Submitted Abstract
For three decades we know that the Ras signalling pathway is a major driver of tumorigenesis. However, it is still not understood why the mutation frequency increases from HRAS, NRAS to KRAS in human cancers. This is puzzling, because Ras proteins are present in all tissues, share the same effectors and on the microscopic level distribute apparently isotropically across the plasma membrane. Intriguingly, K-ras4B but not H-ras has the exquisite potential to drive cancer cell stemness, which can be considered as a prerequisite for the emergence of tumour resistance. However, a molecular cell biological mechanism that explains these isoform specific activities remains to be described. In the past, we elaborated how the apparent degeneracy of Ras is resolved by their isoform specific lateral segregation into distinct, submicroscopic signalling complexes (nanocluster) in the plasma membrane. Here we hypothesize that Ras isoforms exhibit distinct polarized nanoscopic distributions and activities across the plasma membrane of epithelial cells. This fundamentally impacts on polarized/ asymmetric cellular events during cell division, such as centrosome maturation, centrosome- and midbody-inheritance. These processes are fundamentally associated with stemness definition during epithelial differentiation. We postulate that in this context K-ras4B has a particular significance, as it can adopt split-/zwitter-activities by phosphorylation on Ser181. In collaboration with the nanocluster scaffold nucleophosmin and membrane anchorage modulators nucleolin and calmodulin, K-ras may perform non-canonical functions that effect stemness in the cell cycle. With this rationale in mind, oncogenic mutations in Ras do not just lead to an increase e.g. in overall MAPK signaling and cell proliferation, but rather to the mis-localization of active Ras signaling complexes, effectively randomizing their activities across the cell. Ultimately, this leads to a fatal disruption of fundamental polarized signaling and asymmetric apportioning events. Thus, two major aims will be pursued:1. To identify any polarized nanoscale distribution and activity of H-ras, N-ras and K-ras4A/B in polarized epithelial cells.2. To understand non-canonical, cell division associated functions of K-ras4B that promote stemness traits of cancer cells (e.g. centrosome and midbody inheritance). The corresponding two subprojects are mainly performed by two postdoctoral researchers in the Abankwa group in Luxembourg (Ras cell biology) and the Eggeling group in Jena (advanced microscopy) and will synergistically address these aims. We will mainly employ MDCK cells as a well established epithelial cell model to investigate the precise spatial organisation and activity of all Ras proteins, by applying advanced and superresolution microscopy methods. Our work has the potential for a broad impact, as specificity of other protein paralogs/ isoforms that are frequently mutated in cancer, such as B-Raf and PI3Kalpha, could be similarly encoded by their polarized nanoscale activity in epithelia. Given the currently intense efforts to develop Ras drugs, our results will stimulate novel rationales for isoform-specific drug targeting.