Circadian disruption reduces MUC4 expression via the clock molecule BMAL1 during dry eye development.

Circadian disruption, as a result of shiftwork, jet lag, and other lifestyle factors, is a common public health problem associated with a wide range of diseases, such as metabolic disorders, neurodegenerative diseases, and cancer. In the present study, we established a chronic jet lag model using a time shift method every 3 days and assessed the effects of circadian disruption on ocular surface homeostasis. Our results indicated that jet lag increased corneal epithelial defects, cell apoptosis, and proinflammatory cytokine expression. However, the volume of tear secretion and the number of conjunctival goblet cells did not significantly change after 30 days of jet lag. Moreover, further analysis of the pathogenic mechanism using RNA sequencing revealed that jet lag caused corneal transmembrane mucin deficiency, specifically MUC4 deficiency. The crucial role of MUC4 in pathogenic progression was demonstrated by the protection of corneal epithelial cells and the inhibition of inflammatory activation following MUC4 replenishment. Unexpectedly, genetic ablation of BMAL1 in mice caused MUC4 deficiency and dry eye disease. The underlying mechanism was revealed in cultured human corneal epithelial cells in vitro, where BMAL1 silencing reduced MUC4 expression, and BMAL1 overexpression increased MUC4 expression. Furthermore, melatonin, a circadian rhythm restorer, had a therapeutic effect on jet lag-induced dry eye by restoring the expression of BMAL1, which upregulated MUC4. Thus, we generated a novel dry eye mouse model induced by circadian disruption, elucidated the underlying mechanism, and identified a potential clinical treatment.

TRPV4 activation in human corneal epithelial cells promotes membrane mucin production.

Given that the corneal epithelium is situated on the outermost part of the eye, its functions can be influenced by external temperatures and chemical substances. This study aimed to elucidate the expression profile of chemosensory receptors in corneal epithelial cells and analyze their role in eye function regulation. A comprehensive analysis of 425 chemosensory receptors in human corneal epithelial cells-transformed (HCE-T) revealed the functional expression of TRPV4. The activation of TRPV4 in HCE-T cells significantly increased the expression of membrane-associated mucins MUC1, MUC4, and MUC16, which are crucial for stabilizing tear films, with efficacy comparable to the active components of dry eye medications. The present study suggests that TRPV4, which is activated by body temperature, regulates mucin expression and proposes it as a novel target for dry eye treatment.

Clinical Significance of MUC4 and Associated Proteins in Pancreatic and Periampullary Cancers.

OBJECTIVE: This study primarily aimed to assess the expression of MUC4 in patients with pancreatic ductal adenocarcinoma (PDAC) as compared with controls and assess its clinical relevance. MATERIALS AND METHODS: Serum MUC4 levels and MUC4 gene expression in snap-frozen tissue were analyzed through surface plasmon resonance and quantitative polymerase chain reaction, respectively. Tumor tissues and control tissues were analyzed for MUC4 and other mucins through immunohistochemistry. RESULT: MUC4 expression in tumor tissue was found to be significantly elevated in PDAC patients as compared with chronic pancreatitis tissues and normal pancreatic tissues. Periampullary carcinoma and cholangiocarcinoma tissue also showed increased expression of MUC4 and other mucins. CONCLUSIONS: Differential expression of MUC4 in pancreatic tumor tissues can help to differentiate PDAC from benign conditions.

Detection and Analysis of Antidiarrheal Genes and Immune Factors in Various Shanghai Pig Breeds.

