ß-Catenin mutation is selected during malignant transformation in colon carcinogenesis

Yasuhiro Yamada1,3, Takeru Oyama1, Yoshinobu Hirose1, Akira Hara1, Shigeyuki Sugie1, Koujiro Yoshida1, Naoki Yoshimi2 and Hideki Mori1

1 Department of Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705 and
2 Department of Pathology, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Activating mutations in the ß-catenin gene is thought to be responsible for the excessive ß-catenin signaling involved in the majority of colon carcinomas in rodent models. Our recent study which indicated that ß-catenin mutations are present frequently in early dysplastic lesions of rat colon induced by a colon-specific carcinogen, azoxymethane led us to perform more specifically a comparative study regarding types of the ß-catenin mutation as well as K-ras mutations between such early appearing lesions and colon tumors. Male F344 rats, 6 weeks old, received s.c. injections of azoxymethane (15 mg/kg body weight) once a week for 3 weeks, and were killed at 16 and 46 weeks of age. Colons of animals killed at 16 weeks of age were processed for early altered lesions. Colon tumors from animals killed at 46 weeks of age were evaluated histopathologically. Laser capture microdissection system was used to obtain DNA of epithelial cells in both intramucosal lesions and colon tumors. After amplification of exon 3 of the ß-catenin gene and exon 1 of the K-ras gene, the products were then sequenced directly in both directions. Mutations in the exon 3 of ß-catenin gene were detected in 22 of 56 early lesions (39.3%) and 21 of 37 colon cancers (56.8%). Remarkably, all ß-catenin mutations detected in the colon tumors converged at codons encoding functionally important residues that may directly mediate ß-catenin degradation, whereas mutations in the early appearing lesions were found to be scattered in the exon 3 of the gene. K-ras mutations were also detected in 24 of 56 early lesions (42.9%) and 11 of 37 colon cancers (29.7%). All K-ras mutations converged at codon 12 and codon 13, even in the early lesions. The results of this study provide evidence for the first time that ß-catenin mutation is selected during the colon carcinogenesis. Our results also suggest that the activation of ß-catenin signaling pathway is not only an initiating event, but also plays a pivotal role in the promotion stage of colorectal carcinogenesis.

Abbreviations: BCAC, ß-catenin-accumulated crypts; ACF, aberrant crypt foci; AOM, azoxymethane; APC, adenomatous polyposis coli


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The stepwise nature of colorectal carcinogenesis has been well recognized in both histological and genetic terms. The advent of epithelial dysplasia reflects the frequency of genetic alteration in the process of colon carcinogenesis, and numerous reports suggest that colonic dysplasia is a hallmark of malignant potential (1). Alterations in the APC or ß-catenin gene are regarded as early critical events during colon carcinogenesis and are therefore considered to play a gate keeper role in the development of colon cancer in both humans and preclinical models (24). Mutations in the APC or ß-catenin gene were proved to repress the degradation of the protein and generate ß-catenin accumulations (5,6). The excessive ß-catenin functions as a transcriptional activator when complexed with members of the T cell factor (Tcf) family of DNA binding proteins (7,8). Furthermore, target genes of ß-catenin signaling pathway, such as c-myc and cyclin D1, are growth-promoting genes, suggesting that this pathway is potentially an oncogenic pathway (9,10).

The identification of ras gene mutations was the first major breakthrough in the molecular genetics of colorectal tumors (11). Specific point mutations of K-ras are found in ~30–50% of colorectal tumors (1). These findings clearly indicate that ras mutations may play a role during the development of a significant proportion of colorectal tumors. Indeed, the importance of ras in colorectal tumorigenesis has been emphasized by the finding that colon cancer cells in which the mutated ras gene has been removed by homologous recombination lost their tumorigenicity (12).

