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, 1 (5), 229-44

Amphiregulin Mediates Estrogen, Progesterone, and EGFR Signaling in the Normal Rat Mammary Gland and in Hormone-Dependent Rat Mammary Cancers

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Amphiregulin Mediates Estrogen, Progesterone, and EGFR Signaling in the Normal Rat Mammary Gland and in Hormone-Dependent Rat Mammary Cancers

Anastasia Kariagina et al. Horm Cancer.

Abstract

Both estrogen (E) and progesterone (P) are implicated in the etiology of human breast cancer. Defining their mechanisms of action, particularly in vivo, is relevant to the prevention and therapy of breast cancer. We investigated the molecular and cellular mechanisms of E and/or P-induced in vivo proliferation, in the normal rat mammary gland and in hormone-dependent rat mammary cancers which share many characteristics with the normal human breast and hormone-dependent breast cancers. We show that E+P treatment induced significantly greater proliferation in both the normal gland and mammary cancers compared to E alone. In both the normal gland and tumors, E+P-induced proliferation was mediated through the increased production of amphiregulin (Areg), an epidermal growth factor receptor (EGFR) ligand, and the activation of intracellular signaling pathways (Erk, Akt, JNK) downstream of EGFR that regulate proliferation. In vitro experiments using rat primary mammary organoids or T47D breast cancer cells confirmed that Areg and the synthetic progestin, R5020, synergize to promote cell proliferation through EGFR signaling. Iressa, an EGFR inhibitor, effectively blocked this proliferation. These results indicate that mediators of cross talk between E, P, and EGFR pathways may be considered as relevant molecular targets for the therapy of hormone-dependent breast cancers, especially in premenopausal women.

