key: cord-0894655-9erzows3
authors: Wang, Xiaofang; Liu, Zhiyi; Zhang, Li; Yang, Zhaozhi; Chen, Xingxing; Luo, Jurui; Zhou, Zhirui; Mei, Xin; Yu, Xiaoli; Shao, Zhimin; Feng, Yan; Fu, Shen; Zhang, Zhen; Wei, Dongping; Jia, Lijun; Ma, Jinli; Guo, Xiaomao
title: Targeting deubiquitinase USP28 for cancer therapy
date: 2018-02-07
journal: Cell Death Dis
DOI: 10.1038/s41419-017-0208-z
sha: ecf2675fbb7883ca4a8f98c34eadb55c6b857aaa
doc_id: 894655
cord_uid: 9erzows3

As one of the most important post-translational modifications, ubiquitination plays versatile roles in cancer-related pathways, and is involved in protein metabolism, cell-cycle progression, apoptosis, and transcription. Counteracting the activities of the E3 ligases, the deubiquitylating enzymes have been suggested as another important mechanism to modulate the ubiquitination process, and are implicated in cancer as well. In this article, we review the emerging roles of USP28 in cancer pathways as revealed by recent studies. We discuss the major mechanisms by which USP28 is involved in the cancer-related pathways, whereby USP28 regulates physiological homeostasis of ubiquitination process, DNA-damage response, and cell cycle during genotoxic stress. We further review the studies where USP28 was targeted for treating multiples cancers including non-small cell lung cancer, breast cancer, intestinal cancers, gliomas, and bladder cancer. As a result, the clinical significance of targeting USP28 for cancer therapy merits further exploration and demonstration.

• As one of the most prevalent and important posttranslational modifications, ubiquitination is involved in multiple cancer-related pathways, including cell-cycle progression, apoptosis, receptor downregulation, and gene transcription.

• Antagonizing the activities of E3 ubiquitin ligases, the deubiquitylating enzymes (DUBs) are engaged in cancer-related pathways by modulating the ubiquitination process. Open questions • Owing to the versatility of USP28-mediated regulation in the cell, what is the dominant mechanism imposed by USP28 for a given cancer type?

Like phosphorylation, ubiquitination is one of the most prevalent and important post-translational modifications found in the cell, and has multi-faceted roles in normal and pathological cellular processes. In this type of modification, a ubiquitin is attached to the target protein through a covalent isopeptidic bond between the Cterminus of the ubiquitin and a lysine residue of the target protein. Implications of ubiquitination in cancer-related pathways have been unveiled in recent years, and they are mostly related to cell-cycle progression, apoptosis, receptor downregulation, and gene transcription. Three types of enzymes function sequentially to add the ubiquitin to the substrate: the ubiquitin-activating enzyme, E1, catalyzes the formation of a thioester bond on ubiquitin. The E2 ubiquitin-conjugating enzyme carries the ubiquitin. Finally, the E3 ligases attach the ubiquitin from E2 to the substrates (Fig. 1) . Ubiquitination was first reported as a mechanism to mark proteins for proteasome-mediated degradation. Later, more functions of ubiquitination have been revealed, spanning from modulating protein trafficking to regulating activities of enzymes 1 .

It has been appreciated that the E3 ubiquitin ligases are central to the ubiquitin-conjugation system since they directly interact with substrates and determine the substrate specificity. Some E3 ligases have been implicated in cancer development because they may dictate degradation of oncoproteins or tumor suppressors. For example, agonists of FBW7 can promote degradation of c-MYC and cyclin E in the tumor cells 2 .

