Loss of amphiregulin reduces myoepithelial cell coverage of mammary ducts and alters breast tumor growth

Abstract

Background: Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, is not only essential for proper mammary ductal development, but also associated with breast cancer proliferation and growth. In the absence of AREG, mammary ductal growth is stunted and fails to expand. Furthermore, suppression of AREG expression in estrogen receptor-positive breast tumor cells inhibits in-vitro and in-vivo growth.

Methods: We crossed AREG-null (AREG^-/-) mice with the murine luminal B breast cancer model, MMTV-PyMT (PyMT), to generate spontaneous breast tumors that lack AREG (AREG^-/- PyMT). We evaluated tumor growth, cytokeratin-8 (K8)-positive luminal cells, cytokeratin-14 (K14)-positive myoepithelial cells, and expression of AREG, Ki67, and PyMT. Primary myoepithelial cells from nontumor-bearing AREG^+/+ mice underwent fluorescence-activated cell sorting and were adapted to culture for in-vitro coculture studies with AT-3 cells, a cell line derived from C57Bl/6 PyMT mammary tumors.

Results: Intriguingly, PyMT-induced lesions progress more rapidly in AREG^-/- mice than in AREG^+/+ mice. Quantification of K8⁺ luminal and K14⁺ myoepithelial cells in non-PyMT AREG^-/- mammary glands showed fewer K14⁺ cells and a thinner myoepithelial layer. Study of AT-3 cells indicated that coculture with myoepithelial cells or exposure to AREG, epidermal growth factor, or basic fibroblast growth factor can suppress PyMT expression. Late-stage AREG^-/- PyMT tumors are significantly less solid in structure, with more areas of papillary and cystic growth. Papillary areas appear to be both less proliferative and less necrotic. In The Cancer Genome Atlas database, luminal-B invasive papillary carcinomas have lower AREG expression than luminal B invasive ductal carcinomas.

Conclusions: Our study has revealed a previously unknown role of AREG in myoepithelial cell development and PyMT expression. AREG expression is essential for proper myoepithelial coverage of mammary ducts. Both AREG and myoepithelial cells can suppress PyMT expression. We find that lower AREG expression is associated with invasive papillary breast cancer in both the MMTV-PyMT model and human breast cancer.

Keywords: Amphiregulin; Breast cancer; MMTV-PyMT; Mammary ductal development.

Fig. 1

In absence of AREG, growth of lesions accelerated. a–d Representative images of carmine-stained AREG^+/+ PyMT and AREG^−/− PyMT mammary fat pads at 6 weeks and 12 weeks. Lesions marked by arrows. Scale bar shows 2000 μm. e Total lesion area measured for AREG^+/+ PyMT (black bars) and AREG^−/− PyMT (gray bars) mice, respectively. At least seven animals measured for 6-week group and at least three animals for 12-week group. f, g AREG^−/− PyMT palpable lesions reached 1-cm diameter more rapidly. f Kaplan–Meier plot of percentage of mice with palpable lesions less than 1 cm for AREG^−/− PyMT mice (dotted line, N = 33) and AREG^+/+ PyMT mice (solid line, N = 50). g Mean and SEM of data in (f). Statistical analysis performed using a log-rank test (f) and t test (e, g). *p < 0.05, **p < 0.01. AREG amphiregulin, PYMT polyoma middle-T antigen

Fig. 2

Loss of AREG enhances progression to invasive carcinoma. Progression of lesions evaluated based on stages: hyperplasia, ductal carcinoma in situ (DCIS), invasive carcinoma. a, b Low-magnification representative H&E images of MFPs of 12-week-old AREG^+/+ PyMT (a) and AREG^−/− PyMT (b) mice. Scale bar shows 500 μm. c, d High-magnification images of stages of progression as seen in AREG^+/+ PyMT (c) and AREG^−/− PyMT (d) mice. Scale bar shows 50 μm. e Greater proportion of AREG^−/− lesions identified as invasive carcinomas while most AREG^+/+ lesions were hyperplastic. N = 10. AREG amphiregulin, PYMT polyoma middle-T antigen

Fig. 3

AREG not expressed in PyMT lesions. a Tissue sections hybridized in situ with AREG probe (dots in ducts), and PyMT protein detected by immunofluorescence. Individual channels shown in gray scale; merged image shows AREG in red and PyMT in green. Ductal structures or lesions outlined in white, and labeling of surrounding adipose tissue is nonspecific background staining. Scale bar shows 50 μm. b Representative images of PyMT immunofluorescent staining of 6-week-old AREG^+/+ PyMT and AREG^−/− PyMT MFPs, respectively. Scale bar shows 100 μm. c At least 10 mammary glands were analyzed for PyMT staining intensity. Statistical analysis performed using t test. AREG amphiregulin, n.s. not significant, PYMT polyoma middle-T antigen, TEB terminal end bud

