Cancer Chemotherapy and Pharmacology

https://doi.org/10.1007/s00280-020-04046-8

REVIEW ARTICLE

The role of deubiquitinating enzymes in cancer drug resistance

Parthasaradhireddy Tanguturi1 · Kye‑Seong Kim1,2 · Suresh Ramakrishna1,2 

Received: 2 October 2019 / Accepted: 19 February 2020

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Drug resistance is a well-known phenomenon leading to a reduction in the effectiveness of pharmaceutical treatments. Resist-
ance to chemotherapeutic agents can involve various intrinsic cellular processes including drug efflux, increased resistance
to apoptosis, increased DNA damage repair capabilities in response to platinum salts or other DNA-damaging drugs, drug
inactivation, drug target alteration, epithelial–mesenchymal transition (EMT), inherent cell heterogeneity, epigenetic effects,
or any combination of these mechanisms. Deubiquitinating enzymes (DUBs) reverse ubiquitination of target proteins, main-
taining a balance between ubiquitination and deubiquitination of proteins to maintain cell homeostasis. Increasing evidence
supports an association of altered DUB activity with development of several cancers. Thus, DUBs are promising candidates
for targeted drug development. In this review, we outline the involvement of DUBs, particularly ubiquitin-specific proteases,
and their roles in drug resistance in different types of cancer. We also review potential small molecule DUB inhibitors that
can be used as drugs for cancer treatment.

Keywords  DUB inhibitors · Cancer progression · Drug resistance · Cancer cell proliferation · Ubiquitin-specific proteases

Abbreviations Bcl-xL B-cell lymphoma-extra large

USP Ubiquitin-specific protease Bcl-2 B-cell lymphoma 2
UPP Ubiquitin proteosomal pathway CDDP Cis-diaminedichloroplatin (II)
DUBs Deubiquitinases USP Ubiquitin-specific peptidase
UB Ubiquitination UAF1 WD repeat-containing protein 48
UBD Ubiquitin-binding domain LCLs Lymphoblastoid cell lines
ZnF-UBP domain Zinc finger ubiquitin-specific protease PTEN Phosphatase and tensin homolog
domain PARP Poly ADP ribose polymerase
UIM Ubiquitin-interacting motif MDM2 Murine double minute oncogene
UBA domain Ubiquitin-associated domain ER Estrogen receptor
UCHs Ubiquitin C-terminal hydrolases EGFR Epidermal growth factor receptor
OTUs Ovarian tumor proteases RTK Receptor tyrosine kinase
MJD Machado–Joseph domain protease BAG3 BAG family molecular chaperone
FDA Food and drug administration regulator 3
PI Proteasome inhibitor Mcl-1 Myeloid leukemia cell differentiation
MM Multiple myeloma protein
NSCLC Non-small cell lung cancer TAM Tamoxifen
ERα Estrogen receptor α
FLT3-ITD FMS-like tyrosine kinase 3 internal
* Kye‑Seong Kim tandem duplication
[email protected]; [email protected]
USP9x Ubiquitin-specific peptidase 9x
* Suresh Ramakrishna siRNA Small interfering RNA
[email protected]; [email protected]
TKD Tyrosine kinase domain
1
Graduate School of Biomedical Science and Engineering, CAM-DR Cell adhesion-mediated drug
Hanyang University, Seoul 04763, South Korea resistance
2
College of Medicine, Hanyang University, Seoul 04763, WM Waldenstrom macroglobulinemia
South Korea

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 Cancer Chemotherapy and Pharmacology

