Abnormalities of chromosome 8, APC and beta-catenin genes in aggressive fibromatosis

OBJECTIVE: To explore the role of abnormalities of chromosome 8, APC and beta-catenin genes in tumorigenesis of aggressive fibromatosis. METHODS: Trisomy 8 was detected by interphase fluorescence in situ hybridization (FISH). The APC gene and beta-catenin gene mutations were detected by denaturing high performance liquid chromatography (DHPLC) and direct sequence analysis after the PCR transition. RESULTS: The rate of trisomy 8 in recurrent tumors (62.5%, 5/8) was significantly higher than that in the primary tumors (8.3%, 1/12). Somatic substitution of APC gene was found in 18 of 69 (26.1%) aggressive fibrometases. Somatic transition of beta-catenin gene was detected in 13 of 69 (18.8%) and mutation at codon 41 in exon 3 involving threonine residues implicated in the degradation of beta-catenin. The abnormal expression of beta-catenin had no significant correlation with the mutation of APC or beta-catenin gene. The group with positively expressed beta-catenin protein showed a significant higher c-myc protein expression than those without (P = 0.001). The Ki-67 index was extremely low in all the lesions. The apoptosis index (AI) of the groups with positively expressed c-myc and cyclin D1 showed significantly lower AI than those without. CONCLUSION: Trisomy 8 may serve as a useful predictor of recurrence in aggressive fibromatosis. There are somatic mutations of the APC and beta-catenin genes in the aggressive fibromatosis, and there are abnormalities in the Wnt signaling pathway. These abnormalities may result in the aberrances of cell proliferation and apoptosis, which are likely to be import factors in the tumorigenesis.     

Tcf-3 expression and beta-catenin mediated transcriptional activation in aggressive fibromatosis (desmoid tumour).

Aggressive fibromatosis harbours mutations resulting in beta-catenin protein stabilization. Primary cell cultures demonstrate constitutive tcf activation in aggressive fibromatosis. Expression and co-immunoprecipitation studies suggest that beta-catenin binds and activates tcf-3 in this tumour. This is the first demonstration of tcf-3 activation by beta-catenin stabilization in a human neoplastic process.     

The APC/beta-catenin pathway in ulcerative colitis-related colorectal carcinomas: a mutational analysis

BACKGROUND: Although the APC/beta-catenin pathway is known to play a crucial role in sporadic colorectal carcinogenesis, its influence on ulcerative colitis (UC)-related neoplastic progression is unknown. To elucidate the role of the APC-/beta-catenin pathway in UC-related carcinogenesis, the authors identified APC and beta-catenin mutations in a set of UC-related and sporadic colorectal carcinomas. METHODS: The mutational cluster region of APC (codon 1267 to 1529) and exon 3 of the beta-catenin were directly sequenced. RESULTS: Only 1 of 30 UC-related tumors (3%) showed an APC mutation whereas 11 of the 42 sporadic carcinomas (26%) had mutations within the mutational cluster region. Within the sporadic carcinoma group, only 8% of the right-sided carcinomas showed APC mutations whereas 50% of the left-sided carcinomas had mutations within the mutational cluster region. None of the tumors in either group showed a beta-catenin mutation. CONCLUSIONS: Mutations of the APC and beta-catenin are rare in UC-related tumors. These genes may be altered because of mutations outside the regions studied, or by epigenetic silencing. Alternatively, other proteins involved in the APC/beta-catenin signaling cascade may be altered, or this pathway may be involved infrequently in UC-related carcinogenesis. The significant difference in frequency of APC mutations between right- and left-sided sporadic tumors suggests different molecular pathways in these two tumor sites.     

Frequent mutations in the beta-catenin gene in desmoid tumors from patients without familial adenomatous polyposis.

Mutations in the APC gene contribute to development of sporadic desmoid tumors as well as to the hereditary tumors that usually accompany familial adenomatous polyposis (FAP). Adenomatous polyposis coli (APC) mutations cause an intracellular accumulation of beta-catenin that results in abnormal signaling in the wnt/wingless pathway. Mutations of the beta-catenin gene itself have also been noted in several types of tumors. In this study we screened the beta-catenin gene in 13 sporadic desmoid tumors for alterations in exon 3, which encodes several serine/threonine residues that are targets for phosphorylation by GSK-3beta. Somatic substitutions at codons 41 (threonine) and 45 (serine) were identified in seven independent tumors, respectively. Although no APC mutations were detected among the remaining six tumors, we found accumulation of beta-catenin by Western blotting analysis in one such tumor for which frozen tissues were available. Our results have suggested that possible involvement of beta-catenin activation by beta-catenin gene mutation or alteration of other factor(s) can contribute to desmoid tumorigenesis.     

