The Role of Dubs in the Post-Translational Control of Cell Migration

Essays in Biochemistry (2019) 63 579–594 https://doi.org/10.1042/EBC20190022

Review Article The role of DUBs in the post-translational control of cell migration

Guillem Lambies1,2, Antonio Garc´ıade Herreros1,2 and V´ıctor M. D´ıaz1,2,3 1Programa de Recerca en Cancer,` Institut Hospital del Mar d’Investigacions Mediques` (IMIM), Unidad Asociada CSIC, Barcelona, Spain; 2Departament de Ciencies` Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), Barcelona, Spain; 3Faculty of Medicine and Health Sciences, International University of Catalonia, Sant Cugat del Valles,` Barcelona,

Spain Downloaded from https://portlandpress.com/essaysbiochem/article-pdf/63/5/579/859061/ebc-2019-0022c.pdf by guest on 05 November 2019 Correspondence: V.M. Dıaz´ ([email protected])orA.Garcıa´ de Herreros ([email protected])

Cell migration is a multifactorial/multistep process that requires the concerted action of growth and transcriptional factors, motor proteins, extracellular matrix remodeling and proteases. In this review, we focus on the role of transcription factors modulating Epithelial-to-Mesenchymal Transition (EMT-TFs), a fundamental process supporting both physiological and pathological cell migration. These EMT-TFs (Snail1/2, Twist1/2 and Zeb1/2) are labile proteins which should be stabilized to initiate EMT and provide full migratory and invasive properties. We present here a family of enzymes, the deubiquitinases (DUBs) which have a crucial role in counteracting polyubiquitination and proteasomal degradation of EMT-TFs after their induction by TGFβ, inflammatory cytokines and hypoxia. We also describe the DUBs promoting the stabilization of Smads, TGFβ receptors and other key proteins involved in transduction pathways controlling EMT.

Introduction Epithelial-to-mesenchymal transition (EMT) is a cell process allowing epithelial cells to adopt mesenchymal properties and enhance their migratory capability [1]. Although some other types of migration have been described [3,4], in this review, we will focus on that related with EMT and describe how it is modulated by a myriad of labile proteins that need to be stabilized. In particular, we will analyze the role of an emergent class of enzymes called deubiquitinases (DUBs) in several aspects of cell migration.

Typesofcellmigration Physiological cell migration during embryonic development and wound healing EMT is particularly relevant during embryonic development since it allows cells to migrate over extremely long distances in the embryo, giving rise to the formation of the three-dimensional structures and orig- ination of the different organs in the final organism. EMT happens early in embryonic development, in the generation of parietal endoderm and in the mesoderm and neural crest formation [2–4]. For instance, neural crest arises from a population of precursor cells that escape from the neural tube and migrate a long distance in the embryo, promoting the formation of different subtypes of cells [5]. Upon migration of neural crest cells to the different locations, cells become again epithelial undergoing the opposite Received: 21 June 2019 process, the mesenchymal-to-epithelial transition, performing transient epithelial structures such as the Revised: 30 September 2019 somites, the precursors of the urogenital system and the splachnopleure. These structures again undergo Accepted: 01 October 2019 EMT to promote mesenchymal cells with a more restricted differentiation potential [5]. Besides morpho- Version of Record published: genesis, the acquisition of migratory properties through an EMT in physiological conditions plays a key 11 October 2019 role in other physiological processes such as wound healing, the response of the organism to an injury in

the skin. Keratinocytes from the epidermis located at the wound border undergo a partial EMT, acquiring the capabilities to migrate [2].

