Specific ß-containing integrins exert differential control on proliferation and two-dimensional collective cell migration in mammary epithelial cells.

Understanding how cell cycle is regulated in normal mammary epithelia is essential for deciphering defects of breast cancer and therefore for developing new therapies. Signals provided by both the extracellular matrix and growth factors are essential for epithelial cell proliferation. However, the mechanisms by which adhesion controls cell cycle in normal epithelia are poorly established. In this study, we describe the consequences of removing the ß1-integrin gene from primary cultures of mammary epithelial cells in situ, using CreER. Upon ß1-integrin gene deletion, the cells were unable to progress efficiently through S-phase, but were still able to undergo collective two-dimensional migration. These responses are explained by the presence of ß3-integrin in ß1-integrin-null cells, indicating that integrins containing different ß-subunits exert differential control on mammary epithelial proliferation and migration. ß1-Integrin deletion did not inhibit growth factor signaling to Erk or prevent the recruitment of core adhesome components to focal adhesions. Instead the S-phase arrest resulted from defective Rac activation and Erk translocation to the nucleus. Rac inhibition prevented Erk translocation and blocked proliferation. Activated Rac1 rescued the proliferation defect in ß1-integrin-depleted cells, indicating that this GTPase is essential in propagating proliferative ß1-integrin signals. These results show that ß1-integrins promote cell cycle in mammary epithelial cells, whereas ß3-integrins are involved in migration.

Cellular microenvironment influences the ability of mammary epithelia to undergo cell cycle.

The use of cell culture models is a principal and fundamental technology used in understanding how mammalian cells work. However, for some cell types such as mammary epithelia, the lines selected for extended culture are often transformed or have chromosomal abnormalities, while primary cultures have such a curtailed lifespan that their use is restricted. For example, mammary luminal epithelial cells (MECs) are used to study mechanisms of breast cancer, but the proliferation of primary cell cultures is highly limited. Here we describe the establishment of a new culture system to allow extended analysis of cultures of primary mouse MECs. In 2D monolayer culture, primary MECs showed a burst of proliferation 2-3 days post isolation, after which cell cycle decreased substantially. Addition of mammary epithelial growth factors, such as Epidermal Growth Factor, Fibroblast Growth Factor-2, Hepatocyte Growth Factor, and Receptor Activator for Nuclear Factor κB Ligand, or extracellular matrix proteins did not maintain their proliferation potential, neither did replating the cells to increase the mitogenic response. However, culturing MECs directly after tissue extraction in a 3D microenvironment consisting of basement membrane proteins, extended the time in culture in which the cells could proliferate. Our data reveal that the cellular microenvironment has profound effects on the proliferative properties of the mammary epithelia and is dominant over growth factors. Moreover, manipulating the cellular environment using this novel method can maintain the proliferative potential of primary MECs, thus enabling cell cycle to be studied as an endpoint after gene transfer or gene deletion experiments.