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A targeted constitutive mutation in the APC tumor suppressor gene underlies mammary but not intestinal tumorigenesis.

Gaspar C, Franken P, Molenaar L, Breukel C, van der Valk M, Smits R, Fodde R - PLoS Genet. (2009)

Bottom Line: Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers.This hypomorphic mutant allele results in intermediate levels of Wnt/beta-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors.The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.

ABSTRACT
Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/beta-catenin signaling. Notably, genotype-phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/beta-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/beta-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc(+/1572T) mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc(+/1572T) mice suggests that specific dosages of Wnt/beta-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.

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Biochemical characterization of the targeted Apc1572T allele.(A) Schematic representation of the APC tumor suppressor protein, its functional domains, and the truncated proteins resulting from the Apc1572T, Apc1638N, and Apc1638T targeted alleles. Only residual amounts (2%) of the truncated Apc1638N protein are encoded by the targeted allele, as shown by immuno-precipitation analysis of Apc1638N/1638N ES lines [5]. (B) β-catenin/TCF reporter assay (TOP-FLASH) analysis of Apc+/+ (1) and Apc-mutant ES cell lines: Apc1638T/1638T (2); Apc1572T/1572T (3); Apc1638N/1638N (4). Each bar represents the average measurement of the luciferase units from triplicate assays. For each cell line, 3 independent experiments were performed with the TOP (filled bars) and FOP (empty bars) reporter constructs. The bold figures represent the average TOP/FOP ratio of all independent experiments. Depicted error bars correspond to standard deviation. In brief, ES cells were plated on dishes coated with MEFs and subsequently transfected by lipofection with either the TOP-FLASH or FOP-FLASH reporter constructs [10] together with the Renilla luciferase vector for normalization purposes. (C) Immuno-precipitation (IP) analysis of Apc-bound β-catenin in Apc-mutant ES cell lines. For comparative purposes, immuno-precipitates obtained from equal amounts of total cellular lysates were loaded.
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pgen-1000547-g001: Biochemical characterization of the targeted Apc1572T allele.(A) Schematic representation of the APC tumor suppressor protein, its functional domains, and the truncated proteins resulting from the Apc1572T, Apc1638N, and Apc1638T targeted alleles. Only residual amounts (2%) of the truncated Apc1638N protein are encoded by the targeted allele, as shown by immuno-precipitation analysis of Apc1638N/1638N ES lines [5]. (B) β-catenin/TCF reporter assay (TOP-FLASH) analysis of Apc+/+ (1) and Apc-mutant ES cell lines: Apc1638T/1638T (2); Apc1572T/1572T (3); Apc1638N/1638N (4). Each bar represents the average measurement of the luciferase units from triplicate assays. For each cell line, 3 independent experiments were performed with the TOP (filled bars) and FOP (empty bars) reporter constructs. The bold figures represent the average TOP/FOP ratio of all independent experiments. Depicted error bars correspond to standard deviation. In brief, ES cells were plated on dishes coated with MEFs and subsequently transfected by lipofection with either the TOP-FLASH or FOP-FLASH reporter constructs [10] together with the Renilla luciferase vector for normalization purposes. (C) Immuno-precipitation (IP) analysis of Apc-bound β-catenin in Apc-mutant ES cell lines. For comparative purposes, immuno-precipitates obtained from equal amounts of total cellular lysates were loaded.

Mentions: The structure and distribution of β-catenin binding and downregulating motifs along the APC tumor suppressor gene is particularly suited to study the effects of specific dosages of canonical Wnt signaling on the multiplicity and tissue-specific distribution of the resulting tumors (Figure 1A). The vast majority of APC mutations found in hereditary and sporadic colorectal cancers are distributed in the 5′ half of the gene and are predicted to encode for stable truncated proteins encompassing up to 3 β-catenin downregulating (20 a.a.) domains. Stable truncation of the mouse Apc gene at codon 1638 as encoded by the Apc1638T allele, results in a protein retaining a sufficient number of functional domains to ensure wild type β-catenin regulation, namely 3 of the 7 β-catenin down-regulating domains and one Axin-binding SAMP repeat (Figure 1A) [9]. Apc+/1638T animals are tumor-free and even homozygous Apc1638T/1638T mice are viable with no apparent predisposition to tumorigenesis [9], in sharp contrast with the marked tumor predisposition and embryonic lethality characteristic of all Apc-mutant mouse models described to date in hetero- and homozygosity, respectively [6]. Notably, Apc1572T, a targeted allele designed to truncate Apc immediately upstream of the only SAMP (Ser-Ala-Met-Pro) repeat encompassed by Apc1638T (Figure 1A), is characterized by an intermediate level of Wnt/β-catenin signaling activation, higher than wild type Apc and Apc1638T though significantly lower than other Apc targeted alleles known to result in GI tract tumors [9]. Here, we show that Apc+/1572T mice are characterized by a striking predisposition to multifocal mammary adenocarcinomas with no susceptibility to intestinal adenomas.


