Beta-catenin mutations are more frequent in small colorectal adenomas than in larger adenomas and invasive carcinomas

Loss of serine or threonine phosphorylation sites from exon 3 of beta-catenin has been identified in approximately half of colorectal tumors which lack adenomatous polyposis coli (APC) mutations, but the overall contribution of beta-catenin mutations to sporadic colorectal tumorigenesis is unclear. We therefore used PCR to amplify and sequence exon 3 of beta-catenin from 202 sporadic colorectal tumors. Exon 3 beta-catenin mutations were identified in 6 of 48 small (< 1 cm) adenomas, 2 of 82 large (> or =1 cm) adenomas, and 1 of 72 invasive carcinomas. Eight of the nine mutations, including all of those in the small adenomas and the invasive cancer, involved loss of serine or threonine phosphorylation sites. The percentage of beta-catenin mutations in small adenomas (12.5%) was significantly greater than that in large adenomas (2.4%) and invasive cancers (1.4%; P = 0.05 and P = 0.02, respectively). We conclude that mutation of beta-catenin can be an early, perhaps initiating, event in colorectal tumorigenesis. Small adenomas with beta-catenin mutations do not appear to be as likely to progress to larger adenomas and invasive carcinomas as other adenomas, however, with the result that beta-catenin mutations are only rarely seen in invasive cancers. This suggests that APC and beta-catenin mutations are not functionally equivalent, and that the APC gene may have other tumor suppressor functions besides the degradation of beta-catenin.     

Investigation of pathogenic mechanisms in multiple colorectal adenoma patients without germline APC or MYH/MUTYH mutations

Patients with multiple (5-100) colorectal adenomas (MCRAs) often have no germline mutation in known predisposition genes, but probably have a genetic origin. We collected a set of 25 MCRA patients with no detectable germline mutation in APC, MYH/MUTYH or the mismatch repair genes. Extracolonic tumours were absent in these cases. No vertical transmission of the MCRA phenotype was found. Based on the precedent of MYH-associated polyposis (MAP), we searched for a mutational signature in 241 adenomatous polyps from our MCRA cases. Somatic mutation frequencies and spectra at APC, K-ras and BRAF were, however, similar to those in sporadic colorectal adenomas. Our data suggest that the genetic pathway of tumorigenesis in the MCRA patients' tumours is very similar to the classical pathway in sporadic adenomas. In sharp contrast to MAP tumours, we did not find evidence of a specific mutational signature in any individual patient or in the overall set of MCRA cases. These results suggest that hypermutation of APC does not cause our patients' disease and strongly suggests that MAP is not a paradigm for the remaining MCRA patients. Our MCRA patients' colons showed no evidence of microadenomas, unlike in MAP and familial adenomatous polyposis (FAP). However, nuclear beta-catenin expression was significantly greater in MCRA patients' tumours than in sporadic adenomas. We suggest that, at least in some cases, the MCRA phenotype results from germline variation that acts subsequent to tumour initiation, perhaps by causing more rapid or more likely progression from microadenoma to macroadenoma.     

Mutations of the PIK3CA gene in hereditary colorectal cancers

Somatic mutations of the PIK3CA gene have recently been detected in various human cancers, including sporadic colorectal cancer. However, mutations of the PIK3CA gene in hereditary colorectal cancers have not been clarified. To elucidate the mutation status in familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC), which are the most common hereditary colorectal cancers, we investigated PIK3CA mutations in 163 colorectal tumors, including adenomas, intramucosal carcinomas and invasive carcinomas. For comparison, we also analyzed mutations of the same gene in 160 sporadic colorectal tumors at various histopathological stages. Analysis at exons 1, 7, 9 and 20 of the PIK3CA gene revealed somatic mutations in 21% (8 of 39) of FAP invasive carcinomas, 21% (7 of 34) of HNPCC invasive carcinomas, 15% (8 of 52) of sporadic invasive carcinomas, and 14% (7 of 50) of sporadic colorectal metastases in the liver. Mutations in FAP and HNPCC carcinomas predominantly occurred in the kinase domain (exon 20), while the majority of mutations in sporadic cases occurred in the helical domain (exon 9). Adenomas and intramucosal carcinomas from all patients exhibited no mutations (0 of 148). Our data suggest that PIK3CA mutations contribute to the invasion step from intramucosal carcinoma to invasive carcinoma in colorectal carcinogenesis in FAP and HNPCC patients at a similar extent to that seen in sporadic patients.     