In previous studies, we reported the presence of BCAC4 in rat colonic mucosa that was predisposed to colon cancer (13,14). In addition to the accumulation of oncogenic ß-catenin protein, such early lesions harbored frequent mutations in the ß-catenin gene that are involved in the development of colon tumors (15). Histological observations indicate that BCAC showed dysplasia, a hallmark of malignant potential, and increased size with time after the carcinogen exposure. Interestingly, the BCAC are independent lesions from typical ACF that were described as early appearing lesions and putative preneoplastic lesions of colon cancer (16). Importantly, the proliferative activity of BCAC is significantly higher than the activity in typical ACF (14). These observations indicate that BCAC are premalignant lesions of colon cancer. Indeed, expression of BCAC is markedly suppressed by celecoxib, a COX-2 inhibitor (17) which is a potent inhibitor of colon carcinogenesis (1820), whereas dietary cholic acid, a colonic tumor promoter, enhances BCAC formation (Hirose,Y. et al., in preparation), suggesting that BCAC plays an important role in early stages of colon tumorigenesis.

It is noteworthy that treatment with a colon-specific carcinogen AOM induces ~20–30 BCAC/colon (17). Additionally, there are several studies to demonstrate that AOM induces ~120 ACF/colon with several foci containing multiple aberrant crypts (21). However, it should be recognized that the number of colon tumors induced by this regimen amounts to ~1–2/rat which are very low as compared with those early appearing lesions suggesting that not all lesions ultimately be transformed into tumors. In the current study, we investigated the types of ß-catenin mutations as well as K-ras mutations in early appearing lesions of the colon, including BCAC and ACF, and colon cancers induced in rats by AOM. The ultimate goal of this study was to determine which types of the early appearing lesions progress into malignancies.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental procedure
Seventy male F344 rats, 5 weeks old, were obtained from Shizuoka SLC, (Shizuoka, Japan) and quarantined for 1 week and housed in an animal holding room under controlled conditions. They were maintained on a basal diet, CE-2 (CLEA Japan, Tokyo, Japan) until termination of the study. Beginning at 6 weeks of age, 50 rats intended for carcinogen treatment were given subcutaneous injections of AOM (Sigma Chemical Co., St Louis, MO) at a dose rate of 15 mg/kg body weight once a week for 3 weeks, whereas 20 rats intended for the vehicle treatment were given equal volume of normal saline. During the course of the study, groups of rats were killed at 16 and 46 weeks of age by chloroform anesthesia. At each time point, 10 rats treated with normal saline were killed as negative controls. The colons from rats killed at 16 weeks of age were resected, cut open along their longitudinal axis, and the contents were flushed with normal saline. The colons were fixed flat in 10% buffered formalin for 24 h at room temperature. Colon tumors from animals killed at 46 weeks were also fixed in 10% buffered formalin, and processed for histopathological evaluation by routine procedures (22). For the determination of ACF, fixed colons from rats killed at 16 weeks of age were placed in 0.5% solution of methylene blue in distilled water. They were then placed mucosal side up on a microscope slide and observed through a light microscope at a magnification of x40. ACFs were distinguished from the surrounding `normal-appearing’ crypts by their increased size, prominent epithelial cells and the easily discernible pericryptal zone.

Tissue preparation and immunohistochemistry of ß -catenin
To identify BCAC, colonic mucosa from rats killed at 16 weeks of age was embedded in paraffin for histological analysis, and sections were examined by utilizing an en face preparation and 3–5 µm thick serial sections (13,14,17). Immunohistochemical analysis for ß-catenin protein was performed on sections of both early appearing lesions and colon tumors, using the labeled streptavidin biotin method (LSAB KIT; DAKO, Glostrup, Denmark) with microwave accentuation. The paraffin-embedded sections were heated for 30 min at 65°C, deparaffinized in xylene, and rehydrated through graded alcohols at room temperature. A 0.05 M Tris–HCl buffer (pH 7.6) was used to prepare solutions and for washes between various steps. Incubations were performed in a humidified chamber. Sections were treated for 40 min at room temperature with 2% bovine serum albumin and incubated overnight at 4°C with primary antibodies against ß-catenin protein (diluted 1:1000, Transduction Laboratories, Lexington, KY). For each case, negative controls were prepared on serial sections as described above except incubation with the primary antibody was omitted. Horseradish peroxidase activity was visualized by treatment with H2O2 and diaminobenzidine for 5 min. Immunoreactivities were regarded as positive if the stainings were detected in cytoplasm and/or nuclei of early appearing lesions.

