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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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yuko Terada reports financial support was provided by Japan Society for the Promotion of Science. Keisuke Ito reports financial support was provided by Japan Society for the Promotion of Science. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A 70-year-old male presented with diplopia and painless proptosis of the left eye for 5 months. Examination showed 6 mm of axial proptosis and restriction of supraduction, abduction and adduction, and mild limitation of infraduction of the left eye. Magnetic resonance imaging demonstrated a large, moderately well-circumscribed intraconal mass in the left lateral orbit, and excisional biopsy was performed. Histopathologic features of mixed fibrous and myxoid areas in a whorl-like pattern and immunohistochemical staining for MUC4 confirmed the diagnosis of low-grade fibromyxoid sarcoma (LGFMS). Next-generation sequencing revealed genetic fusion of EWSR1-CREB3L1. LGFMS is an extremely rare neoplasm with only two prior documented cases of orbital involvement. Here, we report the third case of orbital LGFMS.

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., (© 2024. The Author(s).)

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.

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., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kwon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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., Competing Interests: Competing interests: RS is a consultant for and a research grant from INmune Bio. All other authors declare they have no competing interests., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Mucin MUC4 is an aberrantly expressed oncogene in pancreatic ductal adenocarcinoma (PDAC), yet no pharmacological inhibitors have been identified to target MUC4. Here, we adapted an in silico screening method using the Cancer Therapeutic Response Database (CTRD) to Identify Small Molecule Inhibitors against Mucins (SMIMs). We identified Bosutinib as a candidate drug to target oncogenic mucins among 126 FDA-approved drugs from CTRD screening. Functionally, Bosutinib treatment alone/and in combination with gemcitabine (Gem)/5' fluorouracil (5FU) reduced in vitro viability, migration, and colony formation in multiple PDAC cell lines as well as human PDAC organoid prolifertaion and growth and in vivo xenograft growth. Further, biochemical and molecular analyses showed that Bosutinib exhibited these functional effects by downregulating MUC4 mucin at both transcript and translation levels in a dose- and time-dependent manner. Mechanistically, global transcriptome analysis in PDAC cells upon treatment with Bosutinib revealed disruption of the Src-ERK/AKT-FosL1 pathway, leading to decreased expression of MUC4 and MUC5AC mucins. Taken together, Bosutinib is a promising, novel, and highly potent SMIMs to target MUC4/MUC5AC mucins. This mucin-targeting effect of Bosutinib can be exploited in the future with cytotoxic agents to treat mucinous tumors., Competing Interests: Declaration of competing interest SKB is a founding member of Sanguine Diagnostics and Therapeutics, Inc. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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.

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.

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, β-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 β-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., Competing Interests: Conflicts of interest None disclosed., (Copyright © 2021 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

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 Apc flox/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/β-catenin signaling pathway that corroborated with an increased nuclear accumulation of β-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.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy demonstrating aberrant and progressive expression of mucins. The contribution of individual mucins has been extensively investigated in PDAC; however, comprehensive mucin profiling including splice variants in PDAC tumors has not been reported., Experimental Design: Using publicly available RNA sequencing (RNA-seq) datasets, we assess the expression of mucin family members and their splice variants (SV) in PDAC tumor samples for the first time. Mucin SVs that are correlated with PDAC patient survival are validated in a cohort of patient tumor samples. Further, we use computational methods to derive novel pancreatic tumor subtypes using mucin expression signatures and their associated activated pathways., Results: Principal component analysis identified four novel mucin-based PDAC subtypes. Pathway analysis implicated specific biological signatures for each subtype, labeled (i) immune activated, (ii) progressive, (iii) pancreatitis-initiated, and (iv) anti-inflammatory/PanIN-initiated. Assessing mucin SVs, significantly longer survival is observed with higher expression of 4 MUC1 and 1 MUC13 SVs, whereas patients expressing 2 MUC4 and 1 MUC16 SVs had shorter survival. Using a whole-transcriptome correlation, a three-gene panel, including ESRP2 , PTK6 , and MAGEH1 , is designated to assess PDAC tumor sample cellularity by PCR. One MUC4 SV and one MUC13 SV are quantified in a separate PDAC patient cohort, and their effects on survival are experimentally validated., Conclusions: Altogether, we demonstrate the unique expression pattern of mucins, four mucin-based PDAC subtypes, and the contribution of MUC1 , MUC4 , and MUC16 SVs in PDAC patient survival., (©2021 American Association for Cancer Research.)

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 β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4β (rMUC4β). Purified rMUC4β 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 β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β 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., (© 2021. The Author(s).)

A major barrier to the emergence of distant metastases is the survival of circulating tumor cells (CTCs) within the vasculature. Lethal stressors, including shear forces from blood flow, anoikis arising from cellular detachment, and exposure to natural killer cells, combine to subvert the ability of primary tumor cells to survive and ultimately seed distant lesions. Further attenuation of this rate-limiting process via therapeutic intervention offers a very attractive opportunity for improving cancer patient outcomes, in turn prompting the need for a deeper understanding of the molecular and cellular mechanisms underlying CTC viability. MUC4 is a very large and heavily glycosylated protein expressed at the apical surfaces of the epithelia of a variety of tissues, is involved in cellular growth signaling and adhesiveness, and contributes to the protection and lubrication of cellular linings. Analysis of patient-matched breast tumor specimens has demonstrated that MUC4 protein levels are upregulated in metastatic lesions relative to primary tumor among all breast tumor subtypes, pointing to a possible selective advantage for MUC4 overexpression in metastasis. Analysis of a genetically engineered mouse model of HER2-positive breast cancer has demonstrated that metastatic efficiency is markedly suppressed with Muc4 deletion and Muc4-knockout tumor cells are poorly associated with platelets and white blood cells known to support CTC viability. In this review, we discuss the diverse roles of MUC4 in tumor progression and metastasis and propose that intervening in MUC4 intercellular interactions with binding partners on blood-borne aggregating cells could potentially thwart breast cancer metastatic efficiency.

