Search

International audience; The serrated neoplasia pathway accounts for 20–30% of colorectal cancers (CRC), which are characterized by extensive methylation (CpG island methylation phenotype, CIMP), frequent BRAF mutation and high microsatellite instability (MSI). We recently identified MUC5AC mucin gene hypomethylation as a specific marker of MSI CRC. The early identification of preneoplastic lesions among serrated polyps is currently challenging. Here, we performed a detailed pathological and molecular analysis of a large series of colorectal serrated polyps and evaluated the usefulness of mucin genes MUC2 and MUC5AC to differentiate serrated polyps and to identify lesions with malignant potential. A series of 330 colorectal polyps including 218 serrated polyps [42 goblet cell‐rich hyperplastic polyps (GCHP), 68 microvesicular hyperplastic polyps (MVHP), 100 sessile serrated adenoma (SSA) and eight traditional serrated adenoma (TSA)] and 112 conventional adenomas was analyzed for BRAF/KRAS mutations, MSI, CIMP, MLH1 and MGMT methylation, and MUC2 and MUC5AC expression and methylation. We show that MUC5AC hypomethylation is an early event in the serrated neoplasia pathway, and specifically detects MVHP and SSA, arguing for a filiation between MVHP, SSA and CIMP‐H/MSI CRC, whereas GCHP and TSA arise from a distinct pathway. Moreover, MUC5AC hypomethylation specifically identified serrated lesions with BRAF mutation, CIMP‐H or MSI, suggesting that it may be useful to identify serrated neoplasia pathway‐related precursor lesions. Our data suggest that MVHP should be recognized among HP and require particular attention.

International audience; During human embryonic and fetal development of the gastrointestinal tract, the gene encoding the MUC5AC mucin has a spatio-temporal pattern of expression restricted to the stomach. In order to better understand the molecular mechanisms responsible for this restricted pattern of expression, we have studied Muc5ac expression in the developing stomach of the mouse and correlated it to that of transcription factors known to be involved in cell differentiation programs during development. Our results indicate that GATA-6 and HNF-4α expression increased concomitantly with the induction of Muc5ac expression in embryonic stomach. We then studied Muc5ac transcriptional regulation by these transcription factors and showed that they all transactivate Muc5ac promoter. We also identified several active GATA-4/-5/-6 and HNF-1/-4 cis-elements using gel shift assays, chromatin immunoprecipitation and site-directed mutagenesis. Among all Muc5ac regulators, only GATA-6 and HNF-4a expression was concomitant to that of Muc5ac in the developing stomach. This is thus in favor of an important role for these two transcription factors as regulators of expression of the Muc5ac mucin during stomach development and in epithelial cancer cells.

International audience; MUC5B is one of the major mucin genes expressed in the respiratory tract. Previous studies in our laboratory have demonstrated that MUC5B is expressed in human lung adenocarcinomas and during lung morphogenesis. Moreover, in human lung adenocarcinoma tissues, a converse correlation between MUC5B and thyroid transcription factor-1 (TTF-1) expression, a lung-specific transcription factor, has been established. However, the molecular mechanisms that govern the regulation of MUC5B expression in the lung are largely unknown. In order to better understand the biological role of MUC5B in lung pathophysiology, we report the characterization of the promoter region of the mouse Muc5b mucin gene. The promoter is flanked by a TATA box (TACATAA) identical to that in the human gene. Human and murine promoters share 67.5% similarity over the first 170 nucleotides. By RT-PCR, co-transfection studies and gel-shift assays, we show that Muc5b promoter activity is completely inhibited by TTF-1, whereas factors of the GATA family (GATA-4/GATA-5/GATA-6) are activators. Together, these results demonstrate, for the first time, that Muc5b is a target gene of transcription factors (TTF-1, GATA-6) involved in lung differentiation programs during development and carcinogenesis, and identify TTF-1 as a strong repressor of Muc5b. The characterization of the structural and functional features of the Muc5b mucin gene will provide us with a strong base to develop studies in murine models aimed at the identification of its biological role in lung pathophysiology.

