Japanese Journal of Clinical Oncology 32:358-362 (2002)
© 2002 Foundation for Promotion of Cancer Research
Mutation and Expression of the ß-Catenin-interacting Protein ICAT in Human Colorectal Tumors
1 Second Department of Surgery, Gunma University School of Medicine, Maebashi, 2 Laboratory of Molecular and Genetic Information, Institution of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo and 3 Biology Division, National Cancer Center Research Institute, Tokyo, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAcknowledgmentsREFERENCES |
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Background: Aberrant activation of Wnt signaling caused by mutations in the tumor suppressor adenomatous polyposis coli or ß-catenin is a critical event in the development of human colorectal tumors. We have recently identified the ICAT gene, which encodes a small protein that interacts with ß-catenin and represses Wnt signaling.
Methods: We examined the prevalence of mutations in the entire ICAT coding sequence and intronic splice donor and acceptor regions of ICAT by PCR–SSCP and also the expression of the ICAT gene by RT-PCR.
Results: The ICAT gene was mapped to chromosome 1p36.1–p36.2, which is implicated in the pathogenesis of various types of cancers. However, no mutations in ICAT were detected among 128 colorectal tumors. Instead, ICAT was found to be overexpressed in almost half of colorectal carcinomas. Cases exhibiting ICAT overexpression showed a significantly higher incidence of well-differentiated adenocarcinoma and positive lymphatic permeation.
Conclusion: Our results suggest that ICAT is not the putative tumor suppressor on 1p36.1–p36.2, although aberrant overexpression of ICAT may play a role in the pathogenesis of colorectal carcinomas.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAcknowledgmentsREFERENCES |
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The Wnt/Wingless signaling transduction pathway plays an important role in both embryonic development and tumorigenesis (1–6). In human colorectal carcinomas, mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are frequently observed (7). Furthermore, in those tumors that retain the wild-type APC gene, mutations in ß-catenin have been observed (8, 9). In addition, Axin1 in addition to ß-catenin is mutated in a certain subset of human hepatocellular carcinomas (10–12). Genetic alterations in the components of Wnt signaling, APC, ß-catenin and Axin1, appear to occur in a mutually exclusive fashion and have a similar result; the deregulated accumulation of ß-catenin (7, 13). ß-catenin, in turn, interacts with the TCF/LEF family of transcription factors and activates transcription of downstream genes such as c-Myc and cyclin D1 (8, 9, 14–17). The accumulation of ß-catenin is also observed in many other types of neoplasms such as colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma pilomatricomas and prostate cancer (18). Thus, constitutive activation of ß-catenin-TCF-mediated transcription may be a critical step in tumorigenesis among divergent types of cancers.
We have recently identified a ß-catenin interacting protein, ICAT, that inhibits the interaction of ß-catenin with TCF, preventing the formation of the transactivator complex and thereby negatively regulating Wnt signaling (19). Although constitutive activation of ß-catenin–TCF-mediated transcription due to inactivation of the tumor suppressor APC or gain-of-function mutations in ß-catenin occurs in the majority of colorectal carcinomas, there is a certain subset of tumors in which no such mutations are found. It is important to elucidate whether these colorectal carcinomas could really develop without aberration in any other components of the Wnt signaling pathway. ICAT is therefore one of the candidate genes to examine this possibility. To clarify the role of ICAT in sporadic colorectal carcinogenesis, we examined the prevalence of mutations in the entire ICAT coding sequence and intronic splice donor and acceptor regions of ICAT by PCR–SSCP and also the expression of the ICAT gene by RT-PCR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAcknowledgmentsREFERENCES |
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Tissue Specimens and DNA and RNA Extraction
A total of 128 sets of colorectal tumors and adjacent non-cancerous tissues (102 colorectal carcinomas, 10 liver metastases and 16 colorectal adenomas) were obtained at surgery from patients admitted to Gunma University Hospital between May 1996 and June 1999. Samples obtained were immediately frozen in liquid nitrogen and stored at –80°C until use. High molecular weight DNA was prepared as described previously (20) from 128 pairs of tumors and adjacent non-cancerous tissues. Total RNA was prepared from 24 pairs of tumors and adjacent non-cancerous tissues by the acid guanidinium–phenol–chloroform (AGPC) method (21).
