Ovarian hormones are not required for PRL-induced mammary tumorigenesis, but estrogen enhances neoplastic processes

Abstract

Epidemiologic studies have demonstrated that increased prolactin (PRL) exposure raises the risk of invasive estrogen receptor alpha (ERalpha)-positive breast cancer in women. However, the mechanism(s) whereby this occurs and the interactions with estrogen itself in this disease remain poorly understood. In order to investigate the role of ovarian hormones in the disease process, we employed a transgenic model neu-related lipocalin (NRL)-PRL in which transgenic PRL is directed to mammary epithelial cells by the PRL- and estrogen-insensitive NRL promoter, mimicking the endogenous PRL expression within the breast observed in women. This high local exposure leads to mammary lesion development and eventually carcinomas. Ovariectomy (ovx), shortly after puberty, did not alter the incidence or latency of PRL-induced mammary carcinomas, consistent with the independence of PRL from circulating estrogens as a risk factor for invasive breast cancer in women. However, chronic estrogen administration to ovx NRL-PRL females decreased the latency of both ERalpha-positive and -negative tumors. We identified multiple mechanisms that may underlie this observation. Elevated estrogen exposure cooperated with PRL to increase epithelial proliferation and myoepithelial abnormalities, increasing the incidence of preneoplastic lesions. Critical components of the extracellular matrix secreted by the myoepithelium were reduced with age, and transgenic PRL raised transcripts for tenascin-C and maspin, both associated with tumor progression and poor prognosis in subclasses of clinical breast tumors. Mammary pERK1/2 and pAkt, but not phosphorylated Stat5, were markedly elevated by local PRL. Together, these findings indicate that PRL employs multiple mechanisms to promote mammary tumorigenesis.

Figure 1

Estrogen decreases latency to tumor development. A. NRL-PRL female mice underwent ovariectomy (ovx), sham surgery, or ovx with 17β-estradiol (E2) administration after puberty, as described in Materials and methods. Ovx females had significantly decreased uterine weights (22±14 mg; mean±s.d.) compared to sham treated females (96±34 mg), while those from E2 treated females were significantly increased (208±47 mg). E2-treated NRL-PRL females developed mammary carcinomas with significantly shorter latencies compared to other treatment groups (p=0.02). B. Estrogen availability did not alter the proportion of ERα positive tumors. The percentage of ERα positive cells in mammary carcinomas from NRL-PRL females was determined as described in Materials and Methods. Each circle denotes an individual tumor.

Figure 2

Aging significantly enhances proliferation and ERα expression in morphologically normal structures in glands of mice. A. Proliferation was significantly enhanced with aging in ducts of nontransgenic FVB/N (FVB) mice regardless of treatment. B. Ducts in glands from 6 month old sham treated NRL-PRL (PRL) females demonstrated similar levels of proliferation as those of 2 year old females. C. Long-term ovx or ovx plus E2 significantly enhanced ERα expression in ducts of nontransgenic females. D. ERα expression was enhanced with E2 treatment in NRL-PRL females, similar to nontransgenic females. BrdU and ERα labeled cells were detected and quantitated as described in Materials and methods, and expressed as mean ± s.d. Data were analyzed by the Kruskal-Wallis test followed by Mann-Whitney post test. Different lower and uppercase letters denote statistical differences among treatments, within 6 month old and end stage animals, respectively, for each genotype (p<0.05). Asterisks denote differences with age for the same treatment of the same genotype (*p<0.05, **p<0.01, ***p<0.001). Proliferation of ducts in NRL-PRL females was higher than all age-matched FVB/N animals with the same treatment, with the exception of the sham-treated elderly females (p<0.005).