Interestingly, acting reversely by removing the tagged ubiquitin from the substrate protein, deubiquitylating enzymes (DUBs) are implicated as an important mechanism to modulate the ubiquitination process as well, and have been related to cancer, too. There are about 100 DUBs in human, which can be categorized into five families, including ubiquitin-specific proteases (USP), ubiquitin carboxy-terminal hydrolases (UCH), ovarian tumor-like proteases (OTU), metalloproteases, and Machado-Jakob-disease proteases. Except for the DUBs of the JAMM/MPN family, which are metalloproteases, all the other DUBs are cysteine proteases (Fig. 2) 3 . Much knowledge has been accumulated for the functions of DUBs, which are not just limited to reversing ubiquitination of the proteins tagged for proteasomal degradation. It is now known that the functions of DUBs are multidimensional, and encompass apoptosis, protein trafficking, cell cycle regulation, DNA damage repair, chromatin remodeling, and modulating signaling pathways 4,5 . Aside Fig. 1 The ubiquitination process. The ubiquitination process is catalyzed by three types of enzymes that function sequentially. The ubiquitinactivating enzyme, E1, promotes the formation of the thioester bond between the C-terminal carboxyl group of ubiquitin and the E1 cysteine sulfhydryl group. This step is dependent on ATP. In the second step, ubiquitin is transferred from E1 to the active site of the conjugating enzyme, E2. In the last step, the E3 ubiquitin ligase catalyzes the attachment of ubiquitin to the substrate through an isopeptide bond between a lysine of the target protein and the glycine of ubiquitin. The E3 enzymes recognize and discriminate various substrates in the cell, thus determining the substrate specificity in a given ubiquitination process. An E3 enzyme typically carries either one of the two domain structures: the homologous to the E6-AP carboxyl terminus (HECT) domain or the really interesting new gene (RING) domain. Depending on the domain it carries, the catalytic mechanisms may differ. The catalytic domain structures of the five subfamilies of deubiquitinases vary significantly. USP, UCH, and MJD catalytic domains share conserved residues around the catalytic core, while OTU domain lacks some of them, and these conserved residues are completely missed in the JAMM domain. (Carbon, gray; nitrogen, blue; oxygen, red; sulfur, orange; zinc, purple. Blue ribbon structures represent the complexed ubiquitin. Reproduced with permission from reference 3 .) from acting on the substrates targeted for degradation, DUBs can also modulate the activities of proteins, therefore affecting the activities of signaling pathways. For instance, in the nuclear factor-κB (NF-κB) pathway, the cylindromatosis-associated DUB, CYLD, negatively regulates NF-κB activation by antagonizing the activity of the E3 ligase tumor necrosis factor receptor-associated factor 2 [6] [7] [8] . The roles of various DUBs in cancer-associated pathways have been reviewed in 5 . In this review, we focus on discussing the specific roles of USP28 in cancer pathways, and their potential therapeutic values in cancer treatment.

The roles of USP28 in cancer-related pathways USP28 was initially identified through homology search for USP25. Like USP25, the USP28 gene contains alternatively spliced exons which can produce tissue-specific isoforms of the enzymes 9 . Crystal structures of several USP family enzymes have been resolved, including USP2, USP7, USP8, USP14, and USP21 [10] [11] [12] [13] [14] [15] . These structural studies suggest that USPs possessing a homologous catalytic site are conferred with a similar ubiquitinrecognition mechanism. Therefore, it is likely that all USP family DUBs may share the same mechanism to recognize ubiquitin. USP28 is highly homologous to USP25, both of which contain ubiquitin-associated domain and ubiquitin-interacting motifs in the Nterminal region 5 . The emerging roles of USP28 in cancer pathways have been revealed by some recent studies. Below, we will review the mechanisms accounting for the involvement of USP28 in these pathways (Fig 3, Table 1 ), hence demonstrating its significance as a therapeutic target for cancer treatment.

Disrupting physiological homeostasis of ubiquitination process USP28 was the first FBW7-antagonizing DUB in an shRNA screen using c-MYC stability as a readout in human tumor cells 16 . USP28 can counteract the activity of FBW7 and promote c-MYC stability in cancer cells. Overexpression of USP28 inhibited FBW7-depedent ubiquitination of c-MYC and cyclin E1, both of which are oncoproteins. Depletion of USP28 in some cancer cell lines phenocopies the effect of MYC depletion. As a result, USP28 is required for c-MYC-mediated tumor cell proliferation. FBW7 is an F-box protein and an important component of the SKP1-CUL1-F-BOX (SCF)-type E3 ubiquitin ligase, and determines the substrate specificity of the SCF complex. FBW7 is considered as a tumor suppressor that controls cell proliferation, differentiation, and apoptosis by targeting key transcriptional factors to ubiquitin-directed proteasome degradation 17 . Its functions can be counteracted by DUBs, where Lys48-linked ubiquitin chain can be removed by DUBs before the proteolysis of the tagged protein. The functions of DUBs may cover a wide range of physiological processes in the cells; but the most prominent scenario can be exemplified by the promotion of oncoprotein stability. Pathological dysregulation of the deubiquitination process would lead to deleterious effects, causing the cancer-driving oncoproteins to be accumulated.