Laser capture microdissection
A total of 103 samples (10 normal-appearing crypts, 28 ACFs, 28 BCACs, and 37 colonic tumors) were microdissected from histological sections. Early lesions and normal-appearing crypts were randomly selected from colons of 10 AOM-treated rats killed at 16 weeks of age. Thirty-seven colonic tumors were picked up from 30 tumor-bearing rats killed at 46 weeks of age. LM100 (Olympus, Tokyo, Japan) was used to obtain laser captures of epithelial cells in normal-appearing crypts, intramucosal lesions, and colon tumors, using an amplitude of 50 mW, a duration of 800 µs and a 7.5 µm beam. DNA was extracted from the Capture lids (Arcturus Engineering, Mountain View, CA) containing microdissected tissue in 25 µl lysis buffer incubated overnight at 37°C.

DNA sequencing and mutation analysis of ß-catenin and K-ras gene
Exon 3 of the ß-catenin gene and exon 1 of the K-ras gene were amplified using Pfu polymerase (Promega, Madison, WI). The primers used here were the same as those in previous studies (13,23) and were designed to PCR-amplify the regions corresponding to exons 2 and 3 (codons 1–57) of ß-catenin including intron 2 and the regions corresponding to exon 1 of K-ras, respectively. Primers (IF4 and R3 for ß-catenin, K1-1 and K1-2 for K-ras) were included in the following PCR reaction mixture containing in a total volume of 20 µl: 20 µM of each primer, 200 µM of each deoxynucleotide triphosphate, 1 unit of Pfu polymerase in 1xPCR buffer (Promega), and template DNA. The mixture was heated at 94°C for 5 min and subjected to 30 cycles of denaturation (94°C, 45 s), annealing (57°C, 45 s) and extension (72°C, 1 min) using a GeneAmp PCR Systems 9700 (Applied Biosystems, Foster City, CA). The products were sequenced directly after gel-purification in both directions using a BigDye Terminator Cycle Sequencing kit (Applied Biosystems) according to the manufacturer’s recommendations. Reactions were analyzed on an ABI Prism 3100 DNA Sequencer (Applied Biosystems). DNAs from colonic tumors, in which ß-catenin and K-ras status have been determined, were used for positive controls. In order to determine the mutation status in each allele, if necessary, PCR products were sequenced after subcloning. All mutations at codon 12 (GGT -> GAT) and codon 13 (GGC -> GAC) of K-ras gene were confirmed by PCR–RFLP method using mismatched 5' primers and Hph I (New England Biolabs, Beverly, MA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Two types of early appearing lesions and ß-catenin immunohistochemistry
In the cross section of AOM-treated rats killed at 16 weeks of age, two populations of altered crypts were histologically detected in colonic mucosa of all animal examined, whereas these lesions were absent in vehicle-treated rats. As shown in Figure 1Go, one population of altered crypts exhibited a typical ACF appearance (arrowhead), which could be easily distinguished from the surrounding `normal-appearing’ crypts by their increased size, prominent epithelial cells, and increase of pericryptal space on whole mount preparations stained with methylene blue. The second population of altered crypts lacked typical ACF appearance, including the size, pericryptal space and raised surface from surrounding mucosa (open arrowhead in Figure 1Go). Analysis of ß-catenin protein was performed by immunohistochemistry. It is noteworthy that, in the present study, the typical ACF did not show obvious immunoreactive ß-catenin in both cytoplasm and nuclei as reported in our previous publications, whereas early lesions without typical ACF appearance, as well as colon tumors, showed the immunoreactivity in their cytoplasm and/or nuclei, which are termed as BCAC (13,14) (Figure 1D,FGo). In histological sections with HE staining, BCAC consisted of adenomatous crypts. Those crypts bore basophilic cytoplasm and hyperchromatic nuclei, and had an increase in the nuclear/cytoplasmic ratio. Mucin production was absent in those crypts (Figure 1EGo). In contrast, typical ACF did not exhibit adenomatous crypts and crypts in ACF kept cellular and nuclear polarity (Figure 1CGo).