Pathological cell migration: ﬁbrosis and cancer metastasis Cell migration is also involved in pathological processes such as fibrosis and cancer invasion. Fibrosis involves organ degeneration and takes place following tissue injuries such as cardiac failure [6]. This results in a massive deposition of collagen fibers mediated by fibroblastic cells derived from a set of epithelial cells via an EMT process. These cells migrate to the injured tissue and secrete a huge amount of fibroblastic proteins such as Collagen I and alpha-smooth muscle actin (α-SMA) [6]. The fibrotic EMT has been observed in many organs, such as liver, lung and intestine [7]. EMT also plays a crucial role in cancer. Even though EMT features had been largely observed in in vitro and in vivo cancer models, the relevance of EMT for cancer metastasis has been a matter of debate for many years [8]. This is due to the similarity between the mesenchymal cells derived from an EMT program and the stromal cells, two cell Downloaded from https://portlandpress.com/essaysbiochem/article-pdf/63/5/579/859061/ebc-2019-0022c.pdf by guest on 05 November 2019 types sharing similar markers [2]. The first relationship between EMT and cancer metastasis was observed in HER2 transgenic mice showing cancer cells with migratory features [9]. On the other hand, detection of cell aggregates detaching from the tumor into the adjacent stroma further confirmed the involvement of EMT in cancer metastasis [10]. Genetic lineage-tracing experiments have formally demonstrated the role of EMT during invasion [11], although other studies have questioned a critical role of EMT in metastasis [12,13]. In any case, besides metastasis formation, the role of EMT in chemo-resistance is beyond any doubt [14,15]. Molecular mechanisms controlling cell migration Epithelial cells maintain cell contacts between them through the arrangement of tight junctions, desmosomes and adherent junctions which are lost during EMT promotion. Occludins, claudins and Zona-Occludens 1 (components of tight junctions) or desmoplakins (of desmosomes) are down-regulated during EMT. However, the most relevant feature of EMT is the loss of the essential homotypic adherent junction E-cadherin [16], encoded by the CDH1 gene, which mediates homophilic intercellular interactions through its extracellular domain. These junctions are main- tained by anchoring its intracellular domain to actin filaments via β-catenin association [17]. This loss of the epithelial markers is accompanied by an up-regulation of mesenchymal proteins such as Fibronectin. Fibronectin mediates extracellular matrix (ECM) assembly through the binding to α5β1 integrins; this stimulates its self-association and organizes the actin cytoskeleton to promote cell contractility. It also allows the assembly of other fibrous proteins such as Collagen I [18]. Another relevant trait in the modulation of cell migration is the positive modulation of the Rho-GTPases, involved in lamelipodium extension at the front of the cell, formation of new adhesions of the cell to surrounding matrix proteins, cell body contraction and tail retraction [19]. Moreover, Rho is essential to mediate actin contractility to promote fibronectin-dependent ECM assembly [20]. Cell migration also requires the up-regulation of proteases that degrade basement membranes, enhancing the capability of cells to invade and migrate [21,22].

Transcription factors modulating EMT and cell migration The expression of epithelial and mesenchymal markers is tightly regulated by different transcription factors collec- tively known as transcription factors modulating EMT (EMT-TFs). Snail1 is considered the main EMT-TF [16]. It binds sequences with a 5-CACCTG-3 core located in the promoters of CDH1 and other epithelial markers, such as occludins and claudins. Besides its role as a repressor, Snail1 also works as an activator, up-regulating the expression of mesenchymal markers such as fibronectin and the EMT-TFs Zeb1 and 2 [23–25]. Similar to Snail1, Zeb1 and 2 repress E-cadherin and are required for EMT [26]. Besides Snail1 and Zeb1/2, other EMT-TFs also promoting migration are the basic helix–loop–helix proteins Twist1 and 2 [27]. Snail1 and EMT-TFs induce all the molecular events required for cell migration such as a high protease secretion and a stimulation of Rho-GTPase and Akt activity, a proteinkinasealsorequiredforcellinvasion[4].

Signaling pathways controlling EMT Different signaling pathways induce EMT being the most studied that are triggered by TGF-β [28]. TGF-β signals through serine/threonine kinase receptors; it binds to the TGF-β receptor type II (TβRII) that associates with the typeI(TβRI), phosphorylating its Ser/Thr kinase domain; this propagates the signal through the recruitment and phosphorylation of the Smad proteins [29]. TGF-β activates Snai1 transcription through Smad–HMGA2 complex that binds to the Snai1 promoter [30]. TGF-β pathway also cross-talks with other signaling pathways, such as Wnt and Notch. Canonical Wnt ligands interact with the transmembrane proteins LRP5/6 and Frizzled promoting the recruitment of the adenomatous polyposis coli (APC)/Axin/Glycogen-synthase kinase 3 complex to the membrane

and preventing β-catenin degradation; consequently, β-catenintranslocatestothenucleusandinducestheexpression of target genes [31]. On the other hand, the Notch pathway is modulated via the nuclear translocation of the Notch receptor Intracellular Domain, which promotes the transcription of target genes [32]. Besides these pathways, others promoting cell migration and EMT are those associated with tyrosine kinase receptors, including the family members epidermalgrowthfactor(EGF)andfibroblastgrowthfactor(FGF)[5],theJak-STAT3pathwaypromotedbycytokines such as interleukin 6 (IL-6) [33] and hypoxia [34].

