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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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Teratoma formation assays indicate an intermediate differentiation defect in Apc1572T/1572T ES cells.(A) HE analysis of normal mammary gland with luminal cells surrounded by a basal layer of myoepithelial cells. (B) IF analysis of normal mammary glands for Ck8 (luminal cells, green) and Sma (myoepithelial, red). (C) HE staining of Apc1572T/1572T teratoma showing the typical mammary gland architecture with lobular and ductal structures. (D) IF analysis of Apc1572T/1572T teratomas for luminal and myoepithelial cell types. The frequency of these structures in teratomas derived from Apc1572T/1572T ES cells is largely increased when compared with (F) teratomas derived from Apc+/+ ES cells. (E) Summary of the results of the teratoma differentiation assays of Apc-mutant ES cells. Antibodies employed to evaluate differentiation are: Glial Fibrillary Acidic Protein (GFAP) for glial cells; 2H3 for neurofilaments; SV2 for synaptic vesicles; A4.1025 for adult myosin. Mammary gland structures were primarily identified by HE and then confirmed by IF as shown in panels a–d; also in the case of cartilage and epithelial structures HE stained section were employed. n.d. not determined. Differentiation levels were scored as: (−) not present; (−*) vestigial presence; (+) present; (++) highly abundant. The shaded areas indicate groups of teratomas for which the corresponding antibody staining was negative.
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pgen-1000547-g002: Teratoma formation assays indicate an intermediate differentiation defect in Apc1572T/1572T ES cells.(A) HE analysis of normal mammary gland with luminal cells surrounded by a basal layer of myoepithelial cells. (B) IF analysis of normal mammary glands for Ck8 (luminal cells, green) and Sma (myoepithelial, red). (C) HE staining of Apc1572T/1572T teratoma showing the typical mammary gland architecture with lobular and ductal structures. (D) IF analysis of Apc1572T/1572T teratomas for luminal and myoepithelial cell types. The frequency of these structures in teratomas derived from Apc1572T/1572T ES cells is largely increased when compared with (F) teratomas derived from Apc+/+ ES cells. (E) Summary of the results of the teratoma differentiation assays of Apc-mutant ES cells. Antibodies employed to evaluate differentiation are: Glial Fibrillary Acidic Protein (GFAP) for glial cells; 2H3 for neurofilaments; SV2 for synaptic vesicles; A4.1025 for adult myosin. Mammary gland structures were primarily identified by HE and then confirmed by IF as shown in panels a–d; also in the case of cartilage and epithelial structures HE stained section were employed. n.d. not determined. Differentiation levels were scored as: (−) not present; (−*) vestigial presence; (+) present; (++) highly abundant. The shaded areas indicate groups of teratomas for which the corresponding antibody staining was negative.

Mentions: Previously, we showed that different levels of β-catenin signaling affect the ability of mouse embryonic stem (ES) cells to differentiate towards specific lineages in a dosage-dependent fashion [5]. To address the same question for the Apc1572T allele, we have subcutaneously injected undifferentiated Apc1572T/1572T ES cells into syngenic mice to induce formation of teratomas, as previously described [5]. The differentiation profiles of the Apc1572T/1572T teratomas were then investigated by histological and immuno-histochemical analysis, and compared with those obtained with wild type (Apc+/+) and other Apc-mutant ES cells. In line with their intermediate level of constitutive Wnt/β-catenin signaling activation, Apc1572T/1572T teratomas show a more heterogeneous spectrum of ecto-, meso-, and endodermal lineages than Apc1638N/1638N (characterized by a higher TopFLASH reporter activity; see Figure 1B), though still more limited in their differentiation capacity than Apc1638T/1638T (characterized by TopFLASH reporter activity comparable with wild type ES cells) (Figure 2E). In agreement with previous observations [5], several differentiation types, namely neural, bone, cartilage and ciliated epithelia were absent in Apc1638N/1638N teratomas. In particular, markers employed to identify neuroectodermal lineages did not stain Apc1638N/1638N sections, in contrast with Apc1572T/1572T teratomas where a limited but significant number of the cells were GFAP positive. Differentiation to striated muscle was also severely affected and detectable in only a minority of the Apc1638N/1638N sections [5], whereas all Apc1572T/1572T teratomas analyzed revealed positive myosin staining. Notably, among the cell types positively identified in Apc+/+ teratomas, mammary epithelia were relatively more abundant in Apc1572T sections, as shown by the combined staining with SMA and CK8 and the typical tissue architecture with luminal cells on top of a myoepithelial basal layer (Figure 2A–2D and Figure 2F). Hence, homozygous Apc1572T ES cells are characterized by an intermediate differentiation defect between Apc1638N/1638N and Apc1638T/1638T, with an unusual enrichment in mammary epithelial differentiation.