In addition to stabilizing the FBW7-substrates, USP28 was also found to directly stabilize FBW7 itself, which leads to reduction of FBW7 substrate proteins. As a result, in contrast to the oncogenic effect of overexpressing USP28 in colorectal cancer 18 , inhibition or deletion of USP28 would otherwise have tumor-promoting effect by destabilizing FBW7 19 . This raises an interesting question regarding the dual functions of USP28: whether it is oncogenic or tumor-suppressive? An accountable explanation is that, the levels of USP28 vary considerably in different cells, which can influence the stability and regulation of FBW7 in these cells. As a result, the dual regulation of USP28 on FBW7 may serve as a mechanism to maintain the physiological levels of proto-oncogenic FBW7 substrates. In this model, USP28 may serve as either a tumor promotor or suppressor depending on the status of autocatalytic ubiquitination of FBW7. In the intestine, USP28 deletion does not affect FBW7 stability and attenuates tumorigenesis, while in tissues where FBW7 autocatalytic turnover is favored, deletion of USP28 resembles FBW7 loss-of-function, thus exhibiting a tumor-promoting effect 20 . It is also worth noting that, in addition to the aforementioned oncogenic proteins, other substrates that are known not be associated with FBW7, are also subject to deubiquitination and stabilization by USP28, including Claspin, CHK2, and LSD1 [21] [22] [23] . This fact adds another layer of complexity regarding the roles of USP28 in cancer-related pathways.

In addition to affecting the turnover of critical oncoproteins like MYC, USP28 may regulate other essential pathways in cancer progression. A study has proposed a novel mechanism by which USP28 upregulates angiogenesis by antagonizing GSK-3β (glycogen synthase kinase-3β) and FBW7-dependent degradation of HIF-1α (hypoxia-inducible factor-1α), a major regulator of angiogenesis, carcinogenesis, and various processes by which cells adapt to hypoxic conditions. HIF-1α is degraded after phosphorylation by GSK-3β and recruitment of FBW7, a process that can be reversed by overexpressing USP28. Importantly, stabilization of HIF-1α by USP28 is dependent on FBW7, and as a result, FBW7 and USP28 reciprocally regulate angiogenesis and metastasis in an HIF-1α-dependent manner 24 . Therefore, modulation of HIF-1 functions by GSK-3/FBW7/USP28 may contribute to a novel adaptive mechanism for the cell to respond to various signals that affect cell proliferation, differentiation, and apoptosis. The finding offers an alternative way to regulate HIF-1-dependent cancer pathways by targeting USP28.

