Repression of the desmocollin 2 gene expression in human colon cancer cells is relieved by the homeodomain transcription factors Cdx1 and Cdx2.

Desmosomes are intracellular junctions that provide strong cell-cell adhesion in epithelia and cardiac muscle. Their disruption causes several human diseases and contributes to the epithelial-to-mesenchymal transition observed in cancer. Desmocollin 2 (DSC2) is a cadherin superfamily member and a critical component of desmosomes found in intestinal epithelium. However, the mechanism regulating DSC2 gene expression in intestinal cells is not known. Cdx1 and Cdx2 are homeodomain transcription factors that regulate intestine-specific gene expression. Cdx expression in the past has been associated with the induction of desmosomes. We now show that the DSC2 gene is a transcriptional target for Cdx1 and Cdx2. Colon cancer cell lines retaining Cdx2 expression typically express DSC2. Restoration of Cdx expression in Colo 205 cells induced DSC2 mRNA and protein and the formation of desmosomes. The 5'-flanking region of the DSC2 promoter contains two consensus Cdx-binding sites. Electrophoretic mobility shift assays show that Cdx1 and Cdx2 bind these sites in vitro, and chromatin immunoprecipitation confirmed Cdx2 binding in vivo. DSC2 promoter truncations established that these regions are Cdx responsive. The truncations also identify a region of the promoter in which potent transcriptional repressors act. This repressor activity is relieved by Cdx binding. We conclude that the homeodomain transcription factors Cdx1 and Cdx2 regulate DSC2 gene expression in intestinal epithelia by reversing the actions of a transcriptional repressor. The regulation of desmosomal junctions by Cdx contributes to normal intestinal epithelial columnar morphology and likely antagonizes the epithelial-to-mesenchymal transition necessary for the metastasis of colon cancer cells in humans.

The homeodomain transcription factors Cdx1 and Cdx2 induce E-cadherin adhesion activity by reducing beta- and p120-catenin tyrosine phosphorylation.

The homeodomain transcription factors Cdx1 and Cdx2 are regulators of intestine-specific gene expression. They also regulate intestinal cell differentiation and proliferation; however, these effects are poorly understood. Previously, we have shown that expression of Cdx1 or Cdx2 in human Colo 205 cells induces a mature colonocyte morphology characterized by the induction of a polarized, columnar shape with apical microvilli and strong cell-cell adhesion. To elucidate the mechanism underlying this phenomenon, we investigated the adherens junction complex. Cdx1 or Cdx2 expression reduced Colo 205 cell migration and invasion in vitro, suggesting a physiologically significant change in cadherin function. However, Cdx expression did not significantly effect E-cadherin, alpha-, beta-, or gamma-catenin, or p120-catenin protein levels. Additionally, no alteration in their intracellular distribution was observed. Cdx expression did not alter the coprecipitation of beta-catenin with E-cadherin; however, it did reduce p120-catenin-E-cadherin coprecipitation. Tyrosine phosphorylation of beta- and p120-catenin is known to disrupt E-cadherin-mediated cell adhesion and is associated with robust p120-catenin/E-cadherin interactions. We specifically investigated beta- and p120-catenin for tyrosine phosphorylation and found that it was significantly diminished by Cdx1 or Cdx2 expression. We restored beta- and p120-catenin tyrosine phosphorylation in Cdx2-expressing cells by knocking down the expression of protein tyrosine phosphatase 1B and noted a significant decline in cell-cell adhesion. We conclude that Cdx expression in Colo 205 cells induces E-cadherin-dependent cell-cell adhesion by reducing beta- and p120-catenin tyrosine phosphorylation. Ascertaining the mechanism for this novel Cdx effect may improve our understanding of the regulation of cell-cell adhesion in the colonic epithelium.

Cdx1 inhibits human colon cancer cell proliferation by reducing beta-catenin/T-cell factor transcriptional activity.

