Overexpression of human Cripto-1 in transgenic mice delays mammary gland development and differentiation and induces mammary tumorigenesis

Abstract

Overexpression of Cripto-1 has been reported in several types of human cancers including breast cancer. To investigate the role of human Cripto-1 (CR-1) in mammary gland development and tumorigenesis, we developed transgenic mice that express the human CR-1 transgene under the regulation of the whey acidic protein (WAP) promoter in the FVB/N mouse background. The CR-1 transgene was detected in the mammary gland of 15-week-old virgin WAP-CR-1 female mice that eventually developed hyperplastic lesions. From mid-pregnancy to early lactation, mammary lobulo-alveolar structures in WAP-CR-1 mice were less differentiated and delayed in their development due to decreased cell proliferation as compared to FVB/N mice. Early involution, due to increased apoptosis, was observed in the mammary glands of WAP-CR-1 mice. Higher levels of phosphorylated AKT and MAPK were detected in mammary glands of multiparous WAP-CR-1 mice as compared to multiparous FVB/N mice suggesting increased cell proliferation and survival of the transgenic mammary gland. In addition, more than half (15 of 29) of the WAP-CR-1 multiparous female mice developed multifocal mammary tumors of mixed histological subtypes. These results demonstrate that overexpression of CR-1 during pregnancy and lactation can lead to alterations in mammary gland development and to production of mammary tumors in multiparous mice.

Figure 1

A: Schematic representation of the WAP promoter-human Cripto-1 (CR-1) expression vector 2B3 is shown. B: RT-PCR analysis using primers specific for CR-1 shows expression of the CR-1 transgene, relative to GAPDH, in the mammary gland obtained at different developmental stages from virgin (V), pregnant (P), lactating (L), and involuting (Inv) WAP-CR-1 transgenic mice. Mammary tissues from multiparous (M) WAP-CR-1 transgenic mice without (NT) or with mammary tumors (T) were also assessed. C: Quantification of RT-PCR by densitometry and normalization for GAPDH expression is shown.

Figure 2

A to F: H&E-stained sections of mouse mammary tissue from FVB/N control (A, C, E) and WAP-CR-1 transgenic (B, D, F) obtained at pregnancy day 20 (P20), lactation day 2 (L2), and lactation day 14 (L14). Reduced formation of lobulo-alveolar structures in WAP-CR-1 transgenic compared to FVB/N control mice is detected at P20 and L2 (A versus B and C versus D). Differences in epithelial content and alveolar development is no longer apparent in mammary glands of FVB/N control (E) and WAP-CR-1 transgenic (F) in later stages of lactation (L14). G and H: A significant reduction in proliferation rate in the WAP-CR-1 transgenic mammary epithelial cells can be detected at lactation day 2 (L2) as compared to FVB/N as demonstrated by the percentage (G) of epithelial cells staining positive for PCNA (H, arrows). Sections are counterstained with hematoxylin. I and J: Reduced synthesis of the milk proteins WAP and β-casein in mammary glands from 20-day pregnant (P20) WAP-CR-1 transgenic as compared to mammary glands from matched FVB/N mice is demonstrated by representative Western blot (I) and summary of densitometric analysis of Western blots from repeat experiments (J). *P < 0.05. Original magnifications: ×20 (A–F); ×63 (H).

Figure 3

A–D: H&E-stained sections of mouse mammary tissue from FVB/N control (A, C) and WAP-CR-1 transgenic mice (B, D) obtained during days 2 and 3 of involution (respectively, Inv2 and Inv3) show reduction in mammary epithelial content in the WAP-CR-1 mice as compared to FVB/N control mice, which is accentuated during Inv3 (C versus D). E and F: Expression of markers for involution, CEBPD, and Stat3, is detected by Western blot (E) of lysates from representative mammary glands of two FVB/N and WAP-CR-1 mice and quantified by densitometric analysis (F). G and H: Immunofluorescent terminal dUTP nick-end labeling assay of mammary glands from FVB (Ga) and WAP/CR-1 (Gb) shows a significant increase in the percentage of apoptotic cells in mammary tissue from WAP-CR-1 during Inv3 compared to matched FVB/N (H). Original magnifications: ×20 (A–D); ×63 (G).