In cancer treatment, radiotherapy complements surgery and chemotherapy. Despite the developments in planning and application of radiotherapy that result in improved cure rate, challenges of radioresistance and radiationinduced damage persist to impair the success of radiotherapy [25] [26] [27] . The major effect of radiotherapy is the ionizing damages caused to the DNA, where base damage, single strand break (SSB), and double strand break (DSB) can occur. The cell has exquisite mechanisms to sense and repair the DNA damages. But if these pathways are disturbed and dysregulated, as observed in the tumor cells, radioresistance can develop and nullify the therapeutic efforts. An early study has shown USP28 plays an important role in the control of the DDR 22 . The DDR signals are sensed and initiated by phosphatidylinositol 3-kinase-like kinases (PIKKs), such as ATM and ATR, transduced by various cellular factors, before activating their downstream checkpoint kinases like CHK1 and CHK2. PIKK specificity factors recruit PIKKs to different forms of DNA damage (i.e., base damage, SSB, and DSB) and regulate their activities. Checkpoint mediators, including 53BP1, serve as scaffolds for PIKK complexes and their substrates. Theses checkpoint mediators are critical in controlling the response to irradiation (IR)induced DDR 28 . USP28 was initially identified as a 53BP1binding partner in a tandem affinity purification experiment. Mechanistically, USP28 deubiquitinates and stabilizes the specificity factors and mediators of ATM and ATR signaling by protecting them from ubiquitinationmediated proteasome degradation. The regulation on these factors and mediators, including 53BP1, Claspin, and MDC1, is dependent on the physical association with USP28. The catalytic domain of USP28 is required for this regulation as well, and a dominant-negative mutant of a Fig. 3 The mechanisms by which USP28 is involved in the cancer-related pathways. (Upper panel) USP28 counteracts the activity of E3 ligases. USP28 antagonizes FBW7, an F-box protein and an important component of the SKP1-CUL1-F-BOX (SCF)-type E3 ubiquitin ligase that targets key transcriptional factors to ubiquitin-directed proteasome degradation. As a result, the stability of some important E3 ligase substrates, such as oncogenic c-MYC, is promoted, leading to enhanced proliferation, carcinogenesis, and metastasis. USP28 also upregulates angiogenesis by antagonizing GSK-3β (glycogen synthase kinase-3β) and FBW7-dependent degradation of HIF-1α (hypoxia-inducible factor-1α), a major regulator of angiogenesis, carcinogenesis, and various processes by which the cell adapts to hypoxic conditions. (Middle panel) USP28 modulates key factors in DNA damage response wherein it forms a complex with PIRH2 and CHK2 and antagonizes PIRH2-mediated poly-ubiquitination and proteasomal degradation of CHK2. Consequently, p53-dependent apoptosis is down-regulated, a process that is favored by the cancer cells. (Lower panel) Genotoxic stress induced by drugs or by ionizing radiation can render the cell to stall before mitosis and repair the resultant DNA damage. USP28 can stabilize Claspin and maintain the G2 arrest, thus promoting the ATR-mediated activation of Chk1. In accordance with its role in checkpoint signaling, Claspin positively regulates DNA replication, and its overexpression can enhance cell proliferation.

catalytic cysteine phenocopies USP28 knockdown cells. Therefore, USP28 may restrain ubiquitination-mediated proteolysis of the components in DDR. Interestingly, in another study, CHK2 is shown to be directly regulated by USP28. The ubiquitin E3 ligase PIRH2 (p53-induced protein with a RING-H2 domain) interacts with CHK2 and mediates its ubiquitination-directed proteasomal degradation. USP28 forms a complex with PIRH2 and CHK2 and counteracts the negative regulation of PIRH2 on CHK2 21 . Although in this system, the phenotype of USP28-deficient cells is unclear, CHK2 is expected to be destabilized by knockdown of USP28, which may lead to the negative regulation of DDR. However, it is worth noting that regulation of DDR by USP28 has another layer of complexity-USP28 is involved in multiple pathways that can have opposing functions. It can either promote apoptotic death by stabilizing factors of the CHK2-p53 pathway, or promote IR resistance by regulating Claspin 22, 29 . Therefore, the effect of USP28 on survival during DDR is determined by the net balance of these opposing pathways in the cell under the physiological milieus, and can be tissue-and cell type-dependent.

During genotoxic stress induced either by drugs or by IR, the cell has to repair the resultant DNA damage before progressing to mitosis. USP28 has been shown to maintain the G2 arrest by stabilizing Claspin, a key regulator of CHK1 30 . Claspin can stimulate ATR-dependent CHK1 activation after genotoxic stress. After the stress is relieved during G2 phase when the cell is recovered from DNA replication stress, Claspin is ubiquitinated and degraded via SCF βTrcp . Therefore, degradation of Claspin serves as a mechanism for the cell to shut off the checkpoint and enter the M phase during the cell cycle. Interestingly, Claspin level oscillates between the previous and next rounds of G1/S transition, and thus is subject to active degradation [31] [32] [33] [34] . Proteolysis of Claspin in G1 phase is required for the cell to maintain the G0/G1 state and prevent premature entry into S phase. Also, in parallel with its role in checkpoint signaling, Claspin positively regulates DNA replication-overexpression of Claspin can enhance both tumor and normal cell proliferation 29 . As a result, it is likely that stabilization of Claspin by USP28 may have further implications in cell cycle regulation that have not yet been identified. Additionally, in this system, USP28 only targets Claspin but not PLK1 (both of which are subject to ubiquitination by the same E3 ligase). This raises another question-while deubiquitinase activity of USP28 is required to revert ubiquitination-mediated protein turnover, how it discriminates the different substrates remains elusive. Answers to this question can promote our understanding of the pathophysiological implications of USP28-mediated Table 1 The reported targets of USP28 Antagonize the ubiquitin-dependent degradation 18 Claspin Deubiquitinates and stabilizes the protein 22 

Deubiquitinates and stabilizes the protein 21, 22 

Deubiquitinates and stabilizes the protein 22 

Deubiquitinates and stabilizes the protein 22 

Deubiquitinates and stabilizes the protein 23 

Antagonizes GSK-3β and FBW7-dependent degradation of HIF-1α 24 regulation on genotoxicity and cell cycle progression, which may shed light on improving strategies for cancer therapy.

