Fascin-dependent control of nuclear plasticity in invading cells

Many clinical studies have identified a protein called fascin at high levels in human breast cancers, however very little is known about how this protein is controlled. This project aims to study the factors that control fascin and how this protein can promote movement of breast cancer cells away from primary tumours and into distant sites of metastases. One of the major gaps in current knowledge is a detailed understanding of the factors regulating breast cancer progression and metastasis. This project will help to identify new proteins that can lead to increased cancer cell invasion and therefore provide new possible targets for designing future treatments. This project aims to study the factors that control fascin function in breast cancer cells with the long-term aim of disrupting this controlling factor and preventing cancer cell invasion. We will use human breast cancer cells in the lab to study the different proteins that are responsible for promoting the invasion of these cells into tissue-like environments. We will then use different strategies to inhibit these newly identified invasion-promoting proteins. Ultimately this study could lead to the identification of new proteins that cause increased breast cancer invasion into tissues. This is very valuable information in the design and development of new drugs and therapeutic agents to prevent breast cancer metastasis.

Cell migration is fundamental in the control of embryonic morphogenesis, tissue homeostasis, repair and inflammatory responses. Consequently, inappropriate migratory movements can result in autoimmune diseases, defective wound repair, or tumor cell metastasis. Cancer cells migrate alone or in small groups to spread from the primary tumour and are able to migrate to distal organs where they can continue to proliferate to form a second tumor mass. Cancer cell migration is regulated by adhesion signalling to drive dynamic actin remodelling. Fascin is an actin-bundling protein that is low or absent in normal human epithelia; its up-regulation correlates with poor prognosis in human carcinomas. Fascin localizes to and stabilizes actin-rich protrusions to promote cell migration. We have previously shown that the actin-bundling and pro-migratory properties of fascin are in part regulated by protein kinase C and the small GTPase Rac. However, very little is known about other regulators of fascin function or activity. Through a mass spec screen we have identified the nuclear envelope protein nesprin-1 as a novel binding partner for fascin. Our preliminary data supports a role for this complex in regulating nuclear shape in cells invading in 3D matrices. We hypothesise that this novel complex acts to control nuclear rigidity and plasticity, thus enabling tumour cells to navigate through complex tissues during invasion. Using a combination of biochemical and state-of-the-art microscopy approaches we aim to characterize the nesprin-fascin interaction further at the molecular level and to determine the upstream regulators responsible for dictating assembly of the complex. We will also define the role of the nesprin-fascin interaction in regulating nuclear shape, actin assembly and cell invasion in 3D matrices. Data arising from this study will provide important novel insight into the regulation of fascin and targets for future design of therapies to target invasive tumour cells.

There is now a wealth of literature suggesting that fascin is an important prognostic marker in cancer and a significant contributing factor to the metastatic potential of primary tumours. However, despite increasing numbers of studies that use fascin as a marker for invasive disease, there are very few known proteins that regulate fascin function. The research outlined here aims to dissect out the signalling mechanisms that co-ordinate fascin-dependent cytoskeletal responses to extracellular matrix (ECM) environments in order to define new targets for future therapeutic intervention. We have used mass spec to screen for novel fascin binding partners and have identified the nuclear envelope scaffold protein nesprin as a possible regulator of fascin function. The study outlined here also aims to systematically identify the nature of this complex and the way in which this contributes towards an invasive phenotype in cells within more physiologically relevant 3D ECM environments. Our use of complex 3D ECM models has two main benefits over traditional and widely used 2D in vitro culture approaches. Firstly, the cells are exposed to a more 'in vivo'-like extracellular milieu and therefore better reflect a complex tumour microenvironment. Secondly, the primary targets identified from this study, as well as future targets of interest, can potentially be analysed in these 3D model systems using semi-high throughput screen approaches. These may include siRNA, antibody or small molecule screens to disrupt tumour cell interactions with ECM proteins or stromal cell derived factors. This not only reduces the requirement for expensive early stage in vivo experiments in animal models, but also provides a reliable and robust alternative strategy for studying tumour cell motility. Importantly, the approaches we propose to adopt will combine direct imaging of tumour cell behaviour during invasion with quantification of longer-term endpoints such as invasive phenotype within 'tuneable' ECM environments. This provides powerful and flexible means to dissect the effects and efficacy of manipulating putative therapeutic targets. Long-term, such approaches are likely to become increasingly important to pharmaceutical companies for the development of novel therapies to treat metastasis as well as other diseases such as fibrosis, where cell-ECM interactions are dysregulated. In addition to the impact and potential benefits of our research to the pharmaceutical sector, we are confident that our work will also be of great interest to the scientific community for a number of reasons. Firstly, there are very few other laboratories in the field worldwide that are currently able to combine the state-of-the-art microscopy, image analysis, biophysics and biochemical approaches that we outline here to dissect and interrogate the molecular details of cell invasion within 3D environments. Thus we feel that our well-rounded approach to understanding these complex protein interactions and networks represents and exciting advance in the field. We anticipate also using the methods outlined here in future studies to investigate cytoskeletal dynamics in other signalling pathways that drive cell migration. Additionally, by adopting a multidisciplinary approach, we have identified novel targets and mechanisms that we believe will be of general interest to a number of biological disciplines including cell adhesion/migration, cytoskeleton, ECM and developmental biologists, and cancer/fibrosis researchers. Our proposed analysis and quantification of cytoskeletal dynamics coupled to nuclear movement, cell navigation through ECM and ECM deformation within defined robust physiologically relevant model systems will be relevant to research questions in all of the above fields. As such, we feel the data sets arising from the questions being tackled in this proposal have the potential to impact upon, and influence, other biological areas.