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

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

[Primary mucinous gland lesions of fallopian tube: a clinicopathological analysis of fourteen cases].

Objective: To investigate the clinical and pathological characteristics of primary mucinous gland lesions of the fallopian tubes. Methods: The clinical data, pathomorphological characteristics and immunophenotype of 14 cases of primary mucinous gland lesions of the fallopian tube diagnosed at Obstetrics and Gynecology Hospital of Fudan University from 2015 to 2023 were analyzed retrospectively. In addition, a comprehensive review of relevant literature was conducted. Results: The age of 14 patients ranged from 53 to 83 years, with an average of 65 years. Among them, 13 cases exhibited unilateral involvement while one case showed bilateral presentation. Nine cases were mucinous metaplasia of the fallopian tube, four cases were invasive mucinous adenocarcinoma and one case was mucinous carcinoma in situ. Morphologically, mucinous metaplasia of the fallopian tube was focal, with or without inflammation. The cells of mucinous adenocarcinoma or mucinous carcinoma in situ exhibited characteristics indicative of gastrointestinal differentiation. Immunohistochemical analysis revealed diffuse positive expression of CK7, and negative expression of SATB2. CDX2 demonstrated positive staining in two cases. One case exhibited diffuse and strongly positive mutant expression of p53, whereas the remaining cases displayed wild-type expression. MUC6 showed diffuse or focally positive staining in mucinous gland lesions characterized by gastric differentiation. Some cases of mucinous adenocarcinoma of fallopian tube were subject to AB-PAS staining, resulting in red to purple cytoplasmic staining. Conclusions: Primary mucinous lesions of the fallopian tube are exceedingly uncommon. All cases of mucinous adenocarcinoma of fallopian tubes in this study exhibit the morphology and immunohistochemical characteristics of gastrointestinal differentiation. Mucinous metaplasia of the fallopian tube is a benign lesion of incidental finding, which is closely related to inflammation or gastric differentiation. Mucinous lesions of cervix, ovary and digestive tract are excluded in all patients, confirming the independent existence of mucinous lesions within fallopian tubes.

Possible association of G6PC2 and MUC6 induced by low­dose­rate irradiation in mouse intestine with inflammatory bowel disease.

Although there are several types of radiation exposure, it is debated whether low­dose­rate (LDR) irradiation (IR) affects the body. Since the small intestine is a radiation­sensitive organ, the present study aimed to evaluate how it changes when exposed to LDR IR and identify the genes sensitive to these doses. After undergoing LDR (6.0 mGy/h) γ radiation exposure, intestinal RNA from BALB/c mice was extracted 1 and 24 h later. Mouse whole genome microarrays were used to explore radiation­induced transcriptional alterations. Reverse transcription­quantitative (RT­q) PCR was used to examine time­ and dose­dependent radiation responses. The histopathological status of the jejunum in the radiated mouse was not changed by 10 mGy of LDR IR; however, 23 genes were upregulated in response to LDR IR of the jejunum in mice after 1 and 24 h of exposure. Upregulated genes were selected to validate the results of the RNA sequencing analysis for RT­qPCR detection and results showed that only Na+/K+ transporting subunit α4, glucose­6­phosphatase catalytic subunit 2 (G6PC2), mucin 6 (MUC6) and transient receptor potential cation channel subfamily V member 6 levels significantly increased after 24 h of LDR IR. Furthermore, G6PC2 and MUC6 were notable genes induced by LDR IR exposure according to protein expression via western blot analysis. The mRNA levels of G6PC2 and MUC6 were significantly elevated within 24 h under three conditions: i) Exposure to LDR IR, ii) repeated exposure to LDR IR and iii) exposure to LDR IR in the presence of inflammatory bowel disease. These results could contribute to an improved understanding of immediate radiation reactions and biomarker development to identify radiation­susceptible individuals before histopathological changes become noticeable. However, further investigation into the specific mechanisms involving G6PC2 and MUC6 is required to accomplish this.

Impaired Glycosylation of Gastric Mucins Drives Gastric Tumorigenesis and Serves as a Novel Therapeutic Target.

