Preclinical Development Evidence for the Ubiquitin Protease UBP43 as an Antineoplastic Target

Newpharmacologic targets are needed for lung cancer. One candidate pathway to target is composed of the E1-like ubiquitin-activating enzyme (UBE1L) that associateswith interferon-stimulated gene 15 (ISG15),which complexes with and destabilizes cyclin D1. Ubiquitin protease 43 (UBP43/USP18) removes ISG15 from conjugated proteins. This study reports that gain of UBP43 stabilized cyclin D1, but not other D-type cyclins or cyclin E. This depended onUBP43 enzymatic activity; an enzymatically inactive UBP43 did not affect cyclin D1 stability. As expected, small interfering RNAs that reduced UBP43 expression also decreased cyclin D1 levels and increased apoptosis in a panel of lung cancer cell lines. Forced cyclin D1 expression rescued UBP43 apoptotic effects, which highlighted the importance of cyclin D1 in conferring this. Short hairpin RNAmediated reduction ofUBP43 significantly increased apoptosis and reducedmurine lung cancer growth in vitro and in vivo after transplantation of these cells into syngeneicmice. These cells also exhibited increased response to all-trans-retinoic acid, interferon, or cisplatin treatments. Notably, gain of UBP43 expression antagonized these effects.Normal-malignant human lung tissue arrayswere examined independently forUBP43, cyclinD1, and cyclin E immunohistochemical expression. UBP43 was significantly (P < 0.01) increased in the malignant versus normal lung. A direct relationship was found between UBP43 and cyclin D1 (but not cyclin E) expression. Differential UBP43 expression was independently detected in a normal-malignant tissue array with diverse human cancers. Taken together, these findings uncovered UBP43 as a previously unrecognized antineoplastic target. Mol Cancer Ther; 11(9); 1–10. 2012 AACR. Introduction Lung cancer is the leading cause of cancer mortality for men and women in the United States (1). The 5-year survival rate for lung cancer patients is only 16% (1). Innovative ways to combat lung cancer are needed. Carcinogenesis is a multistep process. Frequent genetic changes arise in human lung carcinogenesis and some drive this process (2). Previous work highlighted cyclin D1 as a chemopreventive or therapeutic target, as reviewed (3, 4). Increased cyclin D1 expression occurs early during lung carcinogenesis (5, 6) through gene amplification (7) or allele-specific expression imbalance (6), amongothermechanisms.These andotherfindings implicated cyclin D1 as an antineoplastic target in the lung. Lung carcinogenesis is inhibited by induced cyclin D1 proteasomal degradation. This occurred after treatment with retinoid X receptor (RXR, rexinoid) or retinoic acid receptor (RAR, retinoid) agonists (2, 8, 9). Cyclin D1 protein is also repressed by activating a pathway constituted by these retinoid regulated species: ubiquitin activating enzyme E1-like (UBE1L), interferon-stimulated gene 15 (ISG15) and ubiquitin protease UBP43 (UBP43/ USP18; refs. 10 and 11). This study examined whether UBP43 was an antineoplastic target. UBE1L associateswith ISG15, the ubiquitin-like protein familymember; ISG15 conjugation is specifically removed by UBP43, the ubiquitin-specific protease (USP) family member (12, 13). Deregulation of the UBE1L-ISG15UBP43 pathway occurs in diverse tumor types (14–18). All-trans-retinoic acid (RA)-treated human bronchial epithelial (HBE) and acute promyelocytic leukemia (APL) cells increased UBE1L, ISG15, and UBP43 expression (11, 15, 16, anddata not shown).Gain ofUBP43 expression stabilized PML/RARa in APL cells (11) and augmented cyclin D1 expression in HBE cells (10). These findings implicated UBP43 as directly regulating the stability of Authors' Affiliations: Departments of Pharmacology and Toxicology, Medicine, Community and Family Medicine, Pathology; Norris Cotton Cancer Center, Dartmouth Medical School; Dartmouth College, Hanover, New Hampshire; and Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Ethan Dmitrovsky, Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755. Phone: 603-650-1707; Fax: 603-650-1129; E-mail: [email protected] doi: 10.1158/1535-7163.MCT-12-0248 2012 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org OF1 on June 20, 2017. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst July 2, 2012; DOI: 10.1158/1535-7163.MCT-12-0248