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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.

Histologic variant (HV) subtypes of bladder cancer are clinically aggressive tumors that are more resistant to standard therapy compared to conventional urothelial carcinoma (UC). Little is known about the transcriptional programs that account for their biological differences. Here we show using single cell analysis that HVs harbor a tumor cell state characterized by expression of MUC16 (CA125), MUC4 , and KRT24 . This cell state is enriched in metastases, predicted to be highly resistant to chemotherapy, and linked with poor survival. We also find enriched expression of TM4SF1 , a transmembrane protein, in HV tumor cells. Chimeric antigen receptor (CAR) T cells engineered against TM4SF1 protein demonstrated in vitro and in vivo activity against bladder cancer cell lines in a TM4SF1 expression-dependent manner, highlighting its potential as a therapeutic target. Single cell analysis of histologic variant bladder tumors detects a shared CA125+ tumor cell state associated with aggressive clinical features and reveals enriched expression of TM4SF1, a membrane protein that can be targeted with CAR T cells.

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.

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. Chronic jet lag induces dry eye: MUC4 deficiency revealed Dry eye disease, a long-term issue causing discomfort and vision problems, impacts millions globally. In this research, scientists studied how disturbances in our internal clock contribute to DED. Researchers made the mice experience an 8-hour shift in their day-night cycle every 3 days, imitating chronic jet lag. The findings showed that chronic jet lag resulted in a significant decrease in MUC4 expression in the cornea, leading to DED symptoms. Supplementing with MUC4 or treating the mice with melatonin, eased these symptoms. This indicates that disruptions to our internal clock can directly affect eye health by impacting key protective proteins in the eye. Researchers conclude that maintaining a healthy internal clock is vital for eye health and that treatments targeting internal clock disruptions could help DED patients. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

Tubulointerstitial fibrosis (TIF) is the common final outcome of almost all progressive chronic kidney diseases (CKDs). Hypoxia-inducible factor-1 (HIF-1) activation plays an essential role in CKD; however, the specific contribution of tubular HIF-1 to renal TIF remains unclear. In the current study, the mechanism underlying HIF-1-mediated TIF was investigated. Intriguingly, we found that specifically knocking out HIF-1α in tubular cells attenuated unilateral ureteral obstruction-induced TIF, as shown by decreased accumulation of the extracellular matrix (ECM). Then, the results of transcriptomic analysis and and experiments revealed that expression of Mucin 4 (MUC4) was significantly decreased at the transcriptional and protein levels in tubular HIF-1α-knockout mice. Mechanistically, we demonstrated that HIF-1α directly bound to the promoter and enhanced its transcription, as shown by chromatin immunoprecipitation and luciferase reporter assays. Moreover, RNA interference-mediated suppression of MUC4 significantly attenuated transforming growth factor-β induced ECM production by mouse tubular epithelial cells. Furthermore, MUC4 overexpression in the context of HIF-1α knockout was positively correlated with increased ECM protein expression and TIF. Altogether, these results demonstrate that tubular HIF-1α promotes TIF by upregulating transcription. Our findings provide novel insights into the molecular mechanism underlying HIF-1-mediated TIF.

Mucin-4 (Muc4) is a large cell surface glycoprotein implicated in the protection and lubrication of epithelial structures. Previous studies suggest that aberrantly expressed Muc4 can influence the adhesiveness, proliferation, viability and invasiveness of cultured tumor cells, as well as the growth rate and metastatic efficiency of xenografted tumors. Although it has been suggested that one of the major mechanisms by which Muc4 potentiates tumor progression is via its engagement of the ErbB2/HER2 receptor tyrosine kinase, other mechanisms exist and remain to be delineated. Moreover, the requirement for endogenous Muc4 for tumor growth progression has not been previously explored in the context of gene ablation. To assess the contribution of endogenous Muc4 to mammary tumor growth properties, we first created a genetically engineered mouse line lacking functional Muc4 ( Muc4 ko ), and then crossed these animals with the NDL (Neu DeLetion mutant) model of ErbB2-induced mammary tumorigenesis. We observed that Muc4 ko animals are fertile and develop normally, and adult mice exhibit no overt tissue abnormalities. In tumor studies, we observed that although some markers of tumor growth such as vascularity and cyclin D1 expression are suppressed, primary mammary tumors from Muc4 ko /NDL female mice exhibit similar latencies and growth rates as Muc4 wt /NDL animals. However, the presence of lung metastases is markedly suppressed in Muc4 ko /NDL mice. Interestingly, histological analysis of lung lesions from Muc4 ko /NDL mice revealed a reduced association of disseminated cells with platelets and white blood cells. Moreover, isolated cells derived from Muc4 ko /NDL tumors interact with fewer blood cells when injected directly into the vasculature or diluted into blood from wild type mice. We further observed that blood cells more efficiently promote the viability of non-adherent Muc4 wt /NDL cells than Muc4 ko /NDL cells. Together, our observations suggest that Muc4 may facilitate metastasis by promoting the association of circulating tumor cells with blood cells to augment tumor cell survival in circulation.