BACKGROUND & AIMS: Gastric cancer is often accompanied by a loss of mucin 6 (MUC6), but its pathogenic role in gastric carcinogenesis remains unclear. METHODS: Muc6 knockout (Muc6-/-) mice and Muc6-dsRED mice were newly generated. Tff1Cre, Golph3-/-, R26-Golgi-mCherry, Hes1flox/flox, Cosmcflox/flox, and A4gnt-/- mice were also used. Histology, DNA and RNA, proteins, and sugar chains were analyzed by whole-exon DNA sequence, RNA sequence, immunohistochemistry, lectin-binding assays, and liquid chromatography-mass spectrometry analysis. Gastric organoids and cell lines were used for in vitro assays and xenograft experiments. RESULTS: Deletion of Muc6 in mice spontaneously causes pan-gastritis and invasive gastric cancers. Muc6-deficient tumor growth was dependent on mitogen-activated protein kinase activation, mediated by Golgi stress-induced up-regulation of Golgi phosphoprotein 3. Glycomic profiling revealed aberrant expression of mannose-rich N-linked glycans in gastric tumors, detected with banana lectin in association with lack of MUC6 expression. We identified a precursor of clusterin as a binding partner of mannose glycans. Mitogen-activated protein kinase activation, Golgi stress responses, and aberrant mannose expression are found in separate Cosmc- and A4gnt-deficient mouse models that lack normal O-glycosylation. Banana lectin-drug conjugates proved an effective treatment for mannose-rich murine and human gastric cancer. CONCLUSIONS: We propose that Golgi stress responses and aberrant glycans are important drivers of and promising new therapeutic targets for gastric cancer.

Decreased expression of TFF2 and decreased αGlcNAc glycosylation are malignant biomarkers of pyloric gland adenoma of the duodenum.

Pyloric gland adenoma (PGA) is a duodenal neoplasm expressing MUC6 and is often associated with high-grade dysplasia and adenocarcinoma. MUC6 secreted from the pyloric gland cells carries unique O-glycans exhibiting terminal α1,4-linked N-acetylglucosamine residues (αGlcNAc). The small peptide trefoil factor 2 (TFF2) is also secreted from pyloric gland cells and binds to αGlcNAc. We recently demonstrated that αGlcNAc serves as a tumor suppressor for gastric neoplasm including PGA, but the significance of TFF2 expression remains unknown. We examined 20 lesions representing low- and high-grade PGA in 22 cases by immunohistochemistry for αGlcNAc, TFF2, MUC6, MUC5AC, MUC2 and p53. αGlcNAc, TFF2 and MUC6 were co-expressed on the cell surface and a dot-like pattern in the cytosol in low-grade PGA lesions. High-grade PGA also expressed MUC6, but reduced αGlcNAc and TFF2 expression. The ratios of αGlcNAc or TFF2 to MUC6 score in high-grade PGA were significantly lower than low-grade PGA (P < 0.001). Co-expression of αGlcNAc-glycosylated MUC6 and TFF2 in PGA suggests the existence of αGlcNAc/TFF2 form complex in PGA cells, a finding consistent with our observations in non-neoplastic Brunner's gland cells. The decreased αGlcNAc and TFF2 expression are associated with high grade atypical cells, indicative of the malignant potential of PGA.

Evaluation of MUC1, MUC2, MUC5AC, and MUC6 Expression Differences in Lung Adenocarcinoma Subtypes by Using a Final Immunoreactivity Score (FIRS).

