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.

Analysis of MUC6 polymorphisms on the clinicopathologic characteristics of Asian patients with oral squamous cell carcinoma.

Head and neck squamous cell carcinomas (HNSCCs) are generally associated with tobacco consumption, alcohol abuse or both. Mucins (MUCs) are high-molecular-weight glycoproteins produced by many epithelial tissues. Many studies have indicated that MUCs play an important role in cancer metastasis. MUC6 expression has been observed in gastric and oncocytic phenotypes and plays an important role during cancer progression. We found that levels of MUC6 are lower in Asian HNCC patients and affect the disease-free survival of HNCC patients. Next, we investigated the combined effect of MUC6 polymorphisms and exposure to environmental carcinogens on the susceptibility to and clinicopathological characteristics of HNCC. Three single-nucleotide polymorphisms (SNPs) of MUC6 (rs7481521, rs6597947 and rs61869016) were analysed using real-time PCR. After adjusting for other co-variants, we found that carrying a CC genotype at MUC6 rs6597947 led to a lower risk of developing oral squamous cell carcinoma (OSCC) than wild-type carriers among non-betel-quid chewers. Moreover, male oral cancer patients who carried the AA + CC genotype at MUC6 rs6597947 had a lower risk of lymph node metastasis than other genotypes, suggesting a significant functional compromise and decompensated disease. Therefore, our findings suggest that genetic variations in MUC6 may correlate to OSCC and indicate the progression in OSCC patients.

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.

Molecular Alterations in Gastric Preneoplastic Lesions and Early Gastric Cancer.

Prognosis of gastric cancer is dramatically improved by early diagnosis. Correa's cascade correlates the expression of some molecular markers with the progression of preneoplastic lesions toward carcinoma. This article reviews the diagnostic and prognostic values of molecular markers in complete (MUC2) and incomplete (MUC2, MUC5AC, and MUC6) intestinal metaplasia, gastric dysplasia/intra-epithelial neoplasia, and early gastric cancer. In particular, considering preinvasive neoplasia and early gastric cancer, some studies have demonstrated a correlation between molecular alterations and prognosis, for example, mucins phenotype in gastric dysplasia, and GATA6, TP53 mutation/LOH and MUC6 in early gastric cancer. Moreover, this review considers novelties from the literature regarding the (immuno)histochemical characterization of diffuse-type/signet ring cell gastric cancer, with particular attention to clinical outcomes of patients. The aim of this review is the evaluation of the state of the art regarding suitable biomarkers used in the pre-surgical phase, which can distinguish patients with different prognoses and help decide the best therapeutic strategy.

Searching the Evolutionary Origin of Epithelial Mucus Protein Components-Mucins and FCGBP.

The gel-forming mucins are large glycosylated proteins that are essential components of the mucus layers covering epithelial cells. Using novel methods of identifying mucins based on profile hidden Markov models, we have found a large number of such proteins in Metazoa, aiding in their classification and allowing evolutionary studies. Most vertebrates have 5-6 gel-forming mucin genes and the genomic arrangement of these genes is well conserved throughout vertebrates. An exception is the frog Xenopus tropicalis with an expanded repertoire of at least 26 mucins of this type. Furthermore, we found that the ovomucin protein, originally identified in chicken, is characteristic of reptiles, birds, and amphibians. Muc6 is absent in teleost fish, but we now show that it is present in animals such as ghost sharks, demonstrating an early origin in vertebrate evolution. Public RNA-Seq data were analyzed with respect to mucins in zebrafish, frog, and chicken, thus allowing comparison in regard of tissue and developmental specificity. Analyses of invertebrate proteins reveal that gel-forming-mucin type of proteins is widely distributed also in this group. Their presence in Cnidaria, Porifera, and in Ctenophora (comb jellies) shows that these proteins were present early in metazoan evolution. Finally, we examined the evolution of the FCGBP protein, abundant in mucus and related to gel-forming mucins in terms of structure and localization. We demonstrate that FCGBP, ubiquitous in vertebrates, has a conserved N-terminal domain. Interestingly, this domain is also present as an N-terminal sequence in a number of bacterial proteins.

