The control of branching morphogenesis remains one of the most challenging questions in developmental biology. The precise signals that specify new branch points and determine spacing of epithelial ducts remain unclear. In the mammary gland, a variety of genes have been implicated in these processes, and many of these genes are expressed in stromal cells. Many of these genes have also been linked to tumorigenesis.
The mammary gland branches by two mechanistically distinct processes: TEB bifurcation and sprouting of side branches from mature ducts (). During TEB bifurcation, the distal epithelial cells (known as cap cells) abut the fat cells through a sparse basement membrane, and stromal matrix is deposited to form a cleft at the site of bifurcation. In contrast, side branches must extend through the layer of myoepithelial cells, degrade the basement membrane that surrounds the mature epithelial ducts, and invade a periductal layer of fibrous stromal tissue that separates the epithelium from the fat cells of the mammary fat pad ().
Interaction between the epithelium and the ECM plays a major role in mammary gland branching morphogenesis. TEB formation and ductal invasion are disrupted upon inhibition or deletion of factors that regulate the ECM. These factors include two types of receptors for ECM: (i) discoidin domain receptor–1, which can serve as a collagen receptor (5
), and (ii) β1 integrin, which recognizes many ECM proteins (6
). In addition, the ECM protein laminin-1 (6
) and several matrix metalloproteinases (MMPs), which cleave ECM and other proteins in the cellular microenvironment (7
), must function properly. Notably, MMP-mediated cleavage of laminin-5 releases bioactive laminin fragments that induce breast epithelial cells to migrate (9
). This may be an important mechanism for TEB invasion in vivo.
Proper side branching also requires that the ECM and the cellular microenvironment surrounding the ductal epithelium be maintained. Unrestrained side branching often results in tumorigenesis. Indeed, excessive side branching and eventual tumorigenesis occurs when the stromal regulators MMP-3 (10
) and MMP-14 (11
) or the secreted growth/differentiation factor Wnt-1 (12
) are overexpressed in the mouse mammary gland. In contrast, a reduction in side branching occurs in mice deficient in MMP-3 (8
) and Wnt-4 (13
). Wnt-1 or MMP-3 expression also converts the fatty stroma of the mammary gland into a more dense and fibrotic stroma (10
), and human breast hyperplasia, dysplasia, and carcinoma frequently show elevated stromal MMP activity. Wnts are induced by the progesterone receptor (13
), which regulates the branching of neighboring cells (14
), but how this paracrine signal works is unknown. Wnts associate with the ECM, and a cell surface heparan sulfate proteoglycan (HSPG), syndecan-1, is necessary for the phenotype of the Wnt-1 transgenic mice (15
). These observations suggest that Wnts may mediate the paracrine signal from the progesterone receptor through the ECM and this HSPG. Another stromal factor required for side branching is the actin binding/severing protein gelsolin (16
). Interestingly, a high expression level of gelsolin is a feature of early-stage but aggressive non–small-cell lung carcinomas (17
). This suggests a role in invasion; however, gelsolin may regulate other functions as well, because it is markedly down-regulated in ~70% of late-stage human breast cancers (16
Among the stromal factors that function to prevent inappropriate side branching is transforming growth factor β (TGFβ), which is also a key player in tumorigenesis (18
). TGFβ is present in mature periductal ECM in mice and is specifically down-regulated at sites where side branches are being initiated (2
). Furthermore, ducts branch excessively when TGFβ receptor signaling within the mammary stroma is inhibited by the targeted expression of a dominant-negative TGFβ receptor [reviewed in (2
)]. Similarly, mouse studies have shown that the deletion of the myoepithelial cell adhesion molecule P-cadherin causes excessive side branching in addition to mammary hyperplasia and dysplasia later in life (19