Supplemental Fig. S4. (A) Effect of engineered KRAS expression on response to CDK2 inhibition by seliciclib. KRAS mutation sensitized lung cancer cells towards seliciclib-mediated CDK2 inhibition of growth as compared to control-ED-1 cells. (B) Cells transfected with the plasmid expressing HA-tagged human CP110 were used for detection of CP110 using an anti-CP110 antibody (1:1000) and an anti-HA antibody (1:1000), respectively. The upper band detected by the anti-CP110 antibody specifically recognizes CP110. (C) Respective CP110 and RAS protein expression profile in murine lung cancer cell lines is shown. (D) Effect of KRAS knockdown at 48 hours (left panel) and 72 hours (right panel) on CP110 expression in the 344P cell line. (E) Effect of KRAS knockdown at 72 hours (left panel) and 96 hours (right panel) on CP110 expression in the Hop62 cell line.

PDF file, 90K, Repression of UBP43 mRNA levels reduces cyclin D1 protein, but not mRNA expression.

Supplemental File S1. The siRNA sequences and real-time qPCR primer sequences.

PDF file, 76K, Reduction of UBP43 induces apoptosis and decreases cyclin D1 levels in ED-1L lung cancer cells.

The siRNA sequences used in this study.

<p>Supplemental Fig. S3. (A) Schematic diagram showing the relationship between CDK2, CP110, activated KRAS and anaphase catastrophe. (B) Engineered overexpression of CP110 was detected in A549, Hop62, H522 and H460 cells (versus control transfectants) using anti-HA antibody after 24 and 48 hours of transfection. A representative immunoblot for each cell line is displayed.</p>

<p>Supplemental Fig. S1. Effect of wild-type or a phosphorylation sites mutant CP110 species on responses to CDK2 inhibition. (A) Overexpression of CP110 was detected in ED-1 cells with an anti-HA antibody 24 and 48 hours after transfection. (B) Overexpression of wild-type CP110 significantly reduced apoptosis induced by seliciclib treatment. ED-1 cells overexpressing CP110 were treated with the indicated dosages of seliciclib for 24 hours and analyzed for apoptosis. (C and D). Overexpression of phosphorylation-site mutated CP110 did not have a significant effect on anaphase catastrophe or apoptosis induced by CDK2 inhibition. ED-1 cells overexpressing a mutant-CP110 species were treated with the indicated dosages of seliciclib for 24 hours or transfected individually with two different siRNAs targeting CDK2 for 24 hours and (C) scored for multipolar anaphase and (D) analyzed for apoptosis. (E). Seliciclib treatment did not appreciably affect CP110 basal levels in human and murine lung cancer cells. Hop62 and ED-1 were treated with various dosages of seliclcib and CP110 levels were examined after 24 and 48 hours of treatment.</p>

Supplemental File S2. A representative video of a Hop62 cell that undergoes multipolar cell division.

<p>Supplemental Fig. S2. Overexpression of wild-type CP110, but not phosphorylation-sites mutated CP110 in LKR13 murine lung cancer cells that are driven by KRAS rescued anaphase catastrophe and reduced apoptosis induced by pharmacological CDK2 inhibition after seliciclib treatment. LKR13 cells overexpressing wild-type CP110 species or an empty vector, a phosphorylation-sites mutant-CP110 species (MUT-CP110) or an empty vector (Vector) were treated with indicated dosages of seliciclib. After 24 hours treatment, LKR13 cells were (A) scored for multipolar anaphase and (B) analyzed for apoptosis, as detected by Annexin V:FITC and 7-aminoactinomycin D (7-AAD) staining.</p>

PDF file, 75K, Deregulation of UBP43 affects immortalized bronchial epithelial cell growth, apoptosis and cyclin D1 levels.

<p>Supplemental Fig. S5. Representative CP110 immunohistochemistry staining of lung normal (N) versus malignant (T) tissues harvested independently from KRAS wild-type transgenic mice and KRAS mutant transgenic mice.The table displays KRAS mutation information, CP110 scoring and intensity of CP110 immunostaining in 6 lung cancer specimens from cyclin-E transgenic mice and Kras LSL-G12D driven engineered mice respectively.</p>

PDF file, 88K, Model of UBP43 action in tumors and how inhibition of UBP43 activity represents a new therapeutic target.

PDF file, 79K, UBP43 is up-regulated in diverse human cancers as compared to corresponding normal tissues.

PDF file, 166K, Repression of UBP43 mRNA inhibits in vivo and in vitro growth of ED1 lung cancer cells.

PDF file, 677K, The siRNA-mediated repression of UBP43 induced apoptosis which is partially reversed by overexpressing cyclin D1 levels.

