Wnt signaling activation and mammary gland hyperplasia in MMTV-LRP6 transgenic mice: implication for breast cancer tumorigenesis

Abstract

Although Wnt signaling activation is frequently observed in human breast cancer, mutations in genes encoding intracellular components of the Wnt signaling pathway are rare. We found that the expression of Wnt signaling co-receptor, LRP6, is upregulated in a subset of human breast cancer tissues and cell lines. To examine whether the overexpression of LRP6 in mammary epithelial cells is sufficient to activate Wnt signaling and promote cell proliferation, we generated transgenic mice overexpressing LRP6 in mammary epithelial cells driven by the mouse mammary tumor virus (MMTV) promoter. We found that mammary glands from MMTV-LRP6 mice exhibit significant Wnt activation evidenced by the translocation of beta-catenin from membrane to cytoplasmic/nuclear fractions. The expression of several Wnt target genes including Axin2, Cyclin D1 and c-Myc was also increased in MMTV-LRP6 mice. More importantly, mammary glands from virgin MMTV-LRP6 mice exhibit significant hyperplasia, a precursor to breast cancer, when compared with wild-type littermate controls. Several matrix metalloproteinases are upregulated in MMTV-LRP6 mice that could contribute to the hyperplasia phenotype. Our results suggest that Wnt signaling activation at the cell-surface receptor level can contribute to breast cancer tumorigenesis.

Figures

Figure 1

Generation and characterization of MMTV-LRP6 mice. (a) RNA isolated from mammary glands of MMTV-LRP6 virgin mice and WT littermate controls (12 weeks old) was used for RT-PCR to detect myc-tagged human LRP6 transgene in Founder 4. (b) Quantification of LRP6 transgene expression by q-PCR. Founder 4 has the highest level of LRP6 transgene expression, while Founder 5 has the lowest. (c) Mammary glands from MMTV-LRP6 mice or WT littermate controls (12 weeks old, n=4) were dissected and lysates were prepared. The levels of LRP6 in crude membrane fraction were analyzed by Western blotting with a specific LRP6 antibody. Samples were also probed with an anti-actin antibody to verify equal loading. Lower panel, quantification of the Western blot signals of LRP6 from mammary glands, which were normalized to actin levels. Error bars represent SD. *P

Figure 2

LRP6 immunohistochemical staining in the mammary glands of MMTV-LRP6 mice. (a–b) LRP6 expression in mammary glands of MMTV-LRP6 virgin mice and WT littermate controls was shown through immunohistochemical staining using human LRP6 polyclonal antibody. Bar, 200 μm. (c–d) H & E staining of mammary glands from MMTV-LRP6 virgin mice and WT littermate controls. Bar, 400 μm, inset, 200 μm.

Figure 3

Mammary gland hyperplasia in MMTV-LRP6 mice. (a) Mammary glands from MMTV-LRP6 virgin mice or WT littermate controls (Founder 4 line) at 14 or 21 weeks of age were analyzed by whole mount staining. Inset: high magnification of a selected area. Note mammary hyperplasia in MMTV-LRP6 mice. Bar, 500 μm, inset, 400 μm. (b) Quantification of terminal end buds (TEBs) from WT and MMTV-LRP6 mice (Founder 4, n=4) at 14 or 21 weeks of age. Error bars represent SD. *P<0.05 indicates a significant difference compared to mammary glands from WT littermate controls.

Figure 4

Wnt signaling is up-regulated in HMECs by LRP6 overexpression. HMECs were cultured in 24-well plates. For each well, 0.05 μg of Wnt signaling reporter construct TOPFlash (a) or FOPFlash (b), and 0.05 μg of the β-galactosidase-expressing vector were co-transfected with 0.05 μg of LRP6-, Dkk1-expressing vector, or empty pcDNA3 vector. After 24 h, cells were treated with 5% of Wnt3A CM or L cell control CM. After further 24 h incubation, cells were lysed and both luciferase and β-galactosidase activities were determined. The luciferase activity was normalized to the activity of the β-galactosidase. All values are the average of triple determinations with the SD indicated by error bar. *P

Figure 5

Wnt/β-catenin signaling activation in the mammary glands of MMTV-LRP6 mice. (a) Virgin glands from MMTV-LRP6 mice or WT littermate controls (14 weeks old, n=3) were dissected and lysates were prepared. Cytoplasmic free β-catenin was pulled down from 200 μg of cell lysate using pGST-E-Cadherin, and then examined by Western blotting with a specific β-catenin antibody. The levels of total cellular β-catenin and actin were also analyzed by Western blotting. Right panel, quantification of the Western blotting signals of cytoplasmic free β-catenin. (b–c) Nuclear extracts from mammary glands of MMTV-LRP6 mice or WT littermate controls (14 weeks old, n=3) were prepared by fractionation, and examined by Western Blotting with either regular β-catenin antibody (b) or activeβ-catenin (unphosphorated β-catenin) antibody (c). Lamin B1 was blotted as a loading control. Lower panel, quantification of the Western blot signals of β-catenin from mammary gland nuclear extracts. The levels of β-catenin were normalized to the Lamin B1 levels. Error bars represent SD. *P

Figure 6

Axin2, c-Myc, Cyclin D1 and Ki-67 are up-regulated in MMTV-LRP6 mice. (a) Total RNA was isolated from virgin mammary gland of MMTV-LRP6 virgin mice and WT littermate controls (4 weeks, n=3). The levels of Axin2 were measured by q-PCR. Fold changes in expression compared to WT littermate controls were plotted. (b) Virgin glands from MMTV-LRP6 mice or WT littermate controls (14 weeks old, n=4) were dissected, and lysates were prepared. The levels of total cellular c-Myc and Cyclin D1 were analyzed by Western blotting with a specific c-Myc antibody or Cyclin D1 antibody, and the samples were also probed with an anti-actin antibody to verify equal loading. (c) Quantification of the Western blot signals of c-Myc and Cyclin D1 expression described in (b). The levels of c-Myc and Cyclin D1 expression were normalized to the actin levels. (d, e) Ki67 immunostaining of mammary glands from MMTV-LRP6 virgin mice (e) and WT littermate controls (d). Bar, 200 μm. Error bars represent SD. *P

Figure 7

MMPs are up-regulated in MMTV-LRP6 mice. RNA from mammary glands of 14-month old MMTV-LRP6 virgin mice and WT littermate controls were prepared, and the expression levels of 6 MMPs were measured by q-PCR. Fold changes compared to WT littermate controls were plotted. All the values are the average of triple determinations with the SD indicated by error bars. *P