First described in 1995 by Meis-Kindbloom et al. as a variant of fibrosarcoma simulating carcinoma, sclerosing epithelioid fibrosarcoma (SEF) is a malignant soft tissue sarcoma characterized by epithelioid cells in dense sclerotic stroma, frequent immunoreactivity for MUC4 and heterogeneous genetic profile with recurrent EWSR1 gene rearrangement. It typically affects middle-age adults with a predilection for the lower extremity. It is believed that SEF is closely related to low-grade fibromyxoid sarcoma (LGFMS), both tumors show overlapping features in morphology, immunophenotype, and molecular profile. In this review, we discuss the clinical, morphologic, and immunohistochemical features of SEF with particular emphasis on its molecular diversity and relation to LGFMS., (© 2021 The Authors. APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Medical Microbiology and Pathology.)

MUC4 is a predominant membrane-tethered mucin lubricating and protecting the epithelial surface and playing various biological roles in the renewal and differentiation of epithelial cells, cell signaling, cell adhesion, and carcinogenesis. Interestingly, recent studies have demonstrated that MUC4 expression regulates the epithelial-mesenchymal transition (EMT) of cancer cells in ovarian, pancreatic, and lung cancer. However, the effects of MUC4 expression on EMT in human airway epithelial cells are not yet well known. Here, we describe the effects of transforming growth factor beta 1 (TGF-β1)-induced MUC4 expression on EMT and evaluate its downstream signaling pathway in human airway epithelial cells. In human airway epithelial NCI-H292 cells, exposure to TGF-β1 induced expression of MUC4, CDH2, VIM and SNAI1 genes and encoded by them proteins, MUC4, N-cadherin, vimentin and Snail, and reduced the level of CDH1 and its product, E-cadherin. In MUC4-knockdown cells, TGF-β1-induced expression levels of MUC4, CDH2, VIM and SNAI1 and corresponding proteins were suppressed, but CDH1 and E-cadherin levels were not. In addition, TGF-β1-induced phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2) was suppressed, but that of Smad2/3, Akt, and p38 was not. The results of this study suggest that MUC4 silencing inhibits TGF-β1 -induced EMT via the ERK1/2 pathway, and a possible role of MUC4 in the induction of EMT in human airway epithelial cells.

Aims: To present our experience on spinal sclerosing epithelioid fibrosarcoma (SEF) and review the existing literature pertaining to SEF of the spine., Methods and Results: Six cases of spinal SEF were reviewed, and a literature search of all primary SEFs of the spine was performed. All tumours occurred in adults (median age, 41 years) and were located all along the spine, the lumbar vertebrae being the most commonly involved. All patients presented with pain that they had experienced for months. The mean tumour size at diagnosis was 52 mm. Five tumours showed a spectrum of microscopic features consistent with pure SEF, and one showed a hybrid morphology with areas of low-grade fibromyxoid sarcoma. All were diffusely and strongly positive for mucin 4. Two cases were initially misdiagnosed as epithelioid haemangioendothelioma and aggressive chondroblastoma. Fluorescence in-situ hybridisation showed rearrangements of either FUS or EWSR1 in four cases. Reverse transcription polymerase chain reaction showed the presence of FUS-CREB3L1 and EWSR1-CREB3L1 fusion transcripts in two cases and one case, respectively. Of five patients with follow-up data available, two developed one or more local recurrences and three patients had metastatic disease. Distant metastases were mainly to other osseous locations, followed by lungs and lymph nodes. At last follow-up, three patients had died of disease and one was alive with multiple metastases., Conclusions: SEF is an aggressive sarcoma that can involve the spine. It is important to recognise the spine as the primary location of SEF, in order to avoid misdiagnosis as more common primary spinal neoplasms, which can impact on therapeutic approaches., (© 2021 John Wiley & Sons Ltd.)

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal. MUC4 (mucin4) is a heavily glycosylated protein aberrantly expressed in PDAC and promotes tumorigenesis via an unknown mechanism. To assess this, we genetically knocked out (KO) MUC4 in PDAC cells that did not express and did express truncated O-glycans (Tn/STn) using CRISPR/Cas9 technology. We found that MUC4 knockout cells possess less tumorigenicity in vitro and in vivo, which was further reduced in PDAC cells that express aberrant overexpression of truncated O-glycans. Also, MUC4 KO cells showed a further reduction of epidermal growth factor receptors (ErbB) and their downstream signaling pathways in truncated O-glycan expressing PDAC cells. Tn-MUC4 specific 3B11 antibody inhibited MUC4-induced ErbB receptor and its downstream signaling cascades. MUC4 knockout differentially regulates apoptosis and cell cycle arrest in branched and truncated O-glycan expressing PDAC cells. Additionally, MUC4 KO cells were found to be more sensitive to gemcitabine treatment. They possessed the upregulated expression of hENT1 and hCNT3 compared to parental cells, which were further affected in cells with aberrant O-glycosylation. Taken together, our results indicate that MUC4 enhances the malignant properties and gemcitabine resistance in PDAC tumors that aberrantly overexpress truncated O-glycans via altering ErbB/AKT signaling cascades and expression of nucleoside transporters, respectively., (Copyright © 2021 Elsevier B.V. All rights reserved.)