We have previously reported on a novel monoclonal antibody (mAb) we designated F5, which was raised against a glycopeptide derived from the tandem repeat (TR) region of Mucin-4 (MUC4), a heavily O-glycosylated protein that is overexpressed in many pancreatic cancer cells. This mAb was highly specific for the MUC4 glycopeptide antigen in glycan microarrays, ELISA and SPR assays, selectively stained tissue derived from advanced-stage tumors, and bound MUC4+ tumor cells in flow cytometry assays. The mAb was also unique in that it did not cross-react with other commercial anti-MUC4 mAbs that were raised in a similar but non-glycosylated TR sequence. Here we describe the selective conjugation of a novel near-infrared dye to this mAb and in vivo biodistribution of this labeled mAb to various MUC4-expressing tumors in mice. The labeled mAb were selectively distributed to both cell-derived xenograft (CDX) flank tumors and patient-derived xenograft (PDX) tumors that expressed MUC4 compared to those that were MUC4-negative. Organ distribution analysis showed high uptake in MUC4+ relative to MUC4− tumors. These results suggest that mAb F5 may be used to develop MUC4-targeted, passive antibody-based immunotherapies against Pancreatic Ductal Adenocarcinomas (PDACs) which are notorious for being refractory to many chemo- and radiotherapies

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.

Pancreatic cancer (PC) and associated pre-neoplastic lesions have been reported to be hypoxic, primarily due to hypovascular nature of PC. Though the presence of hypoxia under cancerous condition has been associated with the overexpression of oncogenic proteins (MUC1), multiple emerging reports have also indicated the growth inhibitory effects of hypoxia. In spite of being recognized as the top-most differentially expressed and established oncogenic protein in PC, MUC4 regulation in terms of micro-environmental stress has not been determined. Herein, for the first time, we are reporting that MUC4 protein stability is drastically affected in PC, under hypoxic condition in a hypoxia inducible factor 1α (HIF-1α)-independent manner. Mechanistically, we have demonstrated that hypoxia-mediated induction of reactive oxygen species (ROS) promotes autophagy by inhibiting pAkt/mTORC1 pathway, one of the central regulators of autophagy. Immunohistofluorescence analyses revealed significant negative correlation ( P- value=0.017) between 8-hydroxy guanosine (8-OHG) and MUC4 in primary pancreatic tumors ( n =25). Moreover, we found pronounced colocalization between MUC4 and LAMP1/LC3 (microtubule-associated protein 1A/1B-light chain 3) in PC tissues and also observed their negative relationship in their expression pattern, suggesting that areas with high autophagy rate had less MUC4 expression. We also found that hypoxia and ROS have negative impact on overall cell growth and viability, which was partially, though significantly ( P <0.05), rescued in the presence of MUC4. Altogether, hypoxia-mediated oxidative stress induces autophagy in PC, leading to the MUC4 degradation to enhance survival, possibly by offering required metabolites to stressed cells.

The differential diagnosis of epithelioid mesothelioma from lung adenocarcinoma and squamous cell carcinoma requires the positive and negative immunohistochemical markers of mesothelioma. The IMIG guideline has suggested the use of Calretinin, D2–40, WT1, and CK5/6 as mesothelial markers, TTF-1, Napsin-A, Claudin 4, CEA as lung adenocarcinoma markers p40, p63, CK5/6, MOC-31 as squamous cell markers. However, use of other immunohistochemical markers is still necessary. We evaluated 65 epithelioid mesotheliomas, 60 adenocarcinomas, and 57 squamous cell carcinomas of the lung for MUC4 expression by immunohistochemistry and compared with the previously known immunohistochemical markers. MUC4 expression was not found in any of 65 cases of epithelioid mesothelioma. In contrast, MUC4 expression was observed in 50/60(83.3%) cases of lung adenocarcinoma and 50/56(89.3%) cases of lung squamous cell carcinoma. The negative MUC4 expression showed 100% sensitivity, 86.2% specificity and accuracy rate of 91.2% to differentiate epithelioid mesothelioma from lung carcinoma. The sensitivity, specificity, and accuracy of MUC4 are comparable to that of previously known markers of lung adenocarcinoma and squamous cell carcinoma, namely CEA, Claudin 4 and better than that of MOC-31. In conclusion, MUC4 immunohistochemistry is useful for differentiation of epithelioid mesothelioma from lung carcinoma, either adenocarcinoma or squamous cell carcinoma.