Leucine-rich alpha-2-glycoprotein 1 (Lrg1) as a novel modulator of TGFbeta signalling in disease

Diseases of the vasculature constitute a major burden to human wellbeing and their treatment creates a significant economic cost, particularly in western societies. For example, in the eye proliferative diabetic retinopathy is the leading cause of blindness in the working age population, and the exudative (wet) form of age-related macular degeneration (AMD) is the leading cause of loss of vision in the over 65's. Both diseases are common, highly disabling and increasing in prevalence and are characterised by abnormal blood vessel growth and vessel leakage. In both conditions the molecular mechanisms that lead to pathology are poorly understood and the treatment options are limited.

New blood vessel formation also plays a major role in the growth of solid tumours, since a growing mass of cells requires delivery of nutrients and oxygen. Growing tumours promote the formation of new blood vessels in order for them to expand and attempts to destroy or eliminate tumour blood vessels have been the subject of extensive research. To date, however, these approaches have been largely unsuccessful but remain a desirable objective in the pursuit of novel anti-cancer drugs. The blood vessels that form in tumours are also frequently highly disorganised, poorly perfused and leaky, and it is increasingly recognised that if these blood vessels could be 'normalised' this may aid the delivery of therapeutic drugs to the target tumour cells.

In our previous work we discovered a protein named Lrg1, that stimulates blood vessel growth and that is required for pathological blood vessel growth in models of eye disease. We also observed that in mice that lack Lrg1, tumours grow much more slowly. We further showed that blocking the activity of Lrg1 using antibodies prevented the formation of abnormal blood vessels in a mouse model of AMD, demonstrating that Lrg1 is a potential therapeutic target in diseases of the vasculature. We have also discovered that Lrg1 modifies the activity of the growth factor TGFbeta and that it is through this interaction that Lrg1 exerts its effect. We found that Lrg1 is frequently expressed by cancer cells, suggesting that this could be one way in which solid tumours stimulate the formation of a blood supply. However, TGFbeta is not only involved in promoting new blood vessel growth but can also make tumour cells more aggressive and prevent the immune system from attacking the tumour. Preliminary experiments have shown that Lrg1 can indeed modulate the activity of TGFbeta in cancer cells and white blood cells and that this may support tumour cell growth and metastasis.

In this proposal our objectives are to elucidate the mechanism of how Lrg1 works in cancer cells, blood vessels and cells of the immune system. We already have several important clues from studies performed in our current MRC project grant, in that we know the identity of certain cell surface receptors on blood vessels that interact with Lrg1, and we know some of the consequences of these interactions on cell fate. Here, we will define those interactions in greater detail, to work out not only which receptors Lrg1 binds to on blood vessels, cancer cells and immune cells, but also to define which parts of the Lrg1 molecule are required for those interactions. Using a variety of experimental models of disease, we will examine the consequences of Lrg1 binding to its target cells to determine cellular outcomes. Finally, will use Lrg1 blocking antibodies to find out whether, in these models of disease, therapeutic targeting of Lrg1 diminishes or eliminates the pathology.

The objectives of this research are to gain insight into the cellular and molecular mechanisms that mediate the regulatory effects of Lrg1 on TGFbeta signalling. The interconnected cell systems in which we will perform the research are vascular endothelial cells, cancer cells and cells of the immune system.

In preliminary work we have used surface plasmon resonance (Biacore) to demonstrate a direct interaction between Lrg1 and Endoglin, and here will develop this approach to establish the binding parameters and hierarchy of binding of Lrg1 to other endothelial receptors including TbRII and ALK5. We will use mutagenesis to define the binding domains on both Lrg1 and its targets, with expression of mutants both as recombinant proteins for biophysical studies, and in cell culture models for functional studies. We will investigate the effects of Lrg1 on both canonical and non-canonical TGFbeta signalling, and also explore possible interactions between Lrg1 and signalling via bone morphogenetic proteins and their receptors. Studies at the cellular level will be complemented by experiments using in vitro, ex vivo and in vivo models of angiogenesis.

We will also investigate the role of Lrg1 as a modulator of TGFbeta signalling in cancer by using a range of tumour cell lines that vary in their expression of Lrg1, TGFbeta and Endoglin. We will manipulate Lrg1 expression in these cell lines in order to determine the consequences on proliferation, signal transduction and tumour cell characteristics such as anchorage-independent growth and migration. We will extend this work into animal models of tumorigenesis using the RIP-TAG mouse, tumour cell grafting and metastasis, and direct tumour induction in control and Lrg1 KO mice. Since Lrg1 also impacts on cells of the immune system we will investigate leukocyte infiltration of tumours, and follow up preliminary data in which we have shown that Lrg1 modulates lymphocyte and macrophage phenotype.

1. Who will benefit from this research?
The outcomes of this project will benefit i) other workers investigating the molecular mechanisms of angiogenesis, ii) investigators studying cancer and immunology, and iii) pharmaceutical and biotech companies seeking new therapeutic targets for the treatment of neovascular disease and cancer.

2. How will they benefit from this research?
We focus here on group iii) since these are the beneficiaries whose activities are most likely to have a direct impact on the nation's health and wealth. In this regard, diseases associated with aberrant neovascularisation and cancer carry a significant cost in terms of disability, management and therapy and in western societies this socio-economic burden is rising with increased longevity. Among neovascular diseases of the eye, AMD is the most common in the over 60s, and its prevalence is increasing. Similarly, proliferative diabetic retinopathy is also increasing alarmingly. Cancer continues to be a major cause of morbidity in all populations, with >2M living with or beyond cancer (2008 data) in the UK, and 1 in 3 affected at some time in their lives. Therapeutic options for cancer are improving but there remains a significant unmet clinical need. For these reasons there is strong interest in Pharma in the development of therapies aimed at arresting, slowing and curing these diseases. In the eye the best available treatment for AMD is VEGF blockade via biologics such as Lucentis, but this is effective in only around half of the target patient group who have the neovascular exudative form of the disease. Proliferative diabetic retinopathy is the most common blinding disease in the working age population, yet the best available therapy is the essentially destructive application of laser photocoagulation. The front-line drugs for solid tumours comprise chemotherapeutics such as 5-FU and cisplatin, and humanised monoclonal antibodies such as Avastin and Herceptin, but new therapeutic targets and blocking agents are constantly being sought. We already know that Lrg1 blockade inhibits laser-induced choroidal neovascularisation so we anticipate that the outcomes of this project will be of interest to Pharmaceutical companies seeking new therapeutic options in the treatment of vascular disease and cancer.

The wealth implications are substantial with regard to the commercial value of therapeutics in these areas. Thus, the humanized monoclonal antibody Lucentis (Roche-Genentech), which is used to treat wet AMD, is expected to gross ~$2B worldwide in 2013. As Lucentis is effective in only about half of those patients treated, the unmet clinical need creates enormous scope for future revenue-generating drugs. Avastin, which also blocks VEGF but is used in the treatment of cancers, in particular metastatic colorectal cancer, is expected to gross ~$6B in 2013. Therapeutics that target Lrg1 would be attractive because, at least in pathological neovascularisation, Lrg1 blockade provides a means of inhibiting the pathogenic effects of TGFbeta. If the work proposed here demonstrates that Lrg1 blockade is also an option for treating cancer then the impact, in terms of health wealth implications for the UK, would be considerable.