Spatiotemporal mechanisms of eIF5A1/2-mediated metastasis in triple-negative breast cancer

Transforming Growth Factor Beta (TGFbeta) is a secreted protein and the first member of the TGFbeta superfamily of ligands to be described. However, the molecular mechanisms that govern TGFbeta's ability to switch between its paradoxical growth suppressing and epithelial-mesenchymal transition (EMT) promoting functions remain to be fully elucidated. Since TGFbeta elicits pleiotropic functions during normal development, adult tissue homeostasis and pathophysiological processes such as cancer and fibrosis, it is essential that future research efforts focus on producing a complete mechanistic understanding of TGFbeta function. To this end, the objective of this proposal is to investigate the cellular and molecular mechanisms by which PEAK1 (pseudopodium-enriched atypical kinase one) regulates TGFbeta signaling and mediates TGFbeta-induced EMT during disease progression. The proposed work is an extension of our recently published and compelling preliminary data showing that (i) PEAK1 mediates TGFbeta-induced EMT, migration, proliferation and cancer metastasis; (ii) PEAK1 localizes to membrane actin structures and regulates Src/Grb2/MAPK signaling in response to TGFbeta/fibronectin stimulation; and (iii) inhibition of PEAK1 translation blocks the pathophysiological effects of TGFbeta signaling. Thus, the central hypoth- esis of this proposal is that eIF5A-driven PEAK1 translation promotes the assembly of a Src/Grb2/PEAK1 complex in the context of membrane ITGB3 activation to enable TGFbeta-induced MAPK signaling, ZEB1 upregulation and EMT. The approach is innovative because it will employ a combination of state-of-the-art cellular, molecular, biochemical, microscopy, proteomic and model organism methods to elucidate the mechanisms of action for the novel eIF5A/PEAK1 translation and Src/Grb2/PEAK1/MAPK cytoskeletal signaling nodes as novel regulators of TGFbeta-induced EMT. Furthermore, the proposed research is significant because it will address the following two major challenges and needs within the field of TGFbeta research: Specific Aim 1 will characterize translational and post-translational mechanisms of TGFbeta-induced EMT; and Specific Aim 2 will identify context- dependent spatiotemporal dynamics for molecular regulators of TGFbeta responses. The collective knowledge gained from these mechanistic studies will identify and characterize details of the cellular and molecular contexts in which TGFbeta is dysregulated to cause disease, and novel methods for block- ing the negative consequences of TGFbeta signaling.