Abstract 1255: Loss of the ubiquitin protease USP18 represses KRAS mutant lung cancer tumorigenicity in mice by destabilizing KRAS protein
Authors: Mustachio Lisa Maria , Lu Yun , Tafe Laura J. , Andrew Angeline S. , Memoli Vincent , Rodriguez-Canales Jaime , Villalobos Pamela A. , Wistuba Ignacio , Yu Jun , Lee Jack J. , Chinyengetere Fadzai , Sekula David J. , Liu Xi , Freemantle Sarah J. , Dmitrovsky Ethan 
Published: July 2016

Abstract
KRAS is frequently mutated in lung cancers. Innovative strategies are needed to combat KRAS mutant lung cancers because these tumors are often resistant to therapy. This study reports that loss of the deubiquitinase USP18 leads to destabilization of the KRAS oncogenic protein in panels of murine and human lung cancer cells. In marked contrast, engineered gain of USP18 expression using retroviral vectors introduced into the same panel of murine and human lung cancer cells stabilized KRAS protein. Loss of USP18 expression in KRAS mutant-expressing lung cancer cells inhibited their growth while gain of USP18 expression opposed this effect. We sought to identify mechanisms engaged in this KRAS protein destabilization. Intriguingly, immunoprecipitation assays established that KRAS conjugates with the ubiquitin-like protein ISG15. This leads to KRAS protein destabilization. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown was shown to destabilize mutant KRAS more prominently than wild-type KRAS protein. To independently confirm that KRAS directly complexes with ISG15, site-directed mutagenesis was performed to render the C-terminal domain of KRAS lysine-less. This led to stabilization of KRAS, despite knockdown of USP18. These studies were confirmed and extended in the in vivo setting of KRAS-driven lung cancers in KRASLA2/+ mice within the FVB strain background. We crossed these mice with USP18 null (USP18-/-) mice to obtain KRASLA2/+;USP18-/- compound mice. Strikingly, these compound mice had statistically significantly (P &lt; 0.05) reduced lung cancers as compared to parental KRASLA2/+ mice. To establish the biological importance of this finding, USP18 immunohistochemical expression was compared in these engineered mice versus a second engineered mouse lung cancer model that was not driven by KRAS, but by aberrant cyclin E expression. USP18 immunohistochemical expression was substantially higher in the KRAS-driven than cyclin E-dependent lung cancers. To explore the translational relevance of this work, lung cancer tissue arrays were examined in 551 lung cancers where a clinical database was available. USP18 immunohistochemical expression was significantly (P &lt; 0.001) higher in KRAS mutant adenocarcinomas as compared to wild-type cases. There was also a significant increase in USP18 expression in adenocarcinoma versus squamous cell carcinoma cases. Taken together, these studies broaden the role of USP18 as an antineoplastic target to combat lung cancers that harbor KRAS mutations.
Citation Format: Lisa Maria Mustachio, Yun Lu, Laura J. Tafe, Angeline S. Andrew, Vincent Memoli, Jaime Rodriguez-Canales, Pamela A. Villalobos, Ignacio Wistuba, Jun Yu, Jack J. Lee, Fadzai Chinyengetere, David J. Sekula, Xi Liu, Sarah J. Freemantle, Ethan Dmitrovsky. Loss of the ubiquitin protease USP18 represses KRAS mutant lung cancer tumorigenicity in mice by destabilizing KRAS protein. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1255.