Human MUC2 Mucin Gene Is Transcriptionally Regulated by Cdx Homeodomain Proteins in Gastrointestinal Carcinoma Cell Lines

Abstract

In intestinal metaplasia and 30% of gastric carcinomas, MUC2 intestinal mucin and the intestine-specific transcription factors Cdx-1 and Cdx-2 are aberrantly expressed. The involvement of Cdx-1 and Cdx-2 in the intestinal development and their role in transcription of several intestinal genes support the hypothesis that Cdx-1 and/or Cdx-2 play important roles in the aberrant intestinal differentiation program of intestinal metaplasia and gastric carcinoma. To clarify the mechanisms of transcriptional regulation of the MUC2 mucin gene in gastric cells, pGL3 deletion constructs covering 2.6 kb of the human MUC2 promoter were used in transient transfection assays, enabling us to identify a relevant region for MUC2 transcription in all gastric cell lines. To evaluate the role of Cdx-1 and Cdx-2 in MUC2 transcription we performed co-transfection experiments with expression vectors encoding Cdx-1 and Cdx-2. In two of the four gastric carcinoma cell lines and in all colon carcinoma cell lines we observed transactivation of the MUC2 promoter by Cdx-2. Using gel shift assays we identified two Cdx-2 binding sites at ؊177/؊171 and ؊191/؊187. Only simultaneous mutation of the two sites resulted in inhibition of Cdx-2-mediated transactivation of MUC2 promoter, implying that both Cdx-2 sites are active. Finally, stable expression of Cdx-2 in a gastric cell line initially not expressing Cdx-2, led to induction of MUC2 expression. In conclusion, this work demonstrates that Cdx-2 activates the expression of MUC2 mucin gene in gastric cells, inducing an intestinal transdifferentiation phenotype that parallels what is observed both in intestinal metaplasia and some gastric carcinomas.There is consistent data indicating that in human stomach as well as in other organs mucin genes are expressed in a regulated cell-and tissue-specific manner and that altered mucin gene expression occurs in cancer and precancerous lesions (1). In normal gastric mucosa most studies show little or no expression of the intestinal mucin MUC2 (2-9). In intestinal metaplasia, a preneoplastic lesion of the stomach characterized by the transdifferentiation of the gastric mucosa to an intestinal phenotype, there are alterations in the mucin expression pattern including de novo expression of MUC2, mostly in goblet cells (10). Thirty percent of gastric carcinomas, including all carcinomas of the mucinous type, also aberrantly express MUC2 intestinal mucin (11, 12). The molecular mechanisms responsible for the regulation of MUC2 transcription and expression are beginning to be elucidated. The structure of MUC2 promoter was characterized (13, 14) and MUC2 expression was reported to be regulated by methylation of the promoter (15-17) and by the Sp1 family of transcription factors (13,18,19). It has also been described that MUC2 is transcriptionally activated by p53 (20) and, in tracheobronchial epithelial cells, by lipopolysaccharide from Pseudomonas aeruginosa (21, 22) and epidermal growth factor (19). However, information on MUC2 transcriptional regulation in gas...

The intestine-specific homeobox gene Cdx2 decreases mobility and antagonizes dissemination of colon cancer cells

“…None of them inhibited the expression of another homeobox gene, HoxD3 (Figure 5b). Along with Cdx2, two of its targets, LI-cadherin and Muc2 (Hinoi et al, 2002;Mesquita et al, 2003), were also strongly decreased by siRNA@Cdx2 (Figure 5b), attesting the functional relevance of the Cdx2 decline by this approach. To check migration, SW480 cells transfected with siRNA@Cdx2 or with a control siRNA were harvested and seeded into Boyden chambers.…”

“…Thus Cdx2 may exert its inhibitory effect on colon cancer cell migration and spreading by acting on multiple cellular functions. At the molecular level, Cdx2 acts as a transcriptional activator as exemplified by the sucrase-isomaltase, LI-cadherin and Muc2 genes (Suh et al, 1994;Hinoi et al, 2002;Mesquita et al, 2003). However, we have shown that it can also inhibit the transcriptional activity of another homeodomain protein, Cdx1 (Gautier-Stein et al, 2003;Calon et al, 2007).…”

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

“…CDX2 has been identified by Yamamoto et al 27 as a protein able to bind to the MUC2 promoter and initiate transcription, and subsequent studies by Mesquita et al have shown two functionally active CDX2-binding sites. 26 Ectopic expression of CDX2 has also been linked to MUC2 expression in gastric intestinal metaplasia, 69 Barrett's esophagus, 70 and cholangiocarcinoma, 71 suggesting an important regulatory role for CDX2 in MUC2 expression. We and others have reported that CDX2 may be somatically lost in some colorectal cancers either due to out-offrame allelic gains or losses of the microsatellite repeat within the CDX2-coding region, 29,72 epigenetic silencing via methylation, 73 or loss of heterozygosity.…”

“…24 In addition, the regulation of mucin gene expression by various transcription factors such as Sp1, AP-1, and CDX2 has been explored in a range of tissues (reviewed by Andrianifahanana et al 25 ). Of these, the homeobox protein, CDX2, reportedly regulates MUC2 expression in gastrointestinal epithelial goblet cells 26,27 and has shown a loss of expression in microsatellite-unstable colorectal cancers 28,29 and serrated polyps of the colorectum. 30 We report here the results from a large series of colorectal carcinomas demonstrating an association between expression of mucins, MUC2, MUC5AC, MUC5B, and MUC6 and the presence of somatic BRAF p.V600E mutation, CIMP, microsatellite instability, and loss of CDX2 expression.…”

Expression of the intestinal marker Cdx2 in the columnar-lined esophagus with and without intestinal (Barrett's) metaplasia

“…35,36 Cdx2 reactivity in the nongoblet cell population of the columnar esophagus may be an early event in intestinal differentiation, and its presence would indicate that the molecular machinery for intestinal differentiation is in place, even when histologic evidence of such differentiation is lacking. 25 In fact, it has been proposed that the first step in the refluxadenocarcinoma sequence of the columnar-lined esophagus is the development of columnar nongoblet epithelium of 'cardiac-type' which may evolve either to oxynto-cardiac mucosa, with no risk of malignant transformation, or to intestinal-type epithelium, with risk for adenocarcinoma.…”