At present, immunotherapy is widely used for different mismatch repair (dMMR) or highly microsatellite instability (MSI-H) colorectal cancer patients, and tumor mutation burden (TMB) is a valuable independent predictor of response to immunotherapy. However, specific gene mutations and their relationship with TMB and tumor-infiltrating immune cells in colon cancer remains unclear. In the present study, we analyzed somatic mutation data of colon cancer from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) datasets, and found that 17 frequently mutated genes were occurred in both cohorts, including APC, TP53, TNN, KRAS, MUC16, MUC4 (mucin 4), SYNE1, FLG, FAT4, OBSCN, FAT3, RYR2, PIK3CA, FBXW7, DNAH11, MUC5B and ZFHX4. Interestingly, only MUC4 mutation was associated with higher TMB and patient clinical prognosis among the 17 mutated genes. Moreover, according to gene set enrichment analysis (GSEA) and the CIBERSORT algorithm, we revealed that MUC4 mutation activated signaling pathways involved in the immune system and enhanced the antitumor immune response. In conclusion, MUC4 may have important clinical implications for immune therapy of colon cancer.

Several mucins are implicated in idiopathic pulmonary fibrosis (IPF); however, there is no evidence regarding the role of MUC4 in the development of IPF. Here we demonstrated that MUC4 was overexpressed in IPF patients (n = 22) compared with healthy subjects (n = 21) and located in pulmonary arteries, bronchial epithelial cells, fibroblasts, and hyperplastic alveolar type II cells. Decreased expression of MUC4 using siRNA-MUC4 inhibited the mesenchymal/myofibroblast transformations of alveolar type II A549 cells and lung fibroblasts, as well as cell senescence and fibroblast proliferation induced by TGF-β1. The induction of the overexpression of MUC4 increased the effects of TGF-β1 on mesenchymal/myofibroblast transformations and cell senescence. MUC4 overexpression and siRNA-MUC4 gene silencing increased or decreased, respectively, the phosphorylation of TGFβRI and SMAD3, contributing to smad-binding element activation. Immunoprecipitation analysis and confocal immunofluorescence showed the formation of a protein complex between MUC4β/p-TGFβRI and p-SMAD3 in the cell membrane after TGF-β1 stimulation and in lung tissue from IPF patients. Bleomycin-induced lung fibrosis was reduced in mice transiently transfected with siRNA-MUC4. This study shows that MUC4 expression is enhanced in IPF and promotes fibrotic processes in collaboration with TGF-β1 canonical pathway that could be an attractive druggable target for human IPF.

Background: Cholesterol gallstones account for over 80% of gallstones, and the pathogenesis of gallstone formation involves genetic and environmental factors. However, data on the evolution of cholesterol gallstones with various densities are limited. This study aimed to determine the roles of microbiota and mucins on the formation of calcified cholesterol gallstones in patients with cholelithiasis., Methods: Paired gallbladder tissues and bile specimens were obtained from cholelithiasis patients who were categorized into the isodense group and calcified group according to the density of gallstones. The relative abundance of microbiota in gallbladder tissues was detected. Immunohistochemistry and enzyme-linked immunosorbent assay were performed to detect the expression levels of MUC1, MUC2, MUC3a, MUC3b, MUC4, MUC5ac and MUC5b in gallbladder tissues and bile. The correlation of microbiota abundance with MUC4 expression was evaluated by linear regression., Results: A total of 23 patients with gallbladder stones were included. The density of gallstones in the isodense group was significantly lower than that of the calcified group (34.20 ± 1.50 vs. 109.40 ± 3.84 HU, P < 0.0001). Compared to the isodense group, the calcified group showed a higher abundance of gram-positive bacteria at the fundus, in the body and neck of gallbladder tissues. The concentrations of MUC1, MUC2, MUC3a, MUC3b, MUC5ac and MUC5b in the epithelial cells of gallbladder tissues showed no difference between the two groups, while the concentrations of MUC4 were significantly higher in the calcified group than that in the isodense group at the fundus (15.49 ± 0.69 vs. 10.23 ± 0.54 ng/mL, P < 0.05), in the body (14.54 ± 0.94 vs. 11.87 ± 0.85 ng/mL, P < 0.05) as well as in the neck (14.77 ± 1.04 vs. 10.85 ± 0.72 ng/mL, P < 0.05) of gallbladder tissues. Moreover, the abundance of bacteria was positively correlated with the expression of MUC4 (r = 0.569, P < 0.05) in the calcified group., Conclusions: This study showed the potential clinical relevance among biliary microbiota, mucins and calcified gallstones in patients with gallstones. Gram-positive microbiota and MUC4 may be positively associated with the calcification of cholesterol gallstones., (Copyright © 2020. Published by Elsevier B.V.)

