Abstract
Abstract KRAS mutations account for development of approximately 30% of lung adenocarcinomas, yet therapeutic options remain limited. To better understand KRAS-mediated survival mechanisms, we characterized phenotypic effects of KRAS knockdown as well as inhibition of its downstream effectors, including MEK, PI3K and RalB using 11 KRAS mutant non-small cell lung cancer cell lines. We observed only 2 cell lines with clear evidence of apoptosis after shRNA-mediated KRAS knockdown (H23 and Calu6). Interestingly, neither pharmacologic inhibition of MEK and PI3K nor knockdown of RalB fully recapitulated apoptotic phenotypes observed upon KRAS knockdown in these KRAS-sensitive lung cancer cells, suggesting that KRAS promotes survival through additional mechanisms. In order to identify novel KRAS survival pathways, we searched for altered phosphoproteins following shRNA-mediated KRAS knockdown using mass spectrometry (MS)-based phosphoproteomics. Stable isotope labeling by amino acids in cell culture (SILAC) was performed and phosphopeptides (p-Ser/Thr/Tyr) were purified using immobilized metal affinity chromatography (IMAC) resin, then analyzed by LC-MS/MS from sh-control or sh-KRAS-infected H23 lung adenocarcinoma cells. In total, we identified 2,878 phosphopeptides, corresponding to 1375 phosphoproteins, of which 210 phosphopeptides were upregulated and 204 phosphopeptides were downregulated after KRAS knockdown (cutoff: 1.5 fold). Using the altered phosphopeptides, we composed a cohesive network view to KRAS-regulated phosphoproteins (Kim et al. PNAS, 110;12414-12419), which revealed that KRAS regulates phosphorylation of cyclin-dependent kinase 1 and 2 (CDK1/2), and downstream mitotic targets. We also observed that KRAS knockdown activates adaptive resistance mechanisms via increased phosphorylation of RAF, PRAS40 and YAP1. We are currently characterizing mitosis-related KRAS functions. Additionally, we are evaluating adaptive resistance mechanisms followed by loss of KRAS by analyzing KRAS-regulated phosphoproteome in cells harboring KRAS mutations but resistant to KRAS loss. These studies will provide better insights into KRAS-regulated survival signaling mechanisms and adaptive responses, which could offer rational co-targeting strategies. Updated work will be presented. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A61. Citation Format: Jae-Young Kim, Eric A. Welsh, Bin Fang, Jiannong Li, Steven A. Eschrich, John Koomen, Eric B. Haura. Characterization of KRAS-driven survival signaling networks via phosphoproteomics in lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A61.
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