Department of Medicine and
1 Department of Pathology, University of Colorado Health Sciences Center, Denver, CO 80262 and Department of Veterans Affairs Medical Center, Denver, CO 80220, USA
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Abstract |
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Abbreviations: APC, adenomatous polyposis coli; FAP, familial adenomatous polyposis; Lef, lymphoid enhancer factor; MIN, multiple intestinal neoplasia; TBS, Tris-buffered saline; TBX, Tris-buffered saline containing 1% bovine serum albumin and 0.05% Tween 20; Tcf, T cell factor.
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Introduction |
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Linkage analysis followed by positional cloning in patients with familial adenomatous polyposis (FAP) initially identified the APC gene, and germline mutations in this gene are responsible for this inherited, autosomal dominant predisposition to colon cancer (4). FAP patients, who have a germline mutation in one of the APC alleles, develop hundreds to thousands of adenomatous polyps. A role for APC in colon carcinogenesis has been further corroborated by the development of mouse models of FAP neoplasia (610). These models, such as the MIN (multiple intestinal neoplasia) mouse, have mutations in one allele of the murine homolog of the APC gene and exhibit an autosomal dominantly inherited predisposition to intestinal neoplasia.
Almost all of the mutations found in the APC gene are insertions, deletions or nonsense mutations that lead to a stop codon and result in the production of a truncated protein that has lost the C-terminal end (4). Studies of patients with FAP and in MIN mice indicate that loss of the second APC allele by somatic mutation is frequently detected in adenomas and it is thought that bi-allelic loss of APC is required for adenoma formation in these settings (11,12). Similarly, bi-allelic somatic APC mutations are proposed to occur early in the process of sporadic colonic carcinogenesis. Studies of sporadic human adenomas, however, leave open the possibility that complete loss of normal APC may not be obligatory for adenoma formation because bi-allelic loss of APC has not been uniformly detected (2,13).
One of the current mechanistic hypotheses for the role of APC protein in colon carcinogenesis is that it functions as a negative regulator of cytosolic ß-catenin protein expression (14). APC protein has been shown to be present in a multi-protein complex with ß-catenin and two other proteins, axin and glycogen synthase kinase (15,16). Within this multi-protein complex, glycogen synthase kinase phosphorylates ß-catenin (17), leading to its dissociation from the complex and degradation, which occurs via a ubiquitin-dependent proteasomal pathway (18). In cells with two mutant APC alleles, free levels of cytoplasmic ß-catenin increase, leading to nuclear translocation and binding of ß-catenin to a DNA-binding protein called T cell factor (Tcf) or lymphoid enhancer factor (Lef) (19). This ß-cateninTcf /Lef complex then appears to activate the transcription of genes, such as myc and cyclin D1, that may promote the process of colonic carcinogenesis (20,21).
In the present study, immunohistochemical methods were used to examine the relationship between the expression of full-length APC and ß-catenin protein localization in normal, hyperplastic and neoplastic colonic mucosa in both humans and mice. We set out to test the hypothesis that colon carcinogenesis is mediated by loss of functional APC (loss of both alleles) with resultant activation of the ß-catenin transcriptional activation pathway. If this hypothesis were true, one would expect to find a consistent correlation between loss of full-length APC protein expression and nuclear accumulation of ß-catenin during the adenomacarcinoma sequence.
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Materials and methods |
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Three-week-old CF-1 mice were obtained from Charles River Laboratory and maintained in quarantine for 10 days. Four weeks later mice were injected s.c. with 10 mg/kg azoxymethane (Sigma, St Louis, MO) in saline for six consecutive weeks. Mice were weighed weekly and the stool pellets were examined for blood. At 4 and 6 months after the last azoxymethane injection, animals were killed and the colons were examined for the presence of tumors. Colonic tumors and adjacent normal colonic mucosa were removed from the mice and fixed in formalin and embedded in paraffin.