Background: Functional disruptions by large germline genomic structural variants in susceptible genes are known risks for cancer. We used deletion structural variants (DSVs) generated from germline whole-genome sequencing (WGS) and DSV immune-related association tumor microenvironment (TME) to predict cancer risk and prognosis., Methods: We investigated the contribution of germline DSVs to cancer susceptibility and prognosis by silicon and causal inference models. DSVs in germline WGS data were generated from the blood samples of 192 cancer and 499 non-cancer subjects. Clinical information, including family cancer history (FCH), was obtained from the National Cheng Kung University Hospital and Taiwan Biobank. Ninety-nine colorectal cancer (CRC) patients had immune response gene expression data. We used joint calling tools and an attention-weighted model to build the cancer risk predictive model and identify DSVs in familial cancer. The survival support vector machine (survival-SVM) was used to select prognostic DSVs., Results: We identified 671 DSVs that could predict cancer risk. The area under the curve (AUC) of the receiver operating characteristic curve (ROC) of the attention-weighted model was 0.71. The 3 most frequent DSV genes observed in cancer patients were identified as ADCY9, AURKAPS1, and RAB3GAP2 (p < 0.05). The DSVs in SGSM2 and LHFPL3 were relevant to colorectal cancer. We found a higher incidence of FCH in cancer patients than in non-cancer subjects (p < 0.05). SMYD3 and NKD2DSV genes were associated with cancer patients with FCH (p < 0.05). We identified 65 immune-associated DSV markers for assessing cancer prognosis (p < 0.05). The functional protein of MUC4 DSV gene interacted with MAGE1 expression, according to the STRING database. The causal inference model showed that deleting the CEP72 DSV gene affect the recurrence-free survival (RFS) of IFIT1 expression., Conclusions: We established an explainable attention-weighted model for cancer risk prediction and used the survival-SVM for prognostic stratification by using germline DSVs and immune gene expression datasets. Comprehensive assessments of germline DSVs can predict the cancer risk and clinical outcome of colon cancer patients.

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.

Genome-wide association studies of gastric cancer (GC) cases have revealed common gastric cancer susceptibility loci with low effect size. We investigated rare variants with high effect size via whole-exome sequencing (WES) of subjects with familial clustering of gastric cancer. WES of DNAs from the blood of 19 gastric cancer patients and 36 unaffected family members from 14 families with two or more gastric cancer patients were tested. Linkage analysis combined with association tests were performed using Pedigree Variant Annotation, Analysis, and Search Tool (pVAAST) software. Based on the logarithm of odds (LOD) and permutation-based composite likelihood ratio test (CLRT) from pVAAST, MUC4 was identified as a predisposing gene (LOD P-value = 1.9×10-5; permutation-based P-value of CLRT ≤ 9.9×10-9). In a larger cohort consisting of 597 GC patients and 9,759 healthy controls genotyped with SNP array, we discovered common variants in MUC4 regions (rs148735556, rs11717039, and rs547775645) significantly associated with GC supporting the association of MUC4 with gastric cancer. And the MUC4 variants were found in higher frequency in The Cancer Genome Atlas Study (TCGA) germline samples of patients with multiple cancer types. Immunohistochemistry indicated that MUC4 was downregulated in the noncancerous gastric mucosa of subjects with MUC4 germline missense variants, suggesting that loss of the protective function of MUC4 predisposes an individual to gastric cancer. Rare variants in MUC4 can be novel gastric cancer susceptibility loci in Koreans possessing the familial clustering of gastric cancer., Competing Interests: The authors have declared that no competing interests exist.

Lymphatic invasion (LI) is an early event of metastasis and closely associated with overall survival in colon adenocarcinoma (COAD). Our aim was to gain deeper insight into the mechanism of lymphatic invasion in COAD. Subtype-specific somatic mutations and differentially expressed genes (DEGs) screening were based on The Cancer Genome Atlas (TCGA). Gene Ontology (GO) enrichment analysis was utilized to explore the biological function. The condition of tumor-infiltrating lymphocytes was performed by TIMER online database. Survival analysis was based on Kaplan-Meier curve method. Lymphatic invasion was associated with poor prognosis of patients with COAD. Nine mutations were enriched in lymphatic invasion-negative group. A total of 50 were differentially expressed between LI-positive tissues and LI-negative tissues. The DEGs were enriched in lipoprotein-related functions. MUC4 in-frame deletion at A4166-S4181 was associated with favorable prognosis of COAD patients. BMPR2 frameshift mutation g.chr2:202555407delA played cis and trans functions in downregulation of itself and CTLA4 upregulation. And it was associated with higher mutational burden. LAMP5, CUBN and TCHH were DEGs associated with prognosis and abundance of tumor-infiltrating lymphocytes. In conclusion, our study provides LI-associated genetic and transcriptional alterations, which helps to better understand the potential mechanisms and microenvironment in COAD., (Copyright © 2020. Published by Elsevier B.V.)

