The mammary gland develops as a rudimentary ductal structure in the mammary fat pad, during pre- and post-pubertal periods. During pregnancy it undergoes massive development resulting in differentiated epithelial cells which ultimately form the secretory alveolar structures that make milk during lactation. At the end of lactation the vast majority of the epithelial cells die by a process of programmed cell death known as apoptosis. The development and survival of the mammary gland depends upon a variety of hormones and growth factors, but it has been shown that the absence of insulin-like growth factor-I (IGF-I) leads to a dramatic impairment of mammary development. IGF-I is now known to be an important survival factor for many different cell types in the body. Surprisingly, the process of cell death at the end of lactation is not accompanied by a decrease in the concentration of IGF-I in the blood. Instead, we have demonstrated that the epithelial cells produce a suicide protein, IGF-binding protein-5 (IGFBP-5) which binds to and inhibits the actions of IGF-I. The situation is actually more complex in that we, and others, have shown that IGFBP-5 can act independently of IGF-I, but the manner in which it does so is not yet understood. For example, IGFBP-5 can, independently of IGF-I , activate proteases which are involved in degrading the extracellular environment (a crucial part of the re-modelling of the mammary gland which occurs at the end of lactation). We have already successfully generated mutated forms of IGFBP-5 in which the IGF-dependent and /independent effects have been separated. These molecules have been studied in preliminary fashion and shown to exhibit novel properties and a principal objective of the current proposal is to use these mutated forms of IGFBP-5 to provide a clearer insight into the mechanisms of action of IGFBP-5 in inducing mammary apoptosis and tissue remodelling. One of the strengths of this proposal lies in the use of a complex 3D model which more closely resembles the in vivo situation than do typical 2D cultures of cells. Our approach involves co-culture of mammary epithelial cells with fat cells (adipocytes) in a collagen/laminin-based extracellular environment. We believe this co-culture to be crucial as, firstly, the mammary epithelium develops as 3D ducts and alveolar structures which are polarised and have an internal cavity (processes which are not achieved with most cell lines). Secondly, the mammary epithelium interacts extensively with the mammary adipocytes, which secrete factors that influence and 'instruct' epithelial cell morphogenesis and differentiation. Our assessment of these co-cultures, and the effects of IGF-I and IGFBP-5 mutants therein, will involve state-of-the-art technologies including confocal microscopy, adenoviral infection with dominant-negative molecules and mutated proteins, and 96-well rtPCR screening approaches. We will also use more classical approaches of immunohistochemistry and western-immunoblotting techniques to examine intra-cellular signalling events. In addition, we will use rapid screening techniques, utilising established cell lines, in order to focus our studies of the more complex 3D cultures. Finally, we will take advantage of transgenic animals expressing the mutant IGFBP-5 molecules specifically in the mammary gland (provided by a separate project) to compare their phenotype with that induced by over-expression of non-mutated IGFBP-5 (this impairs mammary development in vivo). Thus these studies will identify, in vivo and in co-cultures, the relative importance of the IGF-dependent and IGF-independent effects of IGFBP-5 and explore whether this involves changes in cell surface proteins which influence cell survival and migration. We also aim to determine which intracellular signalling pathway(s) are activated by IGFBP-5.
Maintenance of tissue boundaries is crucial for the control of metastasis, the escape of cells from primary tumours. These cells typically become more aggressive and therefore more difficult to control and it is these metastatic cells that normally kill the patient, rather than the primary tumour. Thus being able to control the process of metastasis is vital in the battle against cancer. The process by which cancer cells become more aggressive includes switching from epithelial (non-migratory) cells into mesenchymal (migratory cells). The cells also lose their normal drive to “stick together” using adhesion molecules (cadherins) on their cell surface. Cancer cells also have to cross “boundaries” which normally exist between the epithelial cells and the mesenchymal cells, as these reside within their own compartments.
Whilst most research effort currently goes into finding drugs to kill tumour cells or to make them less migratory (anti-metastatic), we instead have focussed upon the role of the normal tissue which surrounds the tumour and its ability to “restrain” the tumour and prevent its escape.
In this project, we described a novel signalling pathway in which epithelial cell disruption can be minimised and this restricts epithelial-mesenchymal transgressions. The mechanism involves a protein, IGFBP-5 which is released from injured cells (a process which occurs in normal cells when cancer cells try to escape by “pushing aside” their normal neighbours). IGFBP-5 increased epithelial cell adhesion to the external environment when they encounter the mesenchymal compartment (they are normally excluded from this) and this involved a direct interaction of IGFBP-5 with adhesion molecules on the cell surface known as integrins. In addition, we showed that IGFBP-5 enhanced the interaction of epithelial cells with each other via cadherins. The stimulation of increased adhesion to each other and to the extracellular environment led to a dramatic decrease in epithelial cell migration, which would be anticipated to decrease the metastatic potential of the tumours. We also demonstrated the intracellular pathways that were activated by IGFBP-5, which could ultimately be used as drug targets. In addition, IGFBP-5 facilitated prolonged cell survival in nutrient-poor conditions (this often happens when tumours disrupt the blood supply). Thus the normal cells surrounding the tumour would be better able to resist the aggressive invasion of the tumour (which could kill them by starvation) and retain it within its primary site, where its impact is considerably reduced.
In summary, we showed that, when injured normal cells release IGFBP-5 this has the ability to induce a process of tumour encapsulation which includes spreading of the normal cells to surround the tumour, tight cell-cell contact to prevent tumour cells escaping and in addition, the enhancement of scar tissue, which increases the encapsulation process. These responses to IGFBP-5 would be anticipated to reduce metastatic potential. We have recently shown that high levels of IGFBP-5 are indeed associated with delayed tumour recurrence in breast cancer patients receiving chemotherapy.