Insights into KRAS Biology through its Novel Interactions.

Abstract

A third of all human cancers harbor mutations in the RAS family of genes, which encode members of small GTPases. RAS proteins transduce extracellular growth signals by cycling between a GTP-bound activated state and a GDP-bound basal state, regulating effector pathways through RAF/PI3K binding. Mutations lock RAS in the activated GTP bound state, leading to constitutive signaling through various pathways, leading to neoplastic transformation. In this thesis, KRAS biology was interrogated at three levels. At the chromosomal level, using an integrative genomics approach we identified UBE2L3-KRAS gene fusion in prostatic DU145 cells and functional studies of the fusion suggests proximity of KRAS to the ubiquitin machinery, a recurrent theme in various synthetic lethal screens of KRAS. At the level of the transcript, we identified frequent ‘outlier kinases’ like polo like kinase in KRAS-dependent pancreatic cancer cell lines which show increased sensitivity to PLK inhibition in combination with KRAS knockdown. PLK is part of the Anaphase promoting complex, and is known to associate with the protein degradation machinery. At the level of the protein, an unbiased mass spectrometric analysis identified Argonaute 2 (AGO2) as an unexpected RAS interacting protein. RAS co-sediments with AGO2 in membrane fractions, and co-localizes in intracellular membrane organelles. The N-terminal domain of AGO2 directly binds the Switch II domain of RAS, in a GDP/GTP independent manner. Functionally, knock-down of AGO2 attenuates KRAS-mediated cell proliferation in mutant KRAS-dependent cancer cell lines and KRAS-mediated transformation of normal cells is enhanced upon AGO2 overexpression. The intracellular KRAS-AGO2 interaction increases mutant KRAS levels and PI3K signaling. Additionally, expression of mutant KRAS attenuates the assembly of regulatory messenger ribonucleoprotein particles (mRNPs) in NIH3T3 cells. Employing NIH3T3 AGO2-/- cells, we observed that interaction with AGO2 is required for maximal KRAS-mediated transformation. This most surprising intersection of the signaling networks of KRAS with the RNA silencing machinery through its interaction with its core component protein, AGO2, expands its range of oncogenic activities. Together these studies describe the intersection of KRAS with both the ubiquitin/proteasomal degradation and RNA silencing pathways beyond its well characterized role in signal transduction thus providing new insights into RAS function.