Vitreo-retinal (VR) surgeries induce conjunctival changes. However, there are no study reports regarding prevalence and severity of dry eye after these surgeries. This study evaluated dry eye outcome after VR surgery. Patients undergoing VR surgery classified as scleral buckle and microincision vitrectomy surgery (n = 44, mean age: 56.09±10.2 years) were recruited. Dry eye evaluation was done before and 8 weeks after surgery (2 weeks after omitting topical eye drops). Conjunctival imprint cytology for goblet cell count and tear Mucin 5AC (MUC5AC) protein estimation was done. Gene expressions of MUC5AC, MUC4, MUC16, Aquaporin 4 (AQP4) and AQP5 were analyzed in the conjunctival imprint cells by qPCR. None of the patients exhibited clinical signs of dry eye after VR surgery. But the conjunctival goblet cell density (GCD) was significantly lowered post-VR surgery (63% cases, **p = 0.012) with no alterations in the tear MUC5AC protein. Post-VR surgery, the conjunctival cell gene expression of MUC4, MUC16 and AQP4 were significantly increased (*p = 0.025, *p = 0.05 and *p = 0.02 respectively) and AQP5 was significantly lowered (*p = 0.037), with no change in MUC5AC expression. Tear cytokines were significantly increased post-VR surgery (anti-inflammatory: IL1RA, IL4, IL5, IL9, FGF; PDGFbb and pro-inflammatory: IL2, IL6, IL15, GMCSF and IFNg). Though clinical signs of dry eye were not observed after VR surgery, ocular surface changes in the form of reduced GCD, altered MUC5AC, MUC4, MUC16, AQP4, AQP5 and cytokines are suggestive of dry eye outcome at the molecular level especially inpatients aged above 51 years, especially female gender and those who are diabetic., Competing Interests: The authors have read the journal’s policy and have the following conflicts: Novartis Healthcare Private Limited funded this project. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Mucin 4 (MUC4), a type I membrane-bound mucin, blocks apoptosis, promotes invasion, proliferation and migration and causes chemo-resistance in epithelial cancers. However, the expression profiling and clinical implications of MUC4 alternative splicing during cancer pathogenesis, including melanoma, remain obscure. We examined the mRNA expression profiling of MUC4 isoforms in gastrointestinal cancer cell lines, melanoma cell lines, human epidermal melanocyte cells, as well as 138 cases of human melanoma tissues by RT-qPCR. Then we analyzed the relationship of mRNA expression of MUC4 isoforms to clinicopathological characteristics and survival of patients. The dynamic mRNA expression profiling of MUC4 isoforms was found in melanoma. We identified MUC4 isoform f was highly expressed in melanoma cell lines but negative in gastrointestinal cancer cell lines. Clinical analysis based on 138 cases of human melanomas showed that MUC4 isoform d was related with melanoma subtypes (p = 0.028) and TNM stage (p = 0.036). MUC4 isoform e was related with tumor thickness (p = 0.004) and T stage (p = 0.036). The Kaplan-Meier assay showed that the median overall survival (OS) for patients with MUC4 isoform f high expression was significantly shorter than that of patients with low expression (p = 0.024). And the median PFS of the patients with high expression of MUC4 isoform d or e was significantly shorter than that of with low expression (p = 0.012 and 0.035, respectively). Multivariate analysis indicated that high level of MUC4 isoform f was an independent prognostic factor for OS, and MUC4 isoform d was an independent prognostic factor for PFS of patients treated with chemotherapy. In conclusion, our results indicate that the dynamic MUC4 isoforms expressed in melanoma, and MUC4 isoform d and f might be served as a novel prognostic indicator of melanoma patients.

Pancreatic cancer (PC) is a highly lethal malignancy with a dismal five-year survival rate. This is due to its asymptomatic nature, lack of reliable biomarkers, poor resectability, early metastasis, and high recurrence rate. Limited efficacies of current treatment modalities treatment-associated toxicity underscore the need for the development of immunotherapy-based approaches. For non-resectable, locally advanced metastatic PC, immunotherapy-based approaches including vaccines, antibody-targeted, immune checkpoint inhibition, CAR-T-cells, and adoptive T-cell transfer could be valuable additions to existing treatment modalities. Thus far, the vaccine candidates in PC have demonstrated modest immunological responses in different treatment modalities. The identification of tumor-associated antigens (TAA) and their successful implication in PC treatment is still a challenge. MUC4, a high molecular weight glycoprotein that functionally contributes to PC pathogenesis, is an attractive TAA. It is not detected in the normal pancreas; however, it is overexpressed in mouse and human pancreatic tumors. The recombinant MUC4 domain, as well as predicted immunogenic T-cell epitopes, elicited cellular and humoral anti-MUC4 response, suggesting its ulility as a vaccine candidate for PC therapy. Existence of PC-associated MUC4 splice variants, autoantibodies against overexpressed and aberrantly glycosylated MUC4 and presence of T-cell clones against the mutations present in MUC4 further reinforce its significance as a tumor antigen for vaccine development. Herein, we review the significance of MUC4 as a tumor antigen in PC immunotherapy and discuss both, the development and challenges associated with MUC4 based immunotherapy. Lastly, we will present our perspective on MUC4 antigenicity for the future development of MUC4-based PC immunotherapy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Pituitary stalk interruption syndrome (PSIS) is a rare congenital disease which results in hypopituitarism. Patients with PSIS often exhibit short stature due to their deficiency of growth hormone (GH)., Case Presentation: Here, we present two rare cases of adults males with PSIS, in which the patients were of normal height and did not take any GH supplements. Sterility and multiple hormone deficiencies including GH were seen in both of them. Through whole exome sequencing of their DNA, we found novel mutations they shared, three in MUC4 (c.7815G>T, c.3548C>T, c.3399C>G) and one in NBPF10 (c.536C>A)., Conclusion: The present cases suggest that there are exceptions in GH deficient patients where a select few can attain normal heights without GH therapy. Genetic screening can be a predictor for prognoses of rare types of hypopituitarism.

