Targeting distinct apoptotic pathways in mammary gland involution

Cells grow and divide to produce more cells. During growth of an embryo, certain cells have to be removed for proper function in the adult animal. An interesting example is seen in limb development. Ducks have webbed feet but human embryos also have webbed fingers and toes before they are born. The skin cells between the digits is removed by inducing them to die. This is a normal process. However, sometimes things go wrong and either too many cells, or the wrong type of cells, are made. Occasionally also, mistakes are made in copying the cell's DNA and this could cause cancer to develop. In order to eliminate this problem, and to remove normal cells that are no longer required, all cells have acquired the ability to kill themselves by activating a series of steps that result in the components of the cell being dismantled and packaged into small fragments. This cellular debris is then eaten up, or engulfed, by professional phagocytes whose job is to remove dead cells and clear away debris to prevent further damage to the surrounding tissue. This is called programmed cell death, or apoptosis. Apoptosis is very important in the lactating breast where it is needed to remove the milk producing cells when the baby is weaned and no longer needs breast feeding. We have been studying the apoptotic events that take place in breast cells and how these are controlled. We have found that the transcription factor Stat6, which binds to DNA to induce transcription of specific genes, is important for initiating apoptosis. In this project, we aim to identify genes are controlled by Stat6 and how they are involved in killing mammary cells.

We have developed a number of genetically engineered mouse models of mammary gland involution, which is characterised by extensive apoptosis of the epithelial cells. We have shown that the transcriptional regulators Stat3 and IKKbeta/2 are essential for the induction of apoptosis and that in their absence, caspase-3 is not cleaved and the first phase of involution is abrogated. We have now shown that another member of the Stat family, Stat6, is an important regulator of apoptosis during the first four days of involution. Using Stat6 knockout mice, we have shown that loss of a single allele of Stat6 is sufficient to reduce the number of cells undergoing apoptosis upon forced weaning, and deletion of both alleles results in a failure to induce apoptosis and initiate remodelling of the gland. Analysis of the molecular changes associated with this diminished apoptosis suggested that the receptor for the cytokine LIF is a target of Stat6 during mammary gland involution. Failure to induce LIFR expression is accompanied by loss of Stat3 and ERK1/2 phosphorylation, caspase 3 cleavage and reduced expression of all three isoforms of Bim. We propose that apoptosis of mammary epithelial cells requires Stat6-induced expression of LIFR which mediates activation of Stat3 via LIF, thereby initiating the apoptotic cascade. We hypothesise that a critical downstream component of this pathway is the pro-apoptotic factor Bim and that abrogation of Stat6 and Bim activity would prevent involution. This aims of this project are to identify additional downstream targets of Stat6 using both a proteomic approach and genomewide protein-DNA interaction (Chip on ChIP). We will also define the role of Bim in the two phases of mammary epithelial cell apoptosis by gene-knockdown in vivo using a lentivirus approach to abrogate Bim function. Finally, a cell culture model of mammary gland will be utilised to validate the Stat6 targets and to carry out further molecular analysis.