Molecular genetic analysis of colorectal carcinoma with an aggressive extraintestinal immunohistochemical phenotype.

Colorectal cancer (CRC) is a leading global cause of illness and death. There is a need for identification of better prognostic markers beyond traditional clinical variables like grade and stage. Previous research revealed that abnormal expression of cytokeratin 7 (CK7) and loss of the intestinal-specific Special AT-rich sequence-binding protein 2 (SATB2) are linked to poor CRC prognosis. This study aimed to explore these markers' prognostic significance alongside two extraintestinal mucins (MUC5AC, MUC6), claudin 18, and MUC4 in 285 CRC cases using immunohistochemistry on tissue microarrays (TMAs). CK7 expression and SATB2-loss were associated with MUC5AC, MUC6, and claudin 18 positivity. These findings suggest a distinct "non-intestinal" immunohistochemical profile in CRC, often right-sided, SATB2-low, with atypical expression of CK7 and non-colorectal mucins (MUC5AC, MUC6). Strong MUC4 expression negatively impacted cancer-specific survival (hazard ratio = 2.7, p = 0.044). Genetic analysis via next-generation sequencing (NGS) in CK7 + CRCs and those with high MUC4 expression revealed prevalent mutations in TP53, APC, BRAF, KRAS, PIK3CA, FBXW7, and SMAD4, consistent with known CRC mutation patterns. NGS also identified druggable variants in BRAF, PIK3CA, and KRAS. CK7 + tumors showed intriguingly common (31.6%) BRAF V600E mutations corelating with poor prognosis, compared to the frequency described in the literature and databases. Further research on larger cohorts with a non-colorectal immunophenotype and high MUC4 expression is needed.

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.

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.

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.

Surface plasmon resonance microscopy identifies glycan heterogeneity in pancreatic cancer cells that influences mucin-4 binding interactions.

Membrane proteins are the main targets of therapeutic drugs and most of them are glycosylated. Glycans play pivotal roles in several biological processes, and glycosylation changes are a well-established hallmark of several types of cancer, including pancreatic cancer, that contribute to tumor growth. Mucin-4 (MUC-4) is a membrane glycoprotein which is associated with pancreatic cancer and metastasis, and it has been targeted as a promising vaccine candidate. In this study, Surface Plasmon Resonance Microscopy (SPRM) was implemented to study complex influences of the native N-glycan cellular environment on binding interactions to the MUC-4 receptor as this is currently the only commercially available label-free technique with high enough sensitivity and resolution to measure binding kinetics and heterogeneity on single cells. Such unique capability enables for a more accurate understanding of the "true" binding interactions on human cancer cells without disrupting the native environment of the target MUC-4 receptor. Removal of N-linked glycans in pancreatic cancer cells using PNGase F exposed heterogeneity in Concanavalin (Con A) binding by revealing three new binding populations with higher affinities than the glycosylated control cells. Anti-MUC-4 binding interactions of enzymatically N-linked deglycosylated pancreatic cancer cells produced a 25x faster association and 37x higher affinity relative to the glycosylated control cells. Lastly, four interaction modes were observed for Helix Pomatia Agglutinin (HPA) binding to the glycosylated control cells, but shifted and increased in activity upon removal of N-linked glycans. These results identified predominant interaction modes of glycan and MUC-4 in pancreatic cancer cells, the kinetics of their binding interactions were quantified, and the influence of N-linked glycans in MUC-4 binding interactions was revealed.

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.

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.

Sialylated IVIg promotes clinical improvements in a rabbit dry eye model by regulating inflammatory cytokines.