Human trefoil factor 2 is a lectin that binds α-GlcNAc-capped mucin glycans with antibiotic activity against Helicobacter pylori.

Helicobacter pylori infection is the major cause of gastric cancer and remains an important health care challenge. The trefoil factor peptides are a family of small highly conserved proteins that are claimed to play essential roles in cytoprotection and epithelial repair within the gastrointestinal tract. H. pylori colocalizes with MUC5AC at the gastric surface epithelium, but not with MUC6 secreted in concert with TFF2 by deep gastric glands. Both components of the gastric gland secretome associate non-covalently and show increased expression upon H. pylori infection. Although blood group active O-glycans of the Lewis-type form the basis of H. pylori adhesion to the surface mucin layer and to epithelial cells, α1,4-GlcNAc-capped O-glycans on gastric mucins were proposed to inhibit H. pylori growth as a natural antibiotic. We show here that the gastric glycoform of TFF2 is a calcium-independent lectin, which binds with high specificity to O-linked α1,4-GlcNAc-capped hexasaccharides on human and porcine stomach mucin. The structural assignments of two hexasaccharide isomers and the binding active glycotope were based on mass spectrometry, linkage analysis, (1)H nuclear magnetic resonance spectroscopy, glycan inhibition, and lectin competition of TFF2-mucin binding. Neoglycolipids derived from the C3/C6-linked branches of the two isomers revealed highly specific TFF2 binding to the 6-linked trisaccharide in GlcNAcα1-4Galß1-4GlcNAcß1-6(Fucα1-2Galß1-3)GalNAc-ol(Structure 1). Supposedly, lectin TFF2 is involved in protection of gastric epithelia via a functional relationship to defense against H. pylori launched by antibiotic α1,4-GlcNAc-capped mucin glycans. Lectin-carbohydrate interaction may have also an impact on more general functional aspects of TFF members by mediating their binding to cell signaling receptors.

[Continual self-renewal of the gastric epithelium by cell differentiation: implications for carcinogenesis]. / Kontinuierliche Regeneration des Magenepithels durch Zelldifferenzierung : Bedeutung für die Karzinogenese.

BACKGROUND: The gastric mucosa and its glands represent a close interactive barrier to the outside world. This delicate surface is protected by a multilayered mucus barrier which contains among others the mucins MUC5AC and MUC6 and the trefoil factor family peptide TFF2. Furthermore, two types of gastric glands form delicate homeostatic systems, i.e. the fundic and antral glands, which show continual bidirectional self-renewal via differentiation from stem and progenitor cells. It was the aim of this study to analyze the self-renewal of these gastric units. MATERIAL AND METHODS: Three characteristic regions (i.e. foveolar, proliferative zone and lower gland regions) were isolated from fundic and antral units by the use of laser microdissection and expression profiles concerning known marker genes were generated by reverse transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: The surface mucous cells (SMCs) of fundic and antral units characteristically differed in the expression of certain secretory genes. Furthermore, the maturation of mucous neck cells and their trans-differentiation into chief cells as well as the maturation of antral SMCs and antral gland cells occurred in a stepwise manner. DISCUSSION: The correct maturation particularly of mucous neck cells and their trans-differentiation into chief cells is critical for homeostatic self-renewal of fundic units. Dysregulation of this multistep process can result in generation of the spasmolytic polypeptide-expressing metaplasia (SPEM) lineage which is characterized by its strong ectopic TFF2 expression. Chronic inflammation is known to support SPEM formation. The SPEM lineage is a precancerous lesion which can further differentiate into intestinal metaplasia.

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.

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.

Quantitative MUC5AC and MUC6 mucin estimations in gastric mucus by a least-squares minimization method.

