Abstract IA05: Regulation of Ras proteins and their effectors
Authors: Tina Yuan, Frank McCormick
Journal: Cancer Research (2017)

Abstract Ras proteins regulate multiple phenotypes, including proliferation, contact inhibition, cell motility, metabolism, and genome integrity. This range of phenotypes may relate to the number of different effector pathways that Ras activates. The best validated of these is the Raf/MAPK pathway. Ras proteins can activate PI 3-kinase pathways directly, though this seems to vary between tissue types. Ras proteins bind and activate RalGDS, but this pathway is less well understood. In addition to these three major pathways, Ras proteins in their GTP state interact directly with several other potential effectors. Analysis of the contributions that these pathways make towards cancer phenotypes using genetic approaches or RNA interference has been complicated by redundancies within each effector pathway. For example, there are three Raf isoforms, 3 PI 3-kinase isoforms, etc. This explains why screens for single genes that Ras depends on have not identified classical Ras effectors. Furthermore, each pathway may have several relevant phenotypes, such as effects on cell cycle progression, survival, and metabolic stress. Therefore, screens that depend on one phenotype, such as proliferation or survival, might miss other important functions. In addition to these technical complications, it is important to recognize differences in signaling between different tumor types. For example, in lung adenocarcinoma, the RTK/RAS/MAPK pathway can be activated by mutations in RTKs, activation of SOS, loss of NF1, mutation in KRAS, or activation of BRAF, with similar clinical outcomes. In pancreatic cancer, only mutation in KRAS appears capable of driving malignancy. In lung adenocarcinoma and pancreatic cancer, KRAS mutations are likely initiating events, in colorectal cancer they are definitely not, and exist in the context of un-regulated beta-catenin signaling. To address these issues we analyzed the importance of each canonical effector pathway, plus 37 other potential effectors, in 100 cell lines derived from lung, pancreatic and GI tissues, measuring five different parameters on a single cell basis. We used combinations of highly potent and well characterized siRNAs that enabled us to knock down all the paralogs within an effector node at once. For example, we were able to knock down ARAF, BRAF and RAF1, together, in the “RAF node”. We took advantage of the variable nature of siRNA knockdown to generate dose-response curves for each node, measuring effects on five parameters on each cell. This analysis revealed the expected dependence on tissue of origin, with pancreatic cells being more dependent on Ras signaling than lung adenocarcinoma. Unexpectedly, we found that most KRAS mutant tumor cell lines fell into two major classes: those that depend strongly on KRAS itself, and the Raf/MAPK pathway, and those that had lost dependence on KRAS, but were strongly dependent on RSK p90 S6 kinase. These cells were not dependent on PI 3-kinase genes themselves, but rather on PDK1, loss of LKB1 and other aspects of downstream PI 3-kinase signaling. We also identified a small number of cells that depended strongly on RalGDS. Each of these subsets had remarkably different properties, in terms of energy metabolism, EMT status, and cell-cell interaction. We conclude that oncogenic Ras proteins can indeed activate several downstream pathways, but in individual cell lines, one effector pathway predominates. Citation Format: Tina Yuan, Frank McCormick. Regulation of Ras proteins and their effectors. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA05.