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Estrogen receptor-positive mammary tumorigenesis in TGFalpha transgenic mice progresses with progesterone receptor loss - PubMed

  • ️Mon Jan 01 2007

Estrogen receptor-positive mammary tumorigenesis in TGFalpha transgenic mice progresses with progesterone receptor loss

T A Rose-Hellekant et al. Oncogene. 2007.

Abstract

We characterized the novel NRL-transforming growth factor alpha (NRL-TGFalpha) transgenic mouse model in which growth factor - steroid receptor interactions were explored. The NRL promoter directs transgene expression to mammary ductal and alveolar cells and is nonresponsive to estrogen manipulations in vitro and in vivo. NRL-TGFalpha mice acquire proliferative hyperplasias as well as cystic and solid tumors. Quantitative transcript analysis revealed a progressive decrease in estrogen receptor alpha (ER) and progesterone receptor (PR) mRNA levels with tumorigenesis. However, ER protein was evident in all lesion types and in surrounding stromal cells using immunohistochemistry. PR protein was identified in normal epithelial cells and in very few cells of small epithelial hyperplasias, but never in stromal or tumor cells. Prophylactic ovariectomy significantly delayed tumor development and decreased incidence. Finally, while heterozygous (+/-) p53 mice did not acquire mammary lesions, p53+/- mice carrying the NRL-TGFalpha transgene developed ER negative/PR negative undifferentiated carcinomas. These data demonstrate that unregulated TGFalpha expression in the mammary gland leads to oncogenesis that is dependent on ovarian steroids early in tumorigenesis. Resulting tumors resemble a clinical phenotype of ER+/PR-, and when combined with a heterozygous p53 genotype, ER-/PR-.

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Figures

Figure 1
Figure 1

Photomicrographs of nontransgenic (a and f), NRL-hPAP (b–e), and NRL-TGFα mammary glands (g–u). The hPAP transgene was uniformly expressed throughout epithelia (b–e), including terminal endbuds at 12-weeks (arrowhead, c), while glands from an age-matched wild-type mice did not stain for hPAP (a). H&E staining demonstrates the normal architecture of a virgin adult >12 weeks (f). Glands from NRL-TGFα mice developed small multifocal lobular hyperplasias beginning at 16-weeks (g). At end stage, intraductal hyperlasias (h), squamous hyperplasias (i), large lobular hyperplasias and tumors (j) also were present. Cystic papillary tumors were present in all mice evaluated (k) and some acquired solid adenomatous tumors (l and m). ER (n) and PR (r) staining was evident in normal ducts. ER was evident in hyperplasias (o), infiltrating cells (p), and tumors (q). However, adjacent sections mostly were devoid of PR (s–u, respectively). 10 × : a and b; 15 × : c; 40 × : d, l; 100 × : f, h, j, k; 200 × : g, i, m; 400 × : e, n–u.

Figure 2
Figure 2

Tumor incidence was 100% for three NRL-TGFα lines, and latency was shortened with parity. The asterisk indicates a significant difference in latency between nulliparous and parous mice within each line (P≤0.05; Student’s t-test). Bars represent mean ± s.d.

Figure 3
Figure 3

The NRL promoter is not responsive to estrogen in vitro (a) or in vivo (b). (a) Left: CHO-K1 cells were transiently transfected with either rNRL-Luc, pGL3 Basic (negative control) or pOT-ERE (positive control) along with an expression construct for ER and CMV-β-gal (transfection control), and treated ± 10 nM E for 24 h. Right: T47D cells were transiently transfected with either rNRL-Luc, or pPgRE-Luc (positive control), and CMV-β-gal, and treated ± 10 nM R5020 for 24 h. Open bars indicate vehicle treatment; solid bars indicate hormonal treatment N = 3; mean ±s.e. Asterisks indicates significant differences from vehicle treated controls (*P<0.05; **P<0.001). (b) Mammary gland mRNA was isolated from 4-month-old transgenic NRL-TGFα (left) and WAP-TGFα (right) females in diestrus, d14 post-ovariectomy, d21 post- E implant. TGFα transgene mRNA was measured using RT QPCR. Different letters denote statistical differences among groups (ANOVA; P<0.05).

Figure 4
Figure 4

ER (a) and PR (b) mRNA levels are reduced during tumorigenesis. Mammary transcripts were quantified using RT QPCR normalized to PUM1. Mammary glands were examined from nontransgenic (NonTg), and disease-free TGFα (normal) mice at 4 months, and from end stage mice with hyperplastias or tumors (N = 6–7). Note that ER and PR transcripts were significantly reduced in tumor-containing compared to normal glands. Different letters denote statistical differences among groups.

Figure 5
Figure 5

Ovariectomy reduces tumor incidence and increases latency in NRL-TGFα mice. Sixteen week mice were ovariectomized and monitored for tumor development. Successful ovariectomy was confirmed at necropsy by greatly reduced uterine size. The asterisk denotes a significant increase in latency (P≤0.05, Student’s t-test). Bars represent mean ± s.e.m.

Figure 6
Figure 6

Introduction of NRL-TGFα transgenic mice into the ((FVB/B6)F1) background reduces tumor incidence and increases latency compared to the FVB/N strain, but in the presence of p53 +/−, latency decreases and incidence rises, and tumors demonstrate high grade. Data presented as mean ± s.e. (a) Genetic crosses were generated as described in the Materials and methods. Data for NRL-TGFα in the FVB/N background is from Figure 2. Different letters denote statistical differences among groups (P≤0.05; ANOVA). Bars represent mean ± s.e.m. (b) An aggressive mammary carcinoma from an NRL-TGFα × p53+/− mouse with numerous mitotic cells (arrowheads).

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