The aim of this study was to identify effective genetic markers for the Antigen Processing Associated Transporter 1 (TAP1), α (1,2) Fucosyltransferase 1 (FUT1), Natural Resistance Associated Macrophage Protein 1 (NRAMP1), Mucin 4 (MUC4) and Mucin 13 (MUC13) diarrhea-resistance genes in the local pig breeds, namely Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs, to provide a reference for the characterization of local pig breed resources in Shanghai. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLR) and sequence sequencing were applied to analyze the polymorphisms of the above genes and to explore the effects on the immunity of Shanghai local pig breeds in conjunction with some immunity factors. The results showed that both TAP1 and MUC4 genes had antidiarrheal genotype GG in the five pig breeds, AG and GG genotypes of the FUT1 gene were detected in Pudong white pigs, AA antidiarrheal genes of the NRAMP1 gene were detected in Meishan pigs, the AB type of the NRAMP1 gene was detected in Pudong white pigs, and antidiarrheal genotype GG of the MUC13 gene was only detected in Shanghai white pigs. The MUC13 antidiarrhea genotype GG was only detected in Shanghai white pigs. The TAP1 gene was moderately polymorphic in Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs, among which TAP1 in Shanghai white pigs and Shawutou pigs did not satisfy the Hardy-Weinberg equilibrium. The FUT1 gene of Pudong white pigs was in a state of low polymorphism. NRAMP1 of Meishan pigs and Pudong white pigs was in a state of moderate polymorphism, which did not satisfy the Hardy-Weinberg equilibrium. The MUC4 genes of Shanghai white pigs and Pudong white pigs were in a state of low polymorphism, and the MUC4 genes of Fengjing pigs and Shawutou pigs were in a state of moderate polymorphism, and the MUC4 genes of Fengjing pigs and Pudong white pigs did not satisfy the Hardy-Weinberg equilibrium. The MUC13 gene of Shanghai white pigs and Pudong white pigs was in a state of moderate polymorphism. Meishan pigs had higher levels of IL-2, IL-10, IgG and TNF-α, and Pudong white pigs had higher levels of IL-12 than the other pigs. The level of interleukin 12 (IL-12) was significantly higher in the AA genotype of the MUC13 gene of Shanghai white pigs than in the AG genotype. The indicator of tumor necrosis factor alpha (TNF-α) in the AA genotype of the TAP1 gene of Fengjing pigs was significantly higher than that of the GG and AG genotypes. The indicator of IL-12 in the AG genotype of the Shawutou pig TAP1 gene was significantly higher than that of the GG genotype. The level of TNF-α in the AA genotype of the NRAMP1 gene of Meishan pigs was markedly higher than that of the AB genotype. The IL-2 level of the AG type of the FUT1 gene was obviously higher than that of the GG type of Pudong white pigs, the IL-2 level of the AA type of the MUC4 gene was dramatically higher than that of the AG type, and the IgG level of the GG type of the MUC13 gene was apparently higher than that of the AG type. The results of this study are of great significance in guiding the antidiarrhea breeding and molecular selection of Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs and laying the foundation for future antidiarrhea breeding of various local pig breeds in Shanghai.

A novel fatty acid metabolism-related signature identifies MUC4 as a novel therapy target for esophageal squamous cell carcinoma.

Fatty acid metabolism has been identified as an emerging hallmark of cancer, which was closely associated with cancer prognosis. Whether fatty acid metabolism-related genes (FMGs) signature play a more crucial role in biological behavior of esophageal squamous cell carcinoma (ESCC) prognosis remains unknown. Thus, we aimed to identify a reliable FMGs signature for assisting treatment decisions and prognosis evaluation of ESCC. In the present study, we conducted consensus clustering analysis on 259 publicly available ESCC samples. The clinical information was downloaded from The Cancer Genome Atlas (TCGA, 80 ESCC samples) and Gene Expression Omnibus (GEO) database (GSE53625, 179 ESCC samples). A consensus clustering arithmetic was used to determine the FMGs molecular subtypes, and survival outcomes and immune features were evaluated among the different subtypes. Kaplan-Meier analysis and the receiver operating characteristic (ROC) was applied to evaluate the reliability of the risk model in training cohort, validation cohort and all cohorts. A nomogram to predict patients' 1-year, 3-year and 5-year survival rate was also studied. Finally, CCK-8 assay, wound healing assay, and transwell assay were implemented to evaluate the inherent mechanisms of FMGs for tumorigenesis in ESCC. Two subtypes were identified by consensus clustering, of which cluster 2 is preferentially associated with poor prognosis, lower immune cell infiltration. A fatty acid (FA) metabolism-related risk model containing eight genes (FZD10, TACSTD2, MUC4, PDLIM1, PRSS12, BAALC, DNAJA2 and ALOX12B) was established. High-risk group patients displayed worse survival, higher stromal, immune and ESTIMATE scores than in the low-risk group. Moreover, a nomogram revealed good predictive ability of clinical outcomes in ESCC patients. The results of qRT-PCR analysis revealed that the MUC4 and BAALC had high expression level, and FZD10, PDLIM1, TACSTD2, ALOX12B had low expression level in ESCC cells. In vitro, silencing MUC4 remarkably inhibited ESCC cell proliferation, invasion and migration. Our study fills the gap of FMGs signature in predicting the prognosis of ESCC patients. These findings revealed that cluster subtypes and risk model of FMGs had effects on survival prediction, and were expected to be the potential promising targets for ESCC.

Mutational profile evaluates metastatic capacity of Chinese colorectal cancer patients, revealed by whole-exome sequencing.

