Mechanisms of oncogenesis in colon versus rectal cancer

Observations support the theory that development of left- and right-sided colorectal cancers may involve different mechanisms. This study investigated different genes involved in oncogenesis of colon and rectal cancers and analysed their prognostic value. The study group comprised 35 colon and 42 rectal cancers. Rectal cancer patients had been treated with standardized surgery performed by an experienced rectal cancer surgeon. Mutation analysis was performed for p53 in eight colon cancers and for APC and p53 in 22 rectal cancers. MLH1, MSH2, Bcl-2, p53, E-cadherin and beta-catenin were investigated by immunohistochemistry in all colorectal tumours. APC mutation analysis of the MCR showed truncating mutations in 18 of 22 rectal tumours (82%), but the presence of an APC mutation was not related to nuclear beta-catenin expression (p=0.75). Rectal cancers showed significantly more nuclear beta-catenin than colon cancers (65% versus 40%, p=0.04). p53 mutation analysis corresponded well with p53 immunohistochemistry (p<0.001). Rectal cancers showed significantly more immunohistochemical expression of p53 than colon cancers (64% versus 29%, p=0.003). In rectal cancers, a significant correlation was found between positive p53 expression and worse disease-free survival (p=0.008), but not in colon cancers. Cox regression showed that p53-expression (p=0.03) was an independent predictor for disease-free survival in rectal cancers. This study concluded that rectal cancer may involve more nuclear beta-catenin in the APC/beta-catenin pathway than colon cancer and/or nuclear beta-catenin may have another role in rectal cancer independently of APC. The p53-pathway seems to be more important in rectal cancer, in which it also has independent prognostic value. When prognostic markers are investigated in larger series, differences in biological behaviour between colon and rectal cancer should be considered.     

Somatic Apc mutations are selected upon their capacity to inactivate the beta-catenin downregulating activity

The APC gene, originally identified as the gene for familial adenomatous polyposis (FAP), is now considered as the true "gatekeeper" of colonic epithelial proliferation. Its main tumor suppressing activity seems to reside in the capacity to properly regulate intracellular beta-catenin signaling. Most somatic APC mutations are detected between codons 1286 and 1513, the mutation cluster region (MCR). This clustering can be explained either by the presence of mutation-prone sequences within the MCR, or by the selective advantage provided by the resulting truncated polypeptides. Here, a Msh2-deficient mouse model (Msh2(delta 7N) ) was generated and bred with Apc(1638N) and Apc(Min) that allowed the comparison of the somatic mutation spectra along the Apc gene in the different allelic combinations. Mutations identified in Msh2(delta 7N/delta 7N) tumors are predominantly dinucleotide deletions at simple sequence repeats leading to truncated Apc polypeptides that partially retain the 20 a.a. beta-catenin downregulating motifs. In contrast, the somatic mutations identified in the wild type Apc allele of Msh2(delta 7N/delta 7N) /Apc(+/1638N) and Msh2(delta 7N/delta 7N) /Apc(+/Min) tumors are clustered more to the 5' end, thereby completely inactivating the beta-catenin downregulating activity of APC. These results indicate that somatic Apc mutations are selected during intestinal tumorigenesis and that inactivation of the beta-catenin downregulating function of APC is likely to represent the main selective factor.     

The 'just-right' signaling model: APC somatic mutations are selected based on a specific level of activation of the beta-catenin signaling cascade

According to the classical interpretation of Knudson's 'two-hit' hypothesis for tumorigenesis, the two 'hits' are independent mutation events, the end result of which is loss of a tumor suppressing function. Recently, it has been shown that the APC (adenomatous polyposis coli) gene does not entirely follow this model. Both the position and type of the second hit in familial adenomatous polyposis (FAP) polyps depend on the localization of the germline mutation. This non-random distribution of somatic hits has been interpreted as the result of selection for more advantageous mutations during tumor formation. However, the APC gene encodes for a multifunctional protein, and the exact cellular function upon which this selection is based is yet unknown. In this study, we have analyzed somatic APC point mutations and loss of heterozygosity (LOH) in 133 colorectal adenomas from six FAP patients. We observed that when germline mutations result in truncated proteins without any of the seven beta-catenin downregulating 20-amino-acid repeats distributed in the central domain of APC, the majority of the corresponding somatic point mutations retain one or, less frequently, two of the same 20-amino-acid repeats. Conversely, when the germline mutation results in a truncated protein retaining one 20-amino-acid repeat, most second hits remove all 20-amino-acid repeats. The latter is frequently accomplished by allelic loss. Notably, and in contrast to previous observations, in a patient where the germline APC mutation retains two such repeats, the majority of the somatic hits are point mutations (and not LOH) located upstream and removing all of the 20-amino-acid repeats. These results indicate selection for APC genotypes that are likely to retain some activity in downregulating beta-catenin signaling. We propose that this selection process is aimed at a specific degree of beta-catenin signaling optimal for tumor formation, rather than at its constitutive activation by deletion of all of the beta-catenin downregulating motifs in APC.     