OBJECTIVE: Lung adenocarcinomas are divided into acinar, lepidic, papillary, micropapillary, and solid predominant subtypes according to the current World Health Organization (WHO) classification. We designed this retrospective study to demonstrate profiles of MUC expression (MUC1, MUC2, MUC5AC, and MUC6) of different histologic patterns within the same tumor among pulmonary adenocarcinomas and investigate correlations of MUC expression with clinicopathologic features. MATERIAL AND METHOD: We analyzed the expression of mucins (MUC1, MUC2, MUC5AC, and MUC6) in a series of 99 resected lung adenocarcinomas, which included a total of 193 patterns (71 acinar, 30 lepidic, 25 papillary, 20 micropapillary, 34 solid and 13 mucinous) and calculated a final immune reactivity score (FIRS) per tumor. RESULTS: MUC1 IRS scores were significantly higher in lepidic and solid patterns compared with mucinous patterns (p=0.013). MUC2 expression was seen only in three cases (1 acinar, 2 mucinous). MUC5AC and MUC2 expression was more common in mucinous patterns (p < 0.001 and p=0.028, respectively). MUC6 expression was only detected in seven patterns and the expression was weak. No significant difference was seen among histologic patterns for the staining scores of MUC6. Mucinous adenocarcinoma differed from other histologic subtypes regarding MUC1 and MUC5AC expression. Mucinous adenocarcinoma showed less MUC1 expression with lower IRS scores and higher MUC5AC expression. Tumor size (p=0.006), lymphatic invasion (p=0.018), vascular invasion (p=0.025), perineural invasion (p=0.019), MUC1 IRS scores (p=0.018), and MUC1 IRS scores > 8.5 (p=0.018) were significant predictors for lymph node metastasis. CONCLUSION: An alternative scoring for MUC1 can be used as a predictor for lymph node metastasis regardless of the histologic subtype.

α1,4-Linked N-acetylglucosamine suppresses gastric cancer development by inhibiting Mucin-1-mediated signaling.

Gastric cancer is the second leading cause of cancer deaths worldwide, and more understanding of its molecular basis is urgently needed. Gastric gland mucin secreted from pyloric gland cells, mucous neck cells, and cardiac gland cells of the gastric mucosa harbors unique O-glycans carrying terminal α1,4-linked N-acetylglucosamine (αGlcNAc) residues. We previously reported that αGlcNAc loss correlated positively with poor outcomes for patients with differentiated-type gastric cancer. However, the molecular mechanisms underlying these outcomes remained poorly understood. Here, we examined the effects of upregulated αGlcNAc expression on malignant phenotypes of the differentiated-type gastric cancer cell lines, AGS and MKN7. Upregulation of αGlcNAc following ectopic expression of its biosynthetic enzyme attenuated cell proliferation, motility, and invasiveness of AGS and MKN7 cells in vitro. Moreover, AGS cell tumorigenicity was significantly suppressed by αGlcNAc overexpression in a xenograft model. To define the molecular mechanisms underlying these phenotypes, we investigated αGlcNAc binding proteins in AGS cells and identified Mucin-1 (MUC1) and podocalyxin. Both proteins were colocalized with αGlcNAc on human gastric cancer cells. We also found that αGlcNAc was bound to MUC1 in murine normal gastric mucosa. When we assessed the effects of αGlcNAc binding to MUC1, we found that αGlcNAc blocked galectin-3 binding to MUC1, phosphorylation of the MUC1 C-terminus, and recruitment of Src and ß-catenin to that C-terminus. These results suggest that αGlcNAc regulates cancer cell phenotypes by dampening MUC1 signal transduction.

MUC6 expression is a preferable prognostic marker for invasive mucinous adenocarcinoma of the lung.

Gastric gland mucin consists of core protein MUC6 with residues heavily glycosylated by unique O-glycans carrying α1,4-linked N-acetylglucosamine (αGlcNAc). αGlcNAc-glycosylated MUC6 protein is seen in normal gastric and duodenal glands. Decreased αGlcNAc glycosylation on MUC6-positive tumor cells is often observed in premalignant lesions of the stomach, pancreas, and bile duct, and decreased MUC6 expression is seen in invasive cancer of these organs. Lung cancer (LC) is the most common cause of cancer death worldwide. Recently, the adenocarcinoma subtype has become the most common histological subtype of LC, and one of its invasive forms is invasive mucinous adenocarcinoma (IMA). Currently, prognostic markers of LC IMA are unknown. Here, we analyzed MUC5AC, MUC6, and αGlcNAc expression in 54 IMA LC cases. MUC5AC was positively expressed in 50 (93%), MUC6 in 38 (70%), and αGlcNAc in 19 (35%). Each expression level was scored from 0 to 3. The αGlcNAc expression score was significantly decreased relative to MUC6 (P < 0.001). Interestingly, disease-free survival was significantly higher in MUC6-positive than MUC6-negative cases based on the log-rank test (P = 0.021). For in vitro analysis, we ectopically expressed MUC6 in A549 cells, derived from LC and harboring a KRAS mutation. MUC6-expressing A549 cells showed significantly lower proliferation, motility, and invasiveness than control cells. Finally, F-actin staining in MUC6-expressing cells revealed a decrease or loss of filopodia associated with decreased levels of FSCN transcripts, which encodes an actin-bundling protein fascin1 necessary for cell migration. We conclude that MUC6 expression is a preferable prognostic biomarker in IMA LC.