BCR B-cell receptor downstream signaling. Various reports have shown that
MYD88 Myeloid differentiation primary inhibiting proteosomal cysteine deubiquitinating enzymes
response 88 such as USP14 and UCHL5 has cytotoxic effects on cancer
PBMC Peripheral blood mononuclear cell cells and causes increased concentrations of ubiquitinated
MDR Multidrug resistance proteins.
HCC Hepatocellular carcinoma The isopeptide bond at the C-terminus of the ubiquitin
CSCs Cancer stem cells molecule is broken selectively by DUBs [9]. DUB functions
SIRT1 Silent mating type information regula- include removing ubiquitin from substrates and activation,
tion 2 homolog 1 deactivation, and localization of regulatory proteins. DUBs
MRP1 Multidrug resistance-associated pro- are involved in various cellular processes such as regula-
tein 1 tion of gene expression, apoptosis, and cytokine signaling,
NAFLD Non-alcoholic fatty liver disease and alterations in the activities of DUBs are associated with
TAK1 Transforming growth factor beta- pathological disorders including cancer and multiple neu-
activated kinase 1 rological disorders [10–13]. This suggests that DUBs may
TLS Translesion synthesis repair pathway be explored as potential drug targets. Based on recent data,
DUBs are being explored rigorously as a drug target in can-
cer since they have an important role in maintenance and
Introduction treatment response of multiple cancer types [14–16].
Drug resistance associated with various diseases is of
Cellular homeostasis is an essential process for cellular major concern and can be specific to a particular disease.
health and is maintained through regulation of protein syn- Various data suggest drug resistance as an emerging concern
thesis and degradation. Thirty percent of mammalian pro- in cancer that is thought to be mediated by mechanisms of
teins have a very short half-life and then become degraded drug efflux, epigenetics, or alterations in target molecules.
[1]. This mechanism of protein degradation is very well Other mechanisms of drug resistance include changes in the
regulated. The ubiquitin–proteasome system (UPS) mainly metabolome and mutations.
controls protein function and stabilization. This is a non-lys- This review gives a detailed description of the role of
osomal intracellular protein degradation pathway mediated DUBs, specifically ubiquitin-specific proteases (USPs), in
by proteasome holoenzymes, ubiquitin ligases, and deubiq- the context of drug resistance in cancer.
uitinating enzymes (DUBs) [2]. This UPS has a pivotal role
in various cellular processes [3–6].
Studies have shown that DUBs are differentially Ubiquitination and deubiquitination
expressed in various forms of cancer including non-small
cell lung cancer, breast cancer, lung cancer, and pancreatic A 76-amino acid polypeptide, ubiquitin, when covalently
ductal adenocarcinoma (Table 1) [7, 8]. These enzymes are bound with its target substrate causes multicatalytic 26S pro-
responsible for catalyzing the re-editing of polyubiquitin teasome complex-mediated degradation of protein [6, 17].
chains and/or removing ubiquitin from its target substrates. This process of ubiquitination is important in both degrada-
This causes alterations in the stability of proteins and/or tion of proteins and regulation of their functions [18]. Two

Table 1  DUBs involvement in Cancer type Ubiquitin-specific proteases References

various cancers
Colon USP2, USP5, USP7, USP9X, USP21, USP22 [110–115]
Breast USP4, USP9X, USP10, USP11, USP15, USP25, USP32 [37]
Pancreas USP11, USP39 [116, 117]
Gastric USP42, USP44, UCHL1 [118–120]
Lung USP4, USP7, USP5, USP8, USP13, USP22, USP24, USP37 [38, 77, 121, 122]
Prostate USP1, USP33 [123, 124]
Brain USP1, USP2, USP3, USP4, USP5, USP6, USP8, USP15, USP20, [125–132]
USP22, USP33, USP39, USP44, CYLD
Liver USP2A, USP4, USP7, USP8, USP15, USP21 [36, 114, 133]
Leukemia USP1, USP3, USP7, USP9X, USP20, USP33, USP44, CYLD [134–140]
Fanconi anemia USP1 [141]
Ovarian USP1, USP7, USP5, USP39, UCHL1 [142–146]

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Cancer Chemotherapy and Pharmacology

families of enzymes with contrast activity determine the lysine group of the target protein and the carboxyl terminal
degree of ubiquitination of proteins; ubiquitin ligases cause glycine group of ubiquitin. E3 enzymes serve as substrate
addition of ubiquitin moieties to a protein, while deubiquit- recognition modules for the systems and can interact with
inating enzymes remove these moieties [19]. Ubiquitination both E2 and substrate. After formation of the ubiquitin, E2,
is a well-known phenomenon that has a significant role in E3, and substrate complex, the substrate is bound to ubiqui-
cellular proteins at post-translational levels [20, 21]. tin; this complete process is facilitated by E3 ligase. Later,
Ubiquitination is a process with multiple steps and com- E2 and E3 are removed, and the protein either degrades or
prises three types: (i) mono-ubiquitination, wherein only a is removed from ubiquitin (Fig. 1).
single ubiquitin molecule is attached to the target protein; A very important role of the ubiquitin proteosomal path-
(ii) multi-ubiquitination or poly-mono-ubiquitination, in way (UPP) has been established in the pathogenesis of vari-
which various molecules of ubiquitin are attached to the ous diseases [5]. It has been well established that deregu-
target protein; and (iii) poly-ubiquitination, in which a poly- lation of this UPP pathway has a strong co-relation with
ubiquitin chain is attached to the protein [16, 22, 23]. The various human disease pathologies [17, 24]. Studies suggest
enzymes involved in ubiquitination are ubiquitin-activating that inhibiting this pathway could be an innovative strat-
enzymes (E1), ubiquitin-conjugating enzymes (E2), and egy for therapeutic purposes and can involve inhibition at
ubiquitin ligases or E3 ubiquitin ligases (E3). two levels: the proteosomal level and that of ubiquitinating
Ubiquitination involves activation of ubiquitin by E1 in an E3 ligases. Much research has been performed to develop
ATP-dependent mechanism, followed by thioester bond for- small molecule inhibitors against various other components
mation between cysteine on the active site of enzyme E1 and of the UPP, including design of inhibitors for E1 conjugat-
the glycine residue of ubiquitin on the carboxyl side. Next, ing enzymes or E3 ubiquitin ligases. Studies report that E3
the ubiquitin molecule transfers from E1 to the cysteine ubiquitin ligases are an efficient target for therapeutic impli-
residue of E2. E3 ubiquitin ligases catalyze the final step of cations considering their role in numerous human diseases
ubiquitination by creating an isopeptide bond between the [25].