Abstract Ubiquitin specific peptidase 18 (USP18), previously known as UBP43, is the IFN-stimulated gene 15 (ISG15) deconjugase. USP18 removes ISG15 from substrate proteins. This study reports that USP18-null mice (vs. wild-type mice) exhibited lower lipolysis rates, altered fat to body weight ratios, and cold sensitivity. USP18 is a regulator of lipid and fatty acid metabolism. Prior work established that USP18 promotes lung tumorigenesis. We sought to learn whether this occurs through altered lipid and fatty acid metabolism. Loss of USP18 repressed adipose triglyceride lipase (ATGL) expression; gain of USP18 expression upregulated ATGL in lung cancer cells. The E1-like ubiquitin activating enzyme promoted ISG15 conjugation of ATGL and destabilization. Immunoprecipitation assays confirmed that ISG15 covalently conjugates to ATGL. Protein expression of thermogenic regulators was examined in brown fat of USP18-null versus wild-type mice. Uncoupling protein 1 (UCP1) was repressed in USP18-null fat. Gain of USP18 expression augmented UCP1 protein via reduced ubiquitination. Gain of UCP1 expression in lung cancer cell lines enhanced cellular proliferation. UCP1 knockdown inhibited proliferation. Beta-hydroxybutyrate colorimetric assays performed after gain of UCP1 expression revealed increased cellular fatty acid beta-oxidation, augmenting fatty acid beta-oxidation in Seahorse assays. Combined USP18, ATGL, and UCP1 profiles were interrogated in The Cancer Genome Atlas. Intriguingly, lung cancers with increased USP18, ATGL, and UCP1 expression had an unfavorable survival. These findings reveal that USP18 is a pharmacologic target that controls fatty acid metabolism. Implications: USP18 is an antineoplastic target that affects lung cancer fatty acid metabolism.

Abstract Ubiquitin specific peptidase 18 (USP18) is the Interferon-Stimulated Gene 15 (ISG15) deconjugase that stabilizes target proteins by removing ISG15 from substrate proteins. We found that USP18 null mice have lower lipolysis rates, altered fat to body weight ratios and marked cold sensitivity. These findings implicated USP18 as a regulator of lipid and fatty acid metabolism. Prior work established that USP18 can promote lung tumorigenesis. We examined whether this occurs in lung cancer cells through altered lipid and fatty acid metabolism. The siRNA knockdown of USP18 downregulated Adipose Triglyceride Lipase (ATGL) and this reduced lipolysis. In contrast, engineered gain of USP18 expression upregulated ATGL and increased lipolysis. Notably, Ubiquitin Activating Enzyme E1 Like Protein (UBE1L) promoted ISG15-conjugated destabilization of ATGL protein. Immunoprecipitation experiments confirmed that ISG15 can covalently conjugate to ATGL. Protein expression profiles of thermogenic regulators were examined in brown fat of USP18 null versus wild-type mice. Strikingly, the master thermoregulatory protein Uncoupling Protein 1 (UCP1) was markedly repressed in brown fat of USP18 null mice. In contrast, USP18 reconstitution augmented UCP1 levels. Engineered gain of USP18 expression stabilized UCP1 protein through reduced Ubiquitin conjugation to UCP1 in the examined lung cancer cell lines. Intriguingly, gain of UCP1 expression in both human and murine lung cancer cell lines promoted cellular proliferation and UCP1 knockdown reduced proliferation. The beta-hydroxybutyrate colorimetric assay determined that UCP1 overexpression promoted fatty acid beta-oxidation in lung cancer cells. Seahorse assays independently confirmed that gain of UCP1 expression increased fatty acid beta-oxidation. Expression profiles of USP18, ATGL and UCP1 species were interrogated using The Cancer Genome Atlas (TCGA). Lung adenocarcinoma and squamous cell carcinoma cases having higher USP18, ATGL and UCP1 levels had a statistically-significantly unfavorable survival. This association also occurred in renal chromophobe carcinoma and low grade gliomas. Together, these findings implicate the deubiquitinase USP18 as a novel molecular pharmacologic target that controls fatty acid metabolism as an energy source for lung cancer growth. These observations provide a strong rationale to uncover USP18 pharmacologic inhibitors that would combat lung and potentially other human cancers. Citation Format: Xi Liu, Yun Lu, Zibo Chen, Xiuxia Liu, Weiguo Hu, Lin Zheng, Yulong Chen, Mi Shi, Lisa Mustachio, Jason Roszik, Masanori Kawakami, Sarah Freemantle, Ethan Dmitrovsky. USP18 promotes lipolysis, fatty acid oxidation and lung cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2547.