Crystal structure of a beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex

The "beta-catenin destruction complex" is central to canonical Wnt/beta-catenin signaling. The scaffolding protein Axin and the tumor suppressor adenomatous polyposis coli protein (APC) are critical components of this complex, required for rapid beta-catenin turnover. We determined the crystal structure of a complex between beta-catenin and the beta-catenin-binding domain of Axin (Axin-CBD). The Axin-CBD forms a helix that occupies the groove formed by the third and fourth armadillo repeats of beta-catenin and thus precludes the simultaneous binding of other beta-catenin partners in this region. Our biochemical studies demonstrate that, when phosphorylated, the 20-amino acid repeat region of APC competes with Axin for binding to beta-catenin. We propose that a key function of APC in the beta-catenin destruction complex is to remove phosphorylated beta-catenin product from the active site.     

APC mutations in synovial sarcoma

It has previously been demonstrated that accumulated beta-catenin serves as an oncoprotein in synovial sarcoma and results in a poor overall survival rate, but the frequency of beta-catenin mutation was quite low (8.2%). The present study, using essentially the same study group of cases, screened for genetic alterations in the mutation cluster region (MCR) of the APC gene in 49 cases of synovial sarcoma. SSCP analysis followed by DNA direct sequencing revealed five missense APC mutations in four cases of synovial sarcoma (8.2%). The mutational sites comprised one case each at codons 1299 (GCT to ACT, Ala to Thr), 1412 (GGA to AGA, Gly to Arg), and 1414 (GTA to ATA, Val to Ile), in addition to one case with double point mutations at codon 1398 (AGT to AAT, Ser to Asn) and at codon 1413 (ATG to ATA, Met to Ile), together with beta-catenin mutation at codon 32 (GAC to TAC, Asp to Tyr). All four cases with APC mutations were histologically of the monophasic fibrous type and showed beta-catenin accumulation. All three cases with APC mutations available for follow-up data were long survivors. This study provides the first evidence that APC mutations also occur in the field of sarcoma, especially in synovial sarcoma.     

Analysis of somatic APC mutations in rare extracolonic tumors of patients with familial adenomatous polyposis coli

Patients with familial adenomatous polyposis coli (FAP) carry heterozygous mutations of the APC gene. At a young age, these patients develop multiple colorectal adenomas that consistently display a second somatic mutation in the remaining APC wild-type allele. Inactivation of APC leads to impaired degradation of beta-catenin, thereby promoting continuous cell-cycle progression. The role of APC inactivation in rare extracolonic tumors of FAP patients has not been characterized sufficiently. Among tissue specimen from 174 patients with known APC germ-line mutations, we identified 8 tumors infrequently seen in FAP. To investigate the pathogenic role of APC pathway deregulation in these lesions, they were analyzed for second-hit somatic mutations in the mutational cluster region of the APC gene. Immunohistochemistry was performed to compare the expression pattern of beta-catenin to the mutational status of the APC gene. Exon 3 of the beta-catenin gene (CTNNB1) was analyzed for activating mutations to investigate alternative mechanisms of elevated beta-catenin concentration. Although CTNNB1 mutations were not observed, second somatic APC mutations were found in 4 of the 8 tumors: a uterine adenocarcinoma, a hepatocellular adenoma, an adrenocortical adenoma, and an epidermal cyst. These tumors showed an elevated concentration of beta-catenin. No APC mutations were seen in focal nodular hyperplasia of the liver, angiofibrolipoma, and seborrheic wart. This is the first study reporting second somatic APC mutations in FAP-associated uterine adenocarcinoma and epidermal cysts. Furthermore, our data strengthen a role for impaired APC function in the pathogenesis of adrenal and hepatic neoplasms in FAP patients.     

Genetic inactivation of the APC gene contributes to the malignant progression of sporadic hepatocellular carcinoma: a case report

Although activated Wnt/beta-catenin pathway is considered to be one of the main driving forces of hepatocarcinogenesis, no somatic mutations of the adenomatous polyposis coli (APC) gene have been found in sporadic hepatocellular carcinoma (HCC) to date. Here we present a case of a sporadic nodule-in-nodule-type HCC that provides the first evidence that biallelic genetic inactivation of the APC gene contributed to the development of the tumor. High-density array-based comparative genomic hybridization (aCGH) was performed to clarify genome-wide chromosomal structural alteration profiles of both early and advanced components of this nodule-in-nodule HCC. aCGH analysis revealed a chromosomal loss of the APC gene locus only in the inner advanced component of this tumor. Direct sequencing of the remaining allele revealed a nonsense mutation at codon 682 in the Armadillo repeats, resulting in a truncated protein that lacked all of the beta-catenin-binding motifs. Nonsense mutations at this location are rare among other types of cancer. In conclusion, combined with an immunohistochemical analysis of the beta-catenin protein, this case provides the first evidence that genetic inactivation of the APC gene can play a significant role in the progression of sporadic HCC, probably by activating the Wnt/beta-catenin pathway.     

Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli protein involved in tumorigenesis and development

The adenomatous polyposis coli (APC) gene is considered as the true gatekeeper of colonic epithelial proliferation: It is mutated in the majority of colorectal tumors, and mutations occur at early stages of tumor development in mouse and man. These mutant proteins lack most of the seven 20-amino-acid repeats and all SAMP motifs that have been associated with down-regulation of intracellular beta-catenin levels. In addition, they lack the carboxy-terminal domains that bind to DLG, EB1, and microtubulin. APC also appears to be essential in development because homozygosity for mouse Apc mutations invariably results in early embryonic lethality. Here, we describe the generation of a mouse model carrying a targeted mutation at codon 1638 of the mouse Apc gene, Apc1638T, resulting in a truncated Apc protein encompassing three of the seven 20 amino acid repeats and one SAMP motif, but missing all of the carboxy-terminal domains thought to be associated with tumorigenesis. Surprisingly, homozygosity for the Apc1638T mutation is compatible with postnatal life. However, homozygous mutant animals are characterized by growth retardation, a reduced postnatal viability on the B6 genetic background, the absence of preputial glands, and the formation of nipple-associated cysts. Most importantly, Apc1638T/1638T animals that survive to adulthood are tumor free. Although the full complement of Apc1638T is sufficient for proper beta-catenin signaling, dosage reductions of the truncated protein result in increasingly severe defects in beta-catenin regulation. The SAMP motif retained in Apc1638T also appears to be important for this function as shown by analysis of the Apc1572T protein in which its targeted deletion results in a further reduction in the ability of properly controlling beta-catenin/Tcf signaling. These results indicate that the association with DLG, EB1, and microtubulin is less critical for the maintenance of homeostasis by APC than has been suggested previously, and that proper beta-catenin regulation by APC appears to be required for normal embryonic development and tumor suppression.     

Chromosomal instability and APC gene mutations in human sporadic colorectal adenomas

The mechanisms by which adenomatous polyposis coli (APC) gene mutations contribute to colorectal tumourigenesis and progression are still not fully understood. Using in vitro mouse embryonic stem cells, APC mutations have been proposed to dysregulate the interactions between kinetochores and microtubules during mitosis, leading to chromosomal instability (CIN) and aneuploidy. A link between APC mutations and aneuploidy in vivo among human sporadic colorectal adenomas has not been reported previously and was therefore investigated in the present series of 61 adenomas. Multi-parameter flow cytometry, based on scattering and fluorescence from the DNA-specific 4,6-diamidino-2-phenylindole-2-hydrochloride (DAPI) dye, which separates epithelial from stromal lymphocyte nuclei, was used to evaluate the DNA index (DI) and to sort epithelial nuclei. Additionally, DNA extracted from these sorted nuclei was used to analyse APC mutations by DNA sequencing. Aneuploidy was present in 20 of 61 adenomas (33%), with 15 of these 20 cases (75%) having a near-diploid DI (DI different from 1 and less than 1.3). APC mutations were detected in 19 adenomas (31%): 12 were within or downstream of the mutation cluster region (MCR), roughly defined by codons 1200-1500, and seven were upstream of the MCR. Overall, the prevalence of aneuploidy in APC wild-type and mutated adenomas was 26% and 47%, respectively, and no statistically significant association was found between APC status and DI (p = 0.142). However, when APC mutations were subdivided into two groups, ie occurring within/downstream of the MCR and upstream of the MCR, the association of APC mutations within and downstream of the MCR with aneuploidy was statistically significant (p = 0.017). In conclusion, the present data suggest that the type of APC mutation may play a role in the origin of CIN in vivo in human sporadic colorectal adenomas and that APC mutations within and downstream of the MCR, and large-scale chromosomal alterations, may co-operate in the progression of a subgroup of adenomas.     

Fibromatosis of the breast and mutations involving the APC/beta-catenin pathway

Fibromatoses of the breast are nonmetastasizing tumors, but can be infiltrative and locally recurrent. Breast fibromatoses are rare, and their specific genetic alterations have not been elucidated. However, their occasional occurrence in patients with familial adenomatous polyposis (FAP) and their morphologic identification with other deep fibromatoses (desmoid tumors) suggest that alterations of the APC/beta-catenin pathway might be involved in the pathogenesis of sporadic and FAP-associated breast fibromatoses. We analyzed somatic beta-catenin and APC gene mutations in 33 breast fibromatoses (32 sporadic and 1 FAP-associated) using immunohistochemistry for beta-catenin, 5q allelic loss assays, and direct DNA sequencing for exon 3 of the beta-catenin gene and the mutation cluster region of the APC gene. Nuclear accumulation of beta-catenin was present in the stromal tumor cells in most (82%) cases but not in normal stroma or mammary epithelial cells. Somatic alterations of the APC/beta-catenin pathway were detected in 79% of breast fibromatoses, including activating beta-catenin gene mutations in 15 cases and somatic APC alterations (mutation or 5q allelic loss or both) in 11. These findings indicate that alterations of the APC/beta-catenin pathway with resultant nuclear translocation of beta-catenin are important in the pathogenesis of both sporadic and FAP-associated breast fibromatosis. The spectrum of beta-catenin and APC alterations is similar to that described for desmoid tumors of the abdomen, paraspinal region, and extremities.