Fig. 1  The ubiquitin proteasome pathway. Ubiquitination occurs mediate ligation of ubiquitin to target protein substrates. DUBs
through the actions of E1 (ubiquitin-activating enzyme), E2 (ubiq- reverse E3 ligase-mediated ubiquitination and are involved in recy-
uitin-conjugating enzyme), and E3 enzymes (ubiquitin ligase) that cling of ubiquitin molecules

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DUBs are known to reverse the ubiquitination process modify the ubiquitination status of a protein. Such a pos-
by precisely cleaving the C-terminus isopeptide bond of sibility should be considered while designing novel DUB
ubiquitin [26]. The main function of proteases is to cleave inhibitors and targeted therapeutic strategies [32].
ubiquitin from proteins and other molecules [27, 28]. Pro- Substantial scientific evidence has shown the importance
teases are vital constituents of the ubiquitin–proteasome of UPS in regulating the proliferation and survival of cancer
system and have three different modes by which they act: cells. This system includes a small molecule ubiquitin along
through de novo ubiquitin synthesis by generating or releas- with a proteasome as a multi-subunit proteolytic complex
ing free ubiquitin; by cleaving polyubiquitinated chains; and that leads to breakdown of undesirable cellular proteins into
by removing the ubiquitin sequence from the ubiquitinated peptides that are used for metabolic processes [4, 33]. Vari-
proteins. ous inhibitors of these proteases have been designed, and
Studies have shown that DUBs are highly selective for their activity has been evaluated in in vitro systems as well
their substrates. They also are very specific to a protein as in vivo animal models [17, 34].
and can be even specific for branching pattern or shape of Despite their commendable potential, the available pro-
polyubiquitin chain. The key domain of DUBs [9] is the teasome inhibitors face the issues of drug resistance, toxic-
ubiquitin-binding domain (UBD), which is comprised of a ity, and a small therapeutic index, indicating the need for
ubiquitin-specific zinc finger protease domain (ZnF-UBP careful patient dosing [35]. This might be due to the inter-
domain), a ubiquitin-interacting motif (UIM), and a ubiqui- connection of proteasomes with many pathways within the
tin-associated domain (UBA domain) [29]. Various groups cell. To combat this issue, upstream components of the UPS
have shown that it is the ZnF-UBP domain that defines the have been targeted. Studies performed to date suggest a need
specific target proteins for DUBs [9]. to explore UPS further to identify novel drug targets.
The human genome encodes nearly 100 DUBs [19, 30].
These DUBs can be further classified into five sub-families,
four that are cysteine proteases and one comprised of metal-
loproteases. These sub-families are individually classified Role of ubiquitin‑specific proteases in cancer
as ubiquitin C-terminal hydrolases (UCHs), ubiquitin-spe- development
cific proteases (USPs or UBPs), ovarian tumor proteases
(OTUs), Machado–Joseph disease proteases (MJDs), and Ubiquitin-specific proteases are present in most forms of
JAB1/MPN/Mov34 metalloenzymes (JAMMs) (Table 2) cancer and are involved in regulation of various pathways
[31]. In addition, another class of DUB-like proteases that that have a role in cancer progression (Table 3) [36–39].
do not belong to the same gene family has been identified These proteases may serve as an effective target for thera-
and named M48USP [9]. This indicates that there might be peutic intervention. It has been observed that various dis-
other unexplored gene families that act in the UPS and can eases, including diseases related to the nervous system,