Glycosylation of MUC6 by α1,4-linked N-acetylglucosamine enhances suppression of pancreatic cancer malignancy.

Biomarkers for early diagnosis of pancreatic cancer are greatly needed, as the high fatality of this cancer is in part due to delayed detection. α1,4-linked N-acetylglucosamine (αGlcNAc), a unique O-glycan specific to gastric gland mucus, is biosynthesized by α1,4-N-acetylglucosaminyltransferase (α4GnT) and primarily bound at the terminal glycosylated residue to scaffold protein MUC6. We previously reported that αGlcNAc expression decreases at early stages of neoplastic pancreatic lesions, followed by decreased MUC6 expression, although functional effects of these outcomes were unknown. Here, we ectopically expressed α4GnT, the αGlcNAc biosynthetic enzyme, together with MUC6 in the human pancreatic cancer cell lines MIA PaCa-2 and PANC-1, neither of which expresses α4GnT and MUC6. We observed significantly suppressed proliferation in both lines following coexpression of α4GnT and MUC6. Moreover, cellular motility decreased following MUC6 ectopic expression, an effect enhanced by cotransduction with α4GnT. MUC6 expression also attenuated invasiveness of both lines relative to controls, and this effect was also enhanced by additional α4GnT expression. We found αGlcNAc-bound MUC6 formed a complex with trefoil factor 2. Furthermore, analysis of survival curves of patients with pancreatic ductal adenocarcinoma using a gene expression database showed that samples marked by higher A4GNT or MUC6 mRNA levels were associated with relatively favorable prognosis. These results strongly suggest that αGlcNAc and MUC6 function as tumor suppressors in pancreatic cancer and that decreased expression of both may serve as a biomarker of tumor progression to pancreatic cancer.

Calponin and MUC6 complement inhibin as diagnostic immunomarkers of serous cystadenoma in endoscopic ultrasound-guided aspiration/biopsy specimens.

AIMS: Because serous cystadenoma (SCA) does not usually require excision, it is critical to distinguish it from differential diagnoses which do, especially neuroendocrine tumour (NET). The gold standard for diagnosing SCA is assessment of endoscopic ultrasound-guided fine needle aspiration/biopsy (EUS-FNAB) material. Inhibin immunohistochemistry aids this assessment, but such positivity is not absolutely sensitive or specific to SCA. The following is the largest known study of SCA EUS-FNAB specimens and the first to compare four potential SCA immunomarkers between themselves and inhibin, compared against NET. METHODS AND RESULTS: Immunohistochemistry for calponin, mucin 6 (MUC6), glucose transporter 1 (GLUT1) and vascular endothelial growth factor A (VEGFA) was performed on 30 EUS-FNAB and three resection specimens of SCA and 32 EUS-FNAB specimens of NET. GLUT1 and VEGFA were suboptimal as diagnostic immunomarkers of SCA, being expressed by 10 and 44% of NETs, respectively. Further, their expression by cellular constituents of blood which often contaminate EUS-FNAB specimens hampered identification of neoplastic cells, especially in hypocellular samples. While 19% of NETs showed nuclear MUC6 positivity, cytoplasmic expression of the protein showed 100% specificity and sensitivity as an SCA marker. However, assessing MUC6 in EUS-FNAB specimens must also consider the protein's focal expression in physiological pancreatic, gastric or duodenal tissues, which can contaminate these specimens. Calponin was less sensitive (71% versus 100%) but more specific (100% versus 91%) than inhibin, although easier to assess in EUS-FNAB specimens than MUC6. CONCLUSIONS: Of the four potential immunomarkers of SCA suggested by the existing literature, calponin and MUC6 are useful complementary studies to inhibin for application to EUS-FNAB specimens.