International audience; Background. In esophageal adenocarcinoma, MUC1 mucin expression increases in early stages of the carcinogenetic sequence, during which bile reflux has been identified as a major carcinogen. However, no link between MUC1 overexpression and the presence of bile acids in the reflux has been established so far, and molecular mechanisms regulating MUC1 expression during esophageal carcinogenetic sequence are unknown. Our aim was to identify (1) the bile acids able to upregulate MUC1 expression in esophageal cancer cells and mucosal samples, (2) the regulatory regions in MUC1 promoter responsive to bile acids, and (3) the signaling pathway(s) involved in this regulation. Methods. MUC1 mRNA and mucin expression were studied by the means of real-time reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry, both in the human esophageal OE33 adenocarcinoma cell line and in an ex vivo explant model. MUC1 promoter was cloned and transcription regulation was studied by transient cell transfection to identify the bile acid-responsive regions. Signaling pathways involved were identified using specific pharmacologic inhibitors and siRNA approach. Results. Taurocholic, taurodeoxycholic, taurochenodeoxycholic, glycocholic, sodium glycocholate, and deoxycholic bile acids upregulated MUC1 mRNA and protein expression. The highest induction was obtained with deoxycholic and taurocholic acids in both cellular and explant models. The bile acid-mediated upregulation of MUC1 transcription occurs at the promoter level, with responsive elements located in the-1472/-234 region of the promoter, and involves the phosphatidylinositol 3-kinase signaling pathway. Conclusions. Bile acids induce MUC1 mucin overexpression in human esophageal adenocarcinoma cells and tissues by activating its transcription through a process involving phosphatidylinositol 3-kinase. (Surgery 2008;143:58-71.)

International audience; Membrane-bound mucins belong to an ever-increasing family of O-glycopro-teins that share a structure conserved throughout evolution. Typically, membrane-bound mu-cins contain a long extracellular domain, a hydrophobic transmembrane domain, and a short cytoplasmic tail. They are modular proteins and have a structural organization containing Pro/ Thr/Ser-rich O-glycosylated domains and EGF-like domains. The biological roles of mucins arise from their structures. MUC1 and MUC4 modulate biological properties of the cell, alter its behavior and modulate cell signaling pathways associated with tumorigenesis. Altered expression and post-translational modifi cations confer an important role to MUC1 and MUC4 in tumor progression, metastasis, and cancer cell chimioresistance. Moreover, increasing knowledge about their animal counterparts has made possible a greater understanding of their pathophysiological role in vivo. Most biological functions attributed to MUC4 are based on the structural homology with its rat homologue. From these results, the development of new biological tools targeting mucins has been increasing and the recent attention given to these complex molecules may bring hope for improved cancer treatments in the future. This review discusses the structure/function of MUC1 and MUC4 membrane-bound mucins in relation to cancer cell behavior and cell signaling pathways associated with tumorigenesis, as well as their potential as biological tools for gene therapy and immunotherapy approaches.