Substantial heterogeneity exists within cervical cancer that is generally infected by human papillomavirus (HPV). However, the most common histological subtype of cervical cancer, cervical squamous cell carcinoma (CSCC), is poorly characterized regarding the association between its heterogeneity and HPV oncoprotein expression. We filtered out 138 CSCC samples with infection of HPV16 only as the first step; then we compressed HPV16 E6/E7 expression as HPV pca and correlated HPV pca with the immunological profiling of CSCC based on supervised clustering to discover subtypes and to characterize the differences between subgroups in terms of the HPV pca level, pathway activity, epigenetic dysregulation, somatic mutation frequencies, and likelihood of responding to chemo/immunotherapies. Supervised clustering of immune signatures revealed two HPV16 subtypes (namely, HPV16-IMM and HPV16-KRT) that correlated with HPV pca and clinical outcomes. HPV16-KRT is characterized by elevated expression of genes in keratinization, biological oxidation, and Wnt signaling, whereas HPV16-IMM has a strong immune response and mesenchymal features. HPV16-IMM exhibited much more epigenetic silencing and significant mutation at FBXW7, while MUC4 and PIK3CA were mutated frequently for HPV16-KRT. We also imputed that HPV16-IMM is much more sensitive to chemo/immunotherapy than is HPV16-KRT. Our characterization tightly links the expression of HPV16 E6/E7 with biological and clinical outcomes of CSCC, providing valuable molecular-level information that points to decoding heterogeneity. Together, these results shed light on stratifications of CSCC infected by HPV16 and shall help to guide personalized management and treatment of patients., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Tristetraprolin (TTP), a well-characterized AU-rich element (ARE) binding protein, functions as a tumor suppressor gene. The purpose of this study was to investigate whether a bioactive substance derived from a natural medicinal plant affects the induction of TTP and to elucidate its mechanism. We examined the effects of natural bioactive materials including Resveratrol (RSV), thymoquinone (TQ) and curcumin on the expression of TTP in cancer cell. TQ derived from a natural plant Nigella sativa increased the expression levels of TTP mRNA and proteins in a dose-dependent manner in gastric and breast cancer cells. TQ-induced TTP increased the instability of MUC4 mRNA by direct binding of TTP to ARE in the 3'UTR of MUC4 mRNA. The induction of TTP by TQ also reduced the proliferation, migration and invasion of cancer cells. The expression of the epithelial-mesenchymal (EMT)-related genes, which were target genes of TTP, was also decreased by the TQ treatment. In the in vivo experiments using mouse melanoma cells, TQ-induced TTP inhibited metastasis of tumor cells. We have found that TQ-induced TTP might inhibit metastasis by reducing tumor cell migration and invasion through destabilization of MUC4 mRNA, which suggest the MUC4 as a novel target to TTP.

Mucins and their glycosylation have been suggested to play an important role in colorectal carcinogenesis. We examined potentially functional genetic variants in the mucin genes or genes involved in their glycosylation with respect to colorectal cancer (CRC) risk and clinical outcome. We genotyped 23 single nucleotide polymorphisms (SNPs) covering 123 SNPs through pairwise linkage disequilibrium (r2>0.80) in the MUC1, MUC2, MUC4, MUC5AC, MUC6, and B3GNT6 genes in a hospital-based case-control study of 1532 CRC cases and 1108 healthy controls from the Czech Republic. We also analyzed these SNPs in relation to overall survival and event-free survival in a subgroup of 672 patients. Among patients without distant metastasis at the time of diagnosis, two MUC4 SNPs, rs3107764 and rs842225, showed association with overall survival (HR 1.40, 95%CI 1.08-1.82, additive model, log-rank p = 0.004 and HR 0.64, 95%CI 0.42-0.99, recessive model, log-rank p = 0.01, respectively) and event-free survival (HR 1.31, 95%CI 1.03-1.68, log-rank p = 0.004 and HR 0.64, 95%CI 0.42-0.96, log-rank p = 0.006, respectively) after adjustment for age, sex and TNM stage. Our data suggest that genetic variation especially in the transmembrane mucin gene MUC4 may play a role in the survival of CRC and further studies are warranted., Competing Interests: The authors have declared that no competing interests exist.

Irinotecan-induced mucositis is a major oncological problem. Goblet cells secrete mucus, protecting the intestinal mucosa, with secretion altered during mucositis. The enteric nervous system is involved in regulating gut motility and secretion. The aim of this study was to determine whether enteric neural cells and goblet cells are altered following irinotecan treatment. Tumour-bearing Dark Agouti rats were administered a single dose of 175 mg/kg of irinotecan intraperitoneally and 0.01 mg/kg atropine subcutaneously. Experimental and untreated control rats were killed at times 6, 24, 48, 72, 96 and 120 h after treatment. Jejunum and colon samples were formalin fixed. Haematoxylin and eosin staining, Alcian Blue-PAS staining, and immunohistochemistry with S-100 antibody (neural cell marker) were carried out. Statistical analyses were carried out using Kruskal-Wallis test with Dunns post test, Mann Whitney U test, and nonlinear regression. Total goblet cells decreased at 72 h compared with controls in the colon (p < 0.05). The percentage of cavitated goblet cells decreased compared to all other time points at 120 h in the colon. The number of S-100-positive cells in the submucosal plexus decreased in the colon (p = 0.0046) and in the myenteric plexus of the jejunum and colon (p = 0.0058 and p = 0.0022, respectively), on comparing treated with control. Enteric ganglia in the myenteric plexus of the jejunum decreased at 24 h and 96 h. Irinotecan-induced mucositis is associated with increases in mucus secretion and enteric neural cell change. These changes may contribute to the pathophysiology of mucositis through the dysregulation of neural signalling.

