A recent report by Ewan and colleagues [
1] sheds some light on the mechanism by which steroid receptor-positive cells are prevented from proliferating in the normal adult mammary gland by the expression of activated transforming growth factor (TGF)-β. This report is an extension of earlier studies by the same authors that first demonstrated the ability to detect latent versus activated TGF-β expression
in situ [
2]. Steroid receptor expression is often used as a prognostic indicator and target of endocrine therapy in breast cancer. However, our understanding of the normal distribution and regulation of estrogen receptor (ER)-α and progesterone receptor (PR) expression is still evolving. In normal adult mammary glands from humans, rats, mice, or cows, steroid receptor-positive cells are heterogeneously located in luminal epithelial cells throughout the ducts and rarely co-localize with markers of proliferation [
3-
6]. Growth factors expressed in steroid receptor-positive cells act on neighboring cells to induce proliferation in a paracrine fashion. An elegant series of experiments by Cathrin Brisken and her colleagues [
7,
8] have established the ability of estrogen receptor-positive or progesterone receptor-positive cells to rescue ER-null and PR-null cells, respectively, in chimeric fat-pad transplantation experiments. However, a key question in mammary gland development is what prevents ER/PR-positive cells from dividing and why this proliferative block is lost during breast cancer progression. For example, ER/PR-positive cells are often proliferative in mouse models of mammary hyperplasia and in precancerous lesions of the human breast, such as ductal carcinoma
in situ, suggesting a switch from a paracrine to an autocrine response to proliferative stimuli [
9,
10].
TGF-β is a potent inhibitor of epithelial cell proliferation, but little is known about the process involved in activating the latent form secreted by most cells [
11]. The relationship between TGF-β activation, ER-α expression, and proliferation were the focus of the recent study by Ewan and colleagues [
1]. Using immunostaining to detect the activated form of TGF-β during estrus, they were able to show that the cells positive for active TGF-β also expressed the downstream effector R-SMAD in the nucleus, providing evidence that TGF-β acts in an autocrine manner. Co-immunostaining experiments using double immunofluorescence were performed to show that ER-α co-localized with nuclear R-SMAD staining and activated TGF-β co-localized with both ER-α and PR, supporting their hypothesis that TGF-β activation might inhibit the ability of ER/PR-expressing cells to respond to ovarian hormone-induced proliferation.
Mice heterozygous for the TGF-β1 allele have a 90% reduction in the amount of TGF-β1 protein expressed [
12]. Using mammary glands from these mice, the expression of ER-α and Ki67, a marker of proliferation, was assessed. Mammary glands from TGF-β1
+/- mice exhibited a 24-fold increase in proliferation, and co-localization of ER-α and Ki67 was increased 16-fold. The same results were found when this analysis was performed on outgrowths after transplantation of TGF-β1
+/- mammary epithelial cells, suggesting that epithelial, rather than stromal, TGF-β is responsible for keeping ER/PR-positive cells from proliferating. After ovariectomy, no ER-α/Ki67 double-positive cells were observed in TGF-β1
+/- glands in the absence of ovarian hormones. Only when estrogen and progesterone were added back was there a 17-fold increase in double-labeled cells. Although a loss of TGF-β expression resulted in increased proliferation in the presence of ovarian hormones, the expression of constitutively active TGF-β was able to override hormone-induced proliferation. Mammary glands from mouse mammary tumor virus (MMTV)–TGF-β transgenic mice displayed sixfold fewer ER-α/Ki67 double-positive cells than wild-type glands at estrus.
Fewer proliferating ER-α-positive cells are detected in the mammary glands of parous humans or rats than in glands from nulliparous subjects [
4,
13]. To test whether TGF-β has a function in this phenomenon, parous glands from TGF-β1
+/- mice were analyzed for ER-α and Ki67 co-localization. The frequency of double-labeled cells in parous glands from TGF-β1
+/- mice was fourfold that in parous wild-type mice, indicating that TGF-β is also involved in inhibiting the proliferation of ER-α-positive cells in parous animals. These data also fit with the findings of Boulanger and colleagues, who demonstrated that TGF-β-positive cells from parous glands did not contribute to repopulating the gland, suggesting that they were incapable of 'expansive cellular proliferation' and stem cell self-renewal [
14]. TGF-β expression has also been shown to increase mammary stem cell senescence and to inhibit MMTV-induced mammary carcinogenesis [
15]. Finally, the effect of TGF-β1 depletion was analyzed during puberty, a time of rapid proliferation in the terminal end bud structures. Loss of TGF-β1 did not increase the proliferation of ER-α-positive cells in either the terminal end buds or ducts, although proliferation was increased overall. This supports the idea that proliferation of ER/PR-positive cells during puberty is regulated differently from that in the adult gland.
These data advance our understanding of how steroid receptor-positive cells might be prevented from proliferating in the normal mammary gland, and how this process might become deregulated in breast cancer progression. Further evidence for the role of TGF-β in regulating the proliferation of steroid receptor-expressing cells comes from our studies of CCAAT-enhancer-binding protein (C/EBP)β-null mice. Mammary glands from these mice show increased numbers of ER/PR-positive cells and a 10-fold decrease in proliferation [
5]. Recently, we discovered that these C/EBPβ-null glands also display a significant increase in activated TGF-β along with increased downstream Smad2 expression and signaling [
16]. Another downstream target of TGF-β is the cyclin-dependent kinase inhibitor, p27
Kip1, which in turn affects the activity of other cell cycle components such as cyclin E and cyclin-dependent kinase (cdk)2 [
17]. All of these molecules were altered in C/EBPβ-null glands, resulting in decreased cyclin E expression, loss of cdk2 kinase activity, increased p27 stability and decreased levels of cdc25A phosphatase activity. These studies with mouse models have led us to speculate that loss of active TGF-β expression in precancerous breast lesions might result in increased expression or stability of cdc25A and increased cyclin E/cdk2 activity in steroid receptor-positive cells, allowing them to proliferate. In support of this hypothesis, cdc25A was recently shown to be induced by genomic and non-genomic actions of estrogen in breast cancer cells [
18,
19]. The expression of cdc25a is also regulated both transcriptionally and post-transcriptionally by TGF-β, and thus may be a useful downstream indicator of active TGF-β signaling [
20]. p27 has also been proposed as a prognostic marker in breast cancer [
21]. However, the subcellular localization and phosphorylation state of the protein are critical in regulating its activity, and it would, therefore, be problematic to assess these changes in clinical samples. A hypothetical model summarizing these results with regard to breast cancer progression is shown in Figure .
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