Dry eye disease (DED) is caused by a loss of homeostasis of the tear film, which results in visual disturbance, ocular surface inflammation and damage, and neurosensory abnormalities. Although it is prevalent in 5-50% of the global population, there are limited clinical options for its treatment. This study explored the potential use of human intravenous immunoglobulin (IVIg) and its enriched fractions of sialylation, sialylated IVIg (sIVIg), as a treatment for DED. Fifteen female New Zealand white rabbits were topically instilled with 0.2% benzalkonium chloride (BAC) twice daily for five consecutive days to induce experimental dry eye. Saline, 0.4% IVIg, or 0.04% sIVIg eye drops were instilled twice daily for 20 consecutive days. Clinical evaluations, such as non-invasive tear break-up time (NIBUT) and corneal fluorescein staining (CFS), were conducted. mRNA levels of mucin 4, mucin 16, TNF-α, IL-1ß, MMP9, IL-10, TGF-ß, and CD209 in rabbit conjunctival tissues were examined using reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). The relationships between CD209 family members in rabbits and various mammalian species were analyzed using a phylogenetic tree. IVIg or sIVIg treatment resulted in clinical improvements in the rabbit DED model. The inflammatory cytokines, TNF-α and IL-1ß, were increased and mucin 4 and mucin 16, cell surface-associated mucins, were decreased in BAC-induced dry eye. Following IVIg or sIVIg treatment, inflammatory cytokines decreased, whereas the anti-inflammatory cytokine, IL-10, increased substantially. Moreover, a 10-fold lower sIVIg treatment dose resulted in prolonged IL-10 production, representing a significantly improved DED compared to IVIg. Furthermore, the expression of rabbit CD209 mRNA in the rabbit conjunctiva and its close relationship with primate homologs suggest that it may interact with IVIg or sIVIg to promote IL-10 expression, as previously described in humans. At a lower dosage, sIVIg showed a more efficient improvement in DED, making it a promising new candidate medication for DED.

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.

Oncogenic KRAS, Mucin 4, and Activin A-Mediated Fibroblast Activation Cooperate for PanIN Initiation.

Over 90% of patients with pancreatic ductal adenocarcinoma (PDAC) have oncogenic KRAS mutations. Nevertheless, mutated KRAS alone is insufficient to initiate pancreatic intraepithelial neoplasia (PanIN), the precursor of PDAC. The identities of the other factors/events required to drive PanIN formation remain elusive. Here, optic-clear 3D histology is used to analyze entire pancreases of 2-week-old Pdx1-Cre; LSL-KrasG12D/+ (KC) mice to detect the earliest emergence of PanIN and observed that the occurrence is independent of physical location. Instead, it is found that the earliest PanINs overexpress Muc4 and associate with αSMA+ fibroblasts in both transgenic mice and human specimens. Mechanistically, KrasG12D/+ pancreatic cells upregulate Muc4 through genetic alterations to increase proliferation and fibroblast recruitments via Activin A secretion and consequently enhance cell transformation for PanIN formation. Inhibition of Activin A signaling using Follistatin (FST) diminishes early PanIN-associated fibroblast recruitment, effectively curtailing PanIN initiation and growth in KC mice. These findings emphasize the vital role of interactions between oncogenic KrasG12D/+ -driven genetic alterations and induced microenvironmental changes in PanIN initiation, suggesting potential avenues for early PDAC diagnostic and management approaches.

Utility of MUC4 in the diagnosis of secretory carcinoma of salivary glands.

Salivary gland tumors are diverse in morphology and both benign and malignant tumors may pose diagnostic challenges especially in small biopsies. Secretory carcinoma (SC) is histologically characterized by microcysts, follicles, solid growth pattern and occasional papillary structures, and absence of zymogen granules. SC is molecularly defined by the presence of novel gene fusion ETV6::NTRK3. Among the positive stains (S100 and mammaglobin), MUC4 is now another promising marker for the diagnosis of SC, that would enable the pathologists to exclude other morphologically close simulators. Aim of this study was to report clinicopathological features and assess utility of MUC4 in the diagnosis of SC. MUC4 was performed on 22 cases of SC. Glass slides were reviewed to record morphological patterns and staining of S100, mammaglobin, DOG1 and MUC4. Age ranged from 9 to 63 years with mean age of 34.41 ± 16.28 years. The male: female ratio was 72.7 %:27.3 %. The majority occurred in major salivary glands. A combination of patterns was seen; microfollicles were the most prevalent (90 %) followed by papillary-cystic and macrofollicles. MUC4 was positive in 19/21 (90 %) cases with almost equal number of 2+ and 3+ staining. MUC4 was negative in all cases of acinic cell carcinoma, polymorphous adenocarcinoma, adenoid cystic carcinoma, salivary duct carcinoma, myopepithelioma and myoeithelial carcinoma, cystadenoma and cribriform adenocarcinoma and all except 3 cases of mucoepidermoid carcinoma tested. Overall sensitivity of MUC4 was 95.4 %, specificity 90 %, p-value being <0.01, positive predictive value 87.5 % and negative predictive value 96.4 %. A characteristic cytoplasmic granular pattern was observed in 76.1 % tumors. S100 and mammaglobin were positive in all the performed cases. DOG1 was positive in 6/11 (28.5 %) tumors. In conclusion, MUC4 is a useful addition to a diagnostic immunohistochemical panel for SC, and to distinguish it from close potential mimickers such as acinic cell carcinoma, especially in practice settings where molecular testing is unavailable.

