Characterizing plasma membrane-associated prohibitins in the regulation of RAS activation and tumourigenesis

• Applicant: Professor Dr. Krishnaraj Rajalingam
• Subject Area: Cell Biology
• Term: from 2019 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 417198367

Prohibitins are evolutionarily conserved proteins that are predominantly known for their role in the regulation of cristae morphogenesis and mitochondrial function. They are the flagship member of the SPFH family of proteins and characterized by the presence of a conserved PHB domain. Recent studies revealed a prominent role for the plasma membrane-associated fraction of prohibitins in signal transduction, pathogen entry and immune cell function. We discovered that PHB1 at the plasma membrane is required for the activation of the serine-threonine kinase CRAF. CRAF is the central member of the RAS-RAF-MAPK cascade that controls fundamental cellular processes. RAS proteins are among the major human oncogenes and nearly 30% of human carcinomas carry mutations in RAS. As RAS is considered almost "undruggable" the inhibitors that have been so far developed target the downstream kinases with some clinical success. As PHB1 is required for the activation of CRAF, small molecule inhibitors that disrupt PHB1-CRAF interaction are of significant interest. Recent work revealed that rocaglamides target this interaction and prevent CRAF-MAPK activation in a panel of cell lines. We discovered that PHB1 is highly expressed in NSCLCs and the targeting of PM–associated PHB1 with ligands like rocaglamide and fluorizoline prevented MEK1 activation in RAS-mutated cell lines. By employing animal models including human NSCLC xenografts and allografts, we also demonstrated that rocaglamide treatment prevented KRAS-mediated growth of NSCLC tumours. Interestingly, we discovered that treatment with rocaglamide prevented RAS-GTP loading in cells stimulated with EGF, though initial experiments suggest that this does not occur in a direct way. Altogether, our data suggest that rocaglamide could possibly function as a RAS inhibitor and we propose that targeting PHB with chemical ligands leads to inhibition of KRAS-mediated tumourigenesis. There are several pressing questions that we want to address by employing a highly interdisciplinary approach including chemical biology approaches:1) How does PHB1 contribute to the stability of KRAS-GTP in cells? 2) Are these effects confined to rocaglamides or do other flavaglines show similar effects? 3) Does rocaglamide treatment disrupt RAS dimerization and formation of RAS nanoclusters? 4) Do rocaglamides inhibit other RAS isoforms? 5) How is the PHB1/2 complex targeted to various subcellular compartments? Targeting RAS has been the holy grail of cancer research in the past 30 years. We identified the natural anti-tumour drug rocaglamide, which works in nanomolar concentrations, as a potential tool to target RAS in cells. Thus, apart from providing important insights into PHB and RAS biology the proposed work will open novel therapeutic avenues for treating RAS-driven human cancers.

• DFG Programme: Research Grants