Targeting USP28 for cancer therapy DUBs have long been targeted for cancer treatment. For instance, CYLD, a member of the USP class of DUBs, was identified as the causal gene for the rare familial conditions of cylindromatosis and trichoepithelioma, of which the symptom is the development of multiple skin tumors. Loss of function of CYLD tiptoes the transcriptional activity of the NF-κB pathway to the active side and drives many oncogenic processes [6] [7] [8] 13, [35] [36] [37] [38] . Therefore, targeting CYLD may have important significance in the treatment of multiple cancers in lung, liver, colon, multiple myeloma, and melanoma [39] [40] [41] [42] . Besides CYLD, additional DUBs, including A20, OTUD7B, USP11, H2A, BAP1, USP8, OTUD6B, UCH37, VCPIP1, USP7, COPS5, USP6, USP2, and USP15 have been implicated as potential targets in various cancers [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] . The clinical implications of dysregulated USP28 have been documented for various cancers as well. As a result, the significance of targeting USP28 for cancer treatment has been drawn more attention than before. Below, we will review the advances in USP28-targeted cancer therapy (Table 2) .

Targeting USP28 in non-small cell lung cancer (NSCLC) USP28 has been suggested as a therapeutic target for NSCLC. In one study, USP28 was found to be upregulated in the NSCLC tumors. Importantly, poor survivals were correlated with high levels of USP28 in patients. The cancer-promoting role of USP28 was confirmed by in vitro assays where overexpressing USP28 could enhance NSCLC cell proliferation while downregulating it induced apoptosis. Interestingly, USP28 may be targeted by a microRNA, miR-4295, as USP28 3′UTR-driven luciferase activity was significantly reduced by miR-4295 55 .

In a different study, however, miR-3940-5p was identified to target USP28 and CCND1 (Cyclin D1) in NSCLC. miR-3940-5p level was lower in NSCLC tumors than in matched adjacent normal tissues, and correlated with better clinicopathological features. Coincidently, USP28 was upregulated in NSCLC tumors and associated with poor prognosis of NSCLC patients. Therefore, miR-3940-5p may target CCND1 and USP28 to inhibit NSCLC growth. Indeed, downregulation of miR-3940-5p was concomitant with upregulation of CCND1 and USP28 in NSCLC tissues and cell lines. Importantly, miR-3940-5p suppressed proliferation and promoted apoptosis in NSCLC cells. It was verified that CCND1 and USP28 were direct targets of miR-3940-5p, and the phenotypes of NSCLC cell proliferation and apoptosis imposed by miR-3940-5p could be attenuated by overexpression of CCND1 or USP28. Accordingly, the therapeutic value of targeting USP28 was further demonstrated by in vivo experiments where overexpressing miR-3940-5p inhibited the growth of NSCLC tumors. Therefore, USP28 and CCND1 may be promising diagnostic markers and therapeutic targets for NSCLC 56 .

One study has revealed a critical mechanism underlying the epigenetic regulation by USP28 in breast cancer. An unbiased siRNA screening against all the human DUBs yielded USP28 as a bona fide deubiquitinase of LSD1 (lysine-specific demethylase 1). USP28 can interact with and stabilize LSD1. In clinical tumor samples, USP28 overexpression is correlated with LSD1 upregulation. Interestingly, LSD1 is a critical epigenetic regulator and controls pluripotency and differentiation by demethylating H3K4me1/2. Disrupting USP28 leads to LSD1 destabilization, which suppresses cancer stem cell-like characteristics in vitro and inhibits tumorigenicity in vivo. Therefore, targeting USP28 provides another treatment approach against breast cancer 23 .