Mucin production and secretion by specialized epithelial cells is a common mechanism used by mammals to protect the underlying mucosae against various injuries (pollutants, pathogens, pH). The expression of mucin genes is cell- and tissue-specific but is submitted to variations during cell differentiation, inflammatory process, and is altered during carcinogenesis. The molecular mechanisms responsible for the control of mucin transcription and expression are beginning to be understood as mucin gene promoters and regulatory regions are characterized. The four gel-forming mucin genes, MUC2-MUC5AC-MUC5B-MUC6, are clustered on the p15 arm of chromosome 11. Common regulatory mechanisms (PKA, PKC, PKG and Ca2+ signaling, Sp1/Sp3) may account for the capability of mucous-secreting cells to express several mucin genes simultaneously. In response to an insult or during carcinogenesis, the normal pattern of expression is altered and results from specific answers of the cell by activating different intracellular signaling pathways. 11p15 mucin genes are regulated at the transcriptional level by pro-inflammatory cytokines (IL-1beta, IL-6, TNF-alpha), pleiotropic cytokines (IL-4, IL-13, IL-9), bacterial exoproduct (LPS), growth factors (EGF, TGF-alpha), lipid mediator (PAF), retinoids and hormones. To date, the only downstream cascade known to activate mucin gene transcription is the Src/Ras/MAPK/pp90rsk cascade, which leads to the activation of the transcription factor NF-kappaB. Mucin gene transcription is also regulated by ATF-1, CREB and RAR-alpha transcription factors. Finally, repression of mucin transcription in cancer cells is under the control of the epigenetic mechanism of methylation. As transcriptional regulation of mucin genes begins to be unraveled, it becomes clear that many signaling pathways are involved. Our understanding of mucin gene transcriptional regulation, which awaits more data (identification of the signaling cascades and active cis-elements within promoters and introns), will most certainly lead to the use of mucin genes as molecular markers in cancer and molecular tools in human gene therapy, and to the synthesis of new therapeutic agents in inflammatory diseases of the epithelium.

International audience; Background Helicobacter pylori (Hp), which is one of the causative agents in human gastric adenocarcinoma, is known to interact with mucous gel and alter mucin gene expression. The aim of this work was to study, using an in vitro model of cell infection, the effects of urease, flagellin, and CagA vir-ulence factors on the regulation of the four 11p15 mucin genes (MUC2, MUC5AC, MUC5B, and MUC6). Methods KATO-III and AGS gastric cancer cells were infected for 1, 3 or 6 h with Hp wild-type strains (ATCC 43504, N6, and SS1) or corresponding isogenic mutants deficient for urease subunit B, flagellin subunit A, and CagA. mRNA levels of MUC2, MUC5B, MUC5AC and MUC6 were assessed by RT-PCR, and functional activity of their promoters was measured by transient transfection assays. Results Infection of KATO-III cells with Hp wild-type strains resulted in an early (at 1 h) transient expression of MUC2, MUC5AC, and MUC6 mRNA concomitant with those of interleukin (IL)-1b, IL-8, and TNF-a cytokines. In these cells, the UreB-isogenic mutant induced strong activation of MUC5AC expression, and UreB-responsive elements were located in the-486/-1 region of the promoter. FlaA-and CagA-mutants had no effect on mucin gene mRNA levels in KATO-III cells. In AGS cells, Hp-responsive elements were identified in all promoters, and overexpression of NF-jB induced upregulation of MUC5AC promoter activity when infected with the UreB-isogenic mutant. Conclusion These results indicate that Hp infection of gastric cancer cells alters 11p15 mucin gene transcription and that MUC5AC downregulation is mediated by urease virulence factor.

