Molecular cloning, sequencing and expression of the mRNA encoding human Cdx1 and Cdx2 homeobox. Down-regulation of Cdx1 and Cdx2 mRNA expression during colorectal carcinogenesis

Abstract

Defining the molecular mechanisms involved in cancer formation and progression is still a major challenge in colorectal-cancer research. Our strategy was to characterize genes whose expression is altered during colorectal carcinogenesis. To this end, the phenotype of a colorectal tumour was previously established by partial sequencing of a large number of its transcripts and the genes of interest were selected by differential screening on high-density filters with mRNA of colorectal cancer and normal adjacent mucosa. Fifty-one clones were found over-expressed and 23 were underexpressed in the colorectal-cancer tissues of the 5 analyzed patients. Among the latter, clones 6G2 and 32D6 were found of particular interest, since they had significant homology with several homeodomain-containing genes. The highest degree of similarity was with the murine Cdx1 for 6G2, and with the murine Cdx2 and hamster Cdx3 for 32D6. Using a RT-PCR approach, complete sequence of both types of homeobox-containing cDNA was obtained. The amino-acid sequence of the human Cdx1 is 85% identical to the mouse protein, and human Cdx2 has 94% identity with the mouse Cdx2 and hamster Cdx3. Tissue-distribution analysis of Cdx1 and Cdx2 mRNA showed that both transcripts were specifically expressed in small intestine, in colon and rectum. Colorectal cancer is the second most common tumour in men and the third in women in the Western countries. Although much is known about the epidemiology, morphology and genetics of colorectal tumorigenesis, our knowledge of the perturbations of gene expression that occur in colorectal tumours remains limited. Such tumours may arise from benign adenomatous polyps, which later progress to adenocarcinomas through several molecular events. They thus provide a very useful paradigm for studying the molecular genetic bases of cancer. The multistep process leading to colorectal tumorigenesis probably involves the loss of function of tumour-suppressor genes, as well as the activation of oncogenes. Several important genes have already been identified, but they do not account for the whole process, and other genes are probably involved.Efforts have been made to characterize these genes. Differential hybridization techniques and screening of substracted libraries allowed the elucidation of some of them (Bartsch et al., 1986;Denis et al., 1993;Yow et al., 1988;Schweinfest et al., 1993;Kondoh et al., 1992; Barnard et al., 1992a, b). We developed an alternative strategy, in which the phenotype of a colorectal tumour was established by partial sequencing of a large number of randomly selected transcripts (Frigerio et al., 1995). This repertory of ESTs should therefore contain most of the differentially expressed genes. Recently, Nguyen et al. (1995) have developed an efficient method of differential screening in which cDNA clones are gridded on high-density colony filters and hybridized with complex probes derived from poly (A) 1 RNA from different cells or tissues. The signals observed are measured, providing a ''hybridization s...

Downregulation of the colon tumour-suppressor homeobox gene Cdx-2 by oncogenic ras

“…The ras-dependent decrease in Cdx-2 expression reported here in vitro in human colonic Caco-2 and HT-29 cells is fully consistent with the decline of expression of this tumour-suppressor gene that correlates to the pathological grade in human colorectal cancers and in chemically-induced colonic tumours in the rat (Ee et al, 1995;Mallo et al, 1997). Our results therefore suggest that in vivo, like in cultured cells, Cdx-2 is a target of oncogenic ras.…”

“…The stimulation of Cdx-1 reported in this study by oncogenic ras in colonic Caco-2 cells is consistent with the onset of expression found in metaplasic areas of oesophagus and gastric cancers, since oncogenic ras activation is often associated with well di erentiated tumours of the upper digestive tract (Peddanna et al, 1995). Thus, during early events in the progression of normal colonic epithelium to adenoma, oncogenic ras activation would stimulate Cdx-1; additional genetic alterations may subsequently downregulate this gene during further progression to adenocarcinoma and distant metastasis, as observed by Silberg et al (1997), Mallo et al (1997) and Vider et al (1997). This hypothesis is supported by the fact that the decrease of Cdx-1 in colonic cancers was found not to be related to the pathological grade (Silberg et al, 1997), in contrast to what was reported for Cdx-2 (Ee et al, 1995).…”

“…Beside its role in cell di erentiation, Cdx-2 has recently been identi®ed as a new colonic tumour-suppressor gene, since heterozygous Cdx-2 knock-out mice develop multiple colonic adenomas in which cells fail to express the single resident gene (Chawengsaksophak et al, 1997). Consistently, Cdx-2 expression drops in colon cancers in relation with the severity of displasia (Ee et al, 1995;Mallo et al, 1997). On the contrary, Cdx-1 has oncogenic potential in vitro (Maulbecker and Gruss, 1993).…”

“…No colonic tumour has been reported in Cdx-1-de®cient mice (Subramaniam et al, 1995) and onset of Cdx-1 expression accompanies the development of intestinal metaplasia in the oesophagus and in the stomach (Silberg et al, 1997). However, Cdx-1 downexpression has been observed in some colon cancers (Silberg et al, 1997;Mallo et al, 1997;Vider et al, 1997).…”

The homeobox intestinal differentiation factor CDX2 is selectively expressed in gastrointestinal adenocarcinomas

“…4 Similarly, CDX2 has been detected in human fetal intestine at both the mRNA and protein level. [5][6][7] Conditional expression of CDX2 in an undifferentiated intestinal cell line results in the formation of multicellular structures with demonstrable enterocyte and goblet cell features, supporting a role for CDX2 in intestinal differentiation. 8 This likely reflects direct upregulation of transcription from genes such as liver-intestine cadherin and MUC2 by CDX2, as has been shown in other cell lines.…”