Fig. 4

Myoepithelial layer has fewer cells and is thinner in thickness in AREG^−/− mice. a Representative images of 12-week-old AREG^+/+ (left) and AREG^−/− (right) mammary ducts immunostained with K8 (red) and K14 (green) in merged channel. Arrow indicates discontinuous myoepithelial layer. Scale bar shows 50 μm. At both 6 weeks (b, c) and 12 weeks (d, e), AREG^−/− glands have thinner myoepithelial layer (b, d) and smaller percentage of K14⁺ cells (c, e). At least three animals used in each analysis. f Proportion of K14⁺ cells in ducts and mature duct termini of 12-week-old AREG^+/+ mammary ducts compared (N = 7). g PyMT expression in AT-3 cells suppressed by myoepithelial cells when cocultured. AT-3 cells either cultured alone or with primary myoepithelial cells overnight. RNA extracted and PyMT expression assessed by RT-qPCR. h AT-3 cells cultured without addition of growth factors (control), with 100 ng/ml AREG, 10 ng/ml EGF, or 10 ng/ml bFGF, or with both EGF and bFGF. Statistical analysis performed using t test. *p < 0.05, **p < 0.01, ***p < 0.001. AREG amphiregulin, bFGF basic fibroblast growth factor, EGF epidermal growth factor, PYMT polyoma middle-T antigen

Fig. 5

Late-stage AREG^−/− tumors less solid with greater proportion of papillary tumor features. a One-centimeter tumors from AREG^+/+ PyMT (N = 32) and AREG^−/− PyMT (N = 22) mice stained with H&E. Scale bars for whole tumors and sections 2000 μm (left) and 100 μm (right), respectively. b Proportion of solid and papillary tumor areas determined for each tumor. c Proportion of AREG^+/+ PyMT (N = 32) and AREG^−/− PyMT (N = 22) tumors that have cysts or no cysts. Statistical analysis performed using Mann–Whitney test (b) and chi-square test (c). **p < 0.01, ****p < 0.0001. AREG amphiregulin, PYMT polyoma middle-T antigen

Fig. 6

AREG^−/− tumors are less necrotic and tumor cells in papillary regions are less proliferative. a Representative images of H&E (left column) and Ki67 (right column) staining in AREG^+/+ PyMT and AREG^−/− PyMT 1-cm tumors. Scale bar shows 500 μm for H&E stains and Ki67 stains. b Percentage of necrotic areas calculated as average of five fields per AREG^+/+ (N = 32) and AREG^−/− (N = 22) 1-cm tumors. c, d Ki67⁺ proliferating cells in solid vs papillary areas in 1-cm AREG^+/+ PyMT and AREG^−/− PyMT tumors compared using HistoQuant. Evaluation performed on at least five separate areas from at least three different tumors per genotype. Statistical analysis performed using t test. *p < 0.05, ***p < 0.001, ****p < 0.0001. AREG amphiregulin, H&E hematoxylin and eosin, PYMT polyoma middle-T antigen

Fig. 7

Lower AREG expression associated with papillary breast cancer. a AREG expression compared between 115 luminal-B nonpapillary IDC samples and eight luminal-B IPC samples. b Representative H&E images of luminal-B IDC and luminal-B IPC. Statistical analysis performed using Mann–Whitney test. p < 0.0001. AREG amphiregulin

Fig. 8

Working model to explain increased tumor initiation and morphological changes in AREG^−/− tumors. (a) In mammary duct in absence of PyMT (top), myoepithelial cells (green) form continuous layer around luminal epithelial cells. In mature duct termini, myoepithelial layer is discontinuous. Luminal epithelial cells secrete AREG that binds to EGFR on stromal cells (yellow). Stimulated stromal cells produce FGFR ligands that bind to FGFR luminal epithelial cells and myoepithelial cells [31, 56]. In absence of AREG (bottom), EGFR/FGFR paracrine loop is interrupted and impairs proper mammary ductal development. (b) In AREG^+/+ PyMT animals (top), PyMT initiates transformation of luminal epithelial cells in mature duct termini where there are fewer myoepithelial cells. Myoepithelial cells as well as secreted growth factors such as AREG and bFGF suppress PyMT expression in mammary duct. In AREG^−/− PyMT mice (bottom), PyMT expression is more widespread. Due to global reduction in myoepithelial cells and reduced AREG and FGF expression, oncogenic transformation takes place more broadly in ductal tree. AREG amphiregulin, FGF basic fibroblast growth factor, PYMT polyoma middle-T antigen