Mucin 16 (cancer antigen 125) is a cell surface glycoprotein that plays a role in promoting cancer cell growth in ovarian cancer. The aims of this study were to examine mucin 16 expression in a large number of digestive tract adenocarcinomas and precursors and to determine whether mucin 16 up-regulation is correlated with patient outcome. Tissue microarrays were constructed using surgical resection tissues and included pancreatic (115 normal, 29 precursors, 200 pancreatic ductal adenocarcinomas), esophageal (86 normal, 104 precursors, 95 esophageal adenocarcinomas, 35 lymph node metastases), gastric (211 normal, 8 precursors, 119 gastric adenocarcinomas, 62 lymph node metastases), and colorectal (34 normal, 17 precursors, 39 colorectal adenocarcinomas) tissues. Mucin 16 was detected in 81.5%, 69.9%, 41.2%, and 64.1% of the pancreatic ductal adenocarcinomas, esophageal adenocarcinomas, gastric adenocarcinomas, and colorectal adenocarcinomas, respectively. Mucin 16 was seen in a subset of the precursors. On multivariate analysis, moderate/diffuse mucin 16 in pancreatic ductal adenocarcinomas was strongly associated with poor survival (P < .001), independent of other prognosis predictors. A similar trend was observed for esophageal adenocarcinomas (P = .160) and gastric adenocarcinomas (P = .080). Focal mucin 16 in colorectal adenocarcinomas was significantly correlated (P = .044) with a better patient outcome, when compared with mucin 16–negative cases. Using Western blot analysis, we found mucin 16 expression in 3 of 6 pancreatic ductal adenocarcinoma and 1 of 2 esophageal adenocarcinoma cell lines. We conclude that most of the digestive tract adenocarcinomas and a subset of their precursors express mucin 16. Mucin 16 expression is an independent predictor of poor outcome in pancreatic ductal adenocarcinomas and potentially in esophageal adenocarcinomas and gastric adenocarcinomas. We propose that mucin 16 may function as a prognostic marker and therapeutic target in the future.

International audience; Aims: To study the expression of MUC1 and MUC4 mucins in Barrett-associated oesophageal adenocarcinoma and coexisting lesions of the carcinogenic sequence (normal mucosa, metaplasia, dysplasia) if present, and to investigate their prognostic significance.Methods: The expression profiles of MUC1 and MUC4 were investigated by immunohistochemistry in tissue samples obtained from consecutive patients with primary surgically resected lower third oesophageal adenocarcinoma (OA) between 1997 and 2002. Histopathological parameters, recurrence and long-term survival were correlated with the number of cells stained.Results: All 52 patients exhibited OA, with 25 patients (48.1%) having associated Barrett oesophagus lesions (metaplasia or/and dysplasia). MUC1 and MUC4 were expressed in 52 and 41 of the 52 patients with adenocarcinoma (100% and 78%), respectively. All samples expressed MUC1 strongly. The prevalence of MUC4 staining was significantly decreased in metaplasia compared with normal mucosa (53% versus 92%, p

The gastric mucosal levels of the pro-inflammatory cytokine Interleukin 6 (IL-6) have been reported to be increased in Helicobacter pylori-infected subjects and, in gastric adenocarcinomas, the up-regulation of intestinal mucin genes (MUC2 and MUC4) has been detected. To analyse the regulatory effects of IL-6 on the activation of intestinal mucins, six gastric cancer cell lines were treated for different times with several concentrations of IL-6, and the expression of MUC2 and MUC4 was evaluated. IL-6 induced MUC4 expression, detected by quantitative RT-PCR, Western blot and immunofluorescence, and MUC2 expression was not affected. MUC4 mRNA levels decreased after blocking the gp130/STAT3 pathway at the level of the receptor, and at the level of STAT3 activation using the AG490 specific inhibitor. MUC4 presents two putative binding sites for STAT factors that may regulate MUC4 transcription after a pro-inflammatory stimulus as IL-6. By EMSA, ChIP and site-directed mutagenesis we show that STAT3 binds to a cis-element at −123/−115, that conveys IL-6 mediated up-regulation of MUC4 transcriptional activity. We also demonstrated that p-STAT3 binds to MUC4 promoter and a three-fold increase in p-STAT3 binding was observed after treating GP220 cells with IL-6.In conclusion, IL-6 treatment induced MUC4 expression through the gp130/STAT3 pathway, indicating the direct role of IL-6 on the activation of the intestinal mucin gene MUC4 in gastric cancer cells.