Colorectal cancer (CRC) is the third most common cancer and the prevalence rate of CRC is increasing in the China. In this study, whole-exome sequencing (WES) was performed on primary tissues of 47 CRC Chinese patients including 22 metastatic and 25 non-metastatic patients. By comparison with data from western colorectal cancer patients in the Cancer Genome Atlas (TCGA), we identified a number of genes that are more likely to be mutated in Chinese colorectal cancer patients, such as MUC12, MUC12, MUC2, MUC4, HYDIN and KMT2C. Interestingly, MUC family genes including MUC12, MUC2 and MUC4, have mutation rates of >20%, while the mutation frequency was extremely low in western colorectal cancer patients, which were <3% in TCGA and 0% in Memorial Sloan Kettering Cancer Center (MSKCC). We detected metastasis-specific mutated genes including TCF7L2, MST1L, HRNR and SMAD4, while MUC4, NEB, FLG and RFPL4A alteration is more prevalent in the non-metastasis group. Further analysis reveals mutation genes in metastasis group are more focus in the Wnt and Hippo signaling pathway. APC, SMAD4 and TCF7L2 accounted for the major genetic abnormalities in this pathway. In conclusion, this study identified the unique characteristics of gene mutations in Chinese patients with colorectal cancer, and is a valuable reference for personalized treatment in Chinese CRC patients.

Novel KMT2B gene mutation in MUC4 positive low-grade fibromyxoid sarcoma.

BACKGROUND: Low-grade Fibromyxoid Sarcoma(LGFM)is a rare fibrosarcoma, which mainly occurs in young people and is mostly seen in the trunk and limbs. The tumor is usually FUS-CREB3L2 fusion caused by t(7;16)(q32-34;p11)chromosome translocation, and rarely FUS-CREB3L1 and EWSR1-CREB3L1 fusion. MUC4 diffuse strong positive can be used as a specific index of LGFM. LGFM is similar to Sclerosing Epithelioid Fibrosarcoma(SEF) and may have the same origin. CASE PRESENTATION: We report a case of LGFM in the chest wall. A female who is 59 years old. In 2016, CT showed dense nodule shadow and focal thickening of the left pleura, the patient underwent surgery, Pathological report that low to moderate malignant fibrosarcoma(fibromyxoid type). The CT re-examination in 2021 showed that the tumors on the left chest wall were significantly larger than before. Pathological examination showed the disease is composed of alternating collagen like and mucinous areas. Under high-power microscope, the tumor cells are consistent in shape, spindle or short spindle, and the tumor cells are arranged in bundles. In local areas, the density of tumor cells is significantly increased, mixed with collagen fibers, and small focal SEF appear. The result of immunohistochemistry showed that SMA, Desmin, CD34, STAT6, S100, SOX10, HMB45 and Melan A were negative, EMA was weakly positive, MUC4 was diffuse and strongly positive, and Ki67 index was low (3%). CONCLUSION: Sequencing results showed that MET, EGFR, KMT2B and RET gene were mutated in LGFM, and KMT2B gene had cancer promoting effect, but there was no literature report in LGFM, which may be of certain significance for the diagnosis and treatment of LGFM.

Albumin conjugation promotes arsenic trioxide transport through alkaline phosphatase-associated transcytosis in MUC4 wildtype pancreatic cancer cells.

Pancreatic cancer (PC) has a poor prognosis due to chemotherapy resistance and unfavorable drug transportation. Albumin conjugates are commonly used as drug carriers to overcome these obstacles. However, membrane-bound glycoprotein mucin 4 (MUC4) has emerged as a promising biomarker among the genetic mutations affecting albumin conjugates therapeutic window. Human serum albumin-conjugated arsenic trioxide (HSA-ATO) has shown potential in treating solid tumors but is limited in PC therapy due to unclear targets and mechanisms. This study investigated the transport mechanisms and therapeutic efficacy of HSA-ATO in PC cells with different MUC4 mutation statuses. Results revealed improved penetration of ATO into PC tumors through conjugated with HSA. However, MUC4 mutation significantly affected treatment sensitivity and HSA-ATO uptake both in vitro and in vivo. Mutant MUC4 cells exhibited over ten times higher IC50 for HSA-ATO and approximately half the uptake compared to wildtype cells. Further research demonstrated that ALPL activation by HSA-ATO enhanced transcytosis in wildtype MUC4 PC cells but not in mutant MUC4 cells, leading to impaired uptake and weaker antitumor effects. Reprogramming the transport process holds potential for enhancing albumin conjugate efficacy in PC patients with different MUC4 mutation statuses, paving the way for stratified treatment using these delivery vehicles.