Linking bacterial enterotoxins and alpha defensin 5 expansion in the Crohn's colitis: A new insight into the etiopathogenetic and differentiation triggers driving colonic inflammatory bowel disease.

Evidence link bacterial enterotoxins to apparent crypt-cell like cells (CCLCs), and Alpha Defensin 5 (DEFA5) expansion in the colonic mucosa of Crohn's colitis disease (CC) patients. These areas of ectopic ileal metaplasia, positive for Paneth cell (PC) markers are consistent with diagnosis of CC. Retrospectively, we: 1. Identified 21 patients with indeterminate colitis (IC) between 2000-2007 and were reevaluation their final clinical diagnosis in 2014 after a followed-up for mean 8.7±3.7 (range, 4-14) years. Their initial biopsies were analyzed by DEFA5 bioassay. 2. Differentiated ulcer-associated cell lineage (UACL) analysis by immunohistochemistry (IHC) of the CC patients, stained for Mucin 6 (MUC6) and DEFA5. 3. Treated human immortalized colonic epithelial cells (NCM460) and colonoids with pure DEFA5 on the secretion of signatures after 24hr. The control colonoids were not treated. 4. Treated colonoids with/without enterotoxins for 14 days and the spent medium were collected and determined by quantitative expression of DEFA5, CCLCs and other biologic signatures. The experiments were repeated twice. Three statistical methods were used: (i) Univariate analysis; (ii) LASSO; and (iii) Elastic net. DEFA5 bioassay discriminated CC and ulcerative colitis (UC) in a cohort of IC patients with accuracy. A fit logistic model with group CC and UC as the outcome and the DEFA5 as independent variable differentiator with a positive predictive value of 96 percent. IHC staining of CC for MUC6 and DEFA5 stained in different locations indicating that DEFA5 is not co-expressed in UACL and is therefore NOT the genesis of CC, rather a secretagogue for specific signature(s) that underlie the distinct crypt pathobiology of CC. Notably, we observed expansion of signatures after DEFA5 treatment on NCM460 and colonoids cells expressed at different times, intervals, and intensity. These factors are key stem cell niche regulators leading to DEFA5 secreting CCLCs differentiation 'the colonic ectopy ileal metaplasia formation' conspicuously of pathogenic importance in CC.

Decreased alpha-1,4-linked N-acetylglucosamine glycosylation in biliary tract cancer progression from biliary intraepithelial neoplasia to invasive adenocarcinoma.

Biliary tract cancer (BTC) is typically lethal due to the difficulty of early stage diagnosis. Thus, novel biomarkers of BTC precursors are necessary. Biliary intraepithelial neoplasia (BilIN) is a major precursor of BTC and is classified as low or high grade based on cell atypia. In normal gastric mucosa, gastric gland mucin-specific O-glycans are unique in having α1,4-linked N-acetylglucosamine (αGlcNAc) attached to MUC6. Previously, we reported that αGlcNAc functions as a tumor suppressor of differentiated-type gastric adenocarcinoma and that decreased αGlcNAc glycosylation on MUC6 in gastric, pancreatic, and uterine cervical neoplasms occurs in cancer as well as in their precursor lesions. However, αGlcNAc and MUC6 expression patterns in biliary tract neoplasms have remained unclear. Here, we analyzed MUC5AC, MUC6, and αGlcNAc expression status in 51 BTC cases and compared the expression of each with progression from low-grade BilIN to invasive adenocarcinoma (IAC). The frequency of αGlcNAc-positive and MUC6-positive lesions decreased with tumor progression. When we compared each marker's expression level with tumor progression, we found that the MUC6 expression score in IAC was significantly lower than in low-grade or high-grade BilIN (P < 0.001 or P < 0.01, respectively). However, the αGlcNAc expression score was low irrespective of histological grade, and also lower than that of MUC6 across all histological grades (P < 0.001 for low-grade and high-grade BilIN, and P < 0.01 for IAC). These results suggest that decreased expression of αGlcNAc relative to MUC6 marks the initiation of BTC progression.

