BBA - Reviews on Cancer 1868 (2017) 456–483

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BBA - Reviews on Cancer

journal homepage: www.elsevier.com/locate/bbacan

Review

The roles of ubiquitin modifying enzymes in neoplastic disease MARK

a,b a a,b,c a
Nishi Kumari , Patrick William Jaynes , Azad Saei , Prasanna Vasudevan Iyengar ,
John Lalith Charles Richarda, Pieter Johan Adam Eichhorna,b,⁎
a
Cancer Science Institute of Singapore, National University of Singapore, 117599, Singapore
b
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 117597, Singapore
c
Genome Institute of Singapore, A*STAR, Singapore

A R T I C L E I N F O A B S T R A C T

Keywords: The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific
Ubiquitin degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of
E3 ligases numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and
Deubiquitinating enzymes ubiquitin function over 30 years ago it has become wholly apparent that ubiquitin and their respective ubiquitin
Cancer
modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative
Dub inhibitors
E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in
sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known on-
cogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with spe-
cific focus on PI3K, MAPK, TGFβ, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs
as a novel anticancer therapeutic strategy.

1. Introduction enzymes (DUBs) as tumour suppressors or oncogenes has also been an

The initial concept of protein turnover was viewed as a minor
component in cellular homeostasis for it was thought that proteins in
general were intrinsically stable and were only subjected to degradation
when proteins were damage by prolonged “wear and tear”. It is of
course now recognized that ubiquitination mediated proteosomal de-
gradation of proteins is far more wide-spread and dynamically regu-
lated then was originally imagined. Furthermore, developments over
the last two decades has led to a better understanding of the versatile
functions of ubiquitination. We now understand that ubiquitin is able to
form eight structurally and functionally distinct polymers which can
affect not only protein stability but also affect protein activity and
cellular localization. Accumulating evidence indicates that perturba-
tions affecting the activity of ubiquitin modifying enzymes are im-
portant in tumour cell growth and survival and therefore these enzymes
have been suggested to be potential targets for pharmacological inter-
vention. Clinical validation of this was initially observed with the ap-
proval of the proteasome inhibitor Bortezomib in multiple myeloma
with patients exhibiting significant responses to the treatment [1]. The
involvement of E3 ligases in crucial signalling pathways implicated in
tumour progression is well documented and a number of excellent re-
views have been written on the role of E3 ligases in cancer which we
invite the reviewer to read [2–5]. However, the role of deubiquitinating
area of intense research. In addition to E3 ligase function here we
provide a portrait of DUB mutational profiles and their respective
substrate specificity in contribution to tumorigenesis.
The ubiquitin moiety is a highly conserved 76 amino acid poly-
peptide and ubiquitination of target proteins by ubiquitin and ubiquitin
like molecules serves as mechanism to regulate a myriad of protein
functions including protein stability, subcellular localisation, and ac-
tivity. The ubiquitination cascade comprises of coordinated action of a
E1 activating enzyme, a E2 ubiquitin-conjugating enzyme, and the E3
ubiquitin protein ligase. E1 ligases act as ubiquitin sponges recruiting
free ubiquitin inside the cell and subsequently transferring ubiquitin to
the E2 ligase. The major component of the ubiquitin cascade is the E3
ligases of which the human genome encodes over 600 allowing a high
degree of substrate specificity. Three main families of E3 ligases exist:
homologues to E6-associated protein carboxy terminus (HECT) family,
really interesting new gene (RING) family, which are the most abun-
dant in the genome, and RING-Between-RING (RBR) family. All three
families link E2s with substrates however, ubiquitin transfer is in-
herently unique between the E3s. In the case of HECT domain ligases
ubiquitin transfer involves the formation of a thioester intermediate
formed between ubiquitin and the catalytic cysteine of the HECT E3
prior to transfer of ubiquitin to its recruited substrate [2]. Unlike the
HECT domain ligases, RING ligases appear to act as scaffolds permitting

⁎
Corresponding author at: Cancer Science Institute of Singapore, Centre for Translational Medicine, 14 Medical Drive 12-01, 117599, Singapore.
E-mail address: pieter_eichhorn@nus.edu.sg (P.J.A. Eichhorn).

http://dx.doi.org/10.1016/j.bbcan.2017.09.002
Received 4 August 2017; Received in revised form 11 September 2017; Accepted 12 September 2017
Available online 18 September 2017
0304-419X/ © 2017 Elsevier B.V. All rights reserved.
N. Kumari et al. BBA - Reviews on Cancer 1868 (2017) 456–483

Fig. 1. Copy number variation of deubiquitinating enzymes in cancer. Individual DUB expression in cancer subtypes extrapolated from cBioPortal (www.cBioPortal.org) derived from the
following datasets, [304–317], Adrenocortical Carcinoma (TCGA), Colorectal Adenocarcinoma (TCGA), Cervical Squamous Cell Carcinoma (TCGA), Uveal Melanoma (TCGA), Hepa-
tocellular carcinoma (TCGA), B-cell Lymphoma (TCGA), Ovarian adenocarcinoma (TCGA), Pancreatic Adenocarcinoma (TCGA), Skin Cutaneous Melanoma (TCGA), Testicular Germ Cell
Cancer (TCGA), Uterine Carcinosarcoma (TCGA). Expression was annotated in tumours if DUBs expression was shown to be altered in more than 5 individual cases.

direct transfer of ubiquitin from the E2 to the substrate. However, it has
been proposed that RING ligases may also function by luring ubiquitin
away from the E2 through allosteric mechanisms independent of its
catalytic site [3,4]. The RBR family of ligases share many of the bio-
chemical features present in RING and HECT function including re-
cruiting ubiquitin conjugated E2 to RING1 domains. The bound E2 then
transfers the ubiquitin to the second C-terminal RING domain whereby
the ubiquitin bound at the intrinsic catalytic cysteine is transferred to
the substrate.
The transfer of ubiquitin to the substrate primarily occurs through
the formation of a isopeptide bond between the carboxylic acid group of
glycine 76 (Gly76) of ubiquitin to the epsilon amino group in the target
lysine. Substrates may be modified at a single lysine residues (mono-
ubiquitnation) or at multiple lysine residues simultaneously (multi-
monoubiquitination). Similarly, Gly76 of ubiquitin may attach to lysine
residues on other ubiquitin molecules permitting the formation of poly-
ubiquitin chains. Although the mechanism of chain formation is un-
known the majority of experimental evidence indicates that chains are
built sequentially on the substrate (Reviewed in Ye et al. [6]). However,
a second mechanism of chain formation has been explored whereby
ubiquitin chains are sewn