In a follow-up study, it was further confirmed that USP28 could modulate epigenetic events in breast cancer. Both LSD1 and histone deacetylases (HDACs) facilitate breast cancer proliferation, and interestingly, HDAC5 could promote the stability of USP28. Overexpression of USP28 largely reversed HDAC5-knockdown-induced LSD1 protein degradation, suggesting HDAC5 positively regulates LSD1 by stabilizing USP28. Therefore, targeting USP28 may tune down the HDAC5/LSD1-mediated epigenetic process that drives breast cancer development and progression 57 .

As stated previously, USP28 can stabilize FBW7targeted proteins, exhibiting oncogenic activities. Importantly, USP28 is highly expressed in colon cancers, making it a potential pharmacological target to control c-MYC-driving tumor cells. In a mouse model of colorectal cancer, in contrast to the FBW7-deletion phenotype, intestine-specific deletion of USP28 reduced intestinal tumors. Importantly, in mice carrying the tumors, USP28 deletion reduced tumor size and prolonged lifespan 18 . Interestingly, USP28 can also antagonize the ubiquitindependent degradation of two additional oncogenic proteins, c-Jun and Notch1, expanding the substrate list that is shared by both USP28 and FBW7. The therapeutic benefit of USP28 in colon cancer was further demonstrated in another study, where USP28 knockout could increase the life expectancy of animals harboring FBW7deficient colon tumors. Consistently, FBW7 substrates, including c-MYC, c-JUN, NICD1, and cyclin E1, which were upregulated in FBW7-deficient mice, were downregulated in the double-knockout mouse 2 . Upregulation of USP28 is correlated with poor overall survivals and prognosis in NSCLC patients. It remains to be determined which microRNA targets USP28 in NSCLC (details in the main text). The exact mechanism by which USP28 promotes NSCLC cell proliferation is undetermined yet. 55, 56 Breast cancer USP28 stabilizes LSD1, a critical chromatin modulator that controls cell pluripotency and differentiation, by deubiquitination. USP28 is required for maintaining cancer stem cell (CSC)-like characteristics. Additional mechanism suggests that histone deacetylases 5 (HDAC5) promotes stability of USP28, thus enhancing stability of LSD1.

Overexpression of USP28, HDAC5, and LSD1 is positively correlated in breast tumors. 23, 57 Intestinal cancers USP28 promotes deubiquitination of important oncogenic proteins including c-MYC, c-JUN, and NOTCH1, thus stabilizing them in the intestine. This function is independent of the E3 ligase FBW7 in the intestinal crypt stem cells.

USP28 is prevalently overexpressed in human colorectal carcinomas. USP28 also enhances intestinal tumorigenesis. 2, 18 Gliomas USP28 stimulates glioma cell proliferation both in vitro and in vivo. USP28-indued c-MYC upregulation may contribute to glioma tumorigenecity.

USP28 level is positively correlated with glioma grade and inversely correlated with patient survivals. The mechanism remains unclear for the positive regulation of USP28 on c-MYC in human gliomas. 58 Bladder cancer It remains unclear for the role of USP28 in bladder cancer.

Upregulation of USP28 is significantly correlated with bladder cancer grade, staging, and recurrence. The knowledge of the role of USP28 in bladder cancer is very limited. 59 Targeting USP28 in gliomas

The significance of USP28 in glioma tumorigenesis has been demonstrated by its positive regulation on MYC, which is dysregulated in the majority of gliomas and difficult to target directly. It was determined that USP28 was upregulated in human gliomas but not in normal brain tissues. The USP28 protein level in human glioma tissues was directly correlated with glioma grade, and was inversely correlated with patient survivals. Ectopic USP28 expression promoted proliferation of SW1783 glioma cells both in vitro and in vivo, and conferred enhanced tumorigenicity in a nude mouse model. Disruption of USP28 in glioblastoma U373 cells suppressed anchorageindependent growth in vitro and tumorigenicity in vivo. Importantly, USP28 promoted MYC expression, which was required for USP28-induced cell growth in human glioma. Therefore, USP28 could be a new target of therapy for human malignant gliomas 58 .

