The RAS gene family is among the most commonly mutated genes within cancer. While much research has elucidated its major functions and downstream pathways, little progress has been made in successfully targeting RAS mutations. We recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as a novel partner of the Switch II domain of KRAS. We have found that stable knockdown of AGO2 in KRAS-dependent cell lines lead to a decrease in KRAS protein expression with a subsequent decrease in cellular proliferation. In addition, we observed a decrease in microRNA unwinding in the presence of mutant KRAS, suggesting this interaction inhibits the endogenous RNAi function of AGO2. Despite this clear connection between KRAS and AGO2 in KRAS mediated oncogenesis, the precise function of this interaction remains unclear in both normal and cancer biology.
In order to identify endogenous regulators of AGO2-RAS, we investigated the ability of EGFR signaling to modulate the AGO2-RAS interaction. We established two mouse embryonic fibroblast cell lines (NIH-3T3 and MEF) with complete knockout of AGO2. When compared to normal control cells, we found that knockout of AGO2 resulted in an increase in WT RAS-GTP activation levels, phosphorylation of Y1068-EGFR, and MAPK/ERK and PI3K/AKT signaling. Rescue of AGO2 knockout resulted in a return to normal levels of active RAS-GTP, pEGFR, and downstream signaling.
Recent studies have described EGFR phosphorylation of AGO2 under hypoxic cell conditions, resulting in the inhibition of AGO2 association with RISC members. In order to better characterize the relationship between EGFR-AGO2-RAS, we found that overnight serum starvation followed by stimulation with EGF led to a decrease in AGO2-RAS co-IP in WT KRAS cells. Blocking AGO2 phosphorylation with a Y393F mutant of AGO2 prevented AGO2-RAS dissociation following EGFR stimulation. While WT KRAS cell lines displayed regulation of AGO2-RAS via EGFR, the phosphorylation of AGO2Y393 was unable to disrupt the interaction of AGO2 with mutant KRAS following stimulation with EGF. Together these results suggest a unique EGFR-AGO2-RAS signaling axis, and its dysregulation by mutant KRAS could increase oncogenic growth through promotion of AGO2-RAS interaction in cancer.
Citation Format: Ronald F. Siebenaler, Sunita Shankar, Jean C. Tien, Vijaya L. Dommeti, Malay Mody, Chandan Kumar-Sinha, Arul M. Chinnaiyan. Regulation of AGO2-KRAS interaction through epidermal growth factor receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4370.