Table 2  Classification of DUBs Cysteine proteases Metalloproteases

USP JAMM
USP1, USP2, USP3, USP4, USP5, USP6, USP7 COPS6, EIF3F, PSMD7
USP8, USP9X, USP9Y, USP10, USP11, USP12, AMSH-LP, AMSH,
USP13, USP14, USP15, USP16, USP17, USP18 PRPF8, BRCC36
USP19, USP20, USP21, USP22, USP23, USP24 MPND, MYSM1
USP25, USP26, USP27, USP28, USP28, USP30 EIF3H, COPS5
USP31, USP32, USP33, USP44, USP45, USP46 PSMD14
USP47, USP48, USP49, USP50, USP51, USP52
USP53, USP54, CYLD
OTU
OTUD1, YOD1, OTUB2, OTUB1, TRABID, OTUD7A
OTUD7B, A2O, HIN1L, OTUD4, OTUD6A, OTUD6B
OTUD3, OTUD5, VCPIP1
MJD
ATXN3, ATXN3L, JOSD1, JOSD2
UCH
UCH L1, UCH L3, UCH L5, BAP 1

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Table 3  DUBs and their mechanisms of action

DUBs Expression Pathway References

USP1 Downregulated Plays a vital role in DNA damage, DNA replication, and Fanconi anemia by co- ordinating [141, 147]
DNA repair by promoting the FA pathway and inhibiting the TLS pathway
USP2 Upregulated Deubiquitinates polyubiquitinated target proteins such as fatty acid synthase, murine double [110]
minute 2 (MDM2), MDM4/MDMX, and cyclin D1
USP3 Upregulated Plays a vital role in cell proliferation and spreading by regulating cell cycle control; stabilizes [148, 149]
KLF5 protein
USP4 Upregulated Positive regulator of NF-κB, TGF-β, Wnt/β-catenin, p53, and spliceosome pathways [150]
USP5 Upregulated Efficient repair of DNA double-strand breaks; stabilizes β-catenin protein [122, 151]
USP6 Upregulated Regulates the Wnt signaling pathway [152]
USP7 Upregulated Mediates stabilization of DNMT1 protein and DNA methylation and regulates MDM2/p53 [153]
USP8 Upregulated Promotes trafficking and degradation of chemokine receptor 4 (CXCR4); regulates epidermal [154]
growth factor receptor ubiquitination and trafficking
USP9 Upregulated Regulation of ERα action [84]
USP10 Upregulated Plays a vital role in stabilization of FLT3-ITD oncoprotein [40]
USP11 Upregulated Controls VGLL4 protein stability by promoting its deubiquitination [155]
USP14 Downregulated Downregulated in the apoptotic model and upregulated in the adhesive model of multiple [156]
myeloma in the Bcl-xl apoptotic pathway
USP18 Downregulated Regulatory role in non-alcoholic fatty liver disease [109]
USP20 Upregulated Alters claspin protein level [157]
USP22 Upregulated Interacts with SIRT1 protein and regulates its expression [105]
USP28 Upregulated Stabilizes oncogenic function of LIN28a protein and regulates c-myc protein [158]
USP39 and USP44 Upregulated Maintains mitotic spindle checkpoint and integrity [134, 159]
USP48 Downregulated Promotes mdm2 stability and enhances p53-mediated deubiquitination [160]
UCHL5 Upregulated Plays a vital role in regulation of TGF-β signaling [161]