Tissue preparation and antigen retrieval
APC and ß-catenin immunohistochemistry were performed on formalin-fixed, paraffin-embedded tissue sections using an antigen retrieval protocol. Serial sections (6 µm) of tissue were cut, deparaffinized in Hemo-De (Fisher Scientific, Houston, TX) and rehydrated in a series of graded alcohol:water solutions. Slides were then immersed in a 10x citrate buffer solution (Biogenex, San Ramon, CA) diluted 1:10 in Tris-buffered saline (TBS) and placed in a pressure cooker containing water. Slides were microwaved for 20 min at high power, allowed to cool and then placed in a TBS solution (0.05 M Tris, 0.15 M NaCl, pH 7.6).
APC immunohistochemistry
APC immunohistochemistry was performed using a rabbit polyclonal anti-APC antibody that was raised against a peptide corresponding to the last 20 amino acids of the C-terminus of the human APC protein (Santa Cruz Biotechnology, Santa Cruz, CA) and an avidin/biotin/immunoperoxidase method. After antigen retrieval, endogenous peroxidase activity was blocked by treating sections with 3% hydrogen peroxide in methanol for 10 min. Endogenous biotin was blocked prior to incubation in Powerblock (Biogenex) diluted 1:10 in TBS containing 1% bovine serum albumin (Sigma) and 0.05% Tween 20 (Sigma) (TBX) for 15 min. Sections were then incubated for 1 h with the anti-APC antibody diluted 1:200 in TBX followed by washing in TBS (3x5 min) and then incubated with biotinylated goat anti-rabbit IgG (Zymed, San Francisco, CA) diluted 1:200 in TBX for 30 min. After washing with TBS (3x5 min), the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used for detection with diaminobenzidine tetrahydrochloride (Biogenex) as the substrate. Negative control sections of each sample were incubated with non-immune rabbit IgG (Zymed) in place of the anti-APC antibody. The specificity of the anti-APC antibody was also tested by absorption with a peptide corresponding to the last 20 amino acids of the C-terminus of human APC and by absorption with 1% agarose to exclude non-specific binding of the polyclonal antibody to mucin. Slides were counterstained with hematoxylin, dehydrated, coverslipped and viewed under a light microscope. Tissues were graded positive for APC if definite brown staining was present in >10% of the epithelial cells.
ß-Catenin immunohistochemistry
ß-Catenin, immunohistochemistry was performed using a mouse monoclonal anti-ß-catenin antibody (Transduction Laboratories, Lexington, KY) and an indirect immuno-alkaline phosphatase method. After antigen retrieval, sections were incubated in Powerblock diluted 1:10 in TBX for 15 min followed by incubation with the anti-ß-catenin antibody diluted 1:50 in TBX for 2 h. Sections were then washed with TBS (3x5 min) followed by incubation with the secondary antibody, rabbit anti-mouse Ig (Dako, Denmark), diluted in PBS containing 40% human serum (Gemini, Lexington, CA) for 30 min. Sections were then washed with TBS (3x5 min) and incubated with an alkaline phosphatasemouse anti-alkaline phosphatase complex [3.6 U/ml alkaline phosphatase (Sigma), 1% bovine serum albumin and 10% RU6 mouse antibody (Tissue Culture/Monoclonal Antibody Core Facility, University of Colorado Cancer Center, Denver, CO) in 0.05 M Tris, pH 8.7] for 1 h for detection using the substrate new fuchsin (Sigma). Negative control sections of each sample were incubated with non-immune mouse IgG (Dako) in place of the anti-ß-catenin antibody. Slides were then counterstained with hematoxylin, dehydrated, coverslipped and viewed under a light microscope. Tissues were graded positive for nuclear ß-catenin if nuclear immunoreactivity was present in >10% of the cells.
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Results |
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ß-Catenin immunoreactivity in colonic tissue
In the normal colon ß-catenin expression was predominantly localized to the plasma membrane of normal colonocytes at all levels of the colonic crypt (Figure 1B). There was no gradient of ß-catenin immunoreactivity along the length of the crypt and no nuclear ß-catenin was seen in cells at any level of the crypt (Table I
). Nine hyperplastic polyps that were analyzed showed a pattern of staining similar to that seen in the histologically normal colon. There was no staining observed when the negative control for ß-catenin was used (Figure 1D
).
In all 58 of the adenomatous polyps ß-catenin immunoreactivity was present in both the cytoplasm and the nucleus (Table I and Figure 2B and D
). ß-Catenin immunoreactivity was intense and present in 100% of the cells in all of the adenomatous polyps.