Alteration of O6-methylguanine-DNA methyltransferase in colorectal neoplasms in sporadic and familial adenomatous polyposis patients

DNA repair failure is known to be a critical event during carcinogenesis of colorectal cancers. To investigate whether O(6)-methylguanine-DNA methyltransferase (MGMT) is altered during colorectal carcinogenesis, we performed immunohistochemical staining on 265 sporadic colorectal cancers, 113 sporadic adenomas, 33 familial adenomatous polyposis (FAP) colorectal cancers, and 93 FAP adenomas. Sixty-seven of 265 sporadic colorectal cancer cases and five of 113 sporadic adenoma cases showed loss of MGMT expression (P < 0.001). Among FAP patients, four of 33 cancers and six of 93 adenomas showed loss of MGMT protein expression. When we compared the association between MGMT promoter hypermethylation and protein expression, almost all cases without a methylated allele were positive for the expression of MGMT. In contrast, cases with promoter methylation frequently showed loss of MGMT expression (P < 0.01). Loss of MGMT was correlated with some clinicopathological characteristics, i.e., tumor invasion (P = 0.013) and stage (P = 0.035) in sporadic colorectal cancer, and degree of atypism (P = 0.042) in sporadic adenoma. Our results show that loss of expression of MGMT occurs more frequently in cancer than in adenoma in both sporadic and FAP patients, and that loss of expression of MGMT is associated with hypermethylation of the promoter area of MGMT gene.     

Bottom-up histogenesis of colorectal adenomas: origin in the monocryptal adenoma and initial expansion by crypt fission

The adenoma:carcinoma sequence is well established. Understanding the molecular pathology of the adenoma is therefore important. There is great controversy within the field. The Vogelstein group champions the "top-down" theory (colorectal adenomas arise and grow across the mucosal surface and down into the crypts), whereas other studies, including our own, propose "bottom-up" spread. Serial sections of 40 small (<3 mm) sporadic colorectal adenomas were stained with H&E, MIB-1, and for beta-catenin. 10 early adenomas were Feulgen-stained and microdissected. We also examined the flat mucosa of three patients who had undergone colectomies for familial adenomatous polyposis (FAP) and specimens from a XO/XY individual with FAP, the latter using in situ hybridization for the Y chromosome. In the earliest sporadic adenomas, there were crypts entirely filled with adenomatous epithelium, which showed proliferative activity and nuclear localization of beta-catenin. There was a sharp cutoff between crypt epithelial cells showing nuclear beta-catenin and surface cells with membrane staining. In slightly larger lesions, adenomatous spread from above was seen. Microdissected adenomas showed multiple fission events, with proliferation distributed equally throughout. In FAP tissue, numerous isolated monocryptal adenomas, which were clonal in origin, were seen. Examination of adenomas in the XO/XY individual showed no instances of XY or XO adenomatous epithelium growing down into crypts of the other genotype. Both sporadic and FAP adenomas start as a unicryptal adenomas and grow initially by crypt fission--a bottom-up pattern. Later, in sporadic adenomas, there is evidence of growth down into adjacent crypts (top-down).     

Nuclear accumulation of beta-catenin without an additional somatic mutation in coding region of the APC gene in hepatoblastoma from a familial adenomatous polyposis patient

The APC (adenomatous polyposis coli) gene status in a familial adenomatous polyposis (FAP) patient who developed hepatoblastoma was analyzed by the yeast color assay. Although a single base insertion at codon 462 resulting in truncation of its product was documented in hepatoblastoma cells, no additional somatic mutation was detectable in the whole coding sequence of the APC gene. The nuclear accumulation of beta-catenin without mutation in the exons 2-4 of the beta-catenin gene, however, was observed in the tumor cells by immunohistochemistry. The similar nuclear accumulation of beta-catenin without an additional somatic mutation in its gene, in the absence of somatic mutation in cluster region of the APC gene, has been previously reported in the single FAP case. Moreover, review in the hepatoblastoma cases in the FAP families showed a relatively later onset of the disease when compared with the sporadic cases. These observations suggest that accumulation of beta-catenin without an additional somatic mutation in the APC gene might be a possible mechanism for tumorigenesis of hepatoblastoma in the FAP families.     