cancer, and various viral infections, are related to abnor- and fewer side effects. Although nearly 60 USPs have been
malities in processes mediated through ubiquitin. found in humans, the number of active inhibitors is far
Data reported to date suggest that various USPs includ- smaller.
ing USP1-USP8, USP9X, and many more have significance Genetic mutations and abnormal regulation of growth and
in development and progression of cancer. Two therapeutic survival pathways in cancer lead to its diversity and resist-
strategies could be helpful in treating cancer: inhibiting the ance to existing therapies [41–43]. The FDA has approved
DUBs that act as oncoproteins and activating the DUBs that bortezomib as an inhibitor of 20S proteosome for treating
cause tumor suppression [25]. multiple myeloma and mantle cell lymphoma. This was
Weisberg et al. reported that DUBs help stabilize proteins the first inhibitor targeting UPS to be validated for cancer
responsible for disease, specifically in cancer. FLT3-ITD, an therapy [44–47]. This molecule was also associated with
oncoprotein, is stabilized by USP10, which removes the deg- the problems of toxicity and resistance, which limited its
radative ubiquitin tag in cases of acute myeloid leukemia. usage and efficacy [45]. The reason for this resistance is still
Inhibiting USP10 pharmacologically induced degradation unknown [48, 49]. Ongoing studies have suggested muta-
of FLT3-ITD, but did not degrade FLT3. Oncogenic FLT- tions in the catalytic domain as one reason for resistance to
3-expressing acute myeloid leukemia cells were selectively bortezomib. Another mechanism behind resistance to this
inhibited both in vitro and in vivo under these conditions molecule could be over-expression of anti-apoptotic proteins
[40]. such as BCl2 and BCL-XL [50, 51].
The US FDA has also approved second-generation pro-
teasome inhibitors, such as carfilzomib and ixazomib, for
Drug design for DUBs and the effects treatment of multiple myeloma. Ixazomib is an oral protea-
of DUBs in drug resistance some inhibitor that binds reversibly and inhibits the 20S pro-
teasome with a dissociation rate less than half that of borte-
Studies have shown that USPs serve as promising drug zomib (18 min for ixazomib versus 110 min for bortezomib)
targets for design of small molecule inhibitors as they are and has a higher PK/PD profile [52–56]. Ixazomib showed
potential-specific targets, possibly leading to better activity synergistic anti-multiple myeloma activity in combination

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with dexamethasone and lenelidomide, providing an addi- Cisplatin-resistant cells did not proliferate after synergistic
tional mechanism to overcome resistance to bortezomib. treatment with inhibitors for USP1/UAF1. Therefore, this
Calfilzomib, another proteasome inhibitor, binds irrevers- complex may serve as a prospective combination target for
ibly to the β5 proteasome subunit with greater affinity than overcoming cisplatin resistance in tumor cells.
bortezomib. This proteasome inhibitor is an isolated agent USP7, also known as HAUSP, serves as a deubiquitinat-
used specifically for treatment of relapsed or refractory mul- ing enzyme for various tumor suppressors [61]. Silencing
tiple myeloma patients who have undergone more than two of USP7 reduces CCDC6 level in HCT116 cells to increase
previous therapies including one with bortezomib. Carfil- the sensitivity of these cells to olaparib. Combining cisplatin
zomib has shown an excellent anti-MM effect in combina- with olaparib produced a synergistic effect on the cells.
tion with lenalidomide and dexamethasone [52–56]. Higher expression of USP7 increases the sensitivity of
Marizomib and oprozomib are other notable inhibitors cancer cell lines to tamoxifen (TAM) [62, 63]. Cell growth
that are currently in the first phase of clinical trial. Mari- inhibition by USP7 might be mediated by increased expres-
zomib, an oral irreversible protease inhibitor, binds to a sion of phosphatase and tensin homolog (PTEN). USP7
catalytic subunit of the 20S proteasome isolated from Salin- has also been established as a deubiquitinating enzyme for
ispora tropica. Phase 1 studies of Marizomib confirmed coiled-coil domain-containing protein 6 (CCDC6), a tumor
