This article has Open Peer Review reports available.
Is the gene encoding Chibby implicated as a tumour suppressor in colorectal cancer ?
© Gad et al; licensee BioMed Central Ltd. 2004
Received: 19 December 2003
Accepted: 09 July 2004
Published: 09 July 2004
A novel member of the Wnt signalling pathway, Chibby, was recently identified. This protein inhibits Wnt/β-catenin mediated transcriptional activation by competing with Lef-1 (the transcription factor and target of β-catenin) to bind to β-catenin. This suggests that Chibby could be a tumour suppressor protein. The C22orf2 gene coding Chibby is located on chromosome 22, a region recurrently lost in colorectal cancer. Activation of the Wnt pathway is a major feature of colorectal cancer and occurs through inactivation of APC or activation of β-catenin. All of this led us to analyse the possible implication of Chibby in colorectal carcinogenesis.
First, 36 tumour and matched normal colonic mucosa DNA were genotyped with five microsatellite markers located on chromosome 22 to search for loss of heterozygosity. Then, mutation screening of the C22orf2 coding sequence and splice sites was performed in the 36 tumour DNA. Finally, expression of Chibby was analysed by quantitative RT-PCR on 10 patients, 4 with loss of heterozygosity (LOH) on chromosome 22.
Loss of heterozygosity involving the C22orf2 region was detected in 11 out of 36 patients (30%). Sequencing analysis revealed a known variant, rs3747174, in exon 5: T321C leading to a silent amino acid polymorphism A107A. Allelic frequencies were 0.69 and 0.31 for T and C variants respectively. No other mutation was detected. Among the 10 patients studied, expression analysis revealed that Chibby is overexpressed in 2 tumours and underexpressed in 1. No correlations were found with 22q LOH status.
As no somatic mutation was detected in C22orf2 in 36 colorectal tumour DNA, our results do not support the implication of Chibby as a tumour suppressor in colorectal carcinogenesis. This was supported by the absence of underexpression of Chibby among the tumour samples with 22q LOH. The implication of other Wnt pathway members remains to be identified to explain the part of colorectal tumours without mutation in APC and β-catenin.
Identifying components of the Wnt signalling pathway has been at the forefront of cancer biology since a link was made between Wnt, the mammalian homologue of the fruitfly Wingless (Wg), and the development of cancer. Acting through a core set of proteins that are highly conserved in evolution, this pathway regulates the ability of the oncoprotein β-catenin to activate transcription of specific target genes. This regulation, in turn, results in changes in expression of genes that modulate cell fate, proliferation and apoptosis . Recently, Takemaru et al. identified a novel human protein, named Chibby, that interacts with the carboxy-terminal transcription activation domain of β-catenin . Chibby is a nuclear protein of 126 amino acids with coiled-coil domains and is conserved from Drosophila to Human. It has been shown that Chibby antagonizes the Wnt signalling pathway by inhibition of the transcription protein complex comprising β-catenin. This result suggests that Chibby could act as a tumour suppressor protein.
In colorectal cancer, the activation of the Wnt signalling pathway occurs in more than 60% of tumours through the inactivation of the APC tumour suppressor gene by mutations and allelic losses, or through the presence of β-catenin activating mutations . The C22orf2 gene encoding Chibby is located on 22q13.1. This chromosome region is frequently lost in colorectal cancer suggesting the existence of a tumour suppressor gene that remains to be identified.
The putative function and the location of the C22orf2 gene led us to analyse the possible implication of C22orf2 as a tumour suppressor gene in colorectal carcinogenesis. First, the allelic status of chromosome 22 was established on 36 colorectal tumour and matched normal colonic mucosa DNA, second, mutation analysis of the C22orf2 gene was performed on tumour DNA, and third, expression analysis of Chibby was studied in few patients.
Patients, sample collection and nucleic acid extraction
Tumour samples and matched normal colonic mucosa were collected from 36 patients (23 women, 13 men) hospitalised in the surgical department of Laennec Hospital in Paris between 1997 and 1999. The mean age of patients was 69.9 years old, range [56.4–83.4]. An histological HES staining was performed before DNA extraction and only tumour fragments with more than 70% of tumour cells were retained. Tumours were all classified as adenocarcinoma. Twenty-nine tumours were classified as well differentiated, and 7 as poor differentiated. No tumour samples showed a microsatellite instability phenotype. Tumours were located in proximal colon in 9 cases, in distal colon in 21 cases and 6 were located in rectum. According to TNM classification, tumours were classified in stage I in 1 case, stage II in 17, stage III in 8 and stage IV in 10 cases. Samples were immediately frozen in liquid nitrogen and stored at -80°C. Informed consent was signed according to French laws. DNA extraction was performed using the QIAamp® DNA Mini Kit (Qiagen, Courtaboeuf, France) for all patients in 1999, and stored at -20°C. Tumour samples and matched normal colonic mucosa were available for RNA extraction for 10 patients. RNA isolation was performed recently with RNeasy® Mini Kit (Qiagen) and stored at -80°C. Quality of RNA was determined by electrophoresis through agarose gel stained with ethidium bromide. Intensity of 18S and 28S RNA bands was estimated under UV light. Measuring UV absorbance at 260 nm was performed to quantify RNA.