Figures

Fig. 1
Fig. 1
Hormonal regulation of cell-type-specific proliferation in the normal mammary gland. a Immunofluorescent labeling with anti-BrdU and anti-SMA antibody to identify luminal (BrdU positive/SMA negative) and myoepithelial (BrdU positive/SMA positive) proliferating cells in ducts and lobules of OVX animals treated with vehicle (C), E alone (E), P alone (P), or E+P was performed as described in the “Methods” section. Bars represent mean ± SEM from three to five animals per experimental group and 1,000 cells counted per animal. *P < 0.05 compared to control. b Immunoblot analysis of cell cycle regulatory protein PCNA
Fig. 2
Fig. 2
Hormonal regulation of PR isoform expression and colocalization with ERα or proliferation. ac Immunofluorescent labeling with PRA- and PRB-specific antibody. The percentage of PR-positive cells in ducts (a) or lobules (b) from ovary-intact (OI) or OVX animals treated with vehicle (c), E, P, or E+P. Bars represent the mean ± SEM from three to five animals per experimental group and 1,000 cells counted per animal. *P < 0.05. The percent of PRA+PRB+ cells is reduced compared to OI, E- or E+P-treated groups. c Representative merged images of PRA (teal) and PRB (magenta) expression in lobules from OI, C, E, P, or E+P-treated glands. Yellow arrows indicate cells co-expressing PRA and PRB. White arrows indicate cells expressing only PRB. d Immunoblot analysis of PRB expression in whole mammary gland extracts. Uterine extract (UT) was run as a positive control and was developed with antibody detecting only PRB by immunoblot (hPRa6). e, f Colocalization of ERα with PR isoforms. Representative merged images of immunofluorescent labeling with anti-ERα (green) and e anti-PRA− (magenta) or (f) anti-PRB− (magenta) antibodies in E+P-treated ducts. Yellow arrows indicate cells e co-expressing ERα and PRA or f co-expressing ERα and PRB. White arrows indicate cells expressing only PRB. Nuclei were counterstained with DAPI. Scale bars, 50 μm. g Triple immunolabeling with PRA-, PRB-, and BrdU-specific antibodies and quantitation of PR+BrdU+ cells. Bars represent the mean ± SEM from five animals per experimental group
Fig. 3
Fig. 3
Hormonal regulation of Areg expression and colocalization with PR isoforms. a Real-time RT-PCR analysis of EGFR ligand mRNA expression. Bars represent the mean ± SEM fold increase compared to the level of EGF mRNA in OVX control. *P < 0.05, that hormone treated was greater than control. b Immunoblot analysis of immunoprecipitated Areg protein. c Immunofluorescent colocalization of Areg with PR isoforms. Representative confocal merged images of Areg (green), PRB (red), and PRA (blue) staining in ducts. Yellow arrows indicate cells expressing only PRB (red nuclei). White arrows indicate cells that co-express Areg, PRB, and PRA. Green arrow indicates cells co-expressing PRB and PRA but no Areg. Scale bar, 50 μm
Fig. 4
Fig. 4
Effect of Areg implants plus hormone treatments on proliferation. OVX animals were implanted with Elvax pellets containing Areg and BSA control in the contralateral gland and treated with vehicle control (C), E alone, or P alone. a Immunoperoxidase staining with BrdU-specific antibody. Representative images of ducts adjacent to Areg or control implants. Red arrows indicate BrdU+ proliferating epithelial cells. Yellow arrows indicate BrdU+ proliferating stromal cells. Asterisks indicate location of Elvax implant. Scale bar, 100 μm. b, c Quantitation of proliferating cells in ducts (b) and lobules (c) adjacent to control or Areg implants. Bars represent the mean ± SEM obtained from 3 to 5 animals per treatment group and a minimum of 500 epithelial cells counted. *P < 0.05
Fig. 5
Fig. 5
Hormonal regulation of EGFR signaling. Mammary glands were obtained from OVX animals treated with vehicle (C), with E alone, P alone, or E+P. a Real-time PCR analysis of EGFR mRNA and immunoblot analysis of EGFR protein expression. Bars represent the mean ± SEM fold increase. *P < 0.05 compared to OVX control. b Immunoblot analysis of phospho-Akt, phospho-c-Jun, phospho-Erk, and c-Fos expression in whole mammary gland extracts. c, d Representative merged images of immunofluorescence analysis of c phospho-Akt and d phospho-c-Jun in E- or E+P-treated glands. White arrows indicate nuclear localization of phospho-Akt or phospho-c-Jun. Yellow arrows indicate cytoplasmic phospho-Akt. Nuclei counterstained with DAPI (blue). Scale bar, 50 μm. e Immunoblot analysis of phosphorylated PRB. Total protein was immunoprecipitated with a mixture of anti-PR antibodies (DAKO and hPRa7). Phospho-PRB was detected by immunoblot with phospho-serine-specific antibody (primary mouse mAb and IRDye™800-labeled secondary antibody), followed by incubation with PRB-specific antibody (primary rabbit polyclonalB15, 1:500; IRDye™680-labeled secondary antibody). Simultaneous detection and colocalization of the two antibodies (anti-phospho-serine and anti-PRB) was used to measure the level of phospho-PRB. Total PRB and phospho-PRB are shown separately. Numbers above bands indicate fold change compared to OVX control after normalization to b β-actin or e IgG
Fig. 6
Fig. 6
P and Areg signaling are required for proliferation in primary mammary organoids in vitro. a Merged images of representative PRA and PRB in organoids. Nuclei were counterstained with DAPI (blue). White arrows indicate cells expressing PR. Scale bar, 25 μm. b Effect of EGFR inhibitor, Iressa, and PR inhibitor, RU486, on proliferation of organoids. Quantitation of proliferating myoepithelial (BrdU positive/SMA positive) and luminal (BrdU positive/SMA negative) cells. Bars represent mean ± SEM; 1,000 epithelial cells counted per treatment. *P < 0.05, that Areg+P was greater than all treatment groups. #P < 0.05, that Areg+P combined with either Iressa or RU486 treatment was significantly less than Areg+P
Fig. 7
Fig. 7
PR and EGFR signaling and proliferation in T47D breast cancer cells. T47D breast cancer cell lines expressing only PRA (YA), only PRB (YB), or lacking PR (Y) were treated with basal media (BM), R5020, Areg, or Areg+R5020, and analyzed with a phospho-Akt or c phospho-Erk antibody as described in the “Methods” section. The effect of Iressa or RU486 on b Akt or d Erk phosphorylation in T47D-YB cells. Bars represent the mean ± SEM from a representative experiment. ad *P < 0.05 for comparisons indicated. e Cell cycle analysis in treated T47D-YB cells. *P < 0.05, that the percentage of cells in S phase in Areg+R5020 was greater compared to other treatments. #P < 0.05, that Areg+R5020 combined with either Iressa or RU486 treatment was significantly less than Areg+R5020
Fig. 8
Fig. 8
Hormonal regulation of proliferation and PR isoform expression in hormone-dependent rat mammary tumors. Mammary tumors were obtained from carcinogen-treated OVX animals implanted with E alone or E+P. a Tumor cell proliferation was assessed by BrdU incorporation. Individual tumors (n = 16–22 per treatment group) are represented by a single dot. Mean ± SEM obtained from 1,000 cells counted per tumor. *P < 0.05. b Immunoblot analysis of PCNA in tumor extracts. Numbers above bands indicate normalized intensity of PCNA band. c The percentage of PR-positive cells in E or E+P-treated tumors. Bars represent the mean ± SEM from five tumors per experimental group with 1,000 tumor cells counted. *P < 0.05 that percent of cells expressing only PRA is increased and cells expressing PRB only is decreased in E-treated tumors
Fig. 9
Fig. 9
Hormonal regulation of EGFR signaling in E- or E+P-treated tumors. a, b Real-time RT-PCR analysis of a EGFR and b EGFR ligand mRNA levels. Bars represent the mean ± SEM fold change compared to levels in E-treated tumors (n = 4–5 tumors/group). c Representative merged images of immunolabeling with Areg- (green), PRA- (blue), and PRB- (red) specific antibodies. White arrows indicate cells co-expressing Areg and PRA. Yellow arrows indicate cells co-expressing Areg, PRB, and PRA. Scale bar, 75 μm. d Immunoblot analysis of Areg, phospho-Akt, phospho-JNK, and phospho-Erk for the same E- or E+P-treated tumors. Numbers above bands indicate relative band intensity normalized to β-actin
Fig. 10
Fig. 10
Diagram of convergence of E, P, and EGFR signaling pathways. 1 E acting via ERα and P acting via PRA/PRB induce Areg mRNA expression and protein in ERα+PRA+PRB+ cells. 2 Secreted Areg acts in a paracrine manner and activates EGFR signaling in ERα−PRA−PRB+ cells and 3 in an autocrine manner in ERα+PRA+PRB+ cells and activates EGFR signaling. EGFR signaling activates Akt (4), JNK (5), and Erk (6) leading to formation of AP-1 complex (phospho-c-Jun/c-Fos). 4 PRB may enhance Akt phosphorylation. 7 Activation of Erk leads to phosphorylation of PRB. 8 Activated AP-1 and liganded phospho-PRB induce cell cycle regulatory protein PCNA and proliferation in ERα+PRA+PRB+ and ERα−PRA−PRB+ cells

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