Figure 4

A: Mammary tissue of 15-month-old nulliparous FVB/N mice (Aa, Ab) and nulliparous WAP-CR-1 transgenic mice (Ac, Ad) as compared by whole mount morphology (Aa and Ac, Carnoy stain) and histological analysis (Ab and Ad, H&E). Ductal structures with increased layers of atypical epithelial cells suggestive of MIN were identified more frequently in mammary tissue from ≥15-month-old WAP-CR-1 transgenic nulliparous mice (50%) as compared to the number of similar lesions found in age-matched nulliparous FVB/N mice (14.2%) (Ae). B: Mammary tissue from multiparous (M) WAP-CR-1 mice as compared to multiparous FVB/N expressed increased levels of the active, phosphorylated forms of AKT and MAPK as shown by a representative Western blot analysis (Ba) of the respective mammary tissue lysates and confirmed by densitometric quantification of the Western blot expression bands after normalization against total expression (T-) of the respective signaling molecule (Bb). CK-18 controls for epithelial content in tissues analyzed. C: Mammary gland tumor-free survival is greater in multiparous FVB/N mice compared to multiparous WAP-CR-1 transgenic mice. Overall, ∼50% of WAP-CR-1 transgenic mice developed mammary tumors throughout 13 months. Original magnifications: ×5 (Aa, Ac); ×40 (Ab, Ad).

Figure 5

Representative H&E-stained sections illustrate the major histological subtypes identified in mammary tumors from WAP-CR-1 transgenic mice: A: Mammary intraepithelial neoplasia (arrows); B: glandular; C: papillary; D: adenosquamous lesions surrounded by inflammatory infiltrates (arrows); E: solid; F: myoepithelial. Occasionally, surrounding muscle (G), adipose (H), or subepidermal (I) tissue showed local invasion by tumor cells (arrows). Original magnifications, ×20.

Figure 6

A: Immunohistochemistry results show positive staining for human CR-1 in the different subtypes identified in mammary tumors from WAP-CR-1 transgenic mice. B, top: Staining for smooth muscle actin (SMA) suggests myoepithelial origin of the cells composing myoepithelial-type WAP/CR-1 mammary tumor. B, bottom: In normal FVB/N mammary gland, smooth muscle actin stains the myoepithelial cells (red) (AEC chromogen used) surrounding the acinar unit. C: Western blot analysis of mammary gland tissue lysates demonstrates expression of human CR-1 and expression of DP-β-cat in mammary tumors from WAP-CR-1 multiparous mice as compared to almost no expression in mammary glands from multiparous FVB/N mice. D: Expression of DP-β-cat is also detected in nontumor mammary glands (NMG) of WAP-CR-1 transgenic mice. E: Representative immunohistochemistry shows positive nuclear staining for β-catenin (top, arrows) in sections of mammary tumors from WAP-CR-1 transgenic mice as compared to negative staining in mammary tumor section from FVB/N mouse (bottom). All sections are counterstained with hematoxylin. Original magnifications: ×20 (A; E, bottom); ×40 (B, top; E, top); ×63 (B, bottom).

Figure 7

A: Immunohistochemistry shows positive staining for cytokeratin-1 (CK-1) and cytokeratin-6 (CK-6) detected in the different subtypes in mammary tumors from WAP-CR-1 transgenic mice. B: Little or no staining was observed for either CK-1 or CK-6 in sections of mammary gland from FVB/N mice. C: Positive immunostaining for hair and nail cytokeratin, AE-13, is shown in two squamous metaplastic lesions of the adenosquamous subtype in the WAP-CR-1 mammary tumors. All sections are counterstained with hematoxylin. Original magnifications: ×20 (A, B); ×63 (C).