Progesterone involvement in breast development and tumorigenesis--as revealed by progesterone receptor "knockout" and "knockin" mouse models

In light of recent clinical trials, the debate concerning the risks and benefits of progestin-based postmenopausal hormone replacement therapy (HRT) has reached a renewed level of urgency. Irrespective of the position taken, the consensus is that more basic research needs to be performed to address progesterone's fundamental role in mammary development and tumorigenesis. Towards this end, the progesterone receptor knockout (PRKO) mouse demonstrated that progesterone is essential for pregnancy-associated mammary gland ductal side-branching and alveologenesis and that these morphological changes are dependent on progesterone-induced mammary epithelial proliferation. Importantly, the PRKO mouse showed that the progesterone-proliferative signal significantly contributes to mammary tumor susceptibility in an established mammary tumor model. Insight into the cellular mechanism(s) by which progesterone affects mammary morphogenesis has been disclosed by a new PR-LacZ knockin mouse, which revealed that PR's spatial expression pattern undergoes precise choreographed distributional changes that precede key stages in postnatal mammary development. In the case of early pregnancy, the segregation of cells undergoing progesterone-induced proliferation from those that express PR implicates a paracrine mode of action for progesterone-induced mammary epithelial proliferation, whereas the preparturient decline of PR expression underscores the need to remove this signal for full functional differentiation of this tissue. Our findings support the proposal that the mammary gland's normal response to the progesterone-signal is dependent upon specific spatial organizational patterns of PR expression and that derailment in these cellular processes may contribute to abnormal mammary development, including cancer. This review concludes by emphasizing the need to identify the downstream molecular targets that mediate progesterone's effects in this tissue. Identification of such targets will not only enhance our mechanistic understanding of progesterone's role in mammary development and cancer, but may also facilitate the formulation of new design strategies in breast cancer diagnosis and/or treatment.     

A mouse model to dissect progesterone signaling in the female reproductive tract and mammary gland

Considering the regulatory complexities of progesterone receptor (PR) action throughout the female reproductive axis and mammary gland, we generated a mouse model that enables conditional ablation of PR function in a spatiotemporal specific manner. Exon 2 of the murine PR gene was floxed to generate a conditional PR allele (PR^flox) in mice. Crossing the PR^flox/flox mouse with the ZP3‐cre transgenic demonstrated that the PR^flox allele recombines to a PR null allele (PR^d). Mice homozygous for the recombined null PR allele (PR^d/d) exhibit uterine, ovarian, and mammary gland defects that phenocopy those of our previously described PR knockout (PRKO) model. Therefore, this conditional mouse model for PR ablation represents an invaluable resource with which to further define in a developmental and/or reproductive stage‐specific manner the individual and integrative roles of distinct PR populations resident in multiple progesterone‐responsive target sites. genesis 48:106–113, 2010. © 2009 Wiley‐Liss, Inc.     

A mouse model engineered to conditionally express the progesterone receptor‐B isoform

Using a Rosa26 gene targeting strategy in mouse embryonic stem cells, we have generated a new transgenic mouse (Pgr‐B ^LSL), which is designed to conditionally express the epitope‐tagged mouse progesterone receptor‐B (PGR‐B) isoform when crossed with a specific cre driver mouse. To functionally validate this transgenic mouse, we crossed the Pgr‐B ^LSL mouse with the MMTV‐CREA transgenic mouse to create the MMTV‐CREA/Pgr‐B ^LSL bigenic (termed PR‐B:OE to denote PGR‐B o vere xpressor). As expected, transgene‐derived PGR‐B protein was specifically targeted to the virgin mammary gland epithelium. At a functional level, the PR‐B:OE bigenic exhibited abnormal mammary morphogenesis—dilated epithelial ducts, precocious alveologenesis and lateral side‐branching, along with a prominent proliferative signature—that resulted in pregnant PR‐B:OE mice unable to exhibit mammary gland terminal differentiation at parturition. Because of this developmental failure, the PR‐B:OE mammary gland was incapable of producing milk resulting in early neonatal death of otherwise healthy litters. This first line of analysis demonstrates the utility of the Pgr‐B ^LSL mouse to examine the role of the PGR‐B isoform in different physiologic and pathophysiologic systems that are responsive to progesterone.     