Nuclear localization of immunoreactive beta-catenin is specific to familial adenomatous polyposis in papillary thyroid carcinoma.

Thyroid carcinoma is the first symptom in some patients with familial adenomatous polyposis (FAP). We evaluated the cellular localization of beta-catenin in thyroid carcinomas associated (n = 4) or not associated (n = 173) with FAP, since loss of functional protein of the adenomatous polyposis coli (APC) gene leads to nuclear accumulation of beta-catenin in adenomas and carcinomas of the FAP colon. Immunoreactive beta-catenin was demonstrated at the cell membrane of glandular cells of the non-neoplastic thyroid and non-FAP carcinomas. On the other hand, cytoplasmic and nuclear accumulation of beta-catenin is specific to FAP-associated papillary carcinomas. The abnormality in the APC / beta-catenin pathway is thus also important in FAP-associated thyroid carcinoma, and beta-catenin immunohistochemistry is a feasible screening method to identify occult FAP in young patients with thyroid tumors.     

大肠肿瘤APC基因突变的研究

目的检测ＡＰＣ基因在国人散发性大肠肿瘤中的突变情况,并探讨ＡＰＣ基因突变与大肠肿瘤生物学行为的关系。方法采用ＰＣＲＳＳＣＰ、ＤＮＡ测序对２７例（３３份标本）大肠息肉,３６例大肠癌和相应正常粘膜及３例家族性腺瘤性息肉瘤（ＦＡＰ）标本中ＡＰＣ基因“突变密集区”（ｍｕｔａｔｉｏｎｃｌｕｓｔｅｒｒｅｇｉｏｎＭＣＲ）的突变进行了研究。结果大肠息肉和癌组织中,ＡＰＣ基因ＭＣＲ突变率分别为３０．３％（１０／３３）和３５．９％（１４／３９）,两者差异无显著性,而正常大肠粘膜、炎性息肉和增生性息肉均无突变。３例ＦＡＰ发现有２例突变,其中１例从正常粘膜到腺瘤、到癌变组织均有突变,另１例则在＞１．０ｃｍ的腺瘤和肝转移癌组织有突变,测序结果证实该例在１４２５号密码子存在相同点突变。ＡＰＣ突变与肿瘤的临床病理特征无明显的相关性。结论ＡＰＣ不仅在ＦＡＰ中存在突变,而且在散发性大肠肿瘤中亦存在突变;它的突变至少参与了部分大肠癌的发生发展过程,而且是这一过程中较早发生的事件。     

Difference in characteristics of APC mutations between colonic and extracolonic tumors of FAP patients: variations with phenotype

To clarify the differences in characteristics of adenomatous polyposis coli (APC) mutations between colorectal tumors from various phenotypes of familial adenomatous polyposis (FAP) and between colorectal and extracolonic tumors, we analyzed APC mutations in 86 colorectal tumors from FAP patients including profuse, sparse and attenuated types, 23 sporadic colorectal tumors and 40 FAP extracolonic tumors including duodenal, gastric and desmoid tumors. In all tumors, 1 allele of the truncated APC gene retained armadillo repeats, 15-amino-acid (aa) repeats and various numbers of 20-aa repeats, but lacked SAMP repeats, as a result of germline and somatic mutations. Regarding 20-aa repeats, the truncated APC gene retained 1 repeat due to allele loss in 96% (27/28) of colorectal tumors from profuse-type FAP, 69% (36/52) of sparse-type retained 2 repeats due to somatic mutation, and 100% (6/6) of attenuated-type retained 2 or 3 repeats due to the third or second hit. In sporadic colorectal tumors 74% (17/23) retained 1 or 2 repeats. The truncated APC gene retained 3 repeats in 88% (7/8) of FAP duodenal tumors, 100% (26/26) of gastric tumors retained 2 or 3 repeats and 83% (5/6) of desmoid tumors retained 2 repeats. These data suggest that the number of beta-catenin downregulating 20-aa repeats in truncated APC gene associated with colorectal tumorigenesis is different in profuse, sparse and attenuated types of FAP, and that the association with tumorigenesis is also different between colorectal and extracolonic tumors.     