Microsatellite markers analysed on chromosome 22
5'location on chromosome 22 (bp)
label (on forward primer)
concentration for Multiplex PCR (μM)
PCR product (bp)
PCR conditions for sequencing analysis of the C22orf2 gene
Annealing temperature (°C)
PCR product (bp)
Expression analysis of Chibby by Quantitative RT-PCR
Expression of Chibby in tumour tissues compared to normal tissues
LOH on chromosome 22
Loss of heterozygosity analysis on chromosome 22
Sequencing analysis of C22orf2
The C22orf2 gene spreads over 17.2 kb and comprises five exons. ORF starts at exon 2 leading to a 381 bp cDNA. Coding sequence and intron exon junctions were analysed by direct sequencing on the 36 tumour DNA to search for sequence variations. A known variant, rs3747174, was observed in exon 5 leading to a T321C transition, corresponding to a silent polymorphism A107A. Allelic frequencies were 0.69 and 0.31 for T and C variants respectively. The distribution of the different genotypes is in agreement with Hardy and Weinberg equilibrium. No other somatic mutation was detected either in coding exons or in splice sites.
Expression analysis of Chibby
The difference of the level of expression of Chibby between tumour samples and normal colonic mucosa samples was analysed by quantitative RT-PCR, using ribosomal 18S as internal control. Both samples were available for 10 patients, 4 of which presented LOH on chromosome 22. Q-PCR expression analysis revealed that Chibby is overexpressed in 2 tumours and underexpressed in 1 (Table 3). No correlations were found with 22q LOH status.
The aim of this study was to analyse the possible implication of Chibby in colorectal carcinogenesis. Genotyping analysis was performed on chromosome 22 to search for loss of heterozygosity. Among the 36 patients, 12 (33%) showed LOH including the C22orf2 region. Mutation analysis was performed on C22orf2 coding sequence and splice sites: no somatic mutation was detected. However, a known variant, rs3747174, was observed in exon 5: T321C, corresponding to A107A. In the dbSNP database http://www.ncbi.nlm.nih.gov/SNP/, this variant was detected with a set of 52 chromosomes and allelic frequencies were estimated from 1496 chromosomes at 0.63 and 0.37 respectively which is similar to that observed in this present series, i.e. 0.67 and 0.31 respectively . Furthermore, quantitative RT-PCR expression analysis showed that Chibby is overexpressed in 2 tumours, 1 of which showing LOH at C22orf2 locus, and Chibby is underexpressed in 1 tumour showing no 22q LOH. Taking together, these results do not support a putative epigenetic modification, i.e. methylation of the C22orf2 promoter, that could repress gene expression as another mechanism of gene inactivation than mutation. Thus, Chibby does not seem implicated as a tumour suppressor in colorectal carcinogenesis.
The APC gene was found mutated in several series of colorectal tumours with a frequency of 60%. Furthermore, in a series of tumours lacking APC mutations, 48% presented a mutation in the β-catenin regulatory domain . Thus, more than one gene could be implicated in colorectal carcinogenesis through the activation of the Wnt signalling pathway. The recently identified function of Chibby in this pathway supports the idea that it could act as a tumour suppressor . However, we did not detect mutation in a series of 36 colorectal tumours, suggesting that the role of Chibby in colorectal carcinogenesis is probably weak. Other genes remain to be studied to explain the part of colorectal tumours without mutation in APC and β-catenin.
This study was supported by La Région Ile de France and La Ligue Nationale de Lutte Contre le Cancer.
- Moon RT, Bowerman B, Boutros M, Perrimon N: The promise and perils of Wnt signaling through beta-catenin. Science. 2002, 296: 1644-1646. 10.1126/science.1071549.View ArticlePubMedGoogle Scholar
- Takemaru K, Yamaguchi S, Lee YS, Zhang Y, Carthew RW, Moon RT: Chibby, a nuclear beta-catenin-associated antagonist of the Wnt/Wingless pathway. Nature. 2003, 422: 905-909. 10.1038/nature01570.View ArticlePubMedGoogle Scholar
- Laurent-Puig P, Blons H, Cugnenc PH: Sequence of molecular genetic events in colorectal tumorigenesis. Eur J Cancer Prev. 1999, 8 Suppl 1: S39-47.PubMedGoogle Scholar
- Bluteau O, Legoix P, Bayer J, Bioulac-Sage P, Flejou JF, Capron F, Monges G, Brechot C, Thomas G, Laurent-Puig P, Zucman-Rossi J: [Semi-automated quantitative method for detecting the loss of heterozygosity at the long arm of chromosome 4 in hepatocellular carcinoma]. Gastroenterol Clin Biol. 1999, 23: 1225-1232.PubMedGoogle Scholar
- Haga H, Yamada R, Ohnishi Y, Nakamura Y, Tanaka T: Gene-based SNP discovery as part of the Japanese Millennium Genome Project: identification of 190,562 genetic variations in the human genome. Single-nucleotide polymorphism. J Hum Genet. 2002, 47: 605-610. 10.1007/s100380200092.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/4/31/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.