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All-trans-retinoic acid (ATRA), a pan-agonist for retinoic acid receptors (RARs), regulates diverse cellular functions including growth, differentiation and immune function. We report here that ATRA-treatment represses tumor growth in syngeneic, immunocompetent but not in immunodeficient mice. Tumor immune microenvironment was implicated since depletion of cytotoxic T lymphocytes antagonized these effects in syngeneic mice. Combining ATRA with immune checkpoint blockade did not inhibit lung cancer growth in mice. We sought to augment retinoid anti-tumor effects without affecting its pro-tumorigenicity. We previously reported that CD38 mediated resistance to checkpoint blockade in murine 344SQ lung cancer cells via RARα transcriptional activation of CD38 expression. Yet, combining the RARα antagonist (IRX6696) with anti-PD-L1 did not augment anti-tumorigenicity in transplanted 344SQ cells in syngeneic mice. Prior work implicated RARγ in regulating T cell response. Combining the novel RARγ agonist (IRX4647) with anti-PD-L1 statistically-significantly repressed 344SQ lung cancer cell growth in syngeneic mice. This line is relatively resistant to checkpoint blockade. Immunofluorescent analysis of these treated tumors revealed that combined IRX4647 and anti-PD-L1 treatments reduced CD38 expression in the tumor stroma relative to IRX4647 or anti-PD-L1 treatment alone. Statistically-significantly elevated helper (CD4+) T cells were detected in treated tumors along with increased IL-5 and IL-13 expression observed in plasma and tumors. These cytokines can activate helper T cells, altering lung cancer growth. These microenvironment effects were associated with in vivo anti-tumorigenicity. IRX4647-treatment did not appreciably alter in vitro growth of lung cancer cells although retinoid receptors expression profiles were affected. Pharmacokinetic study of IRX4647 found its plasma half-life was 6 hours. Combining an RARγ agonist with immune checkpoint blockade exerted superior anti-neoplastic efficacy against lung cancer versus an ATRA-based regimen. Given these findings we propose exploring activity of this RARγ agonist with an optimal checkpoint inhibitor in a lung cancer clinical trial. Citation Format: Cheng-Hsin Wei, Lu Huang, Blair Kreh, Xiuxia Liu, Liliya Tyutyunyk-Massey, Zibo Chen, Mi Shi, Vidyasagar Vuligonda, Martin Sanders, Serguei Kozlov, King Chan, Amir Horowitz, Mary Carrington, Patrick Hwu, Weiyi Peng, Ethan Dmitrovsky, Xi Liu. Combining a novel retinoic acid receptor-γ agonist with immune checkpoint blockade represses lung cancer growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1836.

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.

The ubiquitin-like protein FAT10 and the homeobox protein HOXB9 each promote metastatic progression in hepatocellular carcinoma (HCC). In this study, we investigated the clinicopathologic significance of FAT10 and HOXB9 in HCC and investigated a mechanistic role for FAT10 in HOXB9-mediated invasiveness and metastasis. Relative to adjacent normal tissues, FAT10 and HOXB9 were markedly overexpressed in HCC, where a positive correlation in their expression and associated malignant characteristics were found. RNAi-mediated silencing of FAT10 decreased HOXB9 expression and inhibited HCC invasion and metastasis in vitro and in vivo. The effects of FAT10 silencing were reversed by HOXB9 overexpression, whereas RNAi-mediated silencing of HOXB9 decreased HCC invasion and metastasis driven by FAT10 overexpression. Mechanistically, FAT10 regulated HOXB9 expression by modulating the β-catenin/TCF4 pathway, directly binding to β-catenin and preventing its ubiquitination and degradation. Together, our results identified a novel HCC regulatory circuit involving FAT10, β-catenin/TCF4, and HOXB9, the dysfunction of which drives invasive and metastatic character in HCC. Cancer Res; 74(18); 5287–300. ©2014 AACR.