchy, 4 focal). MUC4 was positive in 2 of 64 sarcomatoid mesotheliomas (3%; 1 patchy, 1 focal), and in 12 of 32 sarcomatoid carcinomas (38%; 5 diffuse, 6 patchy, 1 focal). Conclusions.— Diffuse GATA3 expression favors sarcomatoid mesothelioma over sarcomatoid carcinoma, which rarely shows diffuse expression (sensitivity and specificity of diffuse staining 66% and 94%, respectively). Focal and patchy GATA3 expression is observed in both tumor types, and therefore is not helpful in this distinction. Sensitivity of MUC4 for sarcomatoid carcinoma was low in our cohort, positive in only 38% with frequent patchy staining, but it was quite specific.

Members of Prevotella Genus Distinctively Modulate Innate Immune and Barrier Functions in a Human Three-Dimensional Endometrial Epithelial Cell Model

Background Prevotella species are commonly isolated from the reproductive tract of women with obstetric/gynecologic health complications. However, contributions of this genus to changes in local microenvironment are not well characterized. Our objective was to evaluate species-specific effects of Prevotella on the human endometrial epithelium. Methods Thirteen Prevotella strains, originally isolated from the human oral cavity, amniotic fluid, endometrium, or vagina (including women with bacterial vaginosis), were obtained from BEI and ATCC resources. Bacteria were evaluated in silico and in vitro using human endometrial epithelial cells (EEC) grown as monolayers or a 3-dimensional (3D) model. Results Genomic characterization illustrated metabolic and phylogenetic diversity of Prevotella genus. Among tested species, P. disiens exhibited cytotoxicity. Scanning electron microscopy analysis of the 3D EEC model revealed species-specific colonization patterns and alterations of ultracellular structures. Infection with sialidase-producing P. timonensis resulted in elongated microvilli, and increased MUC3 and MUC4 expression. Infections with Prevotella species, including P. bivia, did not result in significant proinflammatory activation of EEC. Conclusions Collectively, findings indicate that Prevotella species are metabolically diverse and overall not cytotoxic or overtly inflammatory in EEC; however, these bacteria can form biofilms, alter barrier properties of the endometrial epithelium, and ultimately impact colonization of secondary colonizers.

The Antioxidant Alpha-Lipoic Acid Inhibits Proliferation and Invasion of Human Gastric Cancer Cells via Suppression of STAT3-Mediated MUC4 Gene Expression

Background. Metastasis and invasion are the main causes of mortality in gastric cancer. To improve the treatment of gastric cancer, the development of effective and innovative antitumor agents toward invasion and proliferation is needed. Alpha-lipoic acid (ALA), a naturally occurring thiol antioxidant, showed antiproliferative and cytotoxic effects on several cancers. So it is feasible to explore whether ALA can be used to inhibit proliferation and invasion in human gastric cancer. Methods. The expression of MUC4 in human gastric cancer tissues was assayed by immunohistochemistry. Then, we performed in vitro cell proliferation and invasion analysis to explore the antitumor effect of ALA using AGS, BGC-823, and MKN-28 cells. To further explore the mechanism of ALA-mediated downregulation of MUC4, we cotransfected human gastric cancer cells with STAT3 siRNA and STAT3 overexpression construct. ChIP assays were carried out to find the relationship between MUC4 and STAT3. Results. We found that the MUC4 gene was strongly expressed in human gastric cancer tissues. Meanwhile, ALA reduced proliferation and invasion of human gastric cancer cells by suppressing MUC4 expression. We also found that STAT3 was involved in the inhibition of MUC4 by ALA. Mechanistically, ALA suppressed MUC4 expression by inhibiting STAT3 binding to the MUC4 promoter region. Conclusion. ALA inhibits both proliferation and invasion of gastric cancer cells by suppression of STAT3-mediated MUC4 gene expression.