Myc is required for β-catenin-mediated mammary stem cell amplification and tumorigenesis - PubMed (original) (raw)
Myc is required for β-catenin-mediated mammary stem cell amplification and tumorigenesis
Mejdi Moumen et al. Mol Cancer. 2013.
Abstract
Background: Basal-like breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Recent studies have linked activation of the Wnt/β-catenin pathway, and its downstream target, Myc, to basal-like breast cancer. Transgenic mice K5ΔNβcat previously generated by our team present a constitutive activation of Wnt/β-catenin signaling in the basal myoepithelial cell layer, resulting in focal mammary hyperplasias that progress to invasive carcinomas. Mammary lesions developed by K5ΔNβcat mice consist essentially of basal epithelial cells that, in contrast to mammary myoepithelium, do not express smooth muscle markers.
Methods: Microarray analysis was used to compare K5ΔNβcat mouse tumors to human breast tumors, mammary cancer cell lines and the tumors developed in other mouse models. Cre-Lox approach was employed to delete Myc from the mammary basal cell layer of K5ΔNβcat mice. Stem cell amplification in K5ΔNβcat mouse mammary epithelium was assessed with 3D-culture and transplantation assays.
Results: Histological and microarray analyses of the mammary lesions of K5ΔNβcat females revealed their high similarity to a subset of basal-like human breast tumors with squamous differentiation. As in human basal-like carcinomas, the Myc pathway appeared to be activated in the mammary lesions of K5ΔNβcat mice. We found that a basal cell population with stem/progenitor characteristics was amplified in K5ΔNβcat mouse preneoplastic glands. Finally, the deletion of Myc from the mammary basal layer of K5ΔNβcat mice not only abolished the regenerative capacity of basal epithelial cells, but, in addition, completely prevented the tumorigenesis.
Conclusions: These results strongly indicate that β-catenin-induced stem cell amplification and tumorigenesis rely ultimately on the Myc pathway activation and reinforce the hypothesis that basal stem/progenitor cells may be at the origin of a subset of basal-like breast tumors.
Figures
Figure 1
K5ΔNβcat mammary tumors display characteristics of basal-like tumors. A. Haematoxylin/eosin staining. B. Double immunofluorescence analysis with anti-K5 (green) and anti α-SMA (red) antibodies; nuclei are stained with DAPI. C, D. Immunohistochemistry with anti-ER (C) and anti-PR (D) antibodies. Arrow in C indicates the presence of normal ductal luminal cells positively stained for ER. Bar: 75 μm (B,C), 150 μm (A,D). E. qPCR analysis of representative basal and luminal genes. The graph shows mean values ± SEM for four control tissue and five K5ΔNβcat tumor samples; p < 0.05. F. Cluster analysis of K5ΔNβcat tumors, for the subset of genes defining the basal cluster in mouse tumor models (upper panel, [24]) and four luminal epithelial genes (lower panel). Data from 4 control mammary tissue, 5 hyperplasia and 11 tumor samples from K5ΔNβcat female are shown.
Figure 2
Myc is necessary for mammary tumor formation in K5ΔNβcat animals. A. Double immunofluorescence analysis of a K5ΔNβcat tumor with anti-HA (red), and anti-Myc (green) antibodies. Arrows indicate the presence of nuclei positively stained for Myc. Bar: 40 μm. B. qPCR analysis of Myc target genes. The graph shows mean ± SEM for four control tissue and five K5ΔNβcat tumor samples; p < 0.05. C. Kaplan-Meier tumor-free survival curve of K5ΔNβcat virgin and parous mice. 17 virgin and 10 parous K5ΔNβcat, 14 virgin and 6 parous K5ΔNβcat;K5Cre;Myc F/+ and 12 virgin and 10 parous K5ΔNβcat; K5Cre;Myc F/F females were analyzed.
Figure 3
Deregulated β-catenin signaling induces the expansion of the basal cell population. A. Whole-mount carmine staining of mammary glands from 10-month-old mice. Bar: 3 mm. B. Separation of luminal (blue ovals, CD24+/CD49low) and basal (red ovals, CD24+/CD49high) epithelial cell populations from 10-month-old mice. C. qPCR analysis of Myc expression in freshly isolated basal (B) and luminal (L) mammary cell populations from 10-month-old virgin mice. D. Percentage of basal cells within the epithelial cell compartment (mean ± SEM of 3 independent experiments; *p < 0.05; **p < 0.001).
Figure 4
Myc is required for stem cell amplification induced by deregulated β-catenin signaling. A. Clonal colonies formed by 1000 sorted mammary basal cells. The graph shows the percentage of clonogenic cells (mean ± SEM of 3 independent experiments; *p < 0.01). B. Sphere formation by sorted basal cells. The graph shows the percentage of sphere-forming cells (mean ± SEM of 3 independent experiments; *p < 0.01). Bar: 100 μm. C. Mammary epithelial outgrowths developed from 1000 mammary basal cells transplanted into the cleared mammary fat pads. Bar: 4 mm. D. qPCR analysis of proliferation-related genes. Basal cell samples from K5ΔNβcat;K5Cre;Myc F/F and control mice obtained in 3 independent sorting experiments were analyzed.
Figure 5
Hypothetical scheme on the effects of β-catenin signaling activation on stem/progenitor population and tumor induction. A. During normal homeostasis, basal and luminal progenitors with limited self-renewal potential, maintain their respective lineages. B. Constitutive activation of β-catenin signaling leads to enhanced basal progenitor self-renewal and expansion preventing myoepithelial differentiation and giving rise to basal-like tumors. Both, basal progenitor amplification and tumorigenesis are Myc-dependent.
References
- Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS. et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA. 2001;98:10869–10874. doi: 10.1073/pnas.191367098. - DOI - PMC - PubMed
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