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Fig. 1. (A) and (B) Topographic view of methylene blue-stained mucosa of whole mount colons (A) and histological view of the corresponding area (B). Two populations of altered crypts are histologically detected at the cross section of cancer-predisposed colonic mucosa. The first type of altered crypts has typical ACF appearance which could be easily distinguished by their increased size, prominent epithelial cells, and increase of pericryptal space from surrounding `normal-appearing’ crypts on whole mount preparations stained with methylene blue (arrowhead). The second type of altered crypts lacked typical ACF appearance (open arrowheads), including the size, pericryptal space and raised surface from surrounding mucosa. *Represents lymph follicles in the colonic mucosa. (C–F) histological sections and ß-catenin immunohistochemistry of early appearing lesions. (C) and (D) ACF exhibit the ß-catenin immunoreactivity only in their membrane. (E) and (F) the increased immunoreactivity of ß-catenin is evident in BCAC. Note that ß-catenin is detected in both cytoplasm and nuclei. Bars, 100 µm.

 
Mutations in ß-catenin and K-ras gene
Histological sections of 37 colon tumors were examined for tumor types and all classified as adenocarcinomas. Our results indicate that most colon tumors were well differentiated tubular adenocarcinomas, except six cases that were poorly differentiated adenocarcinomas. By direct sequencing, all negative controls obtained from adjacent normal-appearing crypts showed wild type sequencing. Tables I and IIGoGo summarize the results of mutational analysis of ß-catenin and K-ras in early appearing lesions and in colon tumors. Mutations in exon 3 of ß-catenin gene were detected in 22 of 56 early lesions (39.3%) and 21 of 37 colon adenocarcinomas (56.8%). As summarized in Figure 2Go, ß-catenin gene mutations in the early altered lesions were scattered at exon 3 of the gene, whereas all mutations detected in colon adenocarcinomas converged at the codons 32–34, 37, 41 and 45 that are suggested to be functionally important codons for ß-catenin degradation. As shown in Table IIGo, ß-catenin mutations were detected in both well differentiated and poorly differentiated adenocarcinomas. K-ras mutations were found in 24 of 56 early lesions (42.9%) and 11 of 37 adenocarcinomas (29.7%) (Tables I and IIGoGo). No K-ras mutations were detected in poorly differentiated adenocarcinomas. All K-ras mutations that were detected in this study converged at codon 12 and codon 13, even in the early lesions. These ras mutations were all confirmed by PCR-RFLP method (data not shown).


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Table I. ß-Catenin mutations in early appearing lesionsa
 

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Table II. ß-Catenin mutations in colon cancersa
 



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Fig. 2. Sequencing analysis and identification of ß-catenin mutations. (a) The specific mutations at the functionally significant sequence in exon 3 of the ß-catenin gene (mutations at codons 32, 34 and 41 of ß-catenin detected in case E37, C26 and C29, respectively). (b) ß-Catenin mutations at the other site (mutations at codons 10, 29 and 50 were recognized in case E33, E25 and E29, respectively). (c) DNA sequence of the rat ß-catenin gene and nucleotide changes. Black capitals indicate DNA sequence of wild type rat ß-catenin gene (23). Yellow boxes represent functionally important codons for ß-catenin degradation (5,24). Nucleotide changes found in this study are shown in blue for early lesions, and red for colon cancers. The numbers adjacent to changed nucleotide indicate the number of lesions with mutations. Figures in parentheses represent nucleotide changes and the number of mutations as reported in previous reports (4,13,23).