comparatively low toxicity with no symptoms of thrombo- suppressor. Reports suggest that NSCLC cells that have low
cytopenia. Marizomib showed great synergy in vitro with expression of CCDC6 protein are more susceptible to the
several other anti-myeloma agents, such as HDACs. Opro- PARP inhibitor olaparib. However, H1975 cells, with high
zomib, an oral irreversible protease inhibitor, is highly selec- expression of CCDC6, were resistant to olaparib. Knocking
tive for the β5 subunit of the 20S proteasome that exhibited down CCDC6 reverted the sensitivity of these cells [64].
similar potency to that of carfilzomib in toxicity assays. The data suggest that inhibitors for USP7 could increase the
Oprozomib has established an acceptable safety profile with response to olaparib and help to enhance response in other
little occurrence of neuropathy in two-phase 1b/2 studies therapeutic agents by downregulating CCDC6 level [65,
[52, 54–59]. 66]. MDM2, a murine oncogene (HDM2, human ortholog),
Cisplatin is a well-known drug used to treat many types acts as the substrate for USP7 and negatively regulates p53
of solid tumors and non-small cell lung cancer. However, protein by ubiquitination and subsequent proteasome-medi-
the response associated with this drug is of a short duration, ated degradation [67–69]. USP7 helps stabilize the level
after which the tumor no longer responds. This resistance of MDM2 during this process. It has been demonstrated
to cisplatin is dependent on various mechanisms that have that siRNA or knockout of USP7 halts deubiquitination of
not been completely elucidated. To dissect the mechanism MDM2 and stabilizes p53 [70, 71]. In addition, p53 mutates
behind this resistance, the effects of cisplatin on transla- in multiple myeloma, and its activation could be a thera-
tion of mRNA in a matched cisplatin-sensitive NSCLC peutic strategy [72]. USP7 is involved in deubiquitination
cell line were compared with those in a resistant cell line. of other onco-targets such as PTEN, FOXO4, and orclaspin
The researchers found that the mRNAs of a few genes were [66].
translated differentially. Among these was the gene encod- A chemical inhibitor of USP7, P5091, causes apoptosis
ing ubiquitin-specific peptidase 1 (USP1), which has signifi- in multiple myeloma cells with resistance to bortezomib
cance in DNA repair mechanisms; its mRNA translation was therapy. These preclinical studies emphasize the need for
inhibited by cisplatin in cisplatin-sensitive cells, but resistant USP7 inhibitors alone or in combination therapy for multi-
cells were unaffected [60]. ple myeloma [66]. A cyano-indenopyrazine derivative found
Sourisseau et al. found that siRNA or ML323 inhibi- by Colland et al. inhibits USP7 at a sub-micromolar IC50.
tion of USP1 expression reversed the resistance of resistant Further studies confirmed that p53 ubiquitination was inhib-
cells, which were surprisingly re-sensitized to cisplatin. The ited by this compound both in vitro and in vivo. The P5091
data suggest that a deficit in downregulation of USP1 at the inhibitor induced dose-dependent activity against various
translational level is the major mechanism for resistance to multiple myeloma cell lines, including cells resistant to
cisplatin in NSCLC, and that this could serve as a marker available therapies such as dexamethasone, doxorubicin, or
for resistance and as a potential drug target in cases with cis- melphalan [73].
platin resistance. This approach of analyzing mRNA at the FT671 is another potent, selective USP7 inhibitor, with an
translational level could aid in identification of new targets ­IC50 of 52 nM and that binds to the USP7 catalytic domain.
for overcoming drug resistance. FT671 destabilizes USP7 substrates such as MDM2, increas-
USP1 has a role in DNA repair as a deubiquitinat- ing the level of p53 protein and inhibiting tumor growth by
ing enzyme for monoubiquitinated FANCD2 [60]. USP1 promoting transcription of p53 target genes and tumor sup-
possesses a low level of deubiquitinating activity but pressor p21. In vitro studies showed that FT671 enhances
shows greater activity in a complex with UAF1 [61]. the p53 protein level in bone osteosarcoma (U2OS) cell