Tepotinib Inhibits the Epithelial-Mesenchymal Transition and Tumor Growth of Gastric Cancers by Increasing GSK3ß, E-Cadherin, and Mucin 5AC and 6 Levels.

Aberrant expression of mucins (MUCs) can promote the epithelial-mesenchymal transition (EMT), which leads to enhanced tumorigenesis. Carcinogenesis-related pathways involving c-MET and ß-catenin are associated with MUCs. In this study, we characterized the expression of EMT-relevant proteins including MET, ß-catenin, and E-cadherin in human gastric cancer (GC) cell lines, and further characterized the differential susceptibility of these cell lines compared with the c-MET inhibitor tepotinib. We assessed the antitumor activity of tepotinib in GC cell lines. The effects of tepotinib on cell viability, apoptotic cell death, EMT, and c-MET and ß-catenin signaling were evaluated by 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium (MTS), flow cytometry, Western blotting, and qRT-PCR. The antitumor efficacy was assessed in MKN45 xenograft mice. Tepotinib treatment induced apoptosis in c-MET-amplified SNU620, MKN45, and KATO III cells, but had no effect on c-MET-reduced MKN28 or AGS cells. Tepotinib treatment also significantly reduced the protein levels of phosphorylated and total c-MET, phosphorylated and total ERK, ß-catenin, and c-MYC in SNU620 and MKN45 cells. In contrast, this drug was only slightly active against KATO III cells. Notably, tepotinib significantly reduced the expression of EMT-promoting genes such as MMP7, COX-2, WNT1, MUC5B, and c-MYC in c-MET-amplified GC cells and increased the expression of EMT-suppressing genes such as MUC5AC, MUC6, GSK3ß, and E-cadherin. In a mouse model, tepotinib exhibited good antitumor growth activity along with increased E-cadherin and decreased phosphorylated c-MET (phospho-c-MET) protein levels. Collectively, these results suggest that tepotinib suppresses tumor growth and migration by negatively regulating c-MET-induced EMT. These findings provide new insights into the mechanism by which MUC5AC and MUC6 contribute to GC progression.

Diffuse MIST1 expression and decreased α1,4-linked N-acetylglucosamine (αGlcNAc) glycosylation on MUC6 are distinct hallmarks for gastric neoplasms showing oxyntic gland differentiation.

AIMS: Gastric neoplasms showing oxyntic gland differentiation (GAOGs) constitute a gastric neoplasm subtype that shows low atypia, thus similar to non-neoplastic gastric oxyntic glands. Therefore, their diagnosis in biopsy specimens is difficult. GAOGs were first described in 2007, and introduced in the latest World Health Organization classification book as gastric adenocarcinoma of the fundic gland type (GA-FG) and oxyntic gland adenoma. Previously, we assessed α1,4-linked N-acetylglucosamine (αGlcNAc) residues attached to the MUC6 scaffold in gastric neoplasms, and observed decreased αGlcNAc glycosylation in both differentiated-type gastric cancer and high-grade pyloric gland adenoma (PGA), a gastric cancer precursor. GA-FG and PGA often harbour the same mutations. However, the αGlcNAc status in GAOGs remained unknown. To elucidate αGlcNAc expression in GAOGs, we performed the study. METHODS AND RESULTS: We assessed the expression of αGlcNAc; the mucin markers MUC6, MUC5AC, and MUC2; the gastric gland cell markers MIST1, pepsinogen 1 (PG1), H/K-ATPase and chromogranin-A (CGA); and the proliferation marker Ki67 in 13 GAOG lesions. All 13 (100%) were MUC6-positive, whereas 10 (76.2%) were αGlcNAc-negative. Moreover, all 13 (100%) were MIST1- and PG1-positive, three (23.1%) were MUC5AC-positive, four (30.8%) were H/K-ATPase-positive, and one (7.7%) was CGA-positive. CONCLUSIONS: GAOGs frequently lost αGlcNAc residues on MUC6, but expressed the gastric gland progenitor marker MIST1 and aberrantly expressed various types of gastric gland cell lineage marker, suggestive of immature differentiation to gastric gland cells. Thus, diffuse MIST1 positivity and decreased αGlcNAc glycosylation on MUC6-positive cells could serve as important biomarkers for the histopathological diagnosis of GAOG.