ß-Catenin expression was also cytoplasmic and nuclear in all 83 of the human adenocarcinomas examined (Table I and Figure 3B and D
). The intensity of ß-catenin staining in the cytoplasm of the cells of the colorectal cancers was generally greater than that seen in the adenomatous polyps. All 12 of the mouse adenocarcinomas had cytoplasmic and nuclear staining of ß-catenin.
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Discussion |
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The hypothesis underlying this study was that loss of full-length, functional APC protein early in the process of colonic carcinogenesis should result in the accumulation of cytoplasmic and nuclear ß-catenin that would be detected by immunohistochemistry. Thus we expected to see a strong correlation between the patterns of expression of the two proteins. We did not expect the correlation to be perfect because other mechanisms that affect the ß-catenin degradation pathway can occur, such as mutations in ß-catenin (23) or activation of the wnt signaling pathway (24). Nonetheless, most colorectal adenomas and cancers are thought to have lost function of both APC alleles, which would be expected to be sufficient to cause loss of full-length APC protein immunoreactivity and nuclear ß-catenin accumulation.
Our immunohistochemical results in the normal colon are similar to previous studies (22,2527). We confirmed the preferential expression of APC protein in the cytoplasm of goblet cells (22) and the basolateral membrane localization of ß-catenin in the normal colonic mucosa (27). Nuclear ß-catenin accumulation was not detected in normal colonocytes. Thus in all the samples the presence of full-length, presumably functional APC protein was associated with cell surface ß-catenin expression.
In the colonic cancers, nuclear accumulation of ß-catenin was universal, suggesting that nuclear localization is a common, potentially important and perhaps essential step in the process of colonic carcinogenesis. As expected, total loss of full-length APC protein was seen in most colon cancers (83%) and regional loss was seen in another 7%. Only 10% of the colonic cancers had expression of full-length APC protein throughout the cancer tissue. Thus almost all of the observed nuclear accumulation of ß-catenin in the cancer cells could have been due to the loss of functional APC protein. The 1017% of cancers with nuclear ß-catenin in the presence of APC immunoreactivity could be explained by other mechanisms of ß-catenin accumulation, such as ß-catenin mutations (23).
The results in the normal colon and in the colon cancers were generally consistent with the working hypothesis that loss of APC function is the major mechanism of nuclear ß-catenin accumulation during the process of colonic carcinogenesis. The findings in adenomas, however, were not. Just as was found in the colorectal cancers, nuclear ß-catenin accumulation was universal in colonic adenomas, suggesting that the ß-cateninTcf/Lef transcriptional activation pathway can occur early in and may be essential for the process of colonic carcinogenesis. In ~30% of the colonic adenomas that we examined there was also loss of full-length APC protein associated with the nuclear accumulation of ß-catenin. Over 60% of the adenomas, however, had prominent APC immunoreactivity throughout the tumor and the pattern of immunoreativity did not differ as a function of the histology of the adenomas. There was no difference in the pattern of ß-catenin expression in the adenomas with abundant full-length APC immunoreactivity compared with those that were totally or regionally devoid of the protein.
The finding of full-length APC immunoreactivity in most colonic adenomas, including those with nuclear ß-catenin accumulation, was inconsistent with two widely held hypotheses about the process of colon carcinogenesis. The first is that inactivation of both alleles of the APC gene occurs early in the adenomacarcinoma sequence and the second is that complete loss of full-length APC protein is responsible for nuclear accumulation of ß-catenin in colonic adenomas.
APC is considered a tumor suppressor gene and it is thought that loss of function of both APC alleles is required to promote colon carcinogenesis. In mice having a germline truncating mutation in the APC gene it has been shown that a second APC mutation due to allelic loss was present in all adenomas (11,12). Consistent with this finding, we have previously reported consistent loss of full-length APC protein in MIN mouse adenomas (25). In humans, however, it is not totally clear whether or not both APC alleles are always mutated in colonic adenomas (2,11,28). Levy et al. (11) were able to detect somatic mutations of the wild-type allele in most but not all (19 of 24) adenomas removed from patients with familial polyposis. Powell et al. (2) examined both alleles in 16 sporadic colonic adenomas and found evidence of mutation of both alleles in only 31%, a frequency similar to the 29% of adenomas we found with total loss of APC immunoreactivity. Thus it remains possible that mutation of one APC allele occurs early and may be the first event in the process of carcinogenesis but that loss of the second allele may occur later, at the stage of adenoma to carcinoma progression. Our finding of a much higher frequency of loss of APC immunoreactivity in cancers than in adenomas is consistent with this concept.