International audience; Abnormal gastro-oesophageal reflux and bile acids have been linked to the presence of Barrett's oesophageal premalignant lesion associated with an increase in mucin-producing goblet cells and MUC4 mucin gene overexpression. However, the molecular mechanisms underlying the regulation of MUC4 by bile acids are unknown. Since total bile is a complex mixture, we undertook to identify which bile acids are responsible for MUC4 up-regulation by using a wide panel of bile acids and their conjugates. MUC4 apomucin expression was studied by immunohistochemistry both in patient biopsies and OE33 oesophageal cancer cell line. MUC4 mRNA levels and promoter regulation were studied by reverse transcriptase-PCR and transient transfection assays respectively. We show that among the bile acids tested, taurocholic, tauro-deoxycholic, taurochenodeoxycholic and glycocholic acids and sodium glycocholate are strong activators of MUC4 expression and that this regulation occurs at the transcriptional level. By using specific pharmacological inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase A and protein kinase C, we demonstrate that bile acid-mediated up-regulation of MUC4 is promoter-specific and mainly involves activation of phosphatidylinositol 3-kinase. This new mechanism of regulation of MUC4 mucin gene points out an important role for bile acids as key molecules in targeting MUC4 overexpression in early stages of oesophageal carcinogenesis.

International audience; Abnormal gastro-oesophageal reflux and bile acids have been linked to the presence of Barrett's oesophageal premalignant lesion associated with an increase in mucin-producing goblet cells and MUC4 mucin gene overexpression. However, the molecular mechanisms underlying the regulation of MUC4 by bile acids are unknown. Since total bile is a complex mixture, we undertook to identify which bile acids are responsible for MUC4 up-regulation by using a wide panel of bile acids and their conjugates. MUC4 apomucin expression was studied by immunohistochemistry both in patient biopsies and OE33 oesophageal cancer cell line. MUC4 mRNA levels and promoter regulation were studied by reverse transcriptase-PCR and transient transfection assays respectively. We show that among the bile acids tested, taurocholic, tauro-deoxycholic, taurochenodeoxycholic and glycocholic acids and sodium glycocholate are strong activators of MUC4 expression and that this regulation occurs at the transcriptional level. By using specific pharmacological inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase A and protein kinase C, we demonstrate that bile acid-mediated up-regulation of MUC4 is promoter-specific and mainly involves activation of phosphatidylinositol 3-kinase. This new mechanism of regulation of MUC4 mucin gene points out an important role for bile acids as key molecules in targeting MUC4 overexpression in early stages of oesophageal carcinogenesis.

The nucleotide sequence of the pMS1 clone was submitted to the GenBank Nucleotide Sequence Database under accession number {"type":"entrez-nucleotide","attrs":{"text":"AF288076","term_id":"33339735","term_text":"AF288076"}}AF288076. Changes in the expression of mucin genes in gastrointestinal cancers is thought to contribute to the development of the disease. In our laboratory we have shown previously that MUC5AC is aberrantly expressed in rectosigmoid villous adenomas. However, the regulatory mechanisms underlying that altered profile of expression is unknown. In order to study its regulation at the transcriptional level, we have isolated and characterized 5.5 kb of the 5'-flanking region of the mouse Muc5ac mucin gene. The promoter is flanked by a TATA box and a transcriptional start site is located 22 bp downstream of the TATA box. Analysis of the sequence showed a high density of binding sites for Smad4, an essential factor in the signalling cascade activated by TGF-beta (transforming growth factor-beta), and Sp1, an important factor in the regulation of MUC5AC. This led us to study Muc5ac regulation by TGF-beta. We show that exogenous addition of TGF-beta to the cells induces Muc5ac endogenous expression, promoter activity and Smad4 binding to the promoter. By co-transfection studies we show that Smad4 is essential for Muc5ac promoter activation and that it does not synergize with Smad2 or Smad3. By gel-retardation and co-transfection assays, we identified Sp1 and Sp3 as important regulators of Muc5ac expression and showed that Smad4 and Sp1 act in a co-operative manner to transactivate Muc5ac promoter activity. Altogether these results bring new insights into the molecular mechanisms of TGF-beta-mediated up-regulation of Muc5ac and enhance our understanding as to how Muc5ac is regulated in certain pathologies of the gastrointestinal tract.