Cyclooxygenase-2 and platelet-derived growth factor receptors as potential targets in treating aggressive fibromatosis.

PURPOSE: To explore the molecular bases of potential new pharmacologic targets in aggressive fibromatosis (desmoid tumor). EXPERIMENTAL DESIGN: Tumor specimens from 14 patients surgically treated for aggressive fibromatosis (6 familial adenomatous polyposis and 8 sporadic cases), analyzed for adenomatous polyposis coli (APC) and CTNNB1 (beta-catenin) mutations, were further investigated for beta-catenin, cyclooxygenase-2 (COX-2), platelet-derived growth factor (PDGF) receptor alpha (PDGFRA)/PDGF receptor beta (PDGFRB), their cognate ligands (PDGFA and PDGFB), and KIT using a comprehensive immunohistochemical, biochemical, molecular, and cytogenetic approach. RESULTS: No CTNNB1 (beta-catenin) mutations were found in the familial adenomatous polyposis patients, but previously reported activating mutations were found in six of the eight sporadic patients. All of the cases carrying an altered WNT pathway showed nuclear and cytoplasmic immunoreactivity for beta-catenin, whereas beta-catenin expression was restricted to the cytoplasm in the sporadic patients lacking CTNNB1 mutations. COX-2 protein and mRNA overexpression was detected in all 14 cases, together with the expression and phosphorylation of PDGFRA and PDGFRB, which in turn paralleled the presence of their cognate ligands. No PDGFRB mutations were found. The results are consistent with PDGFRA and PDGFRB activation sustained by an autocrine/paracrine loop. CONCLUSIONS: Aggressive fibromatosis is characterized by WNT/oncogene pathway alterations triggering COX-2-mediated constitutive coactivation of PDGFRA and PDGFRB, and may therefore benefit from combined nonsteroidal anti-inflammatory drug + tyrosine kinase inhibitor treatment.     

Frequent mutations of the beta-catenin gene in mouse colon tumors induced by azoxymethane

The beta-catenin gene is frequently mutated at codons 33, 41 and 45 of the glycogen synthase kinase-3beta phosphorylation motif in human colon cancers in patients without APC mutations. Frequent mutations at codons 32 and 34, as well as 33 and 41, have been detected in rat colon tumors induced by azoxymethane (AOM), with the second G of CTGGA sequences being considered as a mutational hot-spot. In the present study, exon 3 of the beta-catenin gene in mouse colon tumors induced by AOM was amplified by PCR and mutations were detected by the single strand conformation polymorphism method, restriction enzyme fragment length polymorphism and direct sequencing. All 10 colon tumors tested were found to have beta-catenin mutations, four in codon 34, three in codon 33, two in codon 41 and one in codon 37, nine being G:C-->A:T transitions. However, no mutations were found in codon 32 of the mouse beta-catenin gene. On immmunostaining, beta-catenin was observed in the cytoplasm and nucleus of the tumor cells. The cytoplasmic staining was homogeneous, while both homogeneous and heterogeneous patterns were noted for the nuclei. Highly frequent mutations of the beta-catenin gene in AOM-induced mouse colon tumors suggest that consequent alterations in the stability and localization of the protein may play an important role in this colon carcinogenesis model.     

APC truncation and increased beta-catenin levels in a human breast cancer cell line

Mutations in the Adenomatous Polyposis Coli (APC) tumor suppressor gene or the beta-catenin gene are present in most colon cancers and less frequently in other tumor types. In this study, we screened 24 human breast cancer cell lines and three immortalized human breast epithelial cell lines for alterations in beta- and gamma-catenin and APC by western blotting, protein truncation assay and DNA sequence analysis. In one cell line (DU 4475), an APC mutation was identified (E1577stop) that resulted in expression of truncated APC. This mutation was associated with elevated cytosolic beta-catenin levels, probably due to loss of APC function, as in colon cancers. No mutations were found in exon 3 of the beta- or gamma-catenin genes. We conclude that APC mutations and beta-catenin upregulation may occur with low frequency in human breast cancer cells.     