 
Mutations in BCAC and ACF
Among the early lesions, ACF showed more frequent K-ras mutations (20 of 28, 71%) than BCAC (4 of 28, 14.3%) (P < 0.001). ß-Catenin mutations were detected in both BCAC and ACF. BCAC harbored the ß-catenin mutations more frequently (13/28, 46%), although not statistically significant, than ACF (9/28, 32%). The specific types of ß-catenin mutations were frequently detected in BCAC (7 of 28, 25.0%).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alterations in the APC or ß-catenin gene are considered to play a gate keeper role in the development of colon cancers in both humans and preclinical models (24). It has been demonstrated that ß-catenin levels are regulated by the degradation of protein via ubiquitin-proteasome pathway, and that functionally significant residues for the ubiquitination are shown to be encoded by codons 32–35, 37, 41, and 45 in exon 3 of the gene (5,24). In the current study, we found that ß-catenin gene mutations in the early altered lesions in the colon carcinogenesis are scattered at exon 3 of the gene, whereas all mutations detected in colon adenocarcinomas converge at the specific codons that encode the important residues for ß-catenin degradation. These findings indicate that only early lesions with the specific type of mutations (codons 32–35, 37, 41, and 45) preferentially progress into colon cancers. Results of the present study suggest that a broad spectrum of mutations is selected during the malignant transformation of the colon, and the main selective factor will be an inactivation of the ß-catenin downregulating function. Furthermore, our data also suggest that ß-catenin mutation is not only an initiating event but also plays a critical role in the progression of colon carcinogenesis.

Although the significance of ß-catenin mutations without the specific site (without codons 32–35, 37, 41, and 45) is unclear, it is noteworthy that the residues close to such a functionally important site for ß-catenin degradation also have a slight effect on the ubiquitination of the ß-catenin (5). Mutations at the neighboring codons may result in weak accumulation of the oncogenic ß-catenin, leading to a little growth advantage. Accordingly, it is possible that slight accumulation of ß-catenin protein is sufficient to the formation of early appearing lesions, although tumor development does require sufficient accumulation of ß-catenin. In contrast, K-ras mutations detected in the present study converged at the specific codons (codons 12 and 13), even in the early appearing lesions. The specific K-ras mutations may be due to a genotoxic effect of azoxymethane causing adduct formation at such specific sequences (25). It is also possible that mutations close to codons 12 and 13 of the K-ras do not have any selective advantage, so that we could not detect the neighboring mutations. Further investigations about the precise role of K-ras mutations may elucidate the significance of the mutations in early stages of colon carcinogenesis.

During the last decade, numerous studies have focused on the significance of ACF as early events in colon carcinogenesis, and ACF are now regarded as putative preneoplastic lesions for colon cancers (16,26). It has been widely accepted that the increase in cryptal size on the whole mount preparation is the most distinctive feature of ACF. Previously, we reported the presence of BCAC with increased proliferative activity (13,14). Interestingly, the BCAC frequently consisted of small crypts rather than large crypts on whole mount preparations with methylene blue staining, which are independent lesions from typical ACF. In this study, BCAC frequently harbored the specific type of ß-catenin mutations as recognized in colon cancers. In addition to the ß-catenin accumulations, these findings provide additional evidence that BCAC, which lack typical ACF appearance, are premalignant lesions of colon cancer.

Remarkably, in the current study, we found that K-ras mutations were present in >70% of total ACF. The observation is consistent with previous reports in humans, demonstrating that the majority of non-dysplastic ACF have K-ras mutations (27,28). The results clearly indicate that the K-ras mutations are closely associated with the formation of ACF. It is also interesting to note that ACF, which did not show clear accumulation of ß-catenin, harbored the specific mutation in the ß-catenin gene, suggesting that a part of ACF with such specific mutations possesses neoplastic potential leading to colon cancers. These results also suggest that this mutation is insufficient for the detectable accumulation of ß-catenin. It is conceivable that another genetic and/or epigenetic alterations occur in BCAC, but not in typical ACF, resulting in translocation of ß-catenin into cytoplasm and/or nuclei in BCAC.

In summary, we have shown that ß-catenin mutation is selected during the colon carcinogenesis indicating that a broader spectrum of mutation is selected during the malignant transformation. Our results also provide evidence for the first time that the activation of ß-catenin signaling pathway plays a significant role during initiation and promotion stages of colon carcinogenesis.


    Notes
 
3 To whom correspondence should be addressed Email: y-yamada{at}cc.gifu-u.ac.jp Back


    Acknowledgments
 
We thank M.Yasuda for technical assistance in the sequencing analysis. We also thank K.Takahashi, T.Kajita, H.Shibazaki and K.Kinjyo for assistance. This study was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health, Labor and Welfare and the Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 27, 2002; revised July 18, 2002; accepted August 20, 2002.