Abstract USP18 is the ISG15 (Interferon-Stimulated Gene 15) deconjugase that removes ISG15 from substrate proteins. We found USP18 null mice are markedly cold sensitive versus their wild-type littermates. USP18 null mice had significantly (P < 0.05) greater temperature declines than wild-type mice. These mice also had lower lipolysis rates and altered fat to body weight ratios. This implicated USP18 as a regulator of lipid and fatty acid metabolism. Protein expression profiles of thermogenic regulators were examined in brown fat of USP18 null versus wild-type mice. Strikingly, the thermoregulatory protein UCP-1 was substantially repressed in brown fat of USP18 null mice. To establish that UCP-1 repression was caused by loss of USP18, stable USP18 knock-down was independently achieved in a panel of murine lung cancer cell lines using transfected small hairpin RNAs (shRNAs). USP18 down-regulation by different shRNAs reduced UCP-1 levels as compared to controls. Engineered gain of USP18 expression stabilized UCP-1 expression in these lung cancer cell lines. UCP-1 destabilization followed complex formation with the ubiquitin-like protein ISG15. Immunoprecipitation assays established that complexes formed between ISG15 and UCP-1. Prior work found that USP18 regulates lung tumorigenesis. We therefore examined UCP-1 levels in human lung adenocarcinomas using The Cancer Genome Atlas (TCGA). Lung adenocarcinomas with undetected UCP-1 expression had significantly improved survival versus cases that expressed UCP-1. Notably, gain of UCP-1 expression in both human and murine lung cancer cell lines promoted their growth. In marked contrast, UCP-1 knockdown reduced proliferation. We explored whether UCP-1 expression affected fatty acid metabolism. The beta-hydroxybutyrate (ketone body) colorimetric assay confirmed that UCP-1 overexpression promoted fatty acid beta-oxidation. Gain of UCP-1 expression also increased fatty acid beta-oxidation using seahorse assays. Thus, a direct link exists between USP18 and expression of the thermoregulator UCP-1. This affects UCP-1 stability and lung cancer growth by altering fatty acid metabolism. Together, these findings implicate the deubiquitinase USP18 as a novel molecular pharmacologic target that controls fatty acid metabolism as an energy source for lung cancer growth. Citation Format: Xi Liu, Yun Lu, Weiguo Hu, Zibo Chen, Lisa M. Mustachio, Jason Roszik, Lin Zheng, Masanori Kawakami, Yulong Chen, Sarah J. Freemantle, Ethan Dmitrovsky. Loss of ubiquitin-specific peptidase 18 (USP18) destabilizes the regulator of thermogenesis uncoupling protein-1 (UCP-1) and represses lung cancer growth [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 2399.

Ubiquitination and ubiquitin-like posttranslational modifications (PTM) regulate activity and stability of oncoproteins and tumor suppressors. This implicates PTMs as antineoplastic targets. One way to alter PTMs is to inhibit activity of deubiquitinases (DUB) that remove ubiquitin or ubiquitin-like proteins from substrate proteins. Roles of DUBs in carcinogenesis have been intensively studied, yet few inhibitors exist. Prior work provides a basis for the ubiquitin-specific protease 18 (USP18) as an antineoplastic target. USP18 is the major DUB that removes IFN-stimulated gene 15 (ISG15) from conjugated proteins. Prior work discovered that engineered loss of USP18 increases ISGylation and in contrast to its gain decreases cancer growth by destabilizing growth-regulatory proteins. Loss of USP18 reduced cancer cell growth by triggering apoptosis. Genetic loss of USP18 repressed cancer formation in engineered murine lung cancer models. The translational relevance of USP18 was confirmed by finding its expression was deregulated in malignant versus normal tissues. Notably, the recent elucidation of the USP18 crystal structure offers a framework for developing an inhibitor to this DUB. This review summarizes strong evidence for USP18 as a previously unrecognized pharmacologic target in oncology. Cancer Res; 78(3); 587-92. ©2018 AACR.

The ubiquitin-like modifier interferon-stimulated gene 15 (ISG15) is implicated in both oncogenic and tumor suppressive programs. Yet, few ISGylation substrates are known and functionally validated in cancer biology. We previously found specific oncoproteins were substrates of ISGylation and were stabilized by the ISG15-specific deubiquitinase (DUB) ubiquitin specific peptidase 18 (USP18). Using reverse-phase protein arrays (RPPAs), this study reports that engineered loss of the DUB USP18 destabilized the tumor suppressor protein phosphatase and tensin homologue (PTEN) in both murine and human lung cancer cell lines. In contrast, engineered gain of USP18 expression in these same lung cancer cell lines stabilized PTEN protein. Using the protein synthesis inhibitor cycloheximide (CHX), USP18 knockdown was shown to destabilize PTEN whereas USP18 overexpression stabilized PTEN protein. Interestingly, repression of USP18 decreased cytoplasmic PTEN relative to nuclear PTEN protein levels. We sought to identify mechanisms engaged in this PTEN protein destabilization using immunoprecipitation assays and found ISG15 directly conjugated with PTEN. To confirm translational relevance of this work, USP18 and PTEN immunohistochemical expression were compared in comprehensive lung cancer arrays. There was a significant (P < 0.0001) positive correlation and association between PTEN and USP18 protein expression profiles in human lung cancers. Taken together, this study identified PTEN as a previously unrecognized substrate of the ISGylation post-translational modification pathway. The deconjugase USP18 serves as a novel regulator of PTEN stability. This indicates inhibition of ISGylation is therapeutically relevant in cancers.