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lines and HCT116. FT-6717 is well tolerated at high doses patients with ERα-positive breast cancer [84], and drug
(100 mg/kg and 200 mg/kg) in mice and was administered in resistance to tamoxifen is a major issue, the mechanism of
a dose that significantly correlated with inhibition of tumor which has yet to be elucidated. Studies were performed to
growth [74, 75]. dissect the resistance mechanism, wherein identification
GNE-6640, another novel selective and non-covalent of resistant genes was performed using large-scale loss
inhibitor of USP7, has ­IC50 values of 0.75 μM (full length of function genetic screening in ZR-75–1 luminal breast
USP7), 0.43 μM (USP7 catalytic domain), and 20.3 μM (full cancer cells. This revealed that functional loss of USP9X
length USP43). Ubiquitin-specific protease-7 (USP7) con- inhibited the activity of tamoxifen but not the activity of
trols the stability of the p53 tumor suppressor and various another endocrine agent, fulvestrant, which is an ER down
other proteins critical for tumor cell survival. GNE-6640 regulator. Knockdown of USP9X not only stabilized the
facilitates endogenous MDM2 ubiquitination and targets cel- interaction of ERα and chromatin, but also induced tamox-
lular USP7 and p53 signaling pathways, inducing tumor cell ifen-mediated activation of ERα. In addition, proliferation
death, enhancing cytotoxicity, and decreasing the viability of cells responsive to ERα was stimulated in the presence
of around 108 cell lines with ­IC50 ≤ 10 μM with chemothera- of tamoxifen.
peutic agents such as PIM kinase inhibitors [76]. Various reports suggest that a peptidase specific for
USP8 has a number of substrates including epidermal USP9X regulates various cellular behaviors, including
growth factor receptor (EGFR), binding to which leads tumor growth, invasion, and resistance to available therapies.
to degradation [77]. Inhibition of USP8, either by knock- WP1130 inhibitor exhibits anti-tumor activity by targeting
down or using a synthetic small molecule, led to a decrease a number of DUBs [27]. Increased cisplatin cytotoxicity
in range of receptor tyrosine kinase (RTK) activities and was observed in ER-negative tumor cells, but ER-positive
inhibited cell proliferation in both gefitinib-resistant and cells remained unaffected. Western blotting confirmed that
gefitinib-sensitive non-small cell lung cancer cell lines [77]. expression of USP9X was significantly lower in ER-positive
The BAG3 gene encodes a protein in humans known as BAG cells compared to ER-negative cells. Moreover, expression
family molecular chaperone regulator 3 (BAG3). This pro- of USP9X and Mcl-1 was reduced post-treatment with
tein causes stabilization of Mcl-1, which is a differentiation WP1130 in ER-negative cells but not in ER-positive cells.
protein in stimulated myeloid leukemia cells, leading to drug In breast cancer cells, knockdown of USP9X reduced the
resistance. BAG3 and USP9X interact to stabilize the Mcl-1 chemosensitization activity of WP1130 in the presence
protein; when this interaction was inhibited by WP1130, it of cisplatin. These data indicate that combination therapy
reduced the Mcl-1 level and simultaneously blocked Bcr- of cisplatin with WP1130 increases treatment sensitivity
Abl kinase signaling. This initiated the process of apoptosis, dependent on USP9X in ER-negative breast cancer [85].
overcoming the resistance of cancer cells. Knockdown studies using specific siRNAs of two DUBs,
Another major issue is resistance to tamoxifen, an estro- USP14, and UCHL5, which are highly expressed in multiple
gen receptor (ER) antagonist, in breast cancer. Breast cancer myeloma cells compared to normal plasma cells, resulted
accounts for most of the cancer-related deaths in females in reduced cell viability in multiple myeloma. Both of these
throughout the world [78]. Estrogen helps in growth and DUBs have reversible binding efficacy with 19S regulatory
differentiation of mammary glands [79]. A large percentage proteins and have a role in cancer. Inhibiting these DUBs
of breast cancer patients are ER positive; hence, blocking may lead to reduced uptake of protein substrates to be
this receptor is the ideal therapy for such patients [80]. In degraded [86]. Based on these findings, various other DUB
patients not positive for ER, this therapy fails [81]. This inhibitors were designed. One such inhibitor is b-AP15,
receptor has a very important role in normal growth of the which selectively blocks USP14 and UCHL5 deubiquitinase
breast as well as cancer cell growth in that region [82]. ER activity without affecting 20S core particle activity [16]. A
serves as a prominent biomarker for disease-free survival in potential USP14 inhibitor showed an IC50 dose of 4–5 µM
patients who have had breast cancer. Signaling mediated by and accelerated degradation of protein due to blocking of the
ER has intricate interaction with many other growth signal- proteasome-linked USP14 [87].
ing pathways such as HER2 in breast cancer, and it has a role Another notable mechanism of drug resistance in multiple
in drug resistance that involves various pathways [83]. One myeloma is cell adhesion-mediated drug resistance (CAM-
strategy to combat drug resistance in cancer is to understand DR). This form of cancer occurs due to accumulation of
the methods for drug target alteration. clonal neoplastic cells in the bone marrow and causes bone
Recent studies conducted on USP9X in the context of lesions [88, 89]. Multiple myeloma is also associated with
ERα-positive breast cancer revealed a very interesting drug resistance despite many available treatments [90, 91].
role, wherein loss of USP9X leads to stimulation of tamox- Myeloma cells adhere like glue in the stromal cells of bone
ifen despite resistance due to inhibition of ERα activity. marrow or in the extracellular matrix as the major cause
This drug has been widely used for endocrine therapy for of resistance [92, 93]. This adherence of myeloma cells or