There are several alternative explanations of how loss of APC function could occur without loss of full-length APC protein. It is possible that mutations occur in specific regions of the gene that inactivate APC but still result in full-length protein. This seems unlikely, however, in the light of previous studies that have analyzed many colorectal tumors for mutations at APC, which found that >95% of the mutations in APC result in a truncated product (13,28). Certain missense mutations in the APC gene have been identified that cause few phenotypic consequences (29) so that a spectrum of abnormalities in APC protein function are likely to result from different types of mutations. It remains possible that less severe mutations (point mutations and in-frame deletions) in at least one of the APC alleles could occur in adenomas and not result in loss of APC immunoreacitivity with the C-terminal antibody. If this is so, however, such adenomas do not appear to progress to cancer without further APC mutations, since over 80% of the cancers had total loss of APC protein.
There is evidence that the APC protein can form homodimers (30). A second possible explanation for our APC immunohistochemical results is that a truncated APC protein product could dimerize with full-length APC protein and prevent its normal function but not interfere with anti-APC binding to the C-terminal portion of the protein. If such a dominant negative function of the mutant APC protein occurs, it does not appear to inactivate all functions of the APC protein. If it did, there would be no need for loss of the second allele later in the process, and we found that colorectal cancers generally had complete loss of APC immunoreactivity. Thus, loss of a second APC allele appears to provide a further effect to promote carcinogenesis. APC is a large protein (300 kDa) and may have more than one function. There are, for example, earlier studies that have demonstrated that APC can interact with cytoplasmic microtubules (31). Perhaps these other functions of APC are crucial such that when lost there is progression from adenoma to adenocarcinoma.
It is also likely that there are mechanisms other than loss of APC function that result in ß-catenin accumulation in cells. Mutations in ß-catenin that could prevent its normal degradation have been reported in as many as 12.5% of small human adenomas (32) and very commonly in azoxymethane-induced colon cancers (33). Furthermore, mice with dominant stable mutations of the ß-catenin gene that prevent its degradation develop intestinal polyps similar to animals with germline APC mutations (34). Similarly, other proteins involved in the multi-protein complex with APC and ß-catenin, namely axin and glycogen synthase kinase, could also be mutated or abnormally regulated in adenomas. An alternative explanation for our observations is that loss of a single APC allele could be sufficient to cause dysregulation of ß-catenin expression. This is not the case in the otherwise normal colonic mucosa, since the normal colonocytes of both MIN mice and patients with familial polyposis have truncating mutations of one APC allele but do not accumulate nuclear ß-catenin. It remains possible, however, that in the setting of the other abnormalities that are present in a colonic adenoma the loss of a single APC allele could be sufficient to cause nuclear accumulation of ß-catenin.
In summary, the present study demonstrates that nuclear accumulation of ß-catenin occurs early and consistently in the adenomacarcinoma sequence. However, the results are not consistent with the hypothesis that complete loss of APC function occurs early and is totally responsible for nuclear accumulation of ß-catenin in adenomas. ß-Catenin was universally localized to the cytoplasm and nucleus of colonic adenomas and adenocarcinomas, suggesting that it may be a required step in the process of colonic carcinogenesis. In contrast, APC expression was totally absent in 83% of the human adenocarcinomas but in only 29% of the colonic adenomas. The dissociation between loss of APC and nuclear accumulation of ß-catenin suggests the possibility that other pathways that affect ß-catenin may be dysregulated in many adenomas. Alternatively, in the setting of an adenoma loss of a single APC allele could be sufficient to lead to dysregulation of ß-catenin expression.
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Notes |
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Acknowledgments |
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References |
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