The cessation of proliferation and the induction of differentiation are highly coordinated processes that occur continuously in the intestinal crypts. The homeodomain transcription factors Cdx1 and Cdx2 regulate intestine-specific gene expression and enterocyte differentiation. Their roles in regulating proliferation are recognized but remain poorly understood. Previously, we demonstrated that Cdx1 expression diminished the proliferation of human colon cancer cells in part by reducing cyclin D1 gene expression. In order to elucidate further the molecular mechanisms underlying this phenomenon, we first hypothesized that Cdx1 or Cdx2 expression reduces colon cancer cell proliferation by inhibiting beta-catenin/T-cell factor (TCF) transcriptional activity. We report that Cdx1 or Cdx2 expression does inhibit beta-catenin/TCF transcriptional activity in colon cancer cells. This inhibitory effect is dose-dependent and is observed in different colon cancer cell lines, and the degree of inhibition correlates with the ability of Cdx1 to reduce cell proliferation. Cdx1 expression does not alter beta-catenin protein levels or intracellular distribution nor does it induce an inhibitory TCF isoform. We also find that Cdx1 expression is lost in Min mouse polyps with increased nuclear localization of beta-catenin, suggesting that Cdx1 does not support beta-catenin-mediated transformation. Finally, we show that colon cancer cells effectively reduce Cdx2-mediated inhibition of Wnt/beta-catenin/TCF transcriptional activity when compared with other model systems. This suggests that colon cancer and possibly crypt epithelial cells can modulate the effects of Cdx2 on beta-catenin signaling and proliferation. We conclude that Cdx1 and Cdx2 inhibit colon cancer cell proliferation by blocking beta-catenin/TCF transcriptional activity.

Cdx1 or Cdx2 expression activates E-cadherin-mediated cell-cell adhesion and compaction in human COLO 205 cells.

A mature columnar intestinal epithelium develops late in embryogenesis and is maintained throughout the life of the organism. Although the mechanisms driving intestine-specific gene expression have been well studied, those promoting the acquisition of cell-cell junctions, columnar morphogenesis, and polarization have been less studied. The Cdx homeodomain transcription factors (Cdx1 and Cdx2) regulate intestine-specific gene expression and intestinal epithelial differentiation. We report here that Cdx expression induces E-cadherin activity and cell-cell adhesion in human COLO 205 cancer cells. Within days of Cdx1 or Cdx2 expression, a new homotypic cell-cell adhesion phenotype is induced. This is a specific response to Cdx, inasmuch as a Cdx1 mutant failed to elicit the effect. Additionally, Cdx-expressing COLO 205 cells demonstrate a reduced proliferative capacity and an increase in the mRNA expression of differentiation-associated genes. Electron micrographs of these cells demonstrate induction of tight, adherens, and desmosomal junctions, as well as a columnar shape and apical microvilli. Investigations of the adhesion phenotype determined that it was Ca(2+) dependent and could be blocked by an E-cadherin-blocking antibody. However, E-cadherin protein levels and intracellular distribution were unchanged. Cdx expression restored the ability of the cell membranes to adhere and undergo compaction. We conclude that Cdx1 or Cdx2 expression is sufficient to induce an E-cadherin-dependent adhesion of COLO 205 cells. This adhesion is associated with polarization and cell-cell membrane compaction, as well as induction of a differentiated gene-expression pattern. Ascertaining the mechanism for this novel Cdx effect may yield insight into the development of mature colonic epithelium.

A specific genetic alteration on chromosome 6 in ulcerative colitis-associated colorectal cancers.

Various genetic alterations in ulcerative colitis (UC)-associated colorectal cancers (CRCs) have been reported. However, almost all studies have shown abnormalities of the known genes that have been demonstrated in sporadic CRCs. To identify novel genetic alterations, we selected unintentionally 35 microsatellite markers, and performed allelotype study in CRCs or dysplastic lesions from UC. High frequency of loss of heterozygosity (LOH; 62.5%) was detected on chromosome 6 (D6S468) but not on other chromosomes. With four additional microsatellite markers around the D6S468 locus, we determined the commonly deleted region between two loci, D6S1543 and D6S1580, in UC-associated CRCs and dysplasia. Interestingly, there was no LOH in this region in sporadic CRCs and severely inflamed lesions of longstanding and extensive UC without cancers. These results indicated the presence of novel tumor suppressor genes on chromosome 6 related to the carcinogenesis of UC but not to sporadic CRCs.