Putative genetic markers have been associated with ETEC F4 (Mucine 4 [MUC4]; MUC4 GG;CG as susceptible; MUC4 CC as resistant) and F18 (Fucosyltransferase 1 [FUT1]; FUT1 GG;AG as susceptible; FUT1 AA as resistant) resistances respectively. In this study, 71 post-weaning pigs were followed from d0 (35 days old) to d42 (77 days of age) to investigate the effect of MUC4 or FUT1 genotypes on the mid-jejunal microbiota composition, pigs expression of genes related to inflammation (IL8, GPX2, REG3G, TFF3, CCL20 and LBPI) and glycomic binding pattern profile (Ulex europaeus agglutinin I [UEA] fucose-binding lectin and peanut agglutinin [PNA] galactose-specific), and on blood plasma targeted metabolomics profile, faecal score and performance parameters of growing healthy pigs. The MUC4 and FUT1 resistant genotypes improved the pigs' growth performance and had firmed faecal score susceptible genotypes in d0-d21 period. Pigs with MUC4 GG genotype had a higher jejunal expression of genes relate to immune function (CCL20 and REG3G) than MUC4 CG and MUC4 CC pigs (p < 0.05). MUC4 CG pigs had higher expression of TFF3 (implicated in mucosal integrity) than MUC4 GG and MUC4 CC (p < 0.05). FUT1 influenced the alpha- and beta-jejunal microbial indices. The FUT1 AA group had a higher number of operational taxonomic units (OTUs) belonging to Lactobacillus genus, while FUT1 GG group had a higher number of OTUs belonging to Veillonella genus. MUC4 CC pigs had lower scores for UEA on brush borders and goblet cells in villi than MUC4 GG (p < 0.05). FUT1 AA pigs had lower UEA positivity and higher PNA positivity on brush borders and goblet cells than FUT1 AG and FUT1 GG (p < 0.05). Both FUT1 and MUC4 influenced the metabolic profile of healthy pigs. Results highlight the role of MUC4 and FUT1 on pig intestinal homoeostasis and improved the knowledge regarding the potential interaction between host genetics, gut microbiota composition and host metabolism in a healthy status., (© 2019 Blackwell Verlag GmbH.)

Although type-2-induced (T2-induced) epithelial dysfunction is likely to profoundly alter epithelial differentiation and repair in asthma, the mechanisms for these effects are poorly understood. A role for specific mucins, heavily N-glycosylated epithelial glycoproteins, in orchestrating epithelial cell fate in response to T2 stimuli has not previously been investigated. Levels of a sialylated MUC4β isoform were found to be increased in airway specimens from asthmatic patients in association with T2 inflammation. We hypothesized that IL-13 would increase sialylation of MUC4β, thereby altering its function and that the β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) would regulate the sialylation. Using human biologic specimens and cultured primary human airway epithelial cells (HAECs),we demonstrated that IL-13 increases ST6GAL1-mediated sialylation of MUC4β and that both were increased in asthma, particularly in sputum supernatant and/or fresh isolated HAECs with elevated T2 biomarkers. ST6GAL1-induced sialylation of MUC4β altered its lectin binding and secretion. Both ST6GAL1 and MUC4β inhibited epithelial cell proliferation while promoting goblet cell differentiation. These in vivo and in vitro data provide strong evidence for a critical role for ST6GAL1-induced sialylation of MUC4β in epithelial dysfunction associated with T2-high asthma, thereby identifying specific sialylation pathways as potential targets in asthma.

Background: The transcription factor PR domain containing 16 (PRDM16) is known to play a significant role in the determination and function of brown and beige fat. However, the role of PRDM16 in tumor biology has not been well addressed. Here we investigated the impact of PRDM16 on tumor growth and metastasis in lung cancer., Methods: UALCAN database, immunoblotting and immunohistochemistry analysis were used to assess PRDM16 expression in lung cancer patients. Kaplan-Meier plotter database was used to analyze the overall survival of patients with lung cancer stratified by PRDM16 expression. PRDM16 overexpression and knockdown experiments were conducted to assess the effects of PRDM16 on growth and metastasis in vitro and in vivo, and its molecular mechanism was investigated in lung adenocarcinoma cells by chromatin immunoprecipitation-sequencing (ChIP-Seq), real time-quantitative PCR (RT-qPCR), luciferase assay, xenograft models and rescue experiments., Results: PRDM16 was downregulated in lung adenocarcinomas, and its expression level correlated with key pathological characteristics and prognoses of lung adenocarcinoma patients. Overexpressing PRDM16 inhibited the epithelial-to-mesenchymal transition (EMT) of cancer cells both in vivo and in vitro by repressing the transcription of Mucin-4 (MUC4), one of the regulators of EMT in lung adenocarcinomas. Furthermore, deleting the PR domain from PRDM16 increased the transcriptional repression of MUC4 by exhibiting significant differences in histone modifications on its promoter., Conclusions: Our findings demonstrate a critical interplay between transcriptional and epigenetic modifications during lung adenocarcinoma progression involving EMT of cancer cells and suggest that PRDM16 is a metastasis suppressor and potential therapeutic target for lung adenocarcinomas.

T cells targeting shared oncogenic mutations can induce durable tumor regression in epithelial cancer patients. Such T cells can be detected in tumor infiltrating lymphocytes, but whether such cells can be detected in the peripheral blood of patients with the common metastatic epithelial cancer patients is unknown. Using a highly sensitive in vitro stimulation and cell enrichment of peripheral memory T cells from six metastatic cancer patients, we identified and isolated CD4 + , and CD8 + memory T cells targeting the mutated KRAS G12D and KRAS G12V variants, respectively, in three patients. In an additional two metastatic colon cancer patients, we detected CD8 + neoantigen-specific cells targeting the mutated SMAD5 and MUC4 proteins. Therefore, memory T cells targeting unique as well as shared somatic mutations can be detected in the peripheral blood of epithelial cancer patients and can potentially be used for the development of effective personalized T cell-based cancer immunotherapy across multiple patients.

