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SPATONC Report Summary

Project ID: 322637
Funded under: FP7-IDEAS-ERC
Country: Germany

Final Report Summary - SPATONC (Systems analysis of pancreatic tumor cell phenotype dependence on the spatial regulation of oncogenic Ras signaling)

Publishable brief summary of the project (600words)
Proto-oncogenic Ras is a major intracellular signaling hub for proliferative and survival signals in cells, which displays a very high frequency of over-activating point mutation in various types of cancer. The capacity of Ras for signaling is inextricably linked to its enrichment at the plasma membrane. During the course of this project, we showed that this plasma membrane localization is dynamically maintained by three essential elements: alteration of membrane affinities via lipidation and membrane-interaction motifs; trapping on specific membranes coupled with unidirectional vesicular transport to the plasma membrane; and regulation of diffusion via interaction with the solubilization factor PDEdelta. This system constitutes a cycle that corrects for the entropic equilibration of Ras to all membranes that would impede its signaling capacity. We illuminated how this reaction–diffusion system maintains an out-of-equilibrium localization of Ras GTPases and thereby confers signaling functionality to the plasma membrane. We could show that any interference with this continuous cycle leads to the loss of Ras enrichment at the plasma membrane and thereby its signaling capacity.
Based on the knowledge we gained on this non-equilibrium Ras localization system and the central role of the GDI-like solubilizing factor PDEdelta in it, we were able to block oncogenic KRas signaling by pharmacological interference with the function of PDEdelta in the spatial cycles. In cooperation with the Chemical Biology Department of Herbert Waldmann in our institute, we have identified and characterized several small molecule inhibitors that interfere with the Ras spatial cycle by occupying the farnesyl-binding pocket of the PDEdelta protein and thus preventing PDEdelta from binding Ras. We could show that this affects oncogenic KRas enrichment and signaling at the plasma membrane thereby inhibiting the growth and survival of KRas dependent pancreatic cancer cells in vitro and in vivo. Additionally, we could also show that inhibition of PDEdelta is an efficient way to target colorectal cancer cells that are dependent on the KRas oncogene for their survival and proliferation.
On top of that, we could show that this maintenance of localization by reaction cycles is a principle that is shared by other plasma membrane associated oncogenic proteins, such as Src family kinases (SFKs) that are central to signaling. For myristoylated proteins like SFKs, we identified UNC119 as the GDI-like solubilizing factor (GSF). We could show that knock out of UNC119 proteins affects SFK localization and disrupts oncogenic SFK signaling in cancer cells. These findings offer new opportunities to interfere with (oncogenic) signaling beyond Ras.
Our studies made clear that vesicular dynamics, intracellular diffusion and dynamically maintained spatial organization of proteins are at the center stage in determining growth factor signaling response. To investigate the relation between spatial organization of the phosphatome and growth factor receptor signaling, we resolved how growth factor response originates from dynamically established recursive interactions between epidermal growth factor receptor (EGFR) and spatially organized protein tyrosine phosphatases (PTPs). Using in-house-developed CA-FLIM, we identified for the first time the receptor-like PTPRG/J at the plasma membrane and ER-associated PTPN2 as the major EGFR dephosphorylating activities. Imaging spatial-temporal PTP reactivity revealed that vesicular dynamics establishes a spatially distributed negative feedback with PTPN2 that determines signal duration. Using single-cell dose-response analysis, we uncovered a reactive oxygen species-mediated toggle switch between autocatalytically activated monomeric EGFR and the tumor suppressor PTPRG that governs EGFR's sensitivity to EGF. Vesicular recycling of monomeric EGFR unifies the interactions with these PTPs on distinct membrane systems, dynamically generating a network architecture that can sense and respond to time-varying growth factor signals. This provided the first system level understanding of growth factor receptor response dynamics and changes the paradigm on how cellular signaling takes place.

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