Frequent Mutation of β-Catenin and APC Genes in Primary Colorectal Tumors from Patients with Hereditary Nonpolyposis Colorectal Cancer

Introduction

HNPCC 3 (1) is principally characterized by inactivation of DNA mismatch repair genes, resulting in high replication error at microsatellite loci (2, 3, 4, 5, 6, 7) as well as at repeated sequences in coding regions, such as (A)₁₀ in TGRβRII (8) and (G)₈ in BAX genes (9) . In some patients, a small number of adenomatous polyps can be found in colorectal tissues, in addition to carcinomas (10) . However, the molecular mechanisms by which tumors are developed in HNPCC patients have not yet been fully elucidated. We have previously demonstrated that the molecular nature of HNPCC tumors with severe (high) replication error at microsatellite loci (MSI-H) is quite different from that of FAP and sporadic cases with low MSI or without MSI: the frequency of APC gene mutation was found to be low (∼20%) in tumors from HNPCC patients (6) but high in non-HNPCC tumors, in which >70% of cases had mutation and/or loss of heterozygosity (LOH) (11) .

It is known that wild-type APC protein forms a complex with β-catenin and GSK3β, inducing degradation of β-catenin in normal cells, whereas in tumor cells, mutant APC protein lacks the ability of complex formation and β-catenin accumulates in nuclei (12 , 13) . This causes interaction of β-catenin with the nuclear transcription factor (Lef/Tcf) family, resulting in activation of target genes, including cyclin D1 (14 , 15) . Similar accumulation of β-catenin has been demonstrated in colorectal carcinoma cell lines without APC mutations but with mutations in the regulatory domain (codons 29–48) of the β-catenin gene, suggesting that activation of the β-catenin-Tcf signaling pathway by mutation in either the APC or β-catenin gene contributes to colorectal carcinogenesis (16) . Such activating mutations of the β-catenin gene have occurred in some sporadic primary colorectal tumors (17, 18, 19) and in various other tumors. These results imply that, in addition to its function as a cell adhesion protein associating with E-cadherin, β-catenin acts as a potential oncogene. To assess the possible contribution of this signaling pathway in HNPCC carcinogenesis, we analyzed β-catenin mutation in colorectal tumors from HNPCC patients.

Materials and Methods

Tumor Samples.

Twenty-eight primary colorectal tumors, including 23 invasive carcinomas, 2 intramucosal carcinomas, 2 adenomas, and 1 hyperplastic polyp, were obtained from 18 patients in Japanese HNPCC families (Table 1) ⇓ . These families included 9 cases with germ-line mutations of the hMSH2, hMLH1, and hMSH6 genes (6 , 7 , 20) and 9 cases who had no identified germ-line mutation but fulfilled the Amsterdam criteria for HNPCC, according to the following definitions: (a) at least three family members with colorectal carcinoma, two of whom were first-degree relatives; (b) at least two generations were represented; and (c) at least one individual was younger than 50 years of age at diagnosis (1) . Almost all tumors (27 of 28) exhibited severe (high) replication error at microsatellite loci (MSI-H), as reported previously (6 , 7 , 20) , and one tumor showed low MSI. As summarized in Table 1 ⇓ , six of these tumors had APC gene mutations, as described in our previous report (6) . Forty primary colorectal tumors from FAP, 45 sporadic tumors with APC mutation, and 36 sporadic tumors without APC mutation were included in previous reports (6 , 11) .

Mutation Analysis of β-catenin gene.

Genomic DNA samples from tumors and the corresponding normal tissues were extracted using proteinase K and chloroform-phenol. DNA samples were amplified for single-strand conformation polymorphism (SSCP) analysis of the β-catenin gene using PCR under the same conditions as reported previously for APC and mismatch repair genes (6 , 11) . Primers used to amplify exon 3 of the β-catenin gene were the same as those reported (21) . When abnormal bands were detected in the SSCP analysis, single-stranded DNA fragments were extracted, amplified by asymmetrical PCR, and then subjected to direct sequencing by dideoxy chain-termination reaction as described previously (11) .

Immunohistochemical Analysis.

Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tumor tissues by the avidin-biotin complex method. Sections of tumor tissues on glass slides were dried, deparaffinized, and stained using mouse antihuman β-catenin monoclonal antibody clone 14 (Transduction Laboratories, Lexington, KY).

Results and Discussion

Exon 3 of the β-catenin gene contains the regulatory domain of β-catenin, of which mutations have been detected in various sporadic tumors, resulting in its nuclear accumulation. In this study, mutation of this exon was analyzed in genomic DNA of 28 colorectal tumors from HNPCC patients with germ-line and/or somatic mutations of hMSH2, hMLH1, and hMSH6 genes (6 , 7 , 20) and from patients in families fulfilling the Amsterdam criteria for HNPCC. In the PCR-SSCP analysis, DNA fragments of aberrant bands detected in tumor DNA but not in corresponding normal tissue DNA were further amplified by asymmetrical PCR, followed by direct sequencing (Fig. 1) ⇓ .

