Two recently developed highly specific EZH

Two recently developed, highly specific EZH2 enzymatic inhibitors, GSK126 and EPZ-6438, are currently in clinical trials for treating lymphomas (Kim and Roberts, 2016). Although these EZH2 inhibitors have shown antitumor effects in lymphoma cells with enzyme-activating mutations of EZH2 (Knutson et al., 2012, McCabe et al., 2012, Qi et al., 2012) and in ovarian cancer cells with inactivating mutations of ARID1A (Bitler et al., 2015), certain cancer cells are resistant to the enzymatic inhibition of EZH2 but sensitive to the genetic depletion of EZH2, suggesting that the tumor-promoting function of EZH2 depends on its catalytic and non-catalytic activity. Indeed, independently of its histone methyltransferase activity, EZH2 can promote cancer by stabilizing the PRC2 (Kim et al., 2015) or by acting as a transcriptional coactivator of androgen receptor (Xu et al., 2012), estrogen receptor (Shi et al., 2007), β-catenin (Shi et al., 2007), and nuclear factor κB (NF-κB) (Lee et al., 2011). Consequently, destroying EZH2 protein should be more effective than EZH2 inhibitors in targeting cancers that are dependent on EZH2’s non-catalytic activity. The highly conserved, 76-amino NSC 66811 polypeptide ubiquitin is added to protein substrates through a multi-step process starting with ubiquitin activation by a ubiquitin-activating enzyme (E1), followed by its transfer to a lysine residue on the substrate, which is mediated by ubiquitin-conjugating enzymes (E2) and ubiquitin ligases (E3) (Glickman and Ciechanover, 2002, Pickart, 2001). Ubiquitin contains seven lysines. Whereas lysine 63 (K63)-linked polyubiquitination alters the substrate’s subcellular localization, affects its activity, and modulates its interaction with other proteins (Chen and Sun, 2009, Pickart and Fushman, 2004), all non-K63 ubiquitin linkages can target proteins for degradation via the proteasome (Xu et al., 2009). Ubiquitination is reversed by deubiquitinating enzymes (DUBs, or deubiquitinases), a group of proteases that remove monoubiquitin or polyubiquitin chains from the substrate (Wilkinson, 1997, Xiao et al., 2016). EZH2 protein is subject to ubiquitin-dependent degradation by several E3 ligases, including β-TrCP, SMURF2, and FBW7 (Jin et al., 2017, Sahasrabuddhe et al., 2015, Yu et al., 2013); however, the deubiquitinase that reverses this ubiquitination is unknown. Here we identify an ovarian tumor protease (OTU) family member, ZRANB1 (also known as Trabid), as an EZH2 deubiquitinase and a potential therapeutic target in cancer.
Results
Discussion Genetically engineered mouse models have provided strong evidence that EZH2 drives or accelerates oncogenesis in multiple cancer types, such as lymphoma (Béguelin et al., 2013, Berg et al., 2014, Souroullas et al., 2016), melanoma (Souroullas et al., 2016), breast cancer (Gonzalez et al., 2014, Li et al., 2009), and lung cancer (Zhang et al., 2016). Whereas previous studies attributed the cancer-promoting function of EZH2 to the repression of PRC2 target genes through H3K27me3-mediated epigenetic silencing (Bonasio et al., 2010), accumulating evidence has demonstrated that the non-catalytic activity of EZH2 contributes substantially to tumor formation and progression (Kim et al., 2015, Lee et al., 2011, Shi et al., 2007, Xu et al., 2012), which could explain the resistance of many cancers to EZH2 enzymatic inhibitors. On the other hand, genetic depletion of EZH2 has been shown to suppress tumorigenesis and metastasis. Several ubiquitin ligases promote EZH2 ubiquitination and degradation (Jin et al., 2017, Sahasrabuddhe et al., 2015, Yu et al., 2013). Destabilization of EZH2 by targeting its deubiquitinase may offer an alternative therapeutic approach to treating EZH2-overexpressing tumors, such as TNBC and ovarian cancer. In this study, we identified ZRANB1 as the EZH2 deubiquitinase that regulates the polyubiquitination and protein stability of EZH2. Ablation of ZRANB1 is compatible with the viability of normal tissues, since Zranb1-null mice were viable and did not exhibit phenotypic differences compared with wild-type mice under normal physiological conditions, although they were resistant to the induction of experimental autoimmune encephalomyelitis (Jin et al., 2016). Similarly, our data revealed that ZRNAB1 siRNA treatment was not detrimental to normal human and mouse cells and was not toxic to mice. In contrast, depletion of ZRANB1 in TNBC cells markedly suppressed cell proliferation and migration and induced apoptosis, and systemic delivery of nanoliposome-encapsulated ZRANB1 siRNA led to pronounced anticancer effects in xenograft models of TNBC, suggesting that ZRANB1 could be a novel therapeutic target. Notably, ZRANB1 protein is abundantly expressed in TNBC cell lines, which are responsive to ZRANB1 siRNA or inhibitor, but it is undetectable in normal human mammary epithelial cell lines, which are resistant to knockdown or chemical inhibition of ZRANB1.