Deubiquitinase USP18 Loss Mislocalizes and Destabilizes KRAS in Lung Cancer

Abstract

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 Kras^LA2/+ 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 Kras^LA2/+ /Usp18^-/- mice was found to significantly reduce lung cancers as compared with parental Kras^LA2/+ 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.

Figure 1. KRAS is associated with USP18 expression in murine lung cancer cells

(A) Engineered stable gain of USP18 expression increased lung cancer cell growth and (B) Constitutive loss of USP18 expression by shRNAs decreased cell growth in 393P and LKR13 murine lung cancer cell lines. Proliferation was monitored over 4 days. Growth of these cell lines was normalized to their respective proliferation on day 1. Representative immunoblots confirmed engineered expression and knockdown respectively of USP18 protein in Figure 2A and B. (C) There is no association between Kras and Usp18 mRNA levels in murine lung cancer cell lines. Expression of Kras and Usp18 mRNAs were analyzed by qRT-PCR assays. Kras and Usp18 mRNA expression profiles were quantified relative to respective Gapdh mRNA and normalized to expression in C10 murine lung epithelial cells. (D) There is an association between KRAS and USP18 protein levels in murine lung cancer cell lines. Basal KRAS and USP18 protein expression profiles were quantified relative to respective actin expression and normalized to levels in C10 cells. The representative USP18 immunoblot was exposed for a brief time to avoid overexposure in lanes with abundant USP18 expression.

Figure 2. Modifying USP18 expression alters KRAS protein levels

(A) Stable reduction of endogenous USP18 expression by shRNAs independently introduced into murine and human lung cancer cells decreased endogenous KRAS protein. Murine and human USP18 proteins were detected with mouse and human-specific antibodies, respectively. KRAS expression was quantified relative to actin expression and normalized to vector-transfected lung cancer cell lines. (B) Stable gain of expression of human USP18 by retrovirus introduced independently into murine and human lung cancer cells increased endogenous KRAS protein levels. Human USP18 protein was detected using a human-specific USP18 antibody. Brief exposure times avoided overexposure of exogenous USP18 protein. (C) Decreased KRAS protein conferred by reduced USP18 expression was restored by transiently overexpressing human USP18 in HOP62 lung cancer cells. Immunoblots were individually probed with KRAS and human-specific USP18 antibodies. KRAS was normalized to actin expression and compared between vector control and USP18 shRNA- as well as USP18 shRNA/USP18 expression vector-transfected lung cancer cell lines. Reduced USP18 expression in HOP62 lung cancer cells after introduction of USP18-shRNA appears in panel A. (D) Expression of human enzymatically-inactive USP18 species did not change KRAS expression in ED1 lung cancer cells. Immunoblots were individually probed with KRAS and USP18 specific antibodies. KRAS levels were normalized to actin expression and compared between vector control, enzymatically-inactive USP18 and enzymatically-active (wild-type) USP18 transfected lung cancer cell lines.

Figure 3. USP18 regulates endogenous KRAS protein stability

(A) Endogenous KRAS protein stability decreased with USP18 knockdown. ED1 murine lung cancer cells stably expressing shRNA against USP18 were treated with cycloheximide (CHX) for 15 hours and results were compared to vector-transfected (control) cells. KRAS expression was quantified relative to actin expression at indicated times and normalized to the 0 hour time point (before CHX treatment). Murine USP18 protein expression was detected with a mouse-specific USP18 antibody. (B) Endogenous KRAS protein was stabilized by gain of USP18 expression. HOP62 lung cancer cells transiently overexpressing human GFP-tagged USP18 were treated with CHX for 12 hours and results were compared to vector transfected (control) cells. KRAS protein was quantified relative to actin expression at indicated times and normalized to the 0 hour time point (before CHX treatment). Human USP18 protein expression was detected using a human-specific USP18 antibody.

Figure 4. Knockdown of USP18 protein redistributes KRAS protein from the plasma membrane to the endomembrane compartment

(A) ED1 murine lung cancer cells and H522 human lung cancer cells with constitutive knockdown of USP18 (Figure 2A) were stably transfected with mCherry-CAAX and mGFP-KRAS (wild-type) or (B) mGFP-KRAS G12V (mutant) species. Cells were fixed and imaged by confocal microscopy. Manders coefficients were used to quantify the relative amount of KRAS associated with the endomembrane marker CAAX. Repression of USP18 significantly (P < 0.001) mislocalized KRAS from the plasma membrane to endomembrane compartment. All magnifications are 60X.

Figure 5. USP18 expression is higher in Kras mutant murine lung cancers and USP18 loss reduces tumorigenicity of Kras-driven murine lung cancers

(A) USP18 expression is significantly (P < 0.001) elevated in Kras-driven murine lung cancers relative to cyclin E-driven murine lung cancers. Representative USP18 immunostaining of normal versus malignant lung tissues harvested independently from cyclin E-transgenic mice (n = 3) and Kras^LA2/+ mice (n = 3). All magnifications are 20X. Average USP18 immunostaining in malignant lung was determined. (B) Loss of Usp18 significantly (P < 0.05) decreased the number of Kras-driven murine lung cancers. Average lung tumor numbers of Kras^LA2/+Usp18^+/+ (n = 25), Kras^LA2/+Usp18^+/− (n = 19) and Kras^LA2/+Usp18^−/− (n = 20) mice were determined for each respective group. (C) Loss of Usp18 significantly (P < 0.01) decreased cyclin D1 expression in Kras-driven murine lung cancers. Representative cyclin D1 immunostaining of malignant lung with low versus high cyclin D1 expression are shown. All magnifications are 40X. Average cyclin D1 immunostaining of lung cancer was determined in Kras^LA2/+Usp18^+/+ (n = 16) and Kras^LA2/+Usp18^−/− (n = 15) mice, respectively.

Figure 6. USP18 immunostaining is augmented in human lung adenocarcinomas with activating KRAS mutations

Representative immunostaining of human lung adenocarcinomas with (A) low versus high USP18 expression. (B) Representative USP18 expression profiles are shown for KRAS wild-type versus mutant lung cancers relative to adjacent normal lung tissues. All magnifications are 40X. (C) USP18 immunostaining in normal (n = 76) versus malignant (n = 81) lung was compared and significantly higher (P < 0.001) USP18 levels were detected in the malignant lung. (D) USP18 immunostaining was compared in KRAS wild-type (n = 42) versus mutant (n = 39) lung cancer cases and significantly (P < 0.05) higher USP18 expression was evident in cases where mutant KRAS was detected.