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KRAS is frequently mutated in lung cancers and is associated with aggressive biology and chemotherapy resistance. Therefore, innovative approaches are needed to treat these lung cancers. Prior work implicated the IFN-stimulated gene 15 (ISG15) deubiquitinase (DUB) USP18 as having antineoplastic activity by regulating lung cancer growth and oncoprotein stability. This study demonstrates that USP18 affects the stability of the KRAS oncoprotein. Interestingly, loss of USP18 reduced KRAS expression, and engineered gain of USP18 expression increased KRAS protein levels in lung cancer cells. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown significantly reduced the half-life of KRAS, but gain of USP18 expression significantly increased its stability. Intriguingly, loss of USP18 altered KRAS subcellular localization by mislocalizing KRAS from the plasma membrane. To explore the biologic consequences, immunohistochemical (IHC) expression profiles of USP18 were compared in lung cancers of KrasLA2/+ versus cyclin E engineered mouse models. USP18 expression was higher in Kras-driven murine lung cancers, indicating a link between KRAS and USP18 expression in vivo. To solidify this association, loss of Usp18 in KrasLA2/+/Usp18−/− mice was found to significantly reduce lung cancers as compared with parental KrasLA2/+ mice. Finally, translational relevance was confirmed in a human lung cancer panel by showing that USP18 IHC expression was significantly higher in KRAS-mutant versus wild-type lung adenocarcinomas.Implications: Taken together, this study highlights a new way to combat the oncogenic consequences of activated KRAS in lung cancer by inhibiting the DUB USP18. Mol Cancer Res; 15(7); 905–14. ©2017 AACR.

Contains supplementary methods and supplementary figure legends.

S1. Introduction of enzymatically-active USP18 overcomes growth inhibition of USP18-repressed lung cancer cells. S2. Loss of USP18 expression reduces growth of KRAS mutant human lung cancer cells. S3. Interferon (IFN) treatment of lung cancer cells having repressed USP18 expression promotes growth inhibition. S4. Modifying USP18 expression alters KRAS protein levels. S5. USP18 regulates KRAS post-transcriptionally. S6. Repression of USP18 expression downregulates AKT and MAPK signaling pathways. S7. KRAS is ISG15-modified. S8. USP18 knockdown promotes KRAS localization to the endomembrane compartment. S9. KrasLA2/+ mice were crossed with Usp18-/- mice to generate KrasLA2/+/Usp18-/- compound mice. S10. KRAS mRNA is increased with presence of activating KRAS mutations and USP18 protein expression is augmented in the presence of high KRAS levels.