We have determined the molar proportions of the MUC5AC and MUC6 mucus glycoproteins (mucins) in mucus from the normal and pathological human gastric antrum using a least-squares minimization analysis applied to amino acid compositions. We noted that the content of MUC5AC mucin in mucus from individuals without gastroduodenal disease was very high, suggesting that the integrity and barrier properties of the adherent gastric mucus layer are normally maintained by building-block structures formed from this mucin alone. We observed that the molar content of MUC6 mucin doubled (without significance) in mucus from patients with duodenal ulcer, and increased five times (with high significance) in mucus from patients with gastric ulcer, when compared with that in mucus from individuals without gastroduodenal disease.

Association of MUC6-minisatellite variants with susceptibility to rectal carcinoma.

A secreted MUC6 mucin is reported to be expressed highly in the stomach and gall bladder. In previous our study, the five minisatellites were identified and a significant association between MUC6-MS5 alleles and gastric cancer was reported. Because of aberrant MUC6 expression is often found in gastrointestinal diseases, we evaluated a relationship between MUC6-MS5 and susceptibility to colorectal cancers. Case-control study was performed with 1,103 cancer-free controls and 414 rectal cancer cases. A significant association (OR = 2.70) between short rare MUC6-MS5 alleles (7, 9 repeats) and the occurrence of cancer was observed in rectal cancer [95 % confidence interval (CI), 1.12-6.54; p = 0.022]. Furthermore, a comparison by gender showed the differences in the association ratios between rectal cancer and short rare MUC6-MS5 alleles: male, 3.97 (CI: 1.36-11.5; p = 0.006) versus female 0.91 (CI: 0.18-4.75; p = 0.913). We also examined the association according to lymphovascular invasion (LVI). The frequency of LVI positive rectal cancer was increased in short rare allele cases than in the total rectal cases: 16.2 % versus 42.9 %. Therefore, we suggest that the short rare MUC6-MS5 alleles may be related to cancer development in male and these cancer cases may be related the bad prognosis.

Mucin-microbiome signatures shape the tumor microenvironment in gastric cancer.

BACKGROUND AND AIMS: We aimed to identify mucin-microbiome signatures shaping the tumor microenvironment in gastric adenocarcinomas and clinical outcomes. METHODS: We performed high-throughput profiling of the mucin phenotypes present in 108 gastric adenocarcinomas and 20 functional dyspepsia cases using validated mucin-based RT-qPCRs with subsequent immunohistochemistry validation and correlated the data with clinical outcome parameters. The gastric microbiota was assessed by 16S rRNA gene sequencing, taxonomy, and community composition determined, microbial networks analyzed, and the metagenome inferred in association with mucin phenotypes and expression. RESULTS: Gastric adenocarcinomas with an intestinal mucin environment or high-level MUC13 expression are associated with poor survival. On the contrary, gastric MUC5AC or MUC6 abundance was associated with a more favorable outcome. The oral taxa Neisseria, Prevotella, and Veillonella had centralities in tumors with intestinal and mixed phenotypes and were associated with MUC13 overexpression, highlighting their role as potential drivers in MUC13 signaling in GC. Furthermore, dense bacterial networks were observed in intestinal and mixed mucin phenotype tumors whereas the lowest community complexity was shown in null mucin phenotype tumors due to higher Helicobacter abundance resulting in a more decreased diversity. Enrichment of oral or intestinal microbes was mucin phenotype dependent. More specifically, intestinal mucin phenotype tumors favored the establishment of pro-inflammatory oral taxa forming strong co-occurrence networks. CONCLUSIONS: Our results emphasize key roles for mucins in gastric cancer prognosis and shaping microbial networks in the tumor microenvironment. Specifically, the enriched oral taxa associated with aberrant MUC13 expression can be potential biomarkers in predicting disease outcomes. Video Abstract.

Tn glycosylation of the MUC6 protein modulates its immunogenicity and promotes the induction of Th17-biased T cell responses.