The clinical significance of USP28 was also demonstrated in human bladder cancer. It has been determined that USP28 was overexpressed in bladder cancers compared to adjacent non-cancerous tissues at both the mRNA and protein levels. IHC examination confirmed that a majority of cancerous tissues displayed high USP28 levels. Furthermore, USP28 was strongly correlated with histopathological grade, clinical stage, tumor number, and recurrence rate. Interestingly, USP28 expression level can serve as an independent predictor of survival. Therefore, USP28 can potentially be valuable in the prognostic evaluation of bladder cancer 59 .

To date, pharmacological studies of DUBs have been focused on screening and identifying novel DUB inhibitors or antagonists for clinical regiment use. High throughput screenings in chemical libraries followed by structural-activity relationship studies have yielded fruitful compounds exhibiting selectivity for particular DUBs [60] [61] [62] [63] . Due to the important role of USP7 in stabilizing p53, small molecules selective for USP7 have been a focus of the pharmacological studies for DUBs [64] [65] [66] [67] . HBX 41108 was discovered to inhibit USP7 activity at a submicromolar range. Treatment with this compound leads to stabilization of p53 and subsequent repression of cancer cell growth 66 . A dual inhibitor selective for USP7 and USP47 was also reported to promote p53-induced apoptosis in cancer cells. Its efficacy was documented for a mouse xenograft model of myeloma and B-cell leukemia 64 . In Table 3 , we list some major USP inhibitors [68] [69] [70] [71] [72] [73] . However, despite the advances in DUB-targeting drug discovery, the identified compounds are still in the early stage awaiting clinical approvals. To date, no compounds targeting USP28 have been reported yet, and no DUB-targeted drugs have yet been approved for 

Ubiquitination is one of the important post-translational modifications observed in the cell and plays multifaceted roles in cellular pathways, affecting protein turnover, signaling, and response to various stresses. DUBs, by removing the ubiquitin tags, have been shown to regulate stability of signaling proteins, which in turn affect the outputs of a variety of signaling cascades controlling cell proliferation, survival, DNA damage response, carcinogenesis, and metastasis. Several DUBs have been targeted for therapeutic applications, as exemplified by some promising drug discovery studies. To date, major roles of USP28 are demonstrated by its regulations of DDR and ubiquitinationdependent proteolysis pathways. It is likely that the functions of USP28 are cell/tissue type-dependent, and in a particular system, a specific aspect of these functions can be dominant. Regardless, USP28 may be a potentially beneficial therapeutic target for cancer treatment. Hence, its clinical relatedness and applications should be extensively explored, investigated, and evaluated.

Regulation of USP28 deubiquitinating activity by SUMO conjugation

Usp28 counteracts Fbw7 in intestinal homeostasis and cancer

A genomic and functional inventory of deubiquitinating enzymes

Deubiquitinases in cancer: new functions and therapeutic options

Breaking the chains: structure and function of the deubiquitinases

Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB

CYLD is a deubiquitinating enzyme that negatively regulates NF-kappaB activation by TNFR family members

The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination

Characterization of alternatively spliced products and tissuespecific isoforms of USP28 and USP25

Amino-terminal dimerization, NRDP1-rhodanese interaction, and inhibited catalytic domain conformation of the ubiquitinspecific protease 8 (USP8)

Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde

Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14

The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module

Structural basis of ubiquitin recognition by the deubiquitinating protease USP2

Polyubiquitin binding and cross-reactivity in the USP domain deubiquitinase USP21

The ubiquitin-specific protease USP28 is required for MYC stability

FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation

The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

Dual regulation of Fbw7 function and oncogenic transformation by Usp28

Fbw7 and Usp28 -enemies and allies

The E3 ligase PIRH2 polyubiquitylates CHK2 and regulates its turnover

A role for the deubiquitinating enzyme USP28 in control of the DNA-damage response

The deubiquitinase USP28 stabilizes LSD1 and confers stem-celllike traits to breast cancer cells

GSK-3beta regulates cell growth, migration, and angiogenesis via Fbw7 and USP28-dependent degradation of HIF-1alpha

Advances in radiotherapy

Towards evidence-based guidelines for radiotherapy infrastructure and staffing needs in Europe: the ESTRO QUARTS project

The role of radiotherapy in cancer treatment: estimating optimal utilization from a review of evidencebased clinical guidelines

53BP1, a mediator of the DNA damage checkpoint

Human Claspin works with BRCA1 to both positively and negatively regulate cell proliferation

