Establishing the utility and molecular mechanisms of integrin alphav beta6-directed human-antibody therapy of cancer

The ability of cancers to invade requires that they have certain specialised molecules on their surface and it is this invasive ability that allows cancers to spread around the body and ultimately kill people. We have identified a new invasion-promoting molecule called alphav beta6. It is on the surface of many different types of cancer and our analysis of cancer tissues taken from patients who have died from the disease, show that the presence of alphav beta6 on cancers can significantly shorten life-expectancy. We believe by preventing alphav beta6 from working we can slow down or even stop cancers from invading and, therefore, from killing people so quickly. In this proposal, using our specialised experimental systems, we will test the ability of antibodies, designed to stop alphav beta6 from working properly, to actually prevent the growth and invasion of cancers. We expect that our studies will allow us to treat cancer patients with these antibodies in the near future. Our study will also investigate how the alphav beta6 actually changes the behaviour of cancers to enable them to invade. This information will allow us to create even better therapies for cancer in the future.

The epithelial-specific integrin, alphav beta6, is weak or absent on normal adult tissues but is upregulated on many carcinomas where its strong expression correlates with poor prognosis for those with cancers of the colon, cervix, lung and breast. Our data offer an experimental explanation for these observations. We were the first to show that alphav beta6 promotes invasion of Matrigel in vitro, in a MMP9/plasmin-dependent manner and that antibodies which blocked ligand-binding to alphav beta6 abrogated this invasion. More recently, using organotypic cultures that better replicate the real tumour environment, as they are a composition of cancer cells overlying a stromal collagen matrix embedded with fibroblasts, we confirmed by transplanting organotypic gels into mice, that alphav beta6 also promotes carcinoma invasion in vivo, although the mechanisms remain obscure. Since alphav beta6 is not highly expressed on normal tissues blockade of alphav beta6 ligand-binding is likely to be an effective and safe therapy for the treatment of alphav beta6-positive cancers. Astra Zeneca have generated novel human antibodies that specifically inhibit the ability of alphav beta6 to bind to its ligands (including fibronectin and the latency associated peptide {LAP} of TGFbeta). In this study, using these organotypic gels, we shall confirm that these novel alphav beta6-blocking human antibodies can suppress alphav beta6-dependent carcinoma invasion and growth in vitro and in vivo, as a prelude to their use for the alphav beta6-directed therapy of cancers in humans. We shall also determine the molecular mechanisms of alphav beta6-promoted invasion and growth. It is likely that 1) promotion of increased secretion of proteases, 2) activation of TGFbeta and thus activation of stromal fibroblasts or 3) a combination of both mechanisms, explain why alphav beta6 is such an aggressive component of carcinoma development. Thus we shall generate organotypic gels with carcinoma cell lines (eg VB6, BICR6, CA1) that exhibit alphav beta6-dependent invasion, together with human foetal foreskin fibroblast 2 (HFFF2) cells, where one or both cellular components have been treated with siRNAi to selectively knockdown expression of individual key targets (including MMP9, uPAR, TGFbeta, TGFbeta-receptor). Once the principal pathways of alphav beta6-dependent invasion have been identified in vitro, we shall use shRNA to generate stable variant carcinoma and fibroblast lines, to generate organotypic gels for transplanting into mice and thus confirm the mechanisms in vivo. These studies will confirm that we have effective therapeutic anti-alphav beta6 antibodies and establish their mechanism of action.