The Tn antigen (α-GalNAc-O-Ser/Thr) is one of the most specific human cancer-associated structures. This antigen, together with mucins, the major carriers of O-glycosylated tumor antigens in adenocarcinomas, are being evaluated as anti-cancer immunotherapeutic targets. In particular, the MUC6 protein, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. To develop anti-cancer vaccines based on the Tn antigen, we produced MUC6 proteins with different Tn density by using mixtures of recombinant ppGalNAc-T1, -T2, and -T7. The obtained glycoproteins were characterized and analyzed for their immunological properties, as compared with the non-glycosylated MUC6. We show that these various MUC6:Tn glycoproteins were well recognized by both MUC6 and Tn-specific antibodies. However, Tn glycosylation of the MUC6 protein strongly affected their immunogenicity by partially abrogating Th1 cell responses, and promoting IL-17 responses. Moreover, the non-glycosylated MUC6 was more efficiently presented than MUC6:Tn glycoproteins to specific T CD4(+) hybridomas, suggesting that Tn glycosylation may affect MUC6 processing or MHC binding of the processed peptides. In conclusion, our results indicate that Tn glycosylation of the MUC6 protein strongly affects its B and T cell immunogenicity, and might favor immune escape of tumor cells.

N-3 polyunsaturated fatty acid attenuates cholesterol gallstones by suppressing mucin production with a high cholesterol diet in mice.

BACKGROUND AND AIM: The increasing prevalence of cholesterol gallstone (CG) disease has become an economic burden to the healthcare system. Ursodeoxycholic acid (UDCA) is the only established medical agent used to dissolve gallstones. In investigating novel therapeutics for CG, we assessed the preventive effects of n-3 polyunsaturated fatty acids (n-3PUFA) on the formation of CG induced by feeding a lithogenic diet (LD) containing high cholesterol levels to mice. METHODS: Mice were divided into the following six groups: (A) regular diet (RD); (B) RD+n-3PUFA; (C) LD; (D) LD+n-3PUFA; (E) LD+UDCA; (F) LD+n-3PUFA+UDCA. After RD/LD feeding for 2 weeks, n-3PUFA or UDCA was administered orally and the diet maintained for 8 weeks. The levels of phospholipids and cholesterol in bile, CG formation, gallbladder wall thickness, MUC gene expression in gallbladder were analyzed. RESULTS: No stone or sludge was evident in the RD groups (Groups A, B). Mice in the n-3PUFA treatment (Groups D, F) showed significantly lower stone formation than the other LD groups (Groups C, E). The combination treatment of n-3PUFA and UDCA suppressed stone formation more than mono-therapy with n-3PUFA or UDCA. Bile phospholipid levels were significantly elevated in the Group F. Hypertrophy of the gallbladder wall was evident in mice fed LD. MUC 2, 5AC, 5B and 6 mRNA expression levels were significantly elevated in the LD-fed group, and this was suppressed by n-3PUFA with or without UDCA. CONCLUSIONS: N-3PUFA attenuated gallstone formation in mouse, through increasing the levels of bile phospholipids and suppressing bile mucin formation.

MUC6 mucin expression inhibits tumor cell invasion.

The MUC6 mucin has a critical protective function in the normal stomach, pancreas and duodenum and is aberrantly expressed during the progression of some gastrointestinal cancers. Our aim was to determine whether MUC6 contributes to the etiology or progression of pancreatic cancer and elucidate the molecular basis of its involvement. Expression of MUC6 glycoprotein was examined in pancreatic cancer tissues by immunofluorescence and loss of MUC6 was observed. Next, to determine whether MUC6 inhibits tumor growth and metastasis by altering cell adhesion and invasion, recombinant MUC6 cDNA and separate MUC6 N-terminal and C-terminal domains were transfected into pancreatic, colorectal and breast cancer cell lines. The recombinant N- and C-terminal proteins were each seen to oligomerize under non-reducing conditions. Overexpression of both domains of the MUC6 glycoprotein significantly inhibited cell adhesion to matrix proteins (collagen I, collagen IV, fibronectin and laminin) in LS 180 but not in PANC-1 cells. Moreover, the N- and C-terminal domains of MUC6 inhibited invasion of both LS 180 and PANC-1 cells by 40% and 70%, respectively, in comparison with controls. These results suggest that MUC6 may inhibit invasion of tumor cells through the basement membrane of the pancreatic duct and slow the development of infiltrating carcinoma.