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)

Teratoma formation assays indicate an intermediate differentiation defect in Apc1572T/1572T ES cells.(A) HE analysis of normal mammary gland with luminal cells surrounded by a basal layer of myoepithelial cells. (B) IF analysis of normal mammary glands for Ck8 (luminal cells, green) and Sma (myoepithelial, red). (C) HE staining of Apc1572T/1572T teratoma showing the typical mammary gland architecture with lobular and ductal structures. (D) IF analysis of Apc1572T/1572T teratomas for luminal and myoepithelial cell types. The frequency of these structures in teratomas derived from Apc1572T/1572T ES cells is largely increased when compared with (F) teratomas derived from Apc+/+ ES cells. (E) Summary of the results of the teratoma differentiation assays of Apc-mutant ES cells. Antibodies employed to evaluate differentiation are: Glial Fibrillary Acidic Protein (GFAP) for glial cells; 2H3 for neurofilaments; SV2 for synaptic vesicles; A4.1025 for adult myosin. Mammary gland structures were primarily identified by HE and then confirmed by IF as shown in panels a–d; also in the case of cartilage and epithelial structures HE stained section were employed. n.d. not determined. Differentiation levels were scored as: (−) not present; (−*) vestigial presence; (+) present; (++) highly abundant. The shaded areas indicate groups of teratomas for which the corresponding antibody staining was negative.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2697381&req=5

pgen-1000547-g002: Teratoma formation assays indicate an intermediate differentiation defect in Apc1572T/1572T ES cells.(A) HE analysis of normal mammary gland with luminal cells surrounded by a basal layer of myoepithelial cells. (B) IF analysis of normal mammary glands for Ck8 (luminal cells, green) and Sma (myoepithelial, red). (C) HE staining of Apc1572T/1572T teratoma showing the typical mammary gland architecture with lobular and ductal structures. (D) IF analysis of Apc1572T/1572T teratomas for luminal and myoepithelial cell types. The frequency of these structures in teratomas derived from Apc1572T/1572T ES cells is largely increased when compared with (F) teratomas derived from Apc+/+ ES cells. (E) Summary of the results of the teratoma differentiation assays of Apc-mutant ES cells. Antibodies employed to evaluate differentiation are: Glial Fibrillary Acidic Protein (GFAP) for glial cells; 2H3 for neurofilaments; SV2 for synaptic vesicles; A4.1025 for adult myosin. Mammary gland structures were primarily identified by HE and then confirmed by IF as shown in panels a–d; also in the case of cartilage and epithelial structures HE stained section were employed. n.d. not determined. Differentiation levels were scored as: (−) not present; (−*) vestigial presence; (+) present; (++) highly abundant. The shaded areas indicate groups of teratomas for which the corresponding antibody staining was negative.
Mentions: Previously, we showed that different levels of β-catenin signaling affect the ability of mouse embryonic stem (ES) cells to differentiate towards specific lineages in a dosage-dependent fashion [5]. To address the same question for the Apc1572T allele, we have subcutaneously injected undifferentiated Apc1572T/1572T ES cells into syngenic mice to induce formation of teratomas, as previously described [5]. The differentiation profiles of the Apc1572T/1572T teratomas were then investigated by histological and immuno-histochemical analysis, and compared with those obtained with wild type (Apc+/+) and other Apc-mutant ES cells. In line with their intermediate level of constitutive Wnt/β-catenin signaling activation, Apc1572T/1572T teratomas show a more heterogeneous spectrum of ecto-, meso-, and endodermal lineages than Apc1638N/1638N (characterized by a higher TopFLASH reporter activity; see Figure 1B), though still more limited in their differentiation capacity than Apc1638T/1638T (characterized by TopFLASH reporter activity comparable with wild type ES cells) (Figure 2E). In agreement with previous observations [5], several differentiation types, namely neural, bone, cartilage and ciliated epithelia were absent in Apc1638N/1638N teratomas. In particular, markers employed to identify neuroectodermal lineages did not stain Apc1638N/1638N sections, in contrast with Apc1572T/1572T teratomas where a limited but significant number of the cells were GFAP positive. Differentiation to striated muscle was also severely affected and detectable in only a minority of the Apc1638N/1638N sections [5], whereas all Apc1572T/1572T teratomas analyzed revealed positive myosin staining. Notably, among the cell types positively identified in Apc+/+ teratomas, mammary epithelia were relatively more abundant in Apc1572T sections, as shown by the combined staining with SMA and CK8 and the typical tissue architecture with luminal cells on top of a myoepithelial basal layer (Figure 2A–2D and Figure 2F). Hence, homozygous Apc1572T ES cells are characterized by an intermediate differentiation defect between Apc1638N/1638N and Apc1638T/1638T, with an unusual enrichment in mammary epithelial differentiation.

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