Determining the Role of JNK in Anoikis Using Mammary Epithelial Cells

Mammary Gland Development

The mammary gland is derived from the ectoderm, the outer layer of the embryo, and is formed into a series of ducts from which milk is produced. Although present at birth, the mammary gland undergoes much of its development during puberty when branching morphogenesis occurs. During this process, terminal end buds (the ends of the ducts) grow and branch into the surrounding fat pad to form the mature ductal mammary gland structure.

As they undergo branching morphogenesis, the lumens of the newly formed ducts become hollowed out following epithelial cell anoikis. Anoikis is a specialized process of cell death, initiated when epithelial cells lose contact with the basement membrane and extracellular matrix and become detached. Anoikis is a regulated process that occurs in epithelial tissues like the intestinal tract and during development of the mammary gland.

Lifeline® Mammary Epithelial Cells in Anoikis Research

Multiple groups have debated the role of c-JUN NH2-terminal kinase (JNK) in anoikis; however, in a 2017 study in Cell Reports, Girnius and Davis set out to resolve this debate. They first evaluated the role of JNK in anoikis using Lifeline® normal human mammary epithelial cells treated with a JNK inhibitor and cultured in suspension to mimic epithelial detachment. Using flow cytometry to measure apoptotic markers, they found that cells treated with a JNK inhibitor were protected from anoikis. This effect was also true in murine kidney epithelial cells with genetic ablation of JNK. Additionally, activation of JNK increased the number of cells undergoing apoptosis, suggesting that JNK stimulates anoikis.

To determine the apoptotic pathway members that are required for JNK-induced anoikis, the researchers evaluated BAK- and BAX-null mouse kidney epithelial cells grown in suspension. They found that anoikis in cells lacking BAK and BAX was significantly reduced, and could not be recovered using an activated form of JNK. Through gene expression analysis following anoikis and genetic deletion, the authors found that BIM (a pro-apoptotic protein) expression was dependent on JNK during anoikis. In addition, a second factor, BMF, was implicated, and genetic ablation of both BIM and BMF in the presence of an activated form of JNK significantly reduced anoikis, suggesting these are the major factors involved in JNK-mediated anoikis.

To confirm the roles of these factors in vivo, the authors evaluated mammary gland development in mice lacking BIM, BMF, or both. They found that BMF loss had minor effects on mammary duct extension, while BIM loss had a greater effect. Moreover, mice with double BMF/BIM knock out had the most significant developmental defects, which affected mammary duct extension and increased terminal end bud and duct occlusion, two processes that depend on anoikis. Finally, the authors found that BMF is phosphorylated by JNK on Serine 74, and mutation of this site reduced cell anoikis in vitro. Together, the results of this study suggest that JNK activity induces anoikis and in particular, the effect of JNK is mediated BIM expression and BMF phosphorylation.

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