Requirement of the epithelium-specific Ets transcription factor Spdef for mucous gland cell function in the gastric antrum.

Mucus-secreting cells of the stomach epithelium provide a protective barrier against damage that might result from bacterial colonization or other stimuli. Impaired barrier function contributes to chronic inflammation and cancer. Knock-out mice for the epithelium-specific transcription factor Spdef (also called Pdef) have defects in terminal differentiation of intestinal and bronchial secretory cells. We sought to determine the physiologic function of Spdef in the stomach, another site of significant levels of Spdef expression. We used in situ hybridization and immunohistochemistry to localize Spdef-expressing cells in the mouse stomach; targeted gene disruption to generate mice lacking Spdef; and histologic, immunologic, and transcriptional profiling approaches to determine the requirements of Spdef in stomach epithelial homeostasis. In wild-type mice, Spdef RNA and protein are expressed predominantly in mucous gland cells of the antrum and in mucous neck cells of the glandular corpus. Within 1.5 years, nearly half of homozygous mutant mice developed profound mucosal hyperplasia of the gastric antrum. Submucosal infiltration of inflammatory cells preceded antral hyperplasia by several weeks. The absence of Spdef impaired terminal maturation of antral mucous gland cells, as reflected in reduced expression of Muc6 and Tff2 and reduced numbers of secretory granules. Antral gene expression abnormalities overlapped significantly with those in Spdef(-/-) colon, including genes implicated in secretory granule traffic and functions. Spdef is required for terminal maturation of antral mucous gland cells to protect animals from gastric inflammation and resulting hyperplasia. These requirements parallel Spdef functions in secretory intestinal cells and suggest a common molecular mechanism for maturation of gastrointestinal secretory lineages.

Early weaning accelerates the differentiation of mucous neck cells in rat gastric mucosa: possible role of TGFalpha/EGFR.

The development of the gastric mucosa is controlled by hormones, growth factors and feeding behavior. Early weaning (EW), which means the abrupt interruption of suckling, increases proliferation and differentiation in the rat gastric epithelium. Transforming growth factor alpha (TGFalpha) is secreted in the stomach, binds to the epidermal growth factor receptor (EGFR) and may control cell proliferation, differentiation and migration. Here, we investigated the influence of suckling-weaning transition on the differentiation of mucous neck cells in the stomach and its association to the expression of TGFalpha and EGFR. Fifteen-day-old Wistar rats were divided into two groups: suckling (control), in which pups were kept with the dam, and early weaning (EW), in which rats were separated from their mother and fed with hydrated powdered chow. TGFalpha and EGFR levels were increased at 18 days in EW animals compared to control ones (p<0.05). Histochemical reactions with Periodic Acid-Schiff reagent+Alcian Blue or Bandeiraea simplicifolia II lectin were used to stain the mucous neck cells and showed an increase in this cell population throughout EW, which was more pronounced at 17 days when compared to suckling pups (p<0.05). These morphological results were confirmed by RT-PCR for mucin 6. The levels of mucin 6 mRNA were higher in EW animals from the 16th to the 18th day (1-3 days post-weaning) when compared to the respective control group. Inhibition of EGFR through AG1478 administration to EW animals prevented the expansion of mucous neck cell population induced by EW (p<0.05). Therefore, early weaning up regulated TGFalpha/EGFR expression and induced differentiation of mucous neck cells. Moreover, we showed that EGFR takes part in the maturation of this cell population. We conclude that regular suckling-weaning transition is crucial to guarantee the development of the gastric mucosa.

Low Grade Papillary Sinonasal (Schneiderian) Carcinoma: A Series of Five Cases of a Unique Malignant Neoplasm with Comparison to Inverted Papilloma and Conventional Nonkeratinizing Squamous Cell Carcinoma.