Difference in the role of loss of heterozygosity at 10p15 (KLF6 locus) in colorectal carcinogenesis between sporadic and familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer patients

OBJECTIVES: To clarify the role of the KLF6 (Kruppel-like factor 6) locus in multistep colorectal carcinogenesis, we analyzed loss of heterozygosity (LOH) at 10p15 (KLF6 locus) and mutations of the KLF6gene in 298 colorectal tumors at various pathological stages of sporadic and familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC) patients. METHODS: 10p15 LOH was analyzed using KLF6M1 and KLF6M2, and KLF6 gene mutation was analyzed using PCR-SSCP and sequencing. RESULTS: It was found that the frequencies of LOH (sum of M1 and M2) were 4% in adenomas, 0% in intramucosal carcinomas, 35% in invasive carcinomas, and 33% in liver metastases in sporadic cases. Invasive carcinomas from FAP patients showed only 6% LOH, and invasive carcinomas from HNPCC patients exhibited 0% LOH. Mutation analysis of the KLF6 gene in 298 colorectal tumors detected no somatic mutations. CONCLUSIONS: The present data suggest that LOH of the KLF6 locus at chromosome 10p15 contributes to the invasion step from an intramucosal carcinoma to an invasive carcinoma specifically in sporadic colorectal carcinogenesis, but is rarely involved in the carcinogenesis of FAP and HNPCC cases. Moreover, the absence of somatic mutations suggests the uncertainty of the KLF6 gene as a classical tumor suppressor gene at the lost 10p15 region in colorectal carcinogenesis.     

Cell and molecular biology of gastrointestinal tract cancer.

The gene for familial adenomatous polyposis coli (APC or FAP), which has previously been linked to chromosome 5q21 has been identified. The APC gene has been found to be altered by point mutations in the germ line of both adenomatous polyposis coli and Gardner's syndrome patients and somatically in tumors from sporadic colorectal cancer patients. During the hunt for the APC gene, the closely linked MCC (mutated in colorectal cancer) gene was identified and found to be altered somatically in tumors from sporadic cancer patients. These data suggest that more than one gene on chromosome 5q21 may contribute to colorectal carcinogenesis and that mutations at the APC gene can cause both adenomatous polyposis coli and Gardner's syndrome. The identification of these genes should aid in the counseling of patients with genetic predispositions to colorectal cancer. Progress has also been made in identifying specific genetic changes that occur in other gastrointestinal cancers. A mutational "hotspot" in the p53 gene in human hepatocellular carcinomas has been identified that could reflect exposure to a specific carcinogen, one candidate being aflatoxin B1.     

Increased cyclooxygenase-2 expression in duodenal compared with colonic tissues in familial adenomatous polyposis and relationship to the -765G -> C COX-2 polymorphism.

BACKGROUND: Colorectal cancers arising in patients with familial adenomatous polyposis (FAP) can be largely prevented by polyp surveillance and prophylactic colectomy. As a result, duodenal adenocarcinoma has become a leading cause of death in patients with FAP. Cyclooxygenase 2 (COX-2) inhibition is effective against colorectal polyposis in FAP, but is less effective in treating duodenal polyps. We compared the expression of COX-2 in duodenal and colorectal adenomas from patients with FAP and from patients with sporadic neoplasms and correlated expression to a COX-2 promoter polymorphism (-765G/-->C) that is reported to influence COX-2 expression. METHODS: The study population included 36 FAP patients with colonic adenomas, 22 FAP patients with duodenal adenomas, 22 patients with sporadic duodenal adenomas, and 17 patients with sporadic duodenal adenocarcinoma. Neoplastic and corresponding normal tissue COX-2 expressions were determined using immunohistochemistry on tissue microarrays. The prevalence and ethnic distribution of a polymorphism in the COX-2 promoter that influences COX-2 expression (-765G --> C) were determined in DNA from 274 individuals by real-time quantitative PCR. RESULTS: Among patients with FAP, histologically normal duodenal mucosa showed higher COX-2 expression than normal colonic mucosa (P < 0.02), and duodenal adenomas had higher COX-2 expression than colonic adenomas (P 相似文献   