Activation of beta-catenin in epithelial and mesenchymal hepatoblastomas

Wnt/beta-catenin signaling is frequently activated in cancer cells by stabilizing mutations of beta-catenin or loss-of-function mutations of the APC tumor suppressor gene. We have analysed the role of beta-catenin in the pathogenesis of hepatoblastoma (HB), an embryonic liver tumor occurring mainly in children under 2 years of age. Sequence analysis of the beta-catenin NH2-terminal domain in 18 epithelial and mixed HBs revealed missense mutations in the GSK3beta phosphorylation motif or interstitial deletions in 12 tumors (67%). In the remaining cases, no truncating mutation of APC could be evidenced. Immunohistochemical analysis of beta-catenin in 11 HBs demonstrated nuclear/cytoplasmic accumulation of the protein in all tumors analysed, with predominant nuclear beta-catenin immunostaining in undifferentiated cells. Membranous beta-catenin localization was preserved only in fetal-type tumoral hepatocytes and was associated with E-cadherin expression. Moreover, we show that beta-catenin is aberrantly overexpressed in a large spectrum of tumor components, including hepatocyte-like cells at various differentiation stages and heterologous elements such as squamous, osteoid and chrondroid tissues, and in occasional other mesenchymally-derived cells. These data strongly suggest that activation of beta-catenin signaling is an obligatory step in HB pathogenesis, and raise the possibility that it interferes with developmental signals that specify different tissue types at early stages of hepatic differentiation.     

Somatic mutations of beta-catenin play a crucial role in the tumorigenesis of sporadic hepatoblastoma

Hepatoblastoma (HB) is the most common malignant hepatic tumor during early childhood. Its molecular pathogenesis is still poorly understood. Mutations of adenomatous polyposis coli (APC) gene have been identified in sporadic cases and in individuals associated with familial adenomatous polyposis syndrome. beta-catenin is a key element in the cadherin-mediated cell adhesion system and Wnt/wingless pathway, and is controlled by APC. APC affects the degradation of beta-catenin by its NH(2)-terminal phosphorylation on the serine/threonine residues of exon 3. Mutations of these phosphorylation sites are primary targets for activating mutations in several types of human cancer and lead to nuclear accumulation of beta-catenin protein. In this study, we examined nine patients with HB using immunohistochemistry and direct DNA sequencing. All nine cases showed predominant nuclear expression of beta-catenin. Eight cases (89%) showed mutations involving exon 3 of the beta-catenin gene, including five with deletions and three with missense mutations. All five deletions were in-frame deletions without frameshift. The very high frequency of mutations in the beta-catenin gene suggests that beta-catenin mutations are crucial in the tumorigenesis of HB.     

beta-catenin expression in primary and metastatic colorectal carcinoma.

beta-catenin plays a fundamental role in the regulation of the E-cadherin-catenin cell adhesion complex. It also functions in growth signalling events, independently of the cadherin-catenin complex, and these signalling pathways are disturbed in colorectal cancer. Mutations in either the APC or beta-catenin genes in colorectal cancer cells result in up-regulation of protein expression and subsequent cytoplasmic and nuclear distribution of beta-catenin. In this study, we examined beta-catenin expression in 47 primary colorectal tumors and the corresponding liver metastases. Immunohistochemical studies demonstrated loss of membranous beta-catenin expression in 26% of primary tumors and 60% of liver metastases and a concomitant increase in cytoplasmic and nuclear staining. Widespread nuclear expression of beta-catenin was found in 64% of primary tumors and 21% of liver metastases. No associations were found between any form of beta-catenin expression and either tumor stage or tumor grade. Cellular distribution of beta-catenin was also examined by detergent extraction and Western blot analysis in 16 primary tumors and 23 liver metastases. This analysis showed that most tumors demonstrated reduced beta-catenin in the cytoskeletal fraction and increased beta-catenin in the cytosolic fraction. Furthermore, 3 liver metastases were found to contain a truncated beta-catenin protein of approximately M(r) 80,000. Immunoprecipitation studies showed that the truncated beta-catenin proteins only bound weakly to E-cadherin and beta-catenin compared with non-truncated beta-catenin. These results demonstrate gross alterations in the cellular distribution of beta-catenin in primary colorectal cancers with metastatic potential, as well as in the metastatic tumors. These changes may be the consequence of APC or beta-catenin gene mutations, or possibly result from a post-translational modification of the E-cadherin-catenin complex.     