Interfaz inmunológica de la superficie ocular: expresión de MUC1, MUC4 y MUC16 y producción de citoquinas proinflamatorias en pacientes con síndrome de ojo seco secundario a enfermedad de Sjögren / Ocular surface immunological interface: expression of MUC1, MUC4 and MUC16 and production of proinflammatory cytokines in patients with dry eye syndrome secondary to Sjögren's disease

El síndrome de ojo seco (SOS) constituye una de las patologías oculares más frecuentes y con mayor repercusión en la calidad de vida de pacientes en el mundo actual. A su vez, dado cambios en los hábitos como el mayor uso de pantallas, la mayor polución y el envejecimiento poblacional hacen que la proyección sea en aumento. Se estima que el 10% de los SOS son pacientes con síndrome de Sjögren (SS). Este puede ser primario o secundario cuando se encuentra asociado a otras enfermedades del tejido conectivo como el lupus eritematoso sistémico (LES), la artritis reumatoide (AR) o la esclerosis sistémica (ES). El SOS secundario a SS (SS-SOS) nos resulta de particular interés por ser generalmente severo y de difícil tratamiento. El suero autólogo (SA) constituye una herramienta poco utilizada en nuestro medio hasta la realización de este proyecto. Sus componentes, que incluyen factores de crecimiento epitelial y vitamina A constituyen bloques fundamentales del mantenimiento de las barreras epiteliales haciéndolo una terapia prometedora y accesible. El SS es la tercera enfermedad reumática más prevalente junto con AR y el LES. Se estima que la prevalencia del SSP va del 0.1 al 4.8% de la población dependiendo de la edad, y 90% de estos pacientes son mujeres. A pesar de su alta prevalencia, aún hay gran desconocimiento de los mecanismos moleculares involucrados en el SS-SOS. Tanto la inmunidad innata como adaptativa participan en la patogenia del SOS asociado a SS. Estudios previos encontraron que tanto un perfil Th1 como Th17 serían los principales actores. Por este motivo, elegimos el estudio de las citoquinas IL-17 e IL-22 las cuales son las principales citoquinas efectoras de este perfil linfocitario. Estas citoquinas mantienen la integridad de la barrera epitelial estimulando la formación de proteínas que conforman las uniones estrechas.En especial IL-22 tiene un rol importante en la supervivencia y proliferación celular. Por su parte IL-17 tendría un rol estimulando la producción de péptidos antimicrobianos y quimioquinas que atraen leucocitos a la zona afectada cuando la barrera epitelial es penetrada. En la escasa literatura publicada se ha observado que los niveles de IL-17 e IL­22 se encuentran aumentados significativamente en pacientes con SS-SOS con relación a los no SS y a los controles sanos. A su vez, el nivel de estas citoquinas se correlacionaba positivamente con el cuestionario de queratopatía del índice de enfermedad de la superficie ocular (OSDI por su sigla en inglés) y negativamente con el tiempo de rotura del film lagrimal (TBUT por su sigla en inglés) y el resultado de la prueba de Schirmer. Debemos destacar que estos resultados fueron vistos en un único estudio que incluyó un pequeño número de pacientes. Las mucinas jugarían un rol fundamental en la homeostasis de la superficie ocular (SO). En particular las mucinas transmembrana MUC1, MUC4 y MUC16 estarían involucradas en la activación de factores de transcripción de citoquinas proinflamatorias. Nuestra hipótesis inicial era que se podría observar una disminución en la expresión de estas mucinas en relación con el daño de la SO. Esta hipótesis se basó en el conocido rol protector que las mucinas 12 juegan normalmente en las superficies epiteliales, atrapando agentes patógenos y su estudiada naturaleza inmunorreguladora. A lo largo de este trabajo de doctorado PROINBIO, se utilizó una combinación de diversas estrategias experimentales y clínicas para estudiar la superficie ocular en pacientes con síndrome de Sjögren (SS) y síndrome de ojo seco (SOS) y la correlación de estos hallazgos con la clínica del paciente.Para el desarrollo de este proyecto de investigación, conseguimos articular un servicio dirigido a los pacientes con SOS con la participación de un equipo multidisciplinario integrado por médicos oftalmólogos pertenecientes a la cátedra de oftalmología (valoración- seguimiento - indicación terapéutica), hemoterapia (desarrollo del colirio de suero autólogo) y unidad de enfermedades autoinmunes (captación de los pacientes con síndrome de Sjögren primario o secundario) del Hospital de Clínicas en Montevideo, Uruguay. Al mismo tiempo, desarrollamos y pusimos a punto la toma de biopsias de células conjuntivales mediante impresión conjuntival. Este procedimiento se había