There have been a few case reports and one small series of low grade papillary sinonasal (Schneiderian) carcinomas (LGPSC) which mimic papillomas but have overtly invasive growth and which occasionally metastasize. We describe the morphologic, clinical, immunohistochemical, and molecular features of five patients with LGPSC compared with eight cases each of inverted papilloma (IP) and conventional nonkeratinizing squamous cell carcinoma (SCC) with papillary growth. All LGPSC were nested with predominantly pushing invasion, no stromal reaction, and frequent surface papillary growth. All consisted of one cell type only, with polygonal cells with round nuclei, no (or limited) cytologic atypia, low mitotic activity, and prominent neutrophilic infiltrate. One patient had slightly more infiltrative bone invasion, another lymphovascular, perineural, and skeletal muscle invasion, and a third nodal metastasis after 17 years. By comparison, IPs had bland cytology, neutrophilic microabscesses, mixed immature squamous, goblet cell, and respiratory epithelium, and extremely low mitotic activity. Nonkeratinizing SCCs had basaloid-appearing cells with nuclear pleomorphism, brisk mitotic activity, and apoptosis. All LGPSC were p63 positive. Mitotic activity and Ki67 indices were significantly higher for LGPSCs than IPs and significantly lower than NKSCCs, while p53 immunohistochemistry in LGPSC was identical to nonkeratinizing SCC and higher than for IP. Sequencing showed all five tumors to harbor a MUC6 mutation, one tumor to harbor CDKN2A and PIK3R1 mutations, and one tumor to harbor a NOTCH1 mutation. All LGPSC lacked EGFR and KRAS mutations and lacked copy number variations of any main cancer genes. At a median follow up of 12 months, two LGPSC recurred locally, and one patient died after massive local recurrences and nodal metastases. LGPSC is a distinct, de novo sinonasal carcinoma that can be differentiated from papillomas by morphology and selected immunohistochemistry.

GWAS of peptic ulcer disease implicates Helicobacter pylori infection, other gastrointestinal disorders and depression.

Genetic factors are recognized to contribute to peptic ulcer disease (PUD) and other gastrointestinal diseases, such as gastro-oesophageal reflux disease (GORD), irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Here, genome-wide association study (GWAS) analyses based on 456,327 UK Biobank (UKB) individuals identify 8 independent and significant loci for PUD at, or near, genes MUC1, MUC6, FUT2, PSCA, ABO, CDX2, GAST and CCKBR. There are previously established roles in susceptibility to Helicobacter pylori infection, response to counteract infection-related damage, gastric acid secretion or gastrointestinal motility for these genes. Only two associations have been previously reported for duodenal ulcer, here replicated trans-ancestrally. The results highlight the role of host genetic susceptibility to infection. Post-GWAS analyses for PUD, GORD, IBS and IBD add insights into relationships between these gastrointestinal diseases and their relationships with depression, a commonly comorbid disorder.

Short rare MUC6 minisatellites-5 alleles influence susceptibility to gastric carcinoma by regulating gene.

The human MUC6 gene, which is reported to be expressed in the stomach and gall bladder, is clustered on chromosome 11p15.5 with other secreted mucins. In this study, the genomic structure of MUC6 has been analyzed and five VNTR (minisatellites; MS1-MS5) were identified. These minisatellites were analyzed in genomic DNA extracted from 1,103 controls, 470 gastric cancer patients, and multigenerational families. Five novel minisatellites were found to be polymorphic and transmitted through meiosis by Mendelian inheritance in families. We evaluated allelic variation in these minisatellites to determine if such variation affected the susceptibility to gastric cancer. A significant association (odds ratio [OR]=7.08) between short rare MUC6-MS5 alleles and relative risks were observed for gastric cancer (95% confidence interval [CI], 1.43-35.19; P=0.005). To investigate the function of minisatellite alleles of MUC6-MS5, we examined the effects on gene expression from luciferase reporters when inserted with minisatellites. Interestingly, when the shortest allele (7TR) was inserted in the promoter, the expression level decreased over 20-fold (P<0.001) in normal and cancer cell lines. Furthermore, the cancer-specific rare allele (TR8) also showed decreased expression levels in cancer cells. Therefore, we suggest that the short rare MUC6-MS5 alleles may be related to cancer development by the regulation of MUC6 expression.