The Cdc14B-Cdh1-Plk1 axis controls the G2 DNAdamage-response checkpoint

The DNA damage response: ten years after

Destruction of Claspin by SCFbetaTrCP restrains Chk1 activation and facilitates recovery from genotoxic stress

Polo-like kinase-1 controls proteasome-dependent degradation of Claspin during checkpoint recovery

SCFbetaTrCP-mediated degradation of Claspin regulates recovery from the DNA replication checkpoint response

Familial cylindromatosis (turban tumour syndrome) gene localised to chromosome 16q12-q13: evidence for its role as a tumour suppressor gene

Update of cylindromatosis gene (CYLD) mutations in Brooke-Spiegler syndrome: novel insights into the role of deubiquitination in cell signaling

Tumor suppressor CYLD: negative regulation of NF-kappaB signaling and more

Cylindromatosis and the CYLD gene: new lessons on the molecular principles of epithelial growth control

Reduced expression of CYLD in human colon and hepatocellular carcinomas

Gene mapping and expression analysis of 16q loss of heterozygosity identifies WWOX and CYLD as being important in determining clinical outcome in multiple myeloma

Down-regulation of CYLD expression by Snail promotes tumor progression in malignant melanoma

Pharmacologic inhibition of epigenetic modifications, coupled with gene expression profiling, reveals novel targets of aberrant DNA methylation and histone deacetylation in lung cancer

NF-kappaB suppression by the deubiquitinating enzyme Cezanne: a novel negative feedback loop in pro-inflammatory signaling

DUBs and cancer: the role of deubiquitinating enzymes as oncogenes, non-oncogenes and tumor suppressors

Frequent inactivation of A20 in B-cell lymphomas

Loss of BAP1 function leads to EZH2-dependent transformation

An atlas of altered expression of deubiquitinating enzymes in human cancer

USP6 and CDH11 oncogenes identify the neoplastic cell in primary aneurysmal bone cysts and are absent in so-called secondary aneurysmal bone cysts

Mutations in the deubiquitinase gene USP8 cause Cushing's disease

TNFAIP3 (A20) is a tumor suppressor gene in Hodgkin lymphoma and primary mediastinal B cell lymphoma

The E3 ligase Itch negatively regulates inflammatory signaling pathways by controlling the function of the ubiquitin-editing enzyme A20

Deubiquitylation and regulation of the immune response

USP11 negatively regulates TNFalpha-induced NF-kappaB activation by targeting on IkappaBalpha

Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease

Overexpression of deubiquitinating enzyme USP28 promoted non-small cell lung cancer growth

miR-3940-5p functions as a tumor suppressor in non-small cell lung cancer cells by targeting Cyclin D1 and ubiquitin specific peptidase-28

Functional interaction of histone deacetylase 5 (HDAC5) and lysine-specific demethylase 1 (LSD1) promotes breast cancer progression

Ubiquitin-specific protease 28 is overexpressed in human glioblastomas and contributes to glioma tumorigenicity by regulating MYC expression

USP28 is a potential prognostic marker for bladder cancer

A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

Structure-activity relationship, kinetic mechanism, and selectivity for a new class of ubiquitin Cterminal hydrolase-L1 (UCH-L1) inhibitors

Structure-based design, synthesis, and biological evaluation of a series of novel and reversible inhibitors for the severe acute respiratory syndrome-coronavirus papain-like protease

Targeting ubiquitin specific proteases for drug discovery

Selective dual inhibitors of the cancer-related deubiquitylating proteases USP7 and USP47

Discovery of specific inhibitors of human USP7/HAUSP deubiquitinating enzyme

Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells

A small molecule inhibitor of ubiquitin-specific protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance

The emerging role of deubiquitinating enzymes in genomic integrity, diseases, and therapeutics

A novel small molecule inhibitor of deubiquitylating enzyme USP14 and UCHL5 induces apoptosis in multiple myeloma and overcomes bortezomib resistance

Enhancement of proteasome activity by a small-molecule inhibitor of USP14

Characterization of selective ubiquitin and ubiquitin-like protease inhibitors using a fluorescence-based multiplex assay format

Synthesis and biological evaluation of 9-oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile analogues as potential inhibitors of deubiquitinating enzymes

Deubiquitinase inhibition as a cancer therapeutic strategy