Figure 3

Lifetime administration of 17β-estradiol alters the pattern of ERα expression in nontransgenic ovx nonparous aged females. A. Luminal ERα expression in morphologically normal ducts of a 2 year old ovariectomized (ovx) nontransgenic female (similar to 6 month old ovx, and ovx-E2 treated females). B. Basilar ERα expression in morphologically normal ducts of a 2 year old E2-treated ovx nontransgenic female. ERα-labeled cells (green) colocalize with cytokeratin 8 (K8; red) expression in ducts of ovx (C) and ovx/E2-treated (D) nontransgenic females. ERα labeled cells do not colocalize with cells expressing α-smooth muscle actin (α-SMA; red) in ducts of ovx (E) and ovx/ E2-treated (F) nontransgenic females. Representative micrographs. Original magnification: A, B, 400×; C-F, 600×. Prolonged 17β-estradiol administration reduces laminin 1α (G) and collagen IV α5 (H) transcripts with age. Nontransgenic FVB/N nonparous females were ovx post-pubertally, and thereafter were administered E2, as described in the Materials and Methods. RNA from whole mammary glands of 6 and 24 month old animals was examined for the myoepithelial ECM products by qRT-PCR as described in the Materials and methods. Each symbol denotes a single animal. Asterisks indicate the significant difference in laminin 1α mRNA between the 6 month and 24 month old animals (p=0.007; Student's t test).

Figure 4

PRL and 17β-estradiol diminish the myoepithelial layer surrounding morphologically normal structures. At 6 months of age, ovx (A), sham treated (B), and ovx/E2-treated (C) nontransgenic mice demonstrated normal α-SMA staining around ducts. At 2 years of age, ovx/E2-treated (F) nontransgenic females exhibited decreased α-SMA staining, compared to those subjected to ovx alone (D) or sham surgery (E). Abnormalities in α-SMA staining were present in sham- (H) and ovx/E2- (I) treated NRL-PRL females at 6 months of age, compared to ovx NRL-PRL females (G) of the same age. Pronounced abnormalities were present in all treatment groups at 2 years of age (J, K, L) in NRL-PRL females, with the greatest discontinuities in the E2-treated group (L). Morphologically normal structures were stained for α-SMA (red) and counterstained with DAPI (blue) as described in Materials and methods. Representative micrographs. Original magnification of all images: 600×.

Figure 5

Transgenic PRL reduces the relative level of α-SMA: keratin 8 protein expression, and raises mammary tenascin C and maspin transcripts, without altering fibronectin 1. Protein homogenates and RNA from whole mammary glands of sham-treated 2 year old nontransgenic (FVB) and NRL-PRL nonparous females were examined for levels of α-SMA and keratin 8 protein by western analyses (A), and myoepithelial products by qRT-PCR (B, C, D), as described in the Materials and methods. A. Data are expressed as the ratio between α-SMA and keratin 8 staining (mean ± s.d., N=3). B, C, D. Data are expressed as mean ± s.e.m., N=5. Asterisk indicate a significant difference between genotypes by Student's t test (*, p<0.05; ***, p<0.0001).

Figure 6

NRL-PRL glands exhibit elevated pStat5, pERK1/2 and pAkt. A, B. Phosphorylated Stat5 (pStat5) was examined in morphologically normal structures of age-matched 6 month old nontransgenic and NRL-PRL intact nonparous females (A), and normal structures and epithelial hyperplasias (EH) in glands of 2 year old NRL-PRL females (B). pStat5-labeled cells were detected and quantitated as described in Materials and methods and expressed as mean ± s.d. Lower case letters denote significant differences using ANOVA followed by Student-Newman-Keuls Multiple Comparison test (p<0.05). C. Glands of NRL-PRL females at 12 weeks of age contain elevated pERK1/2 and pAkt, compared to nontransgenic littermates. Each lane represents mammary homogenate from a single 12 week old NRL-PRL female or nontransgenic littermate (FVB), examined by immunoblotting as shown. D. The ratio of phosphorylated: total kinase for each animal shown in (C) was calculated as described in the methods. An asterisk indicates a significant difference between genotypes by a one-tailed Student's t test (*, p<0.05).