Possible involvement of hyperlipidemia in increasing risk of colorectal tumor development in human familial adenomatous polyposis

BACKGROUND: Familial adenomatous polyposis (FAP) results from germline adenomatous polyposis coli (APC) gene mutations and many affected patients die from colorectal cancers which arise from colorectal polyps. We previously reported that two strains of Apc gene-deficient mice developing multiple intestinal polyps exhibit a hyperlipidemic state. The triglyceride (TG) levels were approximately 10-fold higher than the levels observed in wild-type mice. METHODS: To examine whether a positive relationship might exist between hyperlipidemia and colorectal tumor development in FAP patients, as with Apc gene-deficient mice, a pilot experiment was performed using readily available clinical data such as ages, serum lipid levels, number of colorectal polyps and cancer development in 28 FAP patients from the National Cancer Center Hospital, Japan. RESULTS: The overall prevalence of hyperlipidemia in FAP cases was 58%. Average TG levels in the 40-60 year age groups of FAP patients were > or =150 mg/dl (the defined threshold level of hyperlipidemia). Moreover, there was a tendency for higher serum TG levels in patients who developed colorectal cancer, as compared with those without colorectal cancer. CONCLUSIONS: These results show that a hyperlipidemic state occurs in FAP patients. Although it is weaker than that in Apc gene-deficient mice, it may be linked to colon tumor development. These data warrant further studies for wider populations of FAP patients.     

Nuclear translocation of beta-catenin in hereditary and carcinogen- induced intestinal adenomas

The physical interaction between beta-catenin and the adenomatous polyposis coli (APC) gene, and the ability of APC to regulate cytoplasmic levels of beta-catenin suggest a role for beta-catenin in colorectal carcinogenesis. In this study, we found that beta-catenin immunoreactivity was detected exclusively in the cell membrane and cytoplasm of morphologically normal intestinal epithelial cells with predominant distribution in the differentiated nonproliferative cell population. In contrast, beta-catenin was localized predominantly in the nucleus of adenomas from Min/+ mice and transgenic mice expressing a mutant truncated form of the APC gene (Apc(delta716) mice). Beta- catenin was expressed predominantly at the cell membrane and cytoplasm of the nontransformed rat intestinal epithelial (RIE-1) cells in culture, whereas predominantly nuclear localization of beta-catenin was observed in the human colon cancer cell line SW480. In the azoxymethane (AOM) treated rats, overexpression and nuclear localization of beta- catenin was observed in all adenomas. Previous studies have indicated the incidence of APC mutations amongst AOM-induced tumors to be 15% or less. These results demonstrate that nuclear localization of beta- catenin is a common event in colorectal tumorigenesis.      

Interaction between Ku80 protein and a widely used antibody to adenomatous polyposis coli

The adenomatous polyposis coli (APC) gene and its expressed product are highly studied because of its role as a tumour-suppressor protein. Inherited mutations in APC lead to the condition known as familial adenomatous polyposis (FAP), which predisposes the affected individuals to colorectal cancer. Furthermore, mutations in APC are found in the majority of sporadic cases of colon cancer. There have been many published studies concerning the cellular localisation of APC, this being fundamental to our understanding of its function, but there has also been much concern over the specificity of certain commercially available antibodies to APC. Here we report that the widely used antibody APC(N15) demonstrates a strong interaction with the Ku80 subunit of the Ku heterodimer under defined experimental conditions. Based on the data presented here, we suggest that APC(N15) is not suitable for many applications used for the study of APC.