Lunatic Fringe and p53 Cooperatively Suppress Mesenchymal Stem-Like Breast Cancer

Abstract

Claudin-low breast cancer (CLBC) is a poor prognosis molecular subtype showing stemness and mesenchymal features. We previously discovered that deletion of a Notch signaling modulator, Lunatic Fringe (Lfng), in the mouse mammary gland induced a subset of tumors resembling CLBC. Here we report that deletion of one copy of p53 on this background not only accelerated mammary tumor development but also led to a complete penetrance of the mesenchymal stem-like phenotype. All mammary tumors examined in the Lfng/p53 compound mutant mice displayed a mesenchymal/spindloid pathology. These tumors showed high level expressions of epithelial-to-mesenchymal transition (EMT) markers including Vimentin, Twist, and PDGFRα, a gene known to be enriched in CLBC. Prior to tumor onset, Lfng/p53 mutant mammary glands exhibited increased levels of Vimentin and E-cadherin, but decreased expressions of cytokeratin 14 and cytokeratin 8, accompanied by elevated basal cell proliferation and an expanded mammary stem cell-enriched population. Lfng/p53 mutant glands displayed increased accumulation of Notch3 intracellular fragment, up-regulation of Hes5 and down-regulation of Hes1. Analysis in human breast cancer datasets found the lowest HES1 and second lowest LFNG expressions in CLBC among molecular subtypes, and low level of LFNG is associated with poor survival. Immunostaining of human breast cancer tissue array found correlation between survival and LFNG immunoreactivity. Finally, patients carrying TP53 mutations express lower LFNG than patients with wild type TP53. Taken together, these data revealed genetic interaction between Lfng and p53 in mammary tumorigenesis, established a new mouse model resembling CLBC, and may suggest targeting strategy for this disease.

Figure 1

Drastically accelerated mammary tumor development in the Lfng^flox/flox;p53^flox/+;MMTV-Cre mice as compared to the Lfng^flox/flox;MMTV-Cre mice. Kaplan–Meier analysis was performed for mammary tumor-free survival in female mice of these two genotypes. Wild type, Lfng^flox/flox, and Lfng^flox/flox;p53^flox/+ animals were included in the “No Cre” control.

Figure 2

Histological and immunohistochemical analyses of preneoplastic lesions and mammary tumors in the Lfng^flox/flox;p53^flox/+;MMTV-Cre mice. (A-C) Representative photomicrographs of H&E stained mammary gland sections at 2 months of age. (D-F) Representative photomicrographs of H&E stained mammary tumor sections. (G-L) Representative photomicrographs of immunostaining in mammary tumors. Insets: high-magnification images of Notch nuclear staining. Percentage of positively stained cells are quantitated and presented as mean ± standard deviation. Scale bars: 50 μm.

Figure 3

Altered expressions of differentiation markers and increased basal cell proliferation in the Lfng^flox/flox;p53^flox/+;MMTV-Cre mammary epithelium. (A and B) Representative Western blots for epithelial and mesenchymal markers in the mammary glands of littermate mice at 3 months of age. (C) Representative photomicrographs of E-Cadherin immunostaining, Ki67/cytokeratin 8 (K8) and Ki67/cytokeratin 14 (K14) immunofluorescence staining in the mammary glands of 2-month-old Lfng^flox/flox;MMTV-Cre and Lfng^flox/flox;p53^flox/+;MMTV-Cre mice. Scale bars: 50 μm. The percentage of Ki67⁺ cells among K8⁺ luminal cells and K14⁺ basal cells was quantitated and presented as mean ± standard deviation. *P < .05.

Figure 4

Expansion of stem/progenitor cell-enriched population in the Lfng^flox/flox;p53^flox/+;MMTV-Cre mammary gland. (A) Representative flow cytometry analysis of lineage-depleted mammary cells from littermate mice of indicated genotypes at 2 months of age. (B) Quantitation of CD24^MedCD49f^Hi and CD24^HiCD49f^Low cell populations from three experiments. Percentages of the populations were presented as mean ± standard deviation. (C) Representative photomicrographs and quantification of mammospheres formed in 3D cultures of the mammary epithelial cells isolated from the Lfng^flox/flox;p53^flox/+ and Lfng^flox/flox;p53^flox/+;MMTV-Cre mice at 2 months of age. Scale bars: 60 μm. *P < .05.

Figure 5

Altered Notch signaling in the Lfng^flox/flox;p53^flox/+;MMTV-Cre mammary gland. (A) Western blot analysis for Notch receptors and Jagged1 ligand in the mammary glands of littermate mice at 3 months of age. (B) Quantitative RT-PCR for Hes1, Hes5, Hey1 and Hey2 in the mammary glands at 3 months of age. *P < .05.

Figure 6

Human claudin-low breast cancer exhibits low levels of LFNG and HES1 gene expression. (A) Boxplots for expression values of LFNG, NOTCH3, JAG1, HES1 and HEY1 in 6 subtypes of human breast cancer (dataset GSE18229). (B) Heat map for expressions of selected Notch pathways genes in 6 subtypes of human breast cancer (METABRIC dataset). (C) Boxplots for expression values of LFNG, NOTCH3, JAG1, HES1 and HEY1 in 6 subtypes of human breast cancer (METABRIC dataset). Basal: Basal-like. Claudin: Claudin-low. Her2: HER2-positive. LumA: Luminal A. LumB: Luminal B. Normal: Normal breast-like. p values were calculated by comparing expression means across all subtypes.

Figure 7

Low level of LFNG expression is associated with poor survival among breast cancer patients and is enriched in patients carrying TP53 mutation. (A-C) Survival analyses in breast cancer patients using univariate Cox regression and Kaplan–Meier methods. Overall survival rate (A), relapse-free survival rate (B), and post-progression survival rate (C) in patients with high LFNG expression (red line) were all significantly higher than that in patients with low LFNG expression (black line). (D) Representative photomicrographs of LFNG immunostaining on a human breast cancer tissue array. Staining intensity scores are: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). Scale bars: 100 μm. (E) Kaplan–Meier survival analysis for patients with low LFNG expression (staining score 0 or 1) and high LFNG expression (staining score 2 or 3). (F) Boxplot for LFNG expression levels in patients with wild type or mutant TP53 gene.