Hormones exert control in the breast tissue by regulating interactions between different cells
The effect of hormones depends on interactions between the different cell types that make up milk ducts and the surrounding stroma. Cells within milk ducts communicate with each other as well as with cells within the surrounding connective tissue. The highly complex interplay between hormones and the many different cell types of the breast cannot be mimicked by growing cells in plastic culture dishes but requires experiments within a living organism. The mouse is an excellent model because powerful techniques for genetic manipulation of distinct tissues as well as different cell types within a tissue are available, and the mammary glands are readily accessible for experimentation (see scheme 1).
Scheme 1: Scheme of mammary gland development
The use of mutant mice to study breast development
The mouse mammary gland provides a unique experimental system to study in vivo how systemic hormones impinge on molecular determinants of development in the breast and how deregulation of these pathways leads to tumorigenesis. The mammary gland is the only organ to undergo most of its development after birth and can readily be manipulated experimentally. In prepubertal female mice, the part of the inguinal mammary glands that contains the epithelial tree can be surgically removed creating a "cleared fat pad". When epithelial tissue or primary cells are engrafted into a cleared fat pads, they will repopulate it and form a well-organized mammary gland, which responds to all hormonal stimuli (see scheme 2).
Scheme 2: Mammary gland reconstitution: In the 3‑week‑old female mouse the ductal tree growing out from the nipple has only partially penetrated the mammary fat pad (left panel). It can be surgically removed leaving behind a cleared fat pad. Primary mammary epithelial cells injected into this fat pad can form new ducts that grow out to populate the fat pad (right panels).
A rapidly increasing number of mice that lack specific genes are available and the role of these genes in breast development and carcinogenesis can be evaluated. Since the mammary gland is a paired organ it is possible to compare genetically different cells engrafted within the same host, ensuring that both grafts are exposed to the same hormonal milieu (Fig. 1).
Figure 1: Mammary gland development in the absence of estrogen receptor a signaling. The right panel shows a wild type milk duct system penetrating into the mammary fat pad during puberty. In the absence of the estrogen receptor a the ductal system does not grow out at all (Left panel). Both ductal systems are marked with the green fluorescent protein (GFP) that yields the bright green signal under UV light source. Scale bar: 5mm.
We have made extensive use of this model to define the influence of estrogen, progesterone and prolactin signaling on the branching of the milk duct system and the formation of the secretory pouches, alveoli, and to study how these hormones control developmental signaling pathways in the breast. In particular, we have demonstrated that estrogen and progesterone do not act directly on their target cells but affect intercellular communication to induce morphogenesis/proliferation (see scheme 3) (Fig. 2).
Scheme 3: Schematic representation of mammary gland development (black) and our current working model of how various factors control different morphogenetic steps (color) based on our previous work.
Figure 2: Demonstration that estrogen acts in a paracrine fashion. Shown is part of the ductal system of a mammary gland that consists of cells that have the estrogen receptor a (in magenta) and cells that do not have the estrogen receptor a (in blue). Cells lacking the estrogen receptor a can contribute to the milk duct system when they are next to wild type cells.
Normal development and breast cancer
The dissection of the mechanisms underlying the actions of the female sex hormones in breast development will increase our understanding of the origins of breast cancer and provide the basis for developing new preventive and therapeutic strategies. To assess the relevance of our findings to the human situation more directly, we have established strong collaborative links with two clinical departments at the Centre Hospitalier Universitaire Vaudois, Lausanne. We have established primary cultures of human breast cells that we obtain from reduction mammoplasties.
Increased Wnt signaling triggers oncogenic conversion of human breast epithelial cells by a Notch-dependent mechanism
Wnt and Notch signaling have long been established as strongly oncogenic in the mouse mammary gland. Aberrant expression of several Wnts and other components of this pathway in human breast carcinomas has been reported, but evidence for a causative role in the human disease has been missing. We found that increased Wnt signaling, as achieved by ectopic expression of Wnt‑1, triggers the DNA damage response (DDR), and an ensuing cascade of events resulting in tumorigenic conversion of primary human mammary epithelial cells (HMECs). Wnt‑1-transformed cells have high telomerase activity, compromised p53 and Rb function, grow as spheres in suspension, and form tumors in mice, which closely resemble medullary carcinomas of the breast. Notch signaling is up regulated through a mechanism involving increased expression of the Notch ligands Dll1, Dll3 and Dll4 and is required for expression of the tumorigenic phenotype. Increased Notch signaling in primary HMECs is sufficient to reproduce some aspects of Wnt‑induced transformation. The relevance of these findings for human breast cancer is supported by the fact that expression of Wnt‑1 and Wnt‑4 and of established Wnt target genes, such as Axin‑2 and Lef‑1, as well as the Notch ligands, such as Dll3 and Dll4, is upregulated in human breast carcinomas.
Breast cancer, mammary gland development, reproductive hormones, tissue recombination techniques, mouse genetics