Investigating the Role of AGO2-RAS Interaction in Mutant RAS Driven Cancers
Siebenaler, Ronald
2022
Abstract
The RAS genes are among the most commonly mutated genes in cancer. These genes code for GTPases that act as growth factor receptor-regulated molecular switches. Mutations in RAS lead to a loss in GTP hydrolysis and cause constitutive RAS-GTP signaling, ultimately promoting cellular transformation and oncogenic growth in approximately 30% of human cancers. In pursuit of uncovering protein binding partners in RAS mutant cancers, the Chinnaiyan Lab recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as part of a novel interaction with KRAS. Despite showing a role for AGO2-KRAS binding in KRAS driven cancer, the precise function of this interaction remained unclear in both normal and cancer biology. In order to assess the role of AGO2 in KRASG12D driven disease, we developed a mouse model of pancreatic cancer with conditional loss of AGO2. While AGO2 knockout did not prevent development of early precursor pancreatic intraepithelial (PanIN) lesions, AGO2 null lesions displayed increased activation of the EGFR-RAS signaling axis and altered microRNA expression during early PanIN development that led to oncogene induced senescence (OIS). This resulted in a dramatic increase in the survival of mice with AGO2 ablation. Upon loss of AGO2 and p53, progression to PDAC was restored and PanIN lesions bypassed the senescence block. Additionally, we found that EGFR-mediated phosphorylation of AGO2Y393 disrupts the interaction between wild-type (WT) and oncogenic KRAS-AGO2 interaction under different conditions. While KRAS is the most commonly mutated isoform in human cancer, we next extended our observations to explore the role of AGO2 interaction with mutant HRAS and NRAS. We confirmed AGO2-HRAS and AGO2-NRAS interaction, and we observed that AGO2 knockdown led to an induction of OIS that was accompanied with changes in the EGFR-RAS signaling axis in mutant HRAS and NRAS cells. The EGFR-RAS-ERK signaling observed was associated with an increase in reactive oxygen species (ROS). These high ROS levels inhibited the activity of the phosphatase PTP1B and were associated with increasing pEGFR activation stimulating a feed forward loop resulting in OIS. Finally, knockdown of AGO2 led to an inhibition of mutant RAS driven cell migration and metastasis in a zebrafish xenograft model. We also developed a Single Molecule Toolbox for the use of studying AGO2’s interaction with KRAS and other RISC members. Using an in vitro translation (IVT) system, we over-expressed a given protein, such as AGO2, in a HeLa based cell extract system. We studied the in vitro activity of AGO2 demonstrating a requirement for the presence of RISC members and inhibition by GTP-loaded KRAS. Furthermore, we observed the interaction of AGO2 and KRAS at a single molecule resolution demonstrating their binding at a 1 to 1 stoichiometry. Finally, we observed that IVT generated AGO2 formed higher order clusters in vitro that could be disrupted via RNase treatment. This Single Molecule Toolbox represents a new tool to aid in the study of the biochemistry of AGO2 and its interaction with KRAS. Together, this dissertation describes the role of AGO2-RAS interaction in the development and maintenance of mutant RAS driven cancers, uncovering novel insight into these proteins and their role in both normal physiology and cancer biology.Deep Blue DOI
Subjects
RAS Biology RNA Interference and AGO2 Pancreatic Ductal Adenocarcinoma Single Molecule Imaging
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