PCR–SSCP Analysis
All 128 samples were examined for mutations in the entire coding sequence of the ICAT gene by PCR–SSCP analysis. The genomic sequence of ICAT was partially determined by sequencing BAC clones (T. Nakamura et al., unpublished data). We designed three sets of intron-based PCR primers that amplify individual exons 1–3 and boundary intronic regions of ICAT. The primer sequences used were as follows, E1F, 5'-GTC CCA GTA TGT CAC ATC CTG-3'; E1R, 5'-GTG GCT CCA CCC TCC AAT AG-3'; E2F, 5'-GCC TCT AGG GGT GCA GGT G-3'; E2R, 5'-CCT GGG TCT CAT GGA TCA C-3'; E3F, 5'-AAT GCT GCA AGC CCC ATC AG-3'; and E3R, 5'-AAC AGC ATC CAG GGT GTT C-3'. Using these primers, exons 1, 2 and 3 were detected as 265, 281 and 137 bp PCR products, respectively, as predicted. A 50 ng amount of genomic DNA templates was suspended in a total volume of 10 ml of PCR buffer containing 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 100 nM of each primer, 200 mM of each deoxynucleotide triphosphate, 1.5 mCi of [
-32P]dCTP (ICN Biomedicals, Costa Mesa, CA) and 0.5 unit of Taq DNA polymerase (TaKaRa, Ohtsu, Japan). PCR conditions were as follows; 30 s at 95°C, 30 s at 59°C (for exon 1), 60°C (for exon 2) or 57°C (for exon 3) and 60 s at 72°C for 35 cycles, followed by 10 min at 72°C. SSCP analysis was performed in a low-pH buffer system that showed improved separation of long mutant fragments of up to 800 bp (22). PCR products were denatured, loaded on non-denaturing polyacrylamide gels containing 5–10% polyacrylamide (99:1 acrylamide to bisacrylamide) and TPE (30 mM Tris, 20 mM PIPES and 1 mM EDTA; pH6.8) and electrophoresed in TPE buffer at 15°C. The gels were then exposed to Kodak XAR film (Kodak, Rochester, NY) for 24–48 h at –80°C.
Sequence Analysis
PCR fragments were purified using the QIA quick-spin PCR purification kit (QIAGEN, Venlo, Netherlands) and directly sequenced in both directions using Thermo Sequenase II dye terminator cycle sequencing pre-mix kits (Amersham Pharmacia Biotech, Amersham, UK) and an ABI 373A DNA Sequence System (Perkin-Elmer, Foster City, CA).
RT-PCR Analyses
Randomly primed cDNAs were reverse-transcribed from 2 mg of total RNA using murine leukemia virus reverse transcriptase (Perkin-Elmer) in a 40 ml volume according to the manufacturer’s protocol. A 0.5 ml volume of each cDNA conversion mixture was amplified by PCR in a total volume of 10 ml. PCR was performed using forward primer, RT-F, 5'-AAG AGT CCG GAG GAG ATG TAC-3', and reverse primer, RT-R, 5'-TCT TCC GTC TCC GAC CTG GA-3'. PCR conditions were as follows: 30 s at 96°C, 30 s at 55°C and 30 s at 72°C for 25–35 cycles, followed by 10 min at 72°C. For semi-quantitative evaluation, the number of PCR cycles was varied. PCR products were electrophoresed on 2.0% agarose gels and images were obtained with a Gel Print 2000i (Genomic Solutions, Ann Arbor, MI), saved in tagged-image files format (TIFF) and band intensities were quantitated. The ICAT band intensities (25 cycles) were normalized to their respective GAPDH band intensities (20 cycles). Tumors in which the ICAT band intensity was more than double that in adjacent non-cancerous tissues were considered as overexpressing ICAT. Experiments were repeated at least three times independently and the reproducibility was verified.
Statistical Analysis
Statistical analysis was performed using the program StatView J-5.0 (SAS Institute, Cary, NC). The chi-squared test and Fisher’s exact test were used for analysis of clinical data. Overall survival curves were generated by the Kaplan–Meier method, and the log-rank test and the Breslow–Graha–Wilcoxon test were used to compare the curves. Stepwise multivariate regression analysis of a Cox’s proportional hazards model was used to estimate the prognostic factors. P < 0.05 was considered statistically significant.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAcknowledgmentsREFERENCES |
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Chromosome Localization of the ICAT Gene
To determine roughly the chromosome localization of the human ICAT gene, we searched the STS database of the National Center for Biotechnology Information. Three STS markers, WI-16661, WI-9616 and SHGC-30730, were found to be localized within the 3'-untranslated region of the ICAT gene, suggesting that ICAT is localized between D1S214 and D1S244 on human chromosome 1p36.1–p36.2. This chromosomal region is known to be frequently deleted in various types of tumors including neuroblastoma, colorectal cancer, breast cancer and hepatocellular carcinoma (23–26).
ICAT Mutation in Colorectal Tumors
Three sets of intronic PCR primers allowed successful amplification of individual ICAT exons under the conditions described above. DNA was obtained from 102 primary colorectal carcinomas, 10 liver metastases and 16 colon adenomas. The entire coding sequence of each, and also the splicing donor and acceptor sites, were examined for mutation by PCR–SSCP analysis. No tumor specific aberrant bands were detected (data not shown). Furthermore, mutation of ICAT was not detected by sequence analysis of 24 randomly selected tumor samples. These results suggest that mutation of ICAT occurs rarely or does not occur in colorectal carcinoma.