The Tumor Suppressor TFF1 Occurs in Different Forms and Interacts with Multiple Partners in the Human Gastric Mucus Barrier: Indications for Diverse Protective Functions.

TFF1 is a protective peptide of the Trefoil Factor Family (TFF), which is co-secreted with the mucin MUC5AC, gastrokine 2 (GKN2), and IgG Fc binding protein (FCGBP) from gastric surface mucous cells. Tff1-deficient mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas, indicating that Tff1 is a tumor suppressor. As a hallmark, TFF1 contains seven cysteine residues with three disulfide bonds stabilizing the conserved TFF domain. Here, we systematically investigated the molecular forms of TFF1 in the human gastric mucosa. TFF1 mainly occurs in an unusual monomeric form, but also as a homodimer. Furthermore, minor amounts of TFF1 form heterodimers with GKN2, FCGBP, and an unknown partner protein, respectively. TFF1 also binds to the mucin MUC6 in vitro, as shown by overlay assays with synthetic 125I-labeled TFF1 homodimer. The dominant presence of a monomeric form with a free thiol group at Cys-58 is in agreement with previous studies in Xenopus laevis and mouse. Cys-58 is likely highly reactive due to flanking acid residues (PPEEEC58EF) and might act as a scavenger for extracellular reactive oxygen/nitrogen species protecting the gastric mucosa from damage by oxidative stress, e.g., H2O2 generated by dual oxidase (DUOX).

High-fat Diet Alters the Glycosylation Patterns of Duodenal Mucins in a Murine Model.

High-fat diet (HFD) alters the glycosylation patterns of intestinal mucins leading to several health problems. We studied by histochemical and lectin-binding methods mucin alterations in the duodenum of mice fed a HFD for 25 weeks. Histochemical methods included periodic acid-Schiff, alcian blue pH 2.5, and high-iron diamine. Lectin-binding experiments were performed with SBA, PNA, WGA, MAA-II, SNA, ConA, UEA-I, LTA, and AAA. SBA, PNA, WGA, MAA-II, and SNA were tested also after desulfation and ConA after periodate-sodium borohydrate treatments (paradoxical ConA). Duodenal mucins are secreted by Brunner's glands and goblet cells in the villi. Brunner's glands of HFD mice showed increased secreting activity and a general reduction of glycosylated residuals, such as fucose and terminal α1,4-linked GlcNAc. Moreover, a general reduction of glycosylated residuals in the goblet cells of villi such as the fucosylated and sulfated ones was observed. Since the cited residuals are involved in cytoprotective and cytostatic functions, as well as in interactions with the intestinal microbiota and protection against parasites and inflammatory disorders, we conclude that HFD can predispose duodenum to several possible health disorders.

Subcellular Localization of the TFF Peptides xP1 and xP4 in the Xenopus laevis Gastric/Esophageal Mucosa: Different Secretion Modes Reflecting Diverse Protective Functions.

The TFF peptides xP1 and xP4 from Xenopus laevis are orthologs of TFF1 and TFF2, respectively. xP1 is secreted as a monomer from gastric surface mucous cells and is generally not associated with mucins, whereas xP4 is a typical secretory peptide from esophageal goblet cells, and gastric mucous neck and antral gland cells tightly associated as a lectin with the ortholog of mucin MUC6. Both TFF peptides have diverse protective functions, xP1 as a scavenger for reactive oxygen species preventing oxidative damage and xP4 as a constituent of the water-insoluble adherent inner mucus barrier. Here, we present localization studies using immunofluorescence and immunoelectron microscopy. xP1 is concentrated in dense cores of secretory granules of surface mucous cells, whereas xP4 mixes with MUC6 in esophageal goblet cells. Of note, we observe two different types of goblet cells, which differ in their xP4 synthesis, and this is even visible morphologically at the electron microscopic level. xP4-negative granules are recognized by their halo, which is probably the result of shrinkage during the processing of samples for electron microscopy. Probably, the tight lectin binding of xP4 and MUC6 creates a crosslinked mucous network forming a stabile granule matrix, which prevents shrinkage.