Herbal melanin modulates PGE2 and IL-6 gastroprotective markers through COX-2 and TLR4 signaling in the gastric cancer cell line AGS.

We reported a gastric anti-ulcerogenic effect of the Nigella sativa (L.)-derived herbal melanin (HM) using rat models. However, the molecular mechanisms underlying this HM gastroprotective effect remain unknown. Cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E2 (PGE2) and toll-like receptor 4 (TLR4)-mediated interleukin-6 (IL-6) production and secretion play major roles in gastric mucosal protection. In the current study, the human gastric carcinoma epithelial cell line AGS was used as a model to investigate the effect of HM on TLR4, COX-2, glycoprotein mucin 4 protein and gene expression using immuno-cyto-fluorescence staining, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gastroprotective markers PGE2 and IL-6 production and secretion were also assessed using an enzyme-linked immunosorbent assay (ELISA). Bacterial lipopolysaccharides (LPS), well-known inducers of TLR4, COX-2, PGE2 and IL-6 expression, were used as a positive control. We showed that HM upregulated its main receptor TLR4 gene and protein expression in AGS cells. HM increased, in a dose- and time-dependent manner, the secretion of PGE2 and the expression of COX-2 mRNA and protein, which was detected in the nucleus, cytoplasm and predominantly at the intercellular junctions of the AGS cells. In addition, HM enhanced IL-6 production and secretion, and upregulated the mucin 4 gene expression, the hallmarks of gastroprotection. To check whether HM-induced PGE2 and IL-6 through TLR4 signaling and COX-2 generated, AGS cells were pre-treated with a TLR4 signaling inhibitor TAK242 and the COX-2 inhibitor NS-398. A loss of the stimulatory effects of HM on COX-2, PGE2 and IL-6 production and secretion was observed in TAK242 and NS-398-pre-treated AGS cells, confirming the role of TLR4 signaling and COX-2 generated in the HM gastroprotective effects. In conclusion, our results showed that HM enhances TLR4/COX-2-mediated secretion of gastroprotective markers PGE2 and IL-6, and upregulates mucin 4 gene expression in the human gastric epithelial cell line AGS, which may contribute to the promising beneficial gastroprotective effect of HM for human gastric prevention and treatment.

LncRNA BBOX1-AS1 Contributes to the Development of Nasopharyngeal Carcinoma via miR-204-5p/MUC4 Axis.

OBJECTIVE: Dysregulation of long non-coding RNAs (lncRNAs) is common in nasopharyngeal carcinoma (NPC) progression, and it is important to have an in-depth understanding of their functions in NPC. This study is the first to explore the role of the lncRNA BBOX1-AS1 in NPC development. METHODS: The expression of lncRNA BBOX1-AS1, MUC4, or miR-204-5p was measured in NPC cell lines or tissues via RT-qPCR and western blotting. Wound healing assays and CCK-8 were used to identify cell migration and cell viability, respectively. The protein expression of Bax and Bcl-2 was measured by western blotting. The tumorigenic effect of NPC cells in vivo was verified using xenograft tumors in nude mice. Luciferase reporter and RIP assays were conducted to clarify the association between miR-204-5p and lncRNA BBOX1-AS1 or MUC4. RESULTS: lncRNA BBOX1-AS1 upregulation was observed in NPC cells and tissues. Silencing lncRNA BBOX1-AS1 suppressed the migration and viability of C666-1 and TW03 cells while promoting cell apoptosis. Knockdown of the lncRNA BBOX1-AS1 repressed tumor growth in vivo. Moreover, the tumor suppression effect of silenced lncRNA BBOX1-AS1 might be reversed with the help of the miR-204-5p inhibitor. lncRNA BBOX1-AS1 targets miR-204-5p and regulates MUC4 expression in NPC. MUC4 is a miR-204-5p target and exerts a function similar to that of lncRNA BBOX1-AS1. CONCLUSION: These observations highlight that lncRNA BBOX1-AS1 is an essential NPC progression promoter and suggest that the lncRNA BBOX1-AS1/miR-204-5p/MUC4 axis is a potential therapeutic target in NPC.

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.