Frequent mutation of beta-catenin and APC genes in primary colorectal tumors from patients with hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is characterized by defective DNA mismatch repair, which results in genetic instability of tumors; however, only a few target genes have been recognized. Our previous study detected a low frequency of APC gene mutation (21%) in colorectal tumors from HNPCC patients, in contrast to a high frequency of APC gene alteration (>70%) in non-HNPCC tumors. Because both beta-catenin and ACP gene mutations have recently been shown to activate the same signaling pathway, we analyzed beta-catenin mutation in HNPCC tumors. A notable frequency of beta-catenin gene mutation (43%, 12 of 28) was found to occur in HNPCC colorectal tumors. Beta-catenin mutations were not detected in tumors with APC mutations. All beta-catenin mutations detected in HNPCC tumors existed within the regulatory domain of beta-catenin. Immunohistochemical staining of tumors with this mutation showed accumulation of beta-catenin protein in nuclei. These and previous data from our laboratory suggest that activation of the beta-catenin-Tcf signaling pathway, through either beta-catenin or APC mutation, contributes to HNPCC colorectal carcinogenesis in approximately 65% of cases.     

Expression of beta-catenin and APC protein in ovarian epithelial tumor and its implication

Objective： To investigate the expression of beta-catenin, APC protein and its implication in ovarian epithelial tumor. Methods： Immunohistochemical staining with SP method was conducted to determine the expression of beta-catenin and APC protein in 48 cases of ovarian epithelial tumor. Results： The abnormal expression rates of beta-catenin in ovarian malignant and borderline epithelial tumors were higher than that in benign epithelial tumors. The expression of APC protein in benign epithelial tumors was significantly greater than that in malignant epithelial tumors. A significant negative correlation was found between beta-catenin and APC protein in ovarian epithelial tumors. Conclusion： Beta-catenin and APC protein have important effect on pathogenesis and development of ovarian epithelial tumors.     

Agressive fibromatosis: genetic and biological correlations

Aggressive fibromatosis, also known as desmoid tumor, is specific and relatively rarely occuring disease. It belongs to heterogenous group of soft tissue tumors. Originally, it arises from fibroblasts with monoclonal proliferation derived from fibro-aponeurotic tissue with typical local invasive spreading without metastatic tendency. Increased amount of knowledge about the role of the APC gene and its protein product in FAP play an important role in revealing the molecular nature of desmoid tumors. In general, we can conclude that the β-catenin dysregulation is the key player of the FAP associated desmoid tumor onset. The Wingless/Wnt cascade plays a crucial role in the pathogenesis of aggressive fibromatosis. However, it has not been definitely proven that the mutations of APC or β-catenin genes are the trigger mechanisms. The research outcome can pave the way for using target biological therapy in routine practice in patients with aggressive fibromatosis in the future.     

Enhanced colon carcinogenesis induced by azoxymethane in min mice occurs via a mechanism independent of beta-catenin mutation

The multiple intestinal neoplasia (min) mouse is a well-established cancer model in which loss of a single copy of the APC protein predisposes mice to the development of numerous tumors in the intestine. We have developed a novel variation of the min mouse model by using azoxymethane (AOM) to cause an increase in tumor incidence, number and size. Thus, treatment of min mice with AOM resulted in 2.6-, 6.3- and 5.9-fold increases in overall tumor incidence, multiplicity and size, respectively, when compared to wild type C57BL/6J mice treated with AOM. Furthermore, adenocarcinomas of the colon, which are otherwise relatively rare in min mice, increased in incidence (P<0.004), multiplicity (P<0.005), and size (P<0.02) in the AOM-treated min mice when compared to control untreated min mice. Of these adenocarcinomas, the number of poorly plus moderately differentiated adenocarcinomas was also significantly higher in the AOM-treated min mice (P<0.008). Thirty-seven histopathologically verified colon tumors (eight adenomas, five carcinoma in situ and 24 adenocarcinomas) induced in min mice and in C57BL/6J mice after treatment with or without AOM were analyzed for mutations in the beta-catenin gene or de novo mutations in the Apc gene. No mutations in the beta-catenin gene were found in any of colon tumors in min mice with or without treatment with AOM. However, mutations in either the beta-catenin gene or the Apc gene were found in tumors induced in C57BL/6J mice by AOM. These results suggest that mutations in the beta-catenin gene are less contributory to tumor development in min mice, as is the case in familial adenomatous polyposis (FAP) in humans. However, de novo mutations in either the Apc or beta-catenin gene can play a role in tumor development in C57BL/6J mice treated with AOM. The differences in mutation status between min and C57BL/6J mice may indicate different genetic pathways for developing colon tumors. These two experimental systems may, therefore, be useful animal models of human colon carcinomas in patients with FAP and in patients with sporadic colon carcinomas.