Pancreatic cancer is a leading cause of mortality and morbidity worldwide. Due to drug resistance, and the high toxicity and adverse side effects of existing chemotherapeutic drugs, the current treatment of highly aggressive pancreatic cancer is considered inadequate. Allergen‑removed Rhus verniciflua Stokes (aRVS) has a strong antiproliferative effect in various cancer cells, and due to its low toxicity, it has emerged as an attractive candidate for cancer treatment. However, the potential use of aRVS as a treatment for pancreatic cancer is relatively unexplored. The present study examined the effects of aRVS on the invasion and migration of pancreatic cancer cells, and identified the molecular mechanisms underlying its anticancer effects. aRVS inhibited the Janus kinase/signal transducer and activator of transcription pathway in pancreatic cancer cells, and decreased the protein expression of mucin 4. In addition, it inhibited the activation of focal adhesion kinase and Src signaling, and decreased the expression of matrix metalloproteinase 9, which may reduce the migration and invasion of pancreatic cancer cells. In conclusion, the present study suggested that aRVS may be a potential treatment for aggressive pancreatic cancer.

Background: MUC4 is a membrane-bound mucin that promotes carcinogenetic progression and is often proposed as a promising biomarker for various carcinomas. In this manuscript, we analyzed large scale genomic datasets in order to evaluate MUC4 expression, identify genes that are correlated with MUC4 and propose new signatures as a prognostic marker of epithelial cancers., Methods: Using cBioportal or SurvExpress tools, we studied MUC4 expression in large-scale genomic public datasets of human cancer (the cancer genome atlas, TCGA) and cancer cell line encyclopedia (CCLE)., Results: We identified 187 co-expressed genes for which the expression is correlated with MUC4 expression. Gene ontology analysis showed they are notably involved in cell adhesion, cell-cell junctions, glycosylation and cell signaling. In addition, we showed that MUC4 expression is correlated with MUC16 and MUC20, two other membrane-bound mucins. We showed that MUC4 expression is associated with a poorer overall survival in TCGA cancers with different localizations including pancreatic cancer, bladder cancer, colon cancer, lung adenocarcinoma, lung squamous adenocarcinoma, skin cancer and stomach cancer. We showed that the combination of MUC4, MUC16 and MUC20 signature is associated with statistically significant reduced overall survival and increased hazard ratio in pancreatic, colon and stomach cancer., Conclusions: Altogether, this study provides the link between (i) MUC4 expression and clinical outcome in cancer and (ii) MUC4 expression and correlated genes involved in cell adhesion, cell-cell junctions, glycosylation and cell signaling. We propose the MUC4/MUC16/MUC20 high signature as a marker of poor prognostic for pancreatic, colon and stomach cancers.

Alternative splicing is evolving as an eminent player of oncogenic signaling for tumor development and progression. Mucin 4 (MUC4), a type I membrane-bound mucin, is differentially expressed in pancreatic cancer (PC) and plays a critical role in its progression and metastasis. However, the molecular implications of MUC4 splice variants during disease pathogenesis remain obscure. The present study delineates the pathological and molecular significance of a unique splice variant of MUC4, MUC4/X, which lacks the largest exon 2, along with exon 3. Exon 2 encodes for the highly glycosylated tandem repeat (TR) domain of MUC4 and its absence creates MUC4/X, which is devoid of TR. Expression analysis from PC clinical samples revealed significant upregulation of MUC4/X in PC tissues with most differential expression in poorly differentiated tumors. In vitro studies suggest that overexpression of MUC4/X in wild-type-MUC4 (WT-MUC4) null PC cell lines markedly enhanced PC cell proliferation, invasion, and adhesion to extracellular matrix (ECM) proteins. Furthermore, MUC4/X overexpression leads to an increase in the tumorigenic potential of PC cells in orthotopic transplantation studies. In line with these findings, doxycycline-induced expression of MUC4/X in an endogenous WT-MUC4 expressing PC cell line (Capan-1) also displayed enhanced cell proliferation, invasion, and adhesion to ECM, compared to WT-MUC4 alone, emphasizing its direct involvement in the aggressive behavior of PC cells. Investigation into the molecular mechanism suggested that MUC4/X facilitated PC tumorigenesis via integrin-β1/FAK/ERK signaling pathway. Overall, these findings revealed the novel role of MUC4/X in promoting and sustaining the oncogenic features of PC., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The CRISPR/Cas9 system has made significant contributions to genome editing, gene regulation and chromatin studies in recent years. High-throughput and systematic investigations into the multiplexed biological systems require simultaneous expression and coordinated functioning of multiple sgRNAs. However, current cotransfection based sgRNA coexpression systems remain inefficient, and virus-based transfection approaches are relatively costly and labor intensive. Here we established a vector-independent method allowing multiple sgRNA expression cassettes to be assembled in series into a single plasmid. This synthetic biology-based strategy excels in its efficiency, controllability and scalability. Taking the flexibility advantage of this all-in-one sgRNA expressing system, we further explored its applications in single nonrepetitive genomic locus imaging as well as coordinated gene regulation in live cells. With its full potency, our method will facilitate the research in understanding genome structure, function and dynamics.