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.     

A novel LacZ reporter mouse reveals complex regulation of the progesterone receptor promoter during mammary gland development

To further our understanding of progesterone (P) as an endocrine mammogen, a PR(lacz) knockin mouse was generated in which the endogenous progesterone receptor (PR) promoter directly regulated lacZ reporter expression. The PR(lacz) mouse revealed PR promoter activity was restricted to the epithelial compartment during the prenatal and postnatal stages of mammary gland development. At puberty, PR promoter activity was unexpectedly robust and restricted to the body cells within the terminal end buds and to the luminal epithelial cells in the subtending ducts. In the adult, the preferential localization of PR(lacz) positive cells to the distal regions of ductal side branches provided a cellular context to the recognized mandatory role of P in ductal side-branching, and segregation of these cells from cells that undergo proliferation supported an intraepithelial paracrine mode of action for P in branching morphogenesis. Toward the end of pregnancy, the PR(lacz) mouse disclosed a progressive attenuation in PR promoter activity, supporting the postulate that the preparturient removal of the proliferative signal of P is a prerequisite for the emergence of a functional lactating mammary gland. The data suggest that PR expression before pregnancy is to ensure the specification and spatial organization of ductal and alveolar progenitor cell lineages, whereas abrogation of PR expression before lactation is required to enable terminal differentiation of the mammary gland.     

Cre-mediated recombination in cell lineages that express the progesterone receptor

Using gene-targeting methods, a progesterone receptor Cre knockin (PR-Cre) mouse was generated in which Cre recombinase was inserted into exon 1 of the PR gene. The insertion positions the Cre gene downstream (and under the specific control) of the endogenous PR promoter. As for heterozygotes for the progesterone receptor knockout (PRKO) mutation, mice heterozygous for the Cre knockin insertion are phenotypically indistinguishable from wildtype. Crossing the PR-Cre with the ROSA26R reporter revealed that Cre excision activity is restricted to cells that express PR in progesterone-responsive tissues such as the uterus, ovary, oviduct, pituitary gland, and mammary gland. Initial characterization of the PR-Cre mouse underscores the utility of this model to precisely ablate floxed target genes specifically in cell lineages that express the PR. In the wider context of female reproductive tissue ontology, this model will be indispensable in tracing the developmental fate of cell lineages that descend from PR positive progenitors.     

Role of epidermal growth factor in the acquisition of ovarian steroid hormone responsiveness in the normal mouse mammary gland

The purpose of the present studies was to investigate the role of epidermal growth factor (EGF) in the acquisition of estrogen (E) and progestin (P) responsiveness in the mouse mammary gland in vivo. Using the Elvax 40P implant technique to introduce bioactive molecules directly into the mammary gland to produce a localized effect, we have made the novel observation that EGF implanted into glands of pubertal mice followed by E treatment resulted in the precocious acquisition of E-inducible progesterone receptors (PR). In sexually mature mice, EGF implants alone were able to increase PR. A neutralizing antibody specific for EGF blocked E-dependent stimulation of end-bud development and PR induction. Furthermore, the antiestrogen ICI 182,780 blocked the EGF-induced stimulation end-buds and PR induction, indicating that these EGF effects are mediated via estrogen receptors (ER). Immunohistochemical analysis showed that the endogenous EGF content of mammary glands of mature mice was higher than pubertal mice, that E implants caused a localized increase in mammary gland EGF content in both pubertal and mature mice, and that in mature mice E caused an increase in stromal cell EGF content. We have previously shown that the acquisition of E-inducible PR can be modulated by mammary stroma, and the present results indicate that mammary stroma could modulate hormonal responsiveness through control of local growth factor concentration. Taken together, these results provide evidence that E-dependent responses of mouse mammary gland in vivo, such as end-bud proliferation and PR regulation, may be mediated by EGF through an ER-dependent mechanism. J. Cell. Physiol. 174:251–260, 1998. © 1998 Wiley-Liss, Inc.     