realizado anteriormente en nuestro medio, pero nunca para el estudio posterior con técnica de RT-PCR (reacción en cadena de la polimerasa en tiempo real). También conseguimos sistematizar el correcto almacenamiento y procesamiento de las muestras para la realización de esta técnica. Esto facilitará en el futuro continuar con el estudio de nuevos marcadores en esta y otras patologías de la SO. Nuestros resultados nos permitieron arribar a las siguientes conclusiones: Determinamos la primera estimación de prevalencia de SOS en una población de pacientes con diagnóstico de otras patologías autoinmunes en nuestra población la cual fue discretamente menor a la reportada en la literatura. Esta fue de 22% de los pacientes, constituyendo la primera estimación en Uruguay de SOS en pacientes con patología autoinmune. Logramos medir la expresión de las tres principales mucinas transmembrana de la SO en nuestro grupo de pacientes estudiados e individuos sanos. Contrario a nuestra hipótesis, observamos un aumento significativo de la expresión de MUC1 y MUC4 en nuestro grupo de pacientes con SOS.A pesar de que es conocido el rol fundamental de las mucinas en mantener la homeostasis de la SO, se ha descrito el aumento en la expresión de MUC1 y MUC4 frente al daño inflamatorio o en casos de neoplasias tanto en el epitelio digestivo como respiratorio. Detectamos una diferencia en la expresión de estas mucinas entre pacientes con SSP donde observamos un aumento de las tres mucinas de estudio (MUC1, MUC4 y MUC16) y SSS donde únicamente MUC1 se vio significativamente elevada. Asimismo, correlacionamos estos hallazgos con la clínica (síntomas y signos de ojo seco) observando que MUC4 correlaciona significativamente tanto con menor producción del film lagrimal como con un mayor grado de queratopatía superficial. La correlación de MUC4 con los síntomas de SOS no fue significativa lo cual pensamos sí se observaría probablemente incluyendo un mayor número de pacientes en el estudio. Por otra parte, MUC1 y MUC16 se correlacionaron positivamente con una presencia de síntomas más severos de SOS pero su correlación con los signos clínicos de SOS no llegó al nivel de significancia excepto en el caso de MUC1 con relación al grado de queratopatía. Evaluamos la expresión de citoquinas proinflamatorias del perfil Th17 como son IL-17 e IL-22. Observamos como la IL 22 se correlaciona con la expresión de MUC16, la cual a su vez aumenta en pacientes con mayor sintomatología de SOS. Encontramos una correlación significativa entre la expresión de ambas citoquinas como era esperable ya que ambas son citoquinas que corresponden a un mismo perfil de respuesta linfocitaria. No pudimos reproducir los resultados vistos en la literatura, aunque escasa, la cual reporta que los pacientes con SOS presentan un aumento de estas citoquinas en relación a una mayor severidad clínica del SS-SOS.En nuestro estudio no observamos diferencia entre nuestro grupo de pacientes y el grupo control ni una correlación con los síntomas o signos del SOS. Analizamos la expresión de MUC1, MUC4 y MUC16 comparando el grupo de pacientes con SS tratados con inmunomoduladores versus los no tratados observando una diferencia interesante entre estos dos grupos. Mientras ambos grupos sobre expresan MUC1. Los pacientes en tratamiento presentan un aumento de MUC4 y no de MUC16 y lo inverso se observa en los pacientes no tratados. Evidenciamos la mejoría sintomática del tratamiento de estos pacientes con suero autólogo al 20% el cual es una opción terapéutica disponible en nuestro medio que está subutilizada por la falta de evidencia clínica hasta el momento. Estos resultados son un sustento objetivo de la efectividad del tratamiento para este grupo de pacientes. En suma, este trabajo constituye una aproximación a comprender como varia la expresión a nivel de ARN mensajero de mucinas transmembranarias específicas y su asociación con citoquinas de perfil Th17, así como síntomas y signos clínicos del SS-SOS. Constituye un gran paso inicial a la caracterización molecular de la SO de estos pacientes y con esta la potencialidad de desarrollar terapias dirigidas. Según nuestro saber y entender, éste constituye el primer trabajo de estas características en nuestro medio y nos brinda más información de la prevalencia y características de estos pacientes en Uruguay. La puesta a punto de la técnica de impresión conjuntival, conocida en el mundo, pero no utilizada en nuestro país habitualmente para la extracción de ARN, abre las puertas para ampliar nuestras investigaciones focalizándonos en otras moléculas posiblemente cruciales en el desarrollo de SOS como por ejemplo el estudio de glicosil transferasas.

