Mechanism and role of ILEI dimerization in tumor progression

The goal of this proposal is to reveal cellular and molecular mechanisms responsible for the regulation of ILEI activity, a cytokine described as a key player in tumor progression, with particular focus on the mechanistic characterization of disulfide bond-mediated dimerization, its role in the function of the protein and its uniqueness among other ILEI-related molecules. ILEI belongs to the highly homologous FAM3 family of cytokines. It is a facultatively secreted protein, the plasminogen - uPAR system being responsible for the regulation of its secretion. Uniquely within the family, it bears a fifth cysteine in its peptide sequence and this cysteine is responsible for the dimerization of the molecule. As a consequence, ILEI is simultaneously present both intra- and extracellularly in monomers and covalent dimers, stabilized by intra- and intermolecular disulfide bonds, respectively. Our first aim is to validate and identify the extracellular protein disulfide isomerase (PDI) activity responsible for the disulfide-mediated dimerization of ILEI by using inhibitory and in vitro PDI activity assays. Furthermore, we will analyze the reversibility of dimerization and the influence of the ILEI propeptide on dimerization in vitro in biochemical assays using purified proteins and cells with transient or stable overexpression of wild type and mutant ILEI forms. These will help us to identify mechanisms that regulate the balance in the formation of ILEI dimers. Using mutational analysis of other FAM3 members, we will test if the presence of the fifth cysteine is the only requirement for the formation of disulfide bond-mediated dimers in the FAM3 family-type four-helix-bundle secondary structure and how far these dimers can mimic ILEI function. In addition, we will investigate if basal or plasmin-induced ILEI secretion has any selectivity for the different folding forms of the molecule by analyzing the ratio of secreted ILEI monomers and dimers before and after plasmin induction. To analyze the importance of dimerization for the capability of ILEI to induce tumor progression and epithelial-to- mesenchymal-transition (EMT), we will validate preliminary results that point to a requirement for dimerization by performing tumor formation and lung colonization assays with tumor cells overexpressing a dimerization mutant form of the protein and will characterize these cells in vitro for invasiveness and EMT. We will also investigate the reliance of ILEI function on the activity of the identified PDI enzyme in vitro and in vivo using RNAi and inhibitory studies. Finally, we will correlate PDI expression levels in clinical samples of cancer patients with tumor survival to test the relevance of the identified PDI as potential novel therapeutic target.

ILEI is a cytokine described as a key player in tumor progression and metastasis. The goal of this project was to reveal structural requirements of ILEI activity, in particular dimerization, its role in the function of the protein and its uniqueness among other ILEI-related molecules. The most important finding of this work is that ILEI dimerization is essential for tumor progression and metastasis. This study also identifies structural elements unique for ILEI or partially shared with other ILEI-related molecules that are necessary and sufficient for dimerization and ILEI function. Furthermore, it identifies novel factors that regulate the dimerization and thereby the activity of this secreted molecule. These results on the regulation of ILEI activity via dimerization are not only highly relevant for cancer and metastasis research but also for other disease fields, in which the involvement of ILEI has been described, e.g. Alzheimer disease, diabetes, non-alcoholic fatty liver disease and bone density dysregulations, like osteoporosis.Since all of these diseases represent severe public health issues worldwide and belong to the leading diseases in the eldering Western world, a better understanding on their molecular cause and disease development is urgently needed in order to identify new drugable targets and develop efficient and broadly affordable medications and therapies. Our gained knowledge on ILEI function will contribute to this efforts by providing new potential therapeutical targets and approaches.