Controlling the stability of EMT-TFs: ubiquitination and deubiquitination EMT-TFs are very unstable proteins, rapidly degraded in epithelial cells [35,36]. Proteolysis is dependent on ubiquitination, a covalent reversible post-translational modification (PTM) consequence of the action of three en- Downloaded from https://portlandpress.com/essaysbiochem/article-pdf/63/5/579/859061/ebc-2019-0022c.pdf by guest on 05 November 2019 zymes working sequentially: E1 (ubiquitin activating enzyme); E2 (ubiquitin conjugating enzyme) and E3 (ubiquitin protein-ligase) (Figure 1). E3 ubiquitin ligases bind to E2–conjugating enzyme and transfer ubiquitin to a specific lysine of the substrate (monoubiquitination) in a process that can be repeated leading to the elongation of the ubiquitin chain (polyubiquitination) (Figure 1A). In addition, multimonoubiquitination of substrates is also possible (Figure 1B). Polyubiquitination can use ubiquitin N-terminal Methione (M1), which constitute linear elongation [37] or the seven internal lysine residues (K6, K11, K27, K29, K33, K48 and K63) (Figure 1B). The type of chain elongation determines the consequence of substrate ubiquitination: K48 ubiquitination, either homotypic or in heterotypical chains combined with K11 and K29, promotes the proteasomal degradation of substrates (Figure 1B) [38,39]. M1 or K63-mediated polyubiquitin chains are non-degradative and are used as platform for protein–protein interactions [40]. In addition, heterotypic elongation of ubiquitin chains can either be non-degradative, M1/K63-linked chains [41] or degradative, K63/K11-linked combined with K48 (Figure 1B) [42]. Finally, ubiquitin can be conjugated in mixed polymers with ubiquitin-like modifiers such as SUMO giving rise to hybrid chains (Figure 1B) [39]. Specific E3 ligases modulate proteasomal degradation of EMT-TFs. The EMT factors Snail, Twist and Zeb are constantly ubiquitinated and degraded by E3 ligases such β-TrCP1, FBXL14, FBXL5, FBXO11 and Mdm2 [43–48]; therefore, these enzymes act as negative modulators of EMT and cell migration. In contrast, the K63-ubiquitination of Snail1 and 2 by the E3 ligase Pellino1 increases the stability of these factors and boosts migration [49]. In summary, EMT-TF polyubiquitination controls EMT, preventing the acquisition of migratory properties by cells. Ubiquitination is reversed by the action of a family of proteases known as deubiquitinating enzymes (DUBs) pro- ducing not only the stabilization of proteins but non-degradative effects affecting protein localization, signal transduction and many others (Figure 1B) [50,51]. The 100 identified DUBs are divided into two big families: cysteine proteases and Zn-metalloproteases. The cysteine-proteases family is constituted by six subgroups: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), Machado–Joseph Dis- ease (MJD) Proteases, the Monocyte Chemotactic Protein-induced Protein (MCPIP) and the Motif-Interacting with Ubiquitin-containing novel DUB (MINDY). On the other hand, the Zn-metalloprotease family is composed by the JAB1/MPN/MOV34 (JAMM/MPN+) metalloenzymes [50,51]. Members of these families have been described to regulate EMT and migration as we will review below.

DUBs involved in the control of EMT and cell migration UCHs UCH enzymes (UCHL1, UCHL3, UCHL5/UCH37 and the BRCA1-associated protein 1, BAP1) play crucial roles in tumor invasion and migration [52]. There is a clear association between these enzymes and EMT in several types of cancer, mostly reversing transformation, except UCHL1 [53–55]. According to the inhibitory action in EMT, loss of BAP1 is related with poor tumor outcome [56] and the positive expression of UCHL5 with increased survival [57]; in contrast, UCHL1 expression is positively implicated in cell migration and metastasis [58,59]. The mechanism for UCHL1-regulated tumor-cell invasion requires Akt activation [60–62]. Furthermore, β-catenin/TCF pathway activation is required for the stimulation by UCHL1 of cell migration [63]. Unfortunately, specific substrates directly targeted by UCHL1 and related to cell migration have not been characterized yet.

MJD DUBs MJD DUBs contain the catalytic ‘Josephin’ domain. The paradigm is Ataxin3, a protein with a poly-glutamine expansion stretch which causes the neurodegenerative MJD [64]. Other members are the Josephin domain-containing (JosD) proteins (JosD1, JosD2). JosD1 is activated by monoubiquitination and targeted to the cell membrane where it

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Figure 1. The ubiquitin pathway (A) Representation of the process of ubiquitination. Three enzymes acting in cascade are required to accomplish substrate ubiquitination: E1 (E1-activating enzyme), E2 (E2-conjugating enzyme), E3 (E3-ligase). (B) Illustration of the different ubiquitination patterns with their associated roles and their counteraction by DUBs.