Lactational competence and involution of the mouse mammary gland require plasminogen

Urokinase-type plasminogen activator expression is induced in the mouse mammary gland during development and post-lactational involution. We now show that primiparous plasminogen-deficient (Plg(-/-)) mice have seriously compromised mammary gland development and involution. All mammary glands were underdeveloped and one-quarter of the mice failed to lactate. Although the glands from lactating Plg(-/-) mice were initially smaller, they failed to involute after weaning, and in most cases they failed to support a second litter. Alveolar regression was markedly reduced and a fibrotic stroma accumulated in Plg(-/-) mice. Nevertheless, urokinase and matrix metalloproteinases (MMPs) were upregulated normally in involuting glands of Plg(-/-) mice, and fibrin did not accumulate in the glands. Heterozygous Plg(+/-) mice exhibited haploinsufficiency, with a definite, but less severe mammary phenotype. These data demonstrate a critical, dose-dependent requirement for Plg in lactational differentiation and mammary gland remodeling during involution.     

Murine progesterone receptor expression in proliferating mammary epithelial cells during normal pubertal development and adult estrous cycle. Association with eralpha and erbeta status.

The ovarian steroids estrogen and progesterone are important in directing the normal growth and development of the mouse mammary gland. Previously, we have demonstrated that the majority of proliferating mammary epithelial cells do not express estrogen receptor-alpha (ERalpha). In this study we examined the relationship between progesterone receptor (PR) expression and proliferation in mammary epithelial cells using simultaneous immunohistochemistry for progesterone receptor (PR) and tritiated thymidine [(3)H]-Tdr) autoradiography. Results showed that the majority (>80%) of mammary epithelial cells labeled with [(3)H]-Tdr were PR-positive in the terminal end buds (TEBs) of pubertal mice and the ducts of pubertal and adult mice. Whereas the majority of mammary epithelial cells were also PR-positive, the basal cell population, which comprises the minority of mammary epithelial cells in the mammary ducts, was predominantly PR-negative. Nevertheless, the PR-positive phenotype remained the major proliferating cell type in the basal population. These findings suggest that the progesterone signaling pathway is involved in the proliferation of basal cell populations, potentially directing formation of tertiary side branching during pubertal development and alveolar bud formation in adult glands. A proportion of the basal cells exhibited weak expression of ERbeta, suggesting that the role of ERbeta in mediating normal estrogen-induced responses should be further studied. (J Histochem Cytochem: 47:1323-1330, 1999)     

The RANKL signaling axis is sufficient to elicit ductal side-branching and alveologenesis in the mammary gland of the virgin mouse

Receptor of Activated NF-κB Ligand (RANKL) is implicated as one of a number of effector molecules that mediate progesterone and prolactin signaling in the murine mammary epithelium. Using a mouse transgenic approach, we demonstrate that installation of the RANKL signaling axis into the mammary epithelium results in precocious ductal side-branching and alveologenesis in the virgin animal. These morphological changes occur due to RANKL-induced mammary epithelial proliferation, which is accompanied by increases in expression of activated NF-kB and cyclin D1. With age, prolonged RANKL exposure elicits limited mammary epithelial hyperplasia. While these transgenics exhibit RANKL-induced salivary gland adenocarcinomas, palpable mammary tumors are not observed due to RANKL-suppression of its own signaling receptor (RANK) in the mammary epithelium. Together, these studies reveal not only that the RANKL signaling axis can program many of the normal epithelial changes attributed to progesterone and prolactin action in the normal mammary gland during early pregnancy, but underscore the necessity for tight control of this signaling molecule to avoid unwarranted developmental changes that could lead to mammary hyperplasia in later life.