Mucin 4 Confers Gemcitabine Resistance and an Unfavorable Prognosis in Patients with Cholangiocarcinoma via AKT Activation.

Cholangiocarcinoma (CCA) exhibits aggressive biological behavior and a poor prognosis. Gemcitabine (GEM)-based chemotherapy is the first-line chemotherapy for advanced CCA but has a response rate of only 20-30%. Therefore, investigating treatments to overcome GEM resistance in advanced CCA is crucial. Among mucin (MUC) family members, MUC4 showed the greatest increase in the resistant versus parental sublines. MUC4 was upregulated in whole-cell lysates and conditioned media from gemcitabine-resistant (GR) CCA sublines. MUC4 mediated GEM resistance by activating AKT signaling in GR CCA cells. The MUC4-AKT axis induced BAX S184 phosphorylation to inhibit apoptosis and downregulated GEM transporter human equilibrative nucleoside transporter 1 (hENT1) expression. The combination of AKT inhibitors and GEM or afatinib overcame GEM resistance in CCA. In vivo, capivasertib (an AKT inhibitor) increased GEM sensitivity in GR cells. MUC4 promoted EGFR and HER2 activation to mediate GEM resistance. Finally, MUC4 expression in patient plasma correlated with MUC4 expression. Paraffin-embedded specimens from non-responders expressed significantly more MUC4 than did those from responders, and this upregulation was associated with poor progression-free survival and overall survival. In GR CCA, high MUC4 expression promotes sustained EGFR/HER2 signaling and AKT activation. The combination of AKT inhibitors with GEM or afatinib might overcome GEM resistance.

Genetic variants of MUC4 are associated with susceptibility to and mortality of colorectal cancer and exhibit synergistic effects with LDL-C levels.

As a disease with high mortality and prevalence rates worldwide, colorectal cancer (CRC) has been thoroughly investigated. Mucins are involved in the induction of CRC and the regulation of intestinal homeostasis but a member of the mucin gene family MUC4 has a controversial role in CRC. MUC4 has been associated with either decreased susceptibility to or a worse prognosis of CRC. In our study, the multifunctional aspects of MUC4 were elucidated by genetic polymorphism analysis in a case-control study of 420 controls and 464 CRC patients. MUC4 rs1104760 A>G polymorphism had a protective effect on CRC risk (AG, AOR = 0.537; GG, AOR = 0.297; dominant model, AOR = 0.493; recessive model, AOR = 0.382) and MUC4 rs2688513 A>G was associated with an increased mortality rate of CRC (5 years, GG, adjusted HR = 6.496; recessive model, adjusted HR = 5.848). In addition, MUC4 rs1104760 A>G showed a high probability of being a potential biomarker for CRC patients with low-density lipoprotein cholesterol (LDL-C) in the risk range while showing a significant synergistic effect with the LDL-C level. This is the first study to indicate a significant association between MUC4 genetic polymorphisms and CRC prevalence, suggesting a functional genetic variant with the LDL-C level, for CRC prevention.

Superparamagnetic iron oxide nanoparticles target BxPC-3 cells and silence MUC4 for the treatment of pancreatic cancer.

PURPOSE: Superparamagnetic iron oxide nanoparticles (SPION) are excellent magnetic resonance imaging (MRI) contrast agents. Mucin 4 (MUC4) acts as pancreatic cancer (PC) tumor antigen and influences PC progression. Small interfering RNAs (siRNAs) are used as a gene-silencing tool to treat a variety of diseases. METHODS: We designed a therapeutic probe based on polyetherimide-superparamagnetic iron oxide nanoparticles (PEI-SPION) combined with siRNA nanoprobes (PEI-SPION-siRNA) to assess the contrast in MRI. The biocompatibility of the nanocomposite, and silencing of MUC4 were characterized and evaluated. RESULTS: The prepared molecular probe had a particle size of 61.7 ± 18.5 nm and a surface of 46.7 ± 0.8mV and showed good biocompatibility in vitro and T2 relaxation efficiency. It can also load and protect siRNA. PEI-SPION-siRNA showed a good silencing effect on MUC4. CONCLUSION: PEI-SPION-siRNA may be beneficial as a novel theranostic tool for PC.

Mucin 4 protein is expressed in B-acute lymphoblastic leukemia and is restricted to BCR::ABL1-positive and BCR::ABL-like subtypes.

Mucin 4 (MUC4) is a transmembrane mucin that, like most mucins, is not expressed in normal hematopoietic cells, but little is known about its expression in malignant hematopoiesis. B-acute lymphoblastic leukemia (B-ALL) consists of genetically distinct disease subtypes with similarities and differences in gene expression most frequently studied at the mRNA level, which is less amenable to widespread routine clinical use. Here, we demonstrate using immunohistochemistry (IHC) that MUC4 protein is expressed in less than 10% of B-ALL, with expression restricted to BCR::ABL1+ and BCR::ABL1-like (CRLF2 rearranged) subtypes of B-ALL (4/13, 31%). None (0/36, 0%) of the remaining B-ALL subtypes expressed MUC4. We compare clinical and pathologic features of MUC4+ and MUC4- BCR::ABL1+/like cases and most significantly report a possible shorter time to relapse for MUC4+ BCR::ABL1 B-ALL that would need to be validated in larger studies. In conclusion, MUC4 is a specific, albeit insensitive, marker for these high-risk subtypes of B-ALL. We propose that MUC4 IHC may be used diagnostically to rapidly identify these B-ALL subtypes, particularly in resource-limited settings or when an aspirate sample is not available for ancillary genetic studies.

