Aberrant Expression of Human Mucin GeneMUC5B in Gastric Carcinoma and Cancer Cells

Abstract

In gastric cancer, altered expression of MUC1, MUC2, MUC5AC, and MUC6 mucin genes has already been described. We show in this report by the means of in situ hybridization, reverse transcriptase-polymerase chain reaction, and transfection assays that MUC5B is also abnormally expressed in gastric carcinomatous tissues and cell lines. We thus undertook to elucidate the molecular mechanisms that regulate the transcription of MUC5B in gastric cancer cells. To this end, high expressing (KATO-III) and low expressing (AGS) gastric cancer cell lines were chosen to study human mucin gene MUC5B expression and promoter activity. Sequencing of the promoter region revealed a distal TATA box located 1 kilobase upstream of the proximal TATA box. Functional activity of the promoter was addressed by using deletion mutants covering 2044 nucleotides upstream of the MUC5B transcription start site. We identified a distal promoter 10 times more active than the proximal promoter in KATO-III cells. In AGS cells, both promoters, much less active, showed the same range of activity. Binding assays allowed us to show that the transcription factor ATF-1 binds to a cis-element present in the distal promoter. Sp1, which binds to both promoters specifically transactivates the proximal promoter. Treatment of transfected cells with PMA, cholera toxin A subunit, and calcium ionophore A23187 showed that only PMA led to a substantial activation of the distal promoter. MUC5B 5-flanking region having a high GC content, influence of methylation on the MUC5B expression was assessed. Our results indicate that repression of MUC5B expression visualized in AGS cells is due in part to the presence of numerous methylated cytosine residues throughout the 5-flanking region. Altogether these results demonstrate that MUC5B expression in gastric cancer cells is governed by a highly active distal promoter that is up-regulated by protein kinase C and that repression is under the influence of methylation.Mucins are high molecular weight O-glycoproteins synthesized by epithelial cells as large secreted or membrane-bound glycoproteins (1). So far, eight mucin genes have been well characterized (MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, and MUC7) (1), and cDNAs have been proposed for MUC8, MUC9, MUC11, and MUC12 (1, 2). Numerous studies have now demonstrated that the expression of mucin genes is tissue-and cell-specific and that their expression is altered during the pathogenesis of several diseases, which suggests that human mucin gene expression is tightly regulated and that they may play important roles during cell differentiation and carcinogenesis (3-8).In normal stomach, MUC5AC is expressed at the surface/ foveolar epithelium and MUC6 in the mucous neck cells and in the antral glands (9 -11). Other mucin genes expressed in normal gastric mucosae are MUC1 and to a lesser extent MUC2, MUC3, and MUC4. In gastric carcinomas, a decrease of MUC1, MUC5AC and MUC6 expression and an increase of MUC2, MUC3, and MUC4 expression has already been demonstrated (11-15).Human ...

Expression of MUC2, MUC5AC, MUC5B, and MUC6 mucins in colorectal cancers and their association with the CpG island methylator phenotype

“…Several studies have examined the expression of various mucin genes in relation to the nature and extent of CpG island methylation, and have found that MUC2 and MUC5B in particular, show strong correlations between promoter methylation and suppression of mucin protein synthesis. 23,[54][55][56][57] There is currently little or no evidence of methylation-induced silencing of either MUC5AC or MUC6. 23 Furthermore, MUC17, a transmembrane mucin gene, is regulated by promoter CpG island methylation and histone H3-K9 acetylation in pancreatic ductal adenocarcinomas, where promoter hypomethylation is associated with MUC17 expression.…”

Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (MUC2, MUC5AC, MUC5B, MUC6) in epithelial cancer cells

“…Fragments were gel-purified as in Perrais et al (2001a) before being methylated with mSssI for 3 h at 371C (New England Biolabs, OZYME, France). The degree of methylation of the fragment was confirmed by testing its resistance to HpaII digestion.…”

“…The methylated fragments were then ligated into pGL3 basic vector. DNA concentration was measured at 260 nm before being used in transfection experiments as described in Perrais et al (2001a). Influence of methylation on Sp1 transactivation of the promoters was studied by carrying out cotransfections in triplicate in three separate experiments in the presence of pCMV-Sp1 expression vector as described in Van Seuningen et al (2000).…”

“…In pancreatic carcinoma cells (Siedow et al, 2002) and in mucinous gastric carcinomas (Mesquita et al, 2003), on the other hand, it was shown that de novo expression of MUC2 was triggered by promoter demethylation or hypomethylation, respectively. We recently showed that repression of MUC5B in gastric cancer cells was due in part to the presence of methylated cytosines throughout its promoter (Perrais et al, 2001a).…”

Epigenetic Up-Regulation of Leukemia Inhibitory Factor (LIF) Gene During the Progression to Breast Cancer

“…Certain cancer cell types methylate the BubR1, 7-dehydrocholesterol reductase (Dhcr7), aquaporin-5 (AQP5), RUNX3, lysophosphatidic acid receptor-1 (lpa1), galectin-3, CDX1, MUC5B, PDZ-LIM domain-containing protein 2 (PDLIM2), LOT1 (PLAGL1/ZAC1), TNFSF7 (CD70) genes, leading to their transcriptional suppression DNA hypomethylation at CpG islands within the promoters (Abdollahi et al, 2003;Ahmed et al, 2007;Kim et al, 2005;Motegi et al, 2005;Park et al, 2005;Perrais et al, 2001;Qu et al, 2010;Suh et al, 2002;Tsujiuchi et al, 2006;Yu et al, 2010). The upregulation of galectin-7, CDX4, and urokinase-type plasminogen activator (uPA), and MMP-2 genes in several cancer types including breast cancer, ovarian cancer, and lymphoma cells is mediated by DNA hypomethylation (Chernov et al, 2009;Demers et al, 2009;Guo et al, 2002;Honda et al, 2006;Pakneshan et al, 2004).…”