A targeted constitutive mutation in the APC tumor suppressor gene underlies mammary but not intestinal tumorigenesis.

Gaspar C, Franken P, Molenaar L, Breukel C, van der Valk M, Smits R, Fodde R - PLoS Genet. (2009)

Biochemical characterization of the targeted Apc1572T allele.(A) Schematic representation of the APC tumor suppressor protein, its functional domains, and the truncated proteins resulting from the Apc1572T, Apc1638N, and Apc1638T targeted alleles. Only residual amounts (2%) of the truncated Apc1638N protein are encoded by the targeted allele, as shown by immuno-precipitation analysis of Apc1638N/1638N ES lines [5]. (B) β-catenin/TCF reporter assay (TOP-FLASH) analysis of Apc+/+ (1) and Apc-mutant ES cell lines: Apc1638T/1638T (2); Apc1572T/1572T (3); Apc1638N/1638N (4). Each bar represents the average measurement of the luciferase units from triplicate assays. For each cell line, 3 independent experiments were performed with the TOP (filled bars) and FOP (empty bars) reporter constructs. The bold figures represent the average TOP/FOP ratio of all independent experiments. Depicted error bars correspond to standard deviation. In brief, ES cells were plated on dishes coated with MEFs and subsequently transfected by lipofection with either the TOP-FLASH or FOP-FLASH reporter constructs [10] together with the Renilla luciferase vector for normalization purposes. (C) Immuno-precipitation (IP) analysis of Apc-bound β-catenin in Apc-mutant ES cell lines. For comparative purposes, immuno-precipitates obtained from equal amounts of total cellular lysates were loaded.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2697381&req=5

pgen-1000547-g001: Biochemical characterization of the targeted Apc1572T allele.(A) Schematic representation of the APC tumor suppressor protein, its functional domains, and the truncated proteins resulting from the Apc1572T, Apc1638N, and Apc1638T targeted alleles. Only residual amounts (2%) of the truncated Apc1638N protein are encoded by the targeted allele, as shown by immuno-precipitation analysis of Apc1638N/1638N ES lines [5]. (B) β-catenin/TCF reporter assay (TOP-FLASH) analysis of Apc+/+ (1) and Apc-mutant ES cell lines: Apc1638T/1638T (2); Apc1572T/1572T (3); Apc1638N/1638N (4). Each bar represents the average measurement of the luciferase units from triplicate assays. For each cell line, 3 independent experiments were performed with the TOP (filled bars) and FOP (empty bars) reporter constructs. The bold figures represent the average TOP/FOP ratio of all independent experiments. Depicted error bars correspond to standard deviation. In brief, ES cells were plated on dishes coated with MEFs and subsequently transfected by lipofection with either the TOP-FLASH or FOP-FLASH reporter constructs [10] together with the Renilla luciferase vector for normalization purposes. (C) Immuno-precipitation (IP) analysis of Apc-bound β-catenin in Apc-mutant ES cell lines. For comparative purposes, immuno-precipitates obtained from equal amounts of total cellular lysates were loaded.
Mentions: The structure and distribution of β-catenin binding and downregulating motifs along the APC tumor suppressor gene is particularly suited to study the effects of specific dosages of canonical Wnt signaling on the multiplicity and tissue-specific distribution of the resulting tumors (Figure 1A). The vast majority of APC mutations found in hereditary and sporadic colorectal cancers are distributed in the 5′ half of the gene and are predicted to encode for stable truncated proteins encompassing up to 3 β-catenin downregulating (20 a.a.) domains. Stable truncation of the mouse Apc gene at codon 1638 as encoded by the Apc1638T allele, results in a protein retaining a sufficient number of functional domains to ensure wild type β-catenin regulation, namely 3 of the 7 β-catenin down-regulating domains and one Axin-binding SAMP repeat (Figure 1A) [9]. Apc+/1638T animals are tumor-free and even homozygous Apc1638T/1638T mice are viable with no apparent predisposition to tumorigenesis [9], in sharp contrast with the marked tumor predisposition and embryonic lethality characteristic of all Apc-mutant mouse models described to date in hetero- and homozygosity, respectively [6]. Notably, Apc1572T, a targeted allele designed to truncate Apc immediately upstream of the only SAMP (Ser-Ala-Met-Pro) repeat encompassed by Apc1638T (Figure 1A), is characterized by an intermediate level of Wnt/β-catenin signaling activation, higher than wild type Apc and Apc1638T though significantly lower than other Apc targeted alleles known to result in GI tract tumors [9]. Here, we show that Apc+/1572T mice are characterized by a striking predisposition to multifocal mammary adenocarcinomas with no susceptibility to intestinal adenomas.

Bottom Line: Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers.This hypomorphic mutant allele results in intermediate levels of Wnt/beta-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors.The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.

ABSTRACT
Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/beta-catenin signaling. Notably, genotype-phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/beta-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/beta-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc(+/1572T) mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc(+/1572T) mice suggests that specific dosages of Wnt/beta-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.

Show MeSH
Related in: MedlinePlus