Blocking soluble TNFα sensitizes HER2-positive breast cancer to trastuzumab through MUC4 downregulation and subverts immunosuppression.

BACKGROUND: The success of HER2-positive (HER2+) breast cancer treatment with trastuzumab, an antibody that targets HER2, relies on immune response. We demonstrated that TNFα induces mucin 4 (MUC4) expression, which shields the trastuzumab epitope on the HER2 molecule decreasing its therapeutic effect. Here, we used mouse models and samples from HER2+ breast cancer patients to unravel MUC4 participation in hindering trastuzumab effect by fostering immune evasion. METHODS: We used a dominant negative TNFα inhibitor (DN) selective for soluble TNFα (sTNFα) together with trastuzumab. Preclinical experiments were performed using two models of conditionally MUC4-silenced tumors to characterize the immune cell infiltration. A cohort of 91 patients treated with trastuzumab was used to correlate tumor MUC4 with tumor-infiltrating lymphocytes. RESULTS: In mice bearing de novo trastuzumab-resistant HER2+ breast tumors, neutralizing sTNFα with DN induced MUC4 downregulation. Using the conditionally MUC4-silenced tumor models, the antitumor effect of trastuzumab was reinstated and the addition of TNFα-blocking agents did not further decrease tumor burden. DN administration with trastuzumab modifies the immunosuppressive tumor milieu through M1-like phenotype macrophage polarization and NK cells degranulation. Depletion experiments revealed a cross-talk between macrophages and NK cells necessary for trastuzumab antitumor effect. In addition, tumor cells treated with DN are more susceptible to trastuzumab-dependent cellular phagocytosis. Finally, MUC4 expression in HER2+ breast cancer is associated with immune desert tumors. CONCLUSIONS: These findings provide rationale to pursue sTNFα blockade combined with trastuzumab or trastuzumab drug conjugates for MUC4+ and HER2+ breast cancer patients to overcome trastuzumab resistance.

Association Study between Mucin 4 (MUC4) Polymorphisms and Idiopathic Recurrent Pregnancy Loss in a Korean Population.

Recurrent pregnancy loss (RPL) is the loss of two or more consecutive pregnancies before 20 weeks of gestational age. Our study investigated whether mucin 4 (MUC4) polymorphisms are associated with RPL. MUC polymorphisms (rs882605 C>A, rs1104760 A>G, rs2688513 A>G, rs2258447 C>T, and rs2291652 A>G) were genotyped in 374 women with RPL and 239 controls of Korean ethnicity using polymerase chain reaction-restriction fragment length polymorphism analysis and the TaqMan probe SNP genotyping assay. Differences in genotype frequencies between cases of RPL and the controls were compared. MUC4 rs882605 C>A and rs1104760 A>G polymorphisms were associated with increased incidence of RPL in three and four or more pregnancy loss patients. The haplotype analyses showed a tendency for the allelic effect including the association of MUC4 rs882605 A and rs1104760 G alleles with increased incidence of RPL. In addition, the MUC4 rs882605 CA/MUC4 rs2258447 CC genotype combination was associated with increased RPL prevalence. The two exonic polymorphisms lead to amino acid changes of protein and may act as pathogenic variants for RPL. In conclusion, the MUC4 rs882605 C>A and MUC4 rs1104760 A>G polymorphisms were associated with the susceptibility of RPL and we considered them as potential biomarkers for RPL.

Depletion of transmembrane mucin 4 (Muc4) alters intestinal homeostasis in a genetically engineered mouse model of colorectal cancer.