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a fibronectin-coated surface protects myeloma cells from potential drug targets that can combat this issue. A promi-
apoptosis, leading to drug resistance [94]. nent role of USP22 has been found in MDR of HCC cells
USP14 is over-expressed in the glue model of multiple (Fig. 2). Knockdown of USP22 led to cell cycle arrest and
myeloma, while it is downregulated in the apoptotic model. hampered cell growth in HepG2, an HCC cell line. The role
It also contributes to CAM-DR in multiple myeloma by of cancer stem cells, which have the properties of self-repair
interconnecting the BCL-xl and Wnt pathways. Studies and differentiation, has been reported in the context of MDR
report that USP14 is involved in various types of cancer in HCC [105].
including colorectal cancer, intra-hepatic cholangiocarci- There are reports showing that Sirtuin 1 (SIRT1) serves
noma, ovarian serous cystadenocarcinoma, and lung car- as a functional negotiator of USP22, promoting HCC cell
cinoma [95–98]. In leukemia cells, USP14 is upregulated. resistance to chemotherapy. Overexpression of SIRT1 in
Treatment with USP14 and UCHL5 inhibitors can induce HCC leads to HCC cell proliferative capacity and causes
apoptosis in multiple myeloma cells and overcome resist- resistance to existing chemotherapies [106]. Protein expres-
ance to bortezomib [86]. This suggests a clear role of USP14 sion of SIRT1 is positively regulated when it interacts with
in CAM-DR. USP22. The expression of SIRT1 and USP22 has a sig-
Walderstrom macroglobulinemia (WM) is a lymphoma of nificant effect on the AKT pathway and MRP1 expression,
B cells, wherein toll-like receptors along with B-cell recep- both of which are inhibited by LY294002. Both USP22 and
tors and MYD88 aid in tumor survival [99]. Higher expres- MRP1 were expressed in more than 150 clinical samples of
sion levels of USP14 and UCHL5 were found in WM tumor HCC according to multiple myeloma histochemical stain-
cells, which were resistant to drug treatment compared with ing. This demonstrates that USP22 promotes MDR in HCC
normal human peripheral blood monocytes or bone mar- by affecting SIRT1/AKT/MRP1 pathways and suggest that
row cells. An inhibitor of USP14/UCHL5 was used to target could serve as a drug target for reversal of multidrug resist-
their co-disruption, and this caused apoptotic death in WM ance in HCC [105].
cells under both ex vivo and in vivo conditions. This further DUBs are engaged in regulation of cell-signaling path-
showed the importance of USP14/UCHL5 as a potential ways, which are usually distorted in cancers. DUBs also
target for overcoming drug-resistant WM and has a trans- have a role in regulation of p53, which is a well-studied
lational implication [100]. A novel proteosome inhibitory tumor suppressor protein mutated in various types of can-
molecule, b-AP15, selectively inhibited USP14 and UCHL5 cer cells [107]. DUBs such as USP7 and USP15 are mainly
without affecting non-proteosomal DUBs [31]. This mol- involved in stabilization of both P53 and MDM2, whereas
ecule was found to be effective against cancer resistance to USP15, USP2, USP4, USP5, USP10, and USP29 play a role
bortezomib. in regulation of p53 alone [16, 70, 108].
The mode of action of b-AP15 is different from that of DUBs have a wide array of roles in various metabolic
bortezomib [101]. It acts by reducing the cell viability in disorders such as non-alcoholic fatty liver disease (NAFLD),
multiple myeloma cells, even in the presence of bone mar- characterized by hepatic steatosis, impaired insulin sensitiv-
row stromal cells [86, 102]. The b-AP15 acted by arrest- ity, and chronic low-grade inflammation. USP18 protects
ing the cell growth of multiple myeloma by downregulating against these symptoms through its deubiquitinating activity.
CDC2, CDC25C, and cyclin B1, leading to caspase-depend-
ent apoptosis. Tumor growth inhibition and prolonged sur-
vival were observed in a xenograft model of human multi-
ple myeloma treated with b-AP15. This efficacy of b-AP15
against multiple myeloma supports the potential of DUBs as
therapeutic targets. Further clinical studies using inhibitors
of USP14/UCHL5 aim to provide clinical intervention for
patients opposed to proteosomal inhibitors [103].
Since DUBs belong to different classes, small molecule
inhibition of associated enzymes would be possible only in
terms of broad-spectrum DUB inhibition. A number of small
molecules have been designed to act against proteosomal
DUBs. One such molecule is RA-9, which is capable of
inhibiting proteosomal deubiquitinase activity and thereby
causing stress and cell death [31, 104].
The emerging issue of multidrug resistance is of great
concern for hepatocellular carcinoma (HCC). Dissecting
the mechanism for drug resistance will help in identifying Fig. 2  USP22-mediated multidrug resistance in HCC cells

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Cancer Chemotherapy and Pharmacology

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Acknowledgements  This research was supported by the Basic Sci-
enzymes and cancer. Med Oncol 31:93
ence Research Program through the National Research Founda-
21. Voutsadakis IA (2012) Ubiquitination and the ubiquitin-protea-
tion of Korea (NRF), which is funded by the Ministry of Education
some system as regulators of transcription and transcription fac-
(2017M3A9C6061361, 2018M3A9H3022412, 2017R1A2B2008727
tors in epithelial mesenchymal transition of cancer. Tumour Biol
and 2017M3A9B3061830).
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