ICAT Expression in Non-cancerous Colorectal Mucosa and Colon Cancer
To investigate further the association of ICAT with colorectal carcinogenesis, we examined ICAT expression in 24-paired colorectal tumors and adjacent non-cancerous tissues by semi-quantitative RT-PCR analysis. ICAT is known to be expressed at high levels in mouse heart, brain, liver and skeletal muscle, at low levels in kidney, testis and lung and at undetectable levels in spleen (19). However, the expression levels of ICAT mRNA in normal human colorectal tissues have not been evaluated. ICAT expression was detected in all 24 of the normal colorectal tissues by 25 cycles of RT-PCR and band intensities of all samples increased and plateaued by 35 cycles (Fig. 1). These results suggest that ICAT plays a physiological role in normal colorectal cells.
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We next examined the expression of ICAT in the corresponding 24 colorectal tumors. Interestingly, the expression level of ICAT was higher in most of tumors than in the corresponding non-cancerous tissues. In 13 of 24 (54%) cases, ICAT expression was more than twofold higher in tumors than in adjacent non-cancerous tissues (cases C6, 7, 9, 10, 13–15, 17, 20–24). In the other 11 cases, ICAT expression was almost equal to (cases C1–5, 18) or less than twofold higher than those in non-cancerous tissues (cases C8, 11, 12, 16, 19) (Fig. 1).
Association of ICAT Expression with Clinicopathological Features
Since increased mRNA expression of the ICAT gene was observed frequently in colorectal carcinomas, we analyzed the relationship between the ICAT expression and various clinicopathological features such as age, gender, tumor localization, histological grade, JS CCR-stage and the mutational status of p53 and K-ras. The cases were divided into two groups according to their levels of ICAT expression: greater than twofold over the non-cancerous tissues in the first group and between one- and two-fold greater in the second group. Of the 24 cases, 13 were in the first group and 11 were in the second group. The histological grade (P = 0.04) and the status of lymphatic permeation (P = 0.02) were significantly different between the two groups (Table 1). The cases exhibiting ICAT overexpression showed a significantly higher incidence of well-differentiated adenocarcinoma and positive lymphatic permeation. On the other hand, no correlation was found between ICAT expression level and other clinicopathological features including age, gender, tumor localization, vessel permeation, the grade of lymph node metastasis, the grade of distant metastasis and stage classification. There was also no significant difference between ICAT expression and the mutational status of p53 and K-ras. In our data set of 24 patients, the mean observation time was 18.2 (2–42) months, the 75% survival time was 16.8 months and 3-year survival was estimated to be 58%. The 50% survival time could not be determined and the observation time was not long enough to calculate 5-year survival. No significant differences (P = 0.6483) have been observed in survival at present between the two groups (Fig. 2) and ICAT expression level was not a prognostic factor. Consistent with these latter results, the expression of other genes involved in Wnt signaling, such as ß-catenin, APC and GSK3ß, have also been reported to have little correlation with the prognosis of colorectal carcinoma patients (27). However, at present our data set is too small and the observation time is too short to be able to establish conclusively the significance of ICAT expression with regard to the survival of patients. Further observation is necessary to clarify whether expression of ICAT is associated with better or worse prognosis.
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In the present study, no ICAT mutations were found in samples from diverse stages of colorectal carcinomas, including liver metastases and benign adenomas. This finding suggests that mutation of the ICAT gene is not a common event in the etiology of sporadic colorectal cancer. In this regard, it is interesting that the RIZ gene, which encodes a retinoblastoma protein-interacting zinc finger protein, has been reported to be frequently mutated in colorectal cancers and may be a critical gene deleted in the chromosome 1p region (28). In contrast, we found that the ICAT gene is expressed constitutively in normal colorectal tissues and overexpressed in almost half of colorectal carcinomas. Cases exhibiting ICAT overexpression showed a significantly higher incidence of well-differentiated adenocarcinoma and positive lymphatic permeation. It is interesting to speculate that ICAT overexpression may play a role in the pathogenesis of colorectal carcinomas. However, it is also possible that ICAT overexpression is a feedback mechanism that downregulates ß-catenin–TCF-mediated transcription aberrantly activated by inactivation of APC or activation of ß-catenin. Further studies are needed to reveal any relationships between overexpression of ICAT and the regulation of colorectal cancer cell growth.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAcknowledgmentsREFERENCES |
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This work was supported in part by Grants-in-Aid from the Foundation for the Promotion of Cancer Research, Japan and Grants-in-Aid for Scientific Research on Priority Areas.
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FOOTNOTES |
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+ For reprints and all correspondence: Tetsu Akiyama, Department of Molecular and Genetic Information, Institution for Molecular and Cellular Biosciences, The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo, Japan. E-mail: akiyama@imcbns.iam.u-tokyo.ac.jp
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAcknowledgmentsREFERENCES |
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Received January 31, 2002; accepted May 15, 2002
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