Mucins are components of the mucus layer overlying the intestinal epithelial cells, which maintains physiological homeostasis. Altered mucin expression is associated with disease progression. Expression of MUC4 decreases in colorectal cancer (CRC); however, its functional role and implications in the intestinal pathology in CRC are not studied well. Therefore, we generated a genetically engineered Muc4 knockout (Muc4-/-) CRC mouse model by crossing with Muc4-/- and Apcflox/flox mice in the presence of colon-specific inducible Cre. We observed that deficiency of Muc4 results in an increased number of macroscopic tumors in the colon and rectal region and leads to poor survival. Further, the absence of Muc4 was associated with goblet cell dysfunction where the expression of intestinal homeostasis molecules (Muc2 and Fam3D) was downregulated. Next, we also observed that loss of Muc4 showed reduced thickness of mucus layer, leading to infiltration of bacteria, reduction in anti-microbial peptides, and upregulation of pro-inflammatory cytokines. Further, Apc gene mutation results in activation of the Wnt/ß-catenin signaling pathway that corroborated with an increased nuclear accumulation of ß-catenin and activation of its target genes: cyclin D1 and c-Myc in Muc4-/- mice was observed. We conclude that the presence of Muc4 is essential for intestinal homeostasis, reduces tumor burden, and improves overall survival.

MUC4 expression by immunohistochemistry is a specific marker for BCR-ABL1+ and BCR-ABL1-like B-lymphoblastic leukemia.

BCR-ABL1-like B-acute lymphoblastic leukemia (B-ALL) is a genetically heterogeneous group of high-risk B-ALL that benefits from targeted tyrosine kinase inhibitor (TKI) therapy. The incidence of this high-risk B-ALL is relatively low and screening with surrogate markers will be useful to identify patients for further genetic testing. Here we demonstrate that widely available MUC4 protein immunohistochemistry (IHC) is predictive of a BCR-ABL1-like genotype for a subset of patients. Overall, MUC4 expression was observed in 36% (9/25) BCR-ABL1-like, 43% (3/7) BCR-ABL1+ and 9% (2/22) B-ALL other cases (p=.019 for BCR-ABL1 like and BCR-ABL1+ versus B-ALL others). Furthermore, 83% (5/6) of patients with ABL class fusions showed MUC4 expression when compared to 25% (4/16, p=.006) patients with JAK class fusions. Overall, the study demonstrates that MUC4 expression is highly specific (90.9%) for BCR-ABL1+ and BCR-ABL1-like B-ALL with high sensitivity for cases with ABL class fusions.

A flexible summary statistics-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 epithelia (HNE) from 94 CF-affected 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 0.2 Mb region with high linkage disequilibrium (LD) and evidence of allelic heterogeneity. We previously developed the Simple Sum (SS), a powerful colocalization test in regions with allelic 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 priori 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 allelic heterogeneity. Applying SS2 to the MUC20/MUC4 CF lung disease locus with eQTLs from CF HNE revealed significant colocalization with MUC4 (p = 1.31 × 10-5) rather than with 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.

Next-generation sequencing analysis suggests varied multistep mutational pathogenesis for endocrine mucin-producing sweat gland carcinoma with comments on INSM1 and MUC2 suggesting a conjunctival origin.

Endocrine mucin-producing sweat gland carcinoma is a low-grade eyelid tumor. Small biopsies and insensitive immunohistochemistry predispose to misdiagnosis. We aimed to identify clarifying immunohistochemical markers, molecular markers, or both. Clinicopathologic data (22 cases) were reviewed. Immunohistochemistry (insulinoma-associated protein 1, BCL-2, mucin 2 [MUC2], mucin 4, androgen receptor, ß-catenin, and Merkel cell polyomavirus) and next-generation sequencing (Memorial Sloan Kettering integrated mutation profiling of actionable cancer targets, 468 genes) were performed (3 cases). Female patients (n = 15) and male patients (n = 7) (mean age 71.8 years; range 53-88 years) had eyelid or periorbital tumors (>90%) with mucin-containing solid or cystic neuroendocrine pathology. Immunohistochemistry (insulinoma-associated protein 1, BCL2, androgen receptor, retinoblastoma-associated protein 1, and ß-catenin) was diffusely positive (5/5), MUC2 partial, mucin 4 focal, and Merkel cell polyomavirus negative. Memorial Sloan Kettering integrated mutation profiling of actionable cancer targets identified 12 single-nucleotide variants and 1 in-frame deletion in 3 cases, each with DNA damage response or repair (BRD4, PPP4R2, and RTEL1) and tumor-suppressor pathway (BRD4, TP53, TSC1, and LATS2) mutations. Microsatellite instability, copy number alterations, and structural alterations were absent. Insulinoma-associated protein 1 and MUC2 are positive in endocrine mucin-producing sweat gland carcinoma. MUC2 positivity suggests conjunctival origin. Multistep pathogenesis involving DNA damage repair and tumor-suppressor pathways may be implicated.

Characterization of recombinant ß subunit of human MUC4 mucin (rMUC4ß).

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like ß-subunit. Due to the presence of several functional domains, the characterization of MUC4ß is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4ß (rMUC4ß). Purified rMUC4ß was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical ß-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4ß physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4ß that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.