Somatic β-catenin gene mutations were detected in 12 HNPCC colorectal tumors, including 11 carcinomas and 1 adenoma, in which no APC gene mutation was detected (Table 1) ⇓ . These mutations were single-base substitutions that were located within the NH₂-terminal regulatory domain of β-catenin (codons 29–48): TCT (Ser) → TTT (Phe), CCT (Pro), or TGT (Cys) at codon 45; ACC (Thr) → GCC (Ala) at codon 41; TCT (Ser) → TGT (Cys) at codon 37; GGA (Gly) → GAA (Glu) at codon 34; GAC (Asp) → GGC (Gly) or TAC (Tyr) at codon 32; and a 3-bp deletion at codon 43. One carcinoma (HNP15CoCa) had two of these mutations in one allele. All the mutations occurred at the same codons as those previously detected in sporadic colorectal and other tumors, and 75% (9 of 12) of single-base substitutions were transition type.

Immunohistochemical staining of HNPCC carcinomas with β-catenin mutation showed a high level of β-catenin protein in both cytoplasm and nuclei, with stronger staining in the nuclei, although in normal epithelial cells of the colon β-catenin protein was localized in the membrane but not in the nuclei (Fig. 2) ⇓ . This implies that the mutated β-catenin is accumulated in nuclei and may result in activation of transcription of specific genes as proposed (13, 14, 15, 16) .

In this study, β-catenin mutation was not detected in six HNPCC tumors with APC mutations (Tables 1 ⇓ and 2) ⇓ , 45 sporadic colorectal tumors with APC gene alterations (mutation and/or LOH), or 40 colorectal tumors from patients with FAP. Alternatively, APC mutation was not detected in 12 HNPCC colorectal tumors with β-catenin mutations (Table 1 ⇓ and 2) ⇓ . Consistent with this observation, absence of β-catenin mutation has been reported in sporadic colorectal tumors with APC mutation (19) . Such alternate exclusion was also observed between p53 mutation and BAX mutation in these HNPCC tumors (data not shown). However, mutations in the TGFβRIIgene were coexistent in 9 of 12 HNPCC tumors with mutations of β-catenin (Table 1) ⇓ . Coexistence of mutations of TGRβRII and APC was also observed in two of six HNPCC tumors with APC mutations. These results suggest the possibility that TGRβRII mutation, which frequently occurs following replication error in HNPCC tumors at an early stage of tumorigenesis (6 , 7) , does not substitute for the activated β-catenin-Tcf signaling pathway.

HNPCC patients intrinsically have a germ-line mutation in one allele of a mismatch repair gene, and a single hit to the remaining allele inactivates of this gene. Accordingly, HNPCC patients are liable to defects in mismatch repair, which results in MSI and an elevated frequency of mutations in various genes (22) . Spectra of spontaneous mutations have recently been determined by mutation at the hprt locus in colorectal cell lines deficient in mismatch repair and showing the MSI-H phenotype (23 , 24) . Mutation frequency in these cells was 2 orders higher than that in wild-type repair-proficient cells: both single-base substitutions (57 and 66%) and frameshifts (43 and 28%) occurred in hMLH1- and an hPMS2-deficient cell lines, respectively, whereas single-base substitution (92%) was preferentially observed in an hMSH6-deficient cell line. These data suggest that frequent single-base substitution of β-catenin detected in this study (4.3%; 12 of 28) may be due to MSI of HNPCC tumors, although a rather low frequency of this mutation (10%, 1 of 10) has previously been observed in other HNPCC cases (17) . In contrast, sporadic primary colorectal tumors show much lower mutation frequencies of the β-catenin gene, ranging from 1 to 15% (17, 18, 19) ; this value was 4% (3 of 81) in our study.

The frequency of APC gene mutation in HNPCC tumors was lower (∼20%) than alteration of this gene in sporadic tumors (>70%). One possible explanation is that inactivation of the APC gene by mutation plus LOH occurs at a high frequency in sporadic colorectal tumors, but LOH is very rare at various chromosomal regions in HNPCC tumors (3 , 6) . The APC gene has many A-repeat regions, but the number of repeats in each region is not more than six, compared to (A)₁₀ in TGRβRII, which may also be a reason for infrequent frameshift mutation in the APC gene, even in HNPCC tumors with the MSI-H phenotype.

We conclude from these findings that either activating mutation of β-catenin or inactivating mutation of APC may significantly contribute to HNPCC colorectal carcinogenesis. Because mutations in either APC or β-catenin have been known to be associated with adenoma formation, this observation may imply that more than half of HNPCC carcinomas develop via the adenoma-carcinoma sequence. Further analysis of genetic changes in HNPCC may be important for full understanding of the mechanisms of colorectal carcinogenesis in humans.