Lysosomes in the mammary gland : their role in mediating cell death

Lysosomes are small membrane-bound vesicles, called organelles, that contain a number of enzymes that breakdown large molecules such as DNA and proteins in addition to other organelles such as mitochondria, the so-called powerhouse of the cell. Lysosomes also help to destroy bacteria and viruses that invade cells by digesting them after they have been delivered to the lysosome. Lysosomes are essentially recycling centres as they breakdown old and damaged components and recycle small molecules back to the cell cytoplasm where they are used to rebuild DNA, proteins and organelles. There are a number of debilitating inherited diseases that arise from defects in lysosome function and more recently, lysosomes have been associated with neurodegenerative diseases such as Alzheimer's and also with cancer. We have shown that lysosomes leak their contents during post-lactational regression of the breast and that this causes the death of the breast cells. This new discovery has important implications for breast cancer and other diseases where lysosome function is important. The aim of this project is to investigate how lysosomes become leaky and how this is controlled. We will use a range of techniques including innovative ways to identify novel proteins and measure their localisation in the cell using fluorescence and time-lapse microscopy. We will also investigate any role that these proteins may have in damaging lysosomes and causing them to leak their toxic contents into the cell. We hope that this work will provide new ideas for killing breast cancer cells, that are often resistant to other types of cell death.

Recently, we have demonstrated that cell death during involution of the mammary gland is not classical apoptosis but occurs rather by a lysosomal mediated and executioner caspase independent pathway of cell death (LM-PCD). We showed that Stat3 leads to a striking upregulation of the lysosomal proteases cathepsin B and cathepsin L while concomitantly downregulating the expression of an endogenous cathepsin inhibitor Spi2a. This is accompanied by destabilisation of the lysosomal membrane, efflux of cathepsins and subsequent cell death. Importantly, inhibition or deletion of executioner caspases has no effect, while inhibition of cathepsin B abolishes cell death. This is the first time that a mechanism for Stat3 mediated cell death has been elucidated, as well as the first time that LM-PCD has been demonstrated to drive a physiological pathway of cell death. We aim to discover the mechanism of lysosomal leakiness in vivo and how this is controlled. There are two questions that need to be addressed: 1) what is the mechanism by which lysosomes become leaky and 2) which pathways and molecules control LMP? The aim of this programme is to answer these questions. We will utilise two main approaches to uncover the mechanism of LMP: candidate gene analysis and sophisticated proteomics approaches. We have preliminary data on Stat3 targets upregulated during involution and will use the quantitative proteomics techniques, LOPIT and SILAC, to identify novel regulators of LMP both in vivo and in cell culture models of differentiated mammary epithelium. These candidates will be investigated further using fluorescence, time-lapse, and electron microscopy to confirm their lysosomal localisation and we will investigate their function using lentivirus-mediated gene knockdown in culture models and in primary cells transplanted into mammary fat pads. The implications of this work for understanding cell death mechanisms and developing new therapeutic approaches for cancer are considerable

This research will be of immediate benefit to researchers in the fields of cell death, lysosome biology, and cancer biology. It will also be of interest to mammary gland biologists and others interested in the mechanism of lysosome membrane permeabilisation such as those working on cell death in the brain and lysosome storage disorders. Knowledge gained will be informative across all these disciplines.
In addition to furthering knowledge, this work should provide novel approaches for the development of cancer therapeutics, particularly for those cancers that are resistant to apoptosis. Delivery of compounds to the lysosome is relatively straightforward and it can be envisaged that new therapeutics could be developed and tested in animals within the lifetime of this programme grant. This could lead to patent protection and commercialisation of these drugs. Thus, there is the possibility of generating wealth for the UK through the private sector. We are currently in consultation with Cambridge Enterprise with respect to our original findings and are developing a pro-drug for testing.
Other beneficiaries include the junior scientists trained on this project who will have a wide range of skills to take forward into the next stage of their careers. In addition, there will be benefit to undergraduate students and international students undertaking internships who will carry out research projects in the laboratory.
Ultimately, the general public will benefit from this research by way of new treatments for therapy resistant breast cancers and other cancers that are resistant to apoptosis. There may also be longer term benefits to patients suffering from neurodegenerative disorders and stroke. Thus the quality of life in the UK will be enhanced, particularly since cancer and other diseases where LMP plays a role are often extremely debilitating and incurable. These benefits will not occur within the lifetime of this grant but can be envisaged within 10 years.