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Figure 2. Signaling pathways activating DUBs related to EMT-TFs stabilization The diagram shows DUBs deubiquitinating Snail1, Snail2 (Slug), Twist and Zeb1. Known specific transduction pathways are indicated: Hypoxia, that activates USP47 to mediate Snail1 stabilization; TGF-β, USP27X, also related to Snail1; and IL-6, DUB3 that affects Snail1, Snail2 and Twist stability. Also shown other DUBs which the specific activation pathway is unknown (USP11, USP26, OTUB1 and PSDM14). In all cases stabilization of EMT-TFs induces EMT. regulates membrane dynamics, cell motility and endocytosis [65]. The cytoplasmic JosD2 also increases cell proliferation and migration and is related with poor cancer prognosis; JosD2 stabilizes the phosphoglycerate dehydrogenase enzyme which deviates glucose-derived carbons to feed serine synthesis [66].

The OTU proteases The OTU Protease family of DUB contains the OTU domains and comprises Otubains, A-20 like OTUs and OTU proteins [50]. Otubains (OTUB1, OTUB2 and YOD1) are implicated in cancer metastasis; in particular, OTUB1 expression correlates with metastasis and EMT in colorectal cancer [67] and enhances metastasis formation promoting Snail1 deubiquitination and stabilization [68] (Figure 2 and Table 1). The role of OTUB1 in mediating cell migration is particularly crucial after TGFβ induction because, besides Snail1, it also counteracts the ubiquitination and degradation of phospho-SMAD2/3 [69] (Figure 3). The mechanism mediating Smad2/3 stability is highly unusual because OTUB1 acts by blocking the transfer of ubiquitin to the E3 by directly binding to several E2s independently of its catalytic activity [69]. OTUB1-mediated E2 inhibition is a general mechanism affecting other substrates implicated in DNA damage including p53 and RNF168 [70–72]. OTUB2 was also identified by an siRNA inhibition screening of DUBs controlling breast cancer metastasis [73]. The main OTUB2 substrates are YAP/TAZ proteins; in particular, YAP is required for Cancer-associated-Fibroblast (CAF)-driven matrix remodeling and invasion [74]. OTUB2 function requires the previous poly-sumoylation dependent on EGF and K-Ras signaling [73]. YAP/TAZ is also regulated by YOD1 although the mechanism is quite different: YOD1 deubiquitinates and stabilizes ITCH, the E3 ligase of LATS1/2 [75]. As consequence of YOD1 action, LATS1/2 is degraded and YAP/TAZ activates its downstream targets [75]. Another OTU DUB, A20, also called tumor necrosis factor α-induced protein 3 (TNFAIP3), has an important role in inflammatory responses as a potent inhibitor of NFκB signaling [76]. Curiously, A20 has a dual function as an

Table 1 DUBs involved in EMT-TFs stabilization

Overexpression in In vivo phenotype Target DUBs Regulation tumors after depletion Inhibitor Reference

Snail1 DUB3 IL-6 Breast Experimental (tail WP1130 [102,103] vein)/spontaneous PD0332991* metastasis inhibition USP27X TGF-β Breast Experimental (tail vein) - lung metastasis inhibition USP11 Constitutive Ovarian ND - [109] USP26 Constitutive Esophageal squamous Experimental (tail vein) - [110] cell carcinoma lung metastasis

inhibition Downloaded from https://portlandpress.com/essaysbiochem/article-pdf/63/5/579/859061/ebc-2019-0022c.pdf by guest on 05 November 2019 USP47 Hypoxia Colorectal Xenograft tumor - [111] growth inhibition PSMD14 Constitutive Esophageal squamous Experimental (tail vein) - [101] cell carcinoma lung metastasis inhibition OTUB1 Constitutive Esophageal squamous Experimental (tail vein) -[68] cell carcinoma lung metastasis inhibition Snail2 DUB3 IL-6 Breast Experimental (tail WP1130 [116] vein)/spontaneous PD0332991 metastasis inhibition USP5 Constitutive Hepatocellular Xenograft tumor Formononetin [114] growth inhibition USP10 Constitutive NSCLC ND - [115] Twist1 DUB3 IL-6 Breast Experimental (tail WP1130 [102] vein)/spontaneous PD0332991 metastasis inhibition Zeb1 USP51 Constitutive Breast ND - [117]

Abbreviation: ND, not done. The table provides the DUB expression in tumors, the existence of in vivo experiments demonstrating a functional role in tumors, and the inhibitors characterized. *PD0332991 is an inhibitor of the CDK4/6 kinase, an enzyme which phosphorylates and activates DUB3 [103].

OTU DUB or as ubiquitin ligase. As a DUB, A20 removes K63-linked ubiquitin chains from RIP (receptor interacting protein) abolishing the canonical activation of NFκB initiated by TNFα. RIP1 deubiquitination by A20 terminates the signal disassembling the complex; then A20 works as a ubiquitin ligase of RIP1, triggering its K48-polyubiquitinated and proteasomal degradation. Apart from its role in inflammation, A20 is implicated in EMT in basal-like breast cancers by its action multi-monoubiquitinating and stabilizing Snail1, thereby enhancing migration and invasion of tumor cells [77]. Other OTU proteins are OTUD1, OTUD5 (also known as DUBA), OTUD7B and 7A (also known as CEZANNE1 and CEZANNE2, respectively), TRABID (TRAF-binding domain, also known as KEAP) and VCPIP1 (Valosin-containing p97/47 complex-interacting protein p135). Some of these proteins are implicated in cell migration, such as OTUD1, which stabilizes the TGF-β inhibitor Smad7 promoting K48-deubiquitination; therefore, sup- pressing TGF-β signaling, EMT and metastasis [78] (Figure 3). Curiously, OTUD1 also cleaves the atypical K33-chain on Smad7 to expose a phospho-tyrosine binding motif for the SMURF2 E3-ligase. This SMURF2–Smad7 complex binds to TβRI and facilities receptor degradation to further block TGF-β signaling [78] (see below). OTUD7B/CEZANNE1 is up-regulated in invasive and metastatic triple-negative breast cancer (TNBC) [79]. This DUB suppresses the NFκB pathway promoting K63-deubiquitination of TRAF3 [80] and other substrates [81]. Cu- riously, OTUD7B/CEZANNE1 and OTUD7A/CEZANNE2 are the only reported DUBs specifically targeting the atypical K11-ubiquitin chains [82]. Heterotypic chains of K48 and K11 are commonly found in mitotic and misfolded proteins and constitute a signal for their rapid degradation [83,84] (Figure 1). OTUD7B/CEZANNE2 is transcrip- tionally repressed by Snail1 in hepatocellular carcinoma; as a consequence, Snail1 impairs TRAF6 deubiquitination andtriggersaprolongedactivationofNFkBinvolvedintumorgrowth,cellinvasionandmetastasis[85].Curiously, CEZANNE2 depletion also causes Snail1 down-regulation both decreasing its transcription and stability [86] suggesting the existence of an inhibitory feedback loop involving these two proteins.

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Figure 3. DUBs regulate TGF-β signaling pathway Upon TGF-β binding, TβRI is activated by TβRII and phosphorylates Smad2/3 (A). Phosphorylated Smad2/3 associates with Smad4 (B); levels of Smad2/3 and Smad 4 are controlled by DUBs OTUB1 and USP9X, respectively. Smad complex translocates to the nucleus where through the interaction of other specific transcription factors, participates in the activationβ ofTGF- target genes (C). In this compartment USP15 deubiquitinates and stabilizes Smad2/3. The TGF-β target gene Smad7 acts as in inhibitor of the system. Besides preventing Smad2/3 phosphorylation (A), it promotes Smurf ubiquitination and degradation of TβRI (D,E)switching-off the pathway. Deubiquitination of TβRI by UCH37, USP11, USP15 and USP4 is indicated (D,E). Under low concentrations of TGF-β the negative action by Smurf is reversed by DUBs (D), but not after higher stimulations (E). Removal of monoubiquitin from Smad2/and Smad 4 (B) by the corresponding DUBs is also indicated. Smurf deubiquitination by USP9X negatively affects the pathway (F). K63-deubiquitination of Smad7 by CYLD is also indicated (G). (H) OTUD1-mediated Smad7 deubiquitination preventing its degradation. All Dubs and reactions negatively affecting the pathway are indicated in red; those positively increasing signaling are shown in blue. Notice that USP9X is depicted in red or blue, depending on the catalyzed reaction. Similarly, Smad7 stabilization by OTUD1 is shown in red, but deubiquitination by CYLD is shown in blue.

TRABID, also known as ZRANB1, removes the K63-associated polyubiquitin chains from APC to enhance Wnt-dependent transcription [87]. Curiously, it also promotes Twist K63-deubiquitination, enhancing its association with β-TrCP1 and facilitating its K48-ubiquitination and degradation [88]. TRABID is activated by Akt1 phosphorylation, a kinase that also phosphorylates Twist1 to enhance its β-TrCP1-mediated degradation; as a consequence, TRABID and Akt1 cooperate in Twist degradation [88]. Finally, the last member of this OTU subfamily, VCPIP1, controls the reassembly of the Golgi complex during cell cycle progression [89]. Although VCPIP1 has not been reported to be involved in cell migration, its inhibition indirectly decreases Snail1 stability [86].

The JAMM class of DUBs JAMM metalloenzymes are K63-linked specific DUBs [90–93]. Among them, there are epigenetic regulators such as MYSM1 and MPND DUBs that, together with USP16, remove the monoubiquitin repressive mark from histone H2A (H2A-K119Ub) activating transcription during hematopoiesis [94–98]. PSMD7 and PSMD14 (the 26S proteasome non-ATPase regulatory subunits 7 and 14) form part of the 19S proteasome regulatory particle lid; their isopepti- dase activity is fundamental for the removal of polyubiquitin chains prior degradation of incoming substrates [99].

PSMD14 (also known as Rpn11/POH1) is a K63-linked DUB that despite its general role in maintaining protein homeostasis may specifically deubiquitinate and stabilize some substrates such as E2F1 promoting tumor formation in liver cancer [100]. In fact, PSMD14 has also been suggested to target Snail1 to enhance migration and metastasis in esophageal squamous cell carcinoma [101] (Table 1 and Figure 2).

USP class The USP family is the largest group of DUBs and the most involved in cell migration/invasion by directly or indirectly targeting EMT-TFs and also many oncoproteins.

USP DUBs directly regulating EMT-TFs

As explained before, Snail1 and other EMT-TFs are highly unstable proteins [35,36]. Several Snail1 DUBs activated by Downloaded from https://portlandpress.com/essaysbiochem/article-pdf/63/5/579/859061/ebc-2019-0022c.pdf by guest on 05 November 2019 different stimuli stabilize Snail1 in cancer cells: DUB3 (USP17, USP17L2) targets Snail1 after IL-6 treatment [102,103] whereas USP27X does it after TGF-β stimulation [86] (Figure 2). Inhibition of both DUB3 and USP27X strongly decrease Snail1 metastatic potential of breast cancer cells (Table 1). DUB3 activation may require phosphorylation by CDK4/6 as inhibitors of this kinase block metastasis by down-regulating Snail1 [103] (Table 1). Directly related with USP27X, the highly homologous USP22 removes monoubiquitin from histone H2B as a part of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex [104,105]. Interestingly, USP22 expression induces EMT in many different cancer models [106–108] and might stabilize Snail1 [86]. Other DUBs have been suggested to promote Snail1 stability: USP11, USP26 and USP47, apart from the previously mentioned PSMD14 and OTUB1 [68,101,109–111] (Figure 2 and Table 1). No specific regulation by any extracellular signal has been reported for these DUBs, except USP47, which is induced by hypoxia [111] (Figure 2). This high number of Snail1 DUBs ensures a tight control of protein destruction and is common to other labile proteins such as c-Myc (regulated by USP22, USP28, USP36 and USP37) [112,113]. Regarding Snail2, affinity purification of this transcription factor has identified USP5 as a strong stabilizer promoting EMT, invasion and metastasis [114] (Figure 2). A siRNA screening also detected USP10 as a DUB for Snail2 [115] (Figure 2). Besides acting on Snail1, DUB3 also stabilizes Snail2 and Twist1, suggesting a general role of DUB3 in controlling EMT [116]. Much less information is available on DUBs acting on other EMT-TFs; only USP51 targets ZEB1 in mesenchymal-like breast cancer cells [117] (Figure2andTable1).

USP DUBs regulating EMT transduction pathways Some DUBs indirectly regulate cancer cell migration and EMT by targeting key proteins of Wnt/β-catenin, hypoxia and TGF-β transduction pathways. For example, USP7 stimulate EMT through the activation of the Wnt/β-catenin signaling pathway [118]. USP4 and USP20 also deubiquitinate/stabilize β-catenin inducing proliferation, invasion, migration and chemo-resistance of cancer cells [119,120]. Additionally, USP7 targets HIF-1α increasing its stability and inducing EMT and metastasis. Interestingly, hypoxia activates USP7 by inducing its K63-linked polyubiquitination by the HECTH9 E3 ligase [121]. As mentioned before, TGF-β is the main cytokine stimulating EMT and several DUBs control this pathway [122,123]. TGF-β promotes TβRI and TβRII receptor complex heterodimerization and TβRI phosphorylation and activation (Figure 3A, TGF-β ON). Active TβRI phosphorylates receptor-regulated (R-Smads) Smad2 and Smad3 effectors to form complexes with the common mediator (Co)-Smad4 (Figure 3B) which translocate to the nucleus to transcribe several target genes involved in EMT, including Snail1 or its DUB USP27X (Figure 3C) [86]. Moreover, Smads also collaborate with Snail1 and Zeb proteins to repress epithelial genes such as CDH1 [124]. A TGF-β target is Smad7 which negatively regulates the pathway in several ways: (1) interfering with Smad2/3 binding to active TβRI (Figure 3A); (2) recruiting the E3 ubiquitin ligase Smurf1/2 to the TβRI to promote its ubiquitination and degradation(Figure3D),switching-offthepathway.SomeDUBssuchasUSP11(UHX1),USP15andUSP4reverses TβRI ubiquitination. Upon TGF-β stimulation, USP11 and 15 interact with the Smad7-Smurf complex counteracting TβRI ubiquitination at low TGFβ concentrations (Figure 3D, TGF-β OFF/ON), resulting in TGFβ-induced gene transcription and EMT [125–129]. However, at higher TGF-β concentrations, Smurf-dependent TβRI degradation overcomes DUB stabilization (Figure 3E, TGF-β OFF). Curiously, as indicated previously, USP11 also stabilizes Snail1 in ovarian cancer cells, suggesting a dual control of EMT [109]. Another DUB, USP4, requires previous phosphorylation by Akt to be mobilized from the nucleus to the membrane; USP4 directly targets TβRI independently on Smad7 [130] (Figure 3D). Besides acting on TβRI, USP15 deubiquitinates R-Smads, removing monoubiquitin from their DNA-binding domains to favor target promoter recognition [131] (Figure 3C). In addition, USP9X enhances TGF-β activity reversing Smad4 monoubiquitination at K519, which interferes with phospho-Smad2 binding

(Figure 3B) [132]. In contrast with the positive effect, USP9X also interferes with the TGF-β pathway by deubiquitinating Smurf to prevent self-degradation [133] (Figure 3F). Smad7 is also regulated by DUBs such as the K63 DUB CYLD that targets Smad7-K360 and K374 residues required for the activation of TAK1 and p38 kinases (Figure 3G) [134]. As indicated before, OTUD1 also acts on TGF-β signaling directly stabilizing Smad7 by K48-deubiquitination and enhancing TβRI turnover, causing suppression of EMT [78] (Figure 3H). Finally, the long-term activation of mesenchymal markers by TGF-β requires the further participation of other proteins, such as the Gli-1 transcription factor [135] which is also stabilized by DUBs. For example, USP37 targets Gli-1 to enhance cell invasion and EMT in breast cancer cells [136].

USP DUBs directly regulating oncoproteins and tumor suppressors Beyond the role of USP11 and USP15 in TGF-β-stimulatedEMT,theseproteinsactonrelevantoncoproteinsand tumor suppressors. For example, USP11 apart from stabilizing IκBα and inhibiting TNFα-mediated NFκBac- Downloaded from https://portlandpress.com/essaysbiochem/article-pdf/63/5/579/859061/ebc-2019-0022c.pdf by guest on 05 November 2019 tivation [137], also targets some tumor suppressors such the promyelocytic leukemia (PML) protein [138], the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and PTEN [139]. As an activator of tumor suppressors, USP11 expression is commonly reduced in tumors with PML or PTEN destabilization [138,139]. USP15 targets MDM2 [140] and the MDM2 substrate p53 [141]. USP14, together with PSMD14/Rpn11 and UCH37, is one of the three proteasome-associated DUBs. Surprisingly, despite its general role in protein homeostasis, its effects on protein turnover seems to be protein specific as shown for cyclin B [142]. USP14 stabilizes the EMT-marker vimentin in gastric cancer cells [143] and also promotes chemotaxis by stabilizing the chemokine receptor CXCR4 [144]. USP14 expression correlates with bad prognosis in non-small cell lung cancer [145]. USP25 overexpression also causes EMT, enhancing migration/invasion and metastasis [146,147]. USP25 was identified as a SUMO2/3-interacting protein [148] and regulates inflammatory TRAF signaling by removing K63-linked ubiquitination in TRAF5/6 after IL-17 stimulation [146]. USP25 also deubiquitinates and stabilizes tankyrase after Wnt-stimulation; tankyrase mediates the polyADP-ribosylation of Axin, required for its ubiquitination and degradation and the consequent stabilization of β-catenin [146,149]. Axin degradation is also counteracted by USP34 [150]; as a consequence, USP34 inhibition induces EMT [151]. USP25 activity requires its dimerization [152]. By a similar mechanism to USP25, USP28 forms active dimers, which are very efficient in deubiquitinating c-Myc [153,154]. As a c-Myc stabilizer, USP28 is a strong tumor promoter [155] and also stabilizes additional oncogenic factors such as c-Jun and Notch1 [156,157].

USP DUBs regulating other proteins involved in cell migration Apart from the above-mentioned effect of USP14 stabilizing vimentin [143], additional DUBs target other proteins related with cell motility. For example, USP10 acts on integrins β1/β5, up-regulated at the cell surface during wound healing resulting in an activation of TGF-β signaling [158]. USP33 deubiquitinates and stabilizes Robo1, which is required for Slit2-dependent inhibition of cancer cell migration and EMT. USP33 expression is down-regulated in colon and gastric cancer [159,160]. USP45 was found by mass spectrometry to target spindly, a protein forming part of dynein/dynactin complex formed at the leading edge of cells undergoing migration [161].

Inhibitors of DUBs and future studies Several small-molecule inhibitors have been developed targeting all the steps of the ubiquitination cascade [162]. DUBs are potentially ‘druggable’ and pharmacological studies to develop DUB inhibitors are a promising strategy in the treatment of cancer [163,164]. For example, USP7 inhibitors such as HBX 41,108 [165] and its derivative HBX 19,818 [166] lead to p53 stabilization and the inhibition of cancer growth. Formononetin binds and inhibits USP5 decreasing Snail2 stability and consequently EMT and malignant progression [114] (Table 1). A biochemical screen of Federal Drug Administration-approved compounds for their antagonism on USP11 identified mitoxantrone as a potent inhibitor of pancreatic cancer cell survival [167]. Another example is ML323; this compound impairs the formation of the USP1–UAF1 complex, which is essential for USP1 activity. USP1 is a regulator of FANCD2, a key modulator of DNA repair response and USP1 inhibition has emerged as a promising anticancer therapy [51]. Proteasome-associated DUBs are also an attractive target of inhibition [168]. Anticancer activity has been reported for the USP14 inhibitors IU1 and IU1-147 [169,170], the metal-based inhibitors copper (II) pyrithione CuPT and gold (I) complex auranofin and the UCHL5 and USP14 small antagonist β-AP15 [168,171–173]. However, despite theadvancesinDUB-targetingdrugs,theidentifiedcompoundsarestillverypromiscuousandmostofthemare ‘multi-DUB’ inhibitors, as WP1130 that acts on USP5, USP9X, USP14, UCH37, UCHL1 and, recently on DUB3 [102,174] (Table 1). To date, no compounds targeting USP27X (and therefore Snail1) or USP28 (c-Myc) have been

reported. Currently, auranofin is the unique DUB inhibitor in clinical trials and its anticancer properties are enhanced when synergistically combined with disulfiram [175]. Actually, auranofin is studied for treatment of recurrent or ad- vanced lung cancer and chronic lymphocytic leukemia; in vitro it also overcomes resistance to the tyrosine kinase inhibitor imatinib mesylate, which is the current treatment for chronic myelocytic leukemia expressing the fusion oncoprotein Bcr-Abl [176]. Considering the wide involvement of DUBs in EMT, invasion and metastasis, the characterization of new drugs targeting specific DUBs offer new opportunities for development of novel antitumor drugs.

Summary • EMT provides tumor cells with higher migratory and invasive capabilities. Downloaded from https://portlandpress.com/essaysbiochem/article-pdf/63/5/579/859061/ebc-2019-0022c.pdf by guest on 05 November 2019 • EMT is controlled by extracellular factors, such as TGF-β that stimulate the expression of EMT-TFs, such as Snail1.

• EMT-TFs are very unstable proteins controlled by the coordinated action of E3 ubiquitin ligases and a specific type of proteases, the DUBs.

• The characterization of DUBs acting on EMT-TFs or the pathways activating them opens the possi- bility to the design of specific protease inhibitors that block EMT and act as antineoplastic agents.

Funding This work in A. Garc´ıa de Herreros’ laboratory was supported by the Ministerio de Econom´ıa y Competitividad (MINECO) co-funded by Fondo Europeo de Desarrollo Regional-FEDER-UE [grant number SAF2016-76461-R].

Competing Interests The authors declare that there are no competing interests associated with the manuscript.

Abbreviations APC, adenomatous polyposis coli; α-SMA, alpha smooth muscle actin; BAP1, BRCA1-associated protein 1; CAF, cancer-associated fibroblast; DUB, deubiquitinase; ECM, extracellular matrix; EGF, epidermal growth factor; EMT, epithelial-to-mesenchymal transition; EMT-TF, transcription factor modulating EMT; FGF, fibroblast growth factor; IL-6, interleukin 6; JAMM, JAB1/MPN/MOV34; JosD, Josephin domain-containing protein; MJD, Machado–Joseph disease; OTU, ovarian tumor protease; PML, promyelocytic leukemia; PTM, post-translational modification; RIP, receptor interacting protein; R-Smads, receptor-regulated Smads; SAGA, Spt-Ada-Gcn5 acetyltransferase; TNBC, triple negative breast cancer; TRABID, TRAF-binding domain; TβRI, TGF-β receptor type I; TβRII, TGF-β receptor type II; UCH, ubiquitin C-terminal hydrolase; USP, ubiquitin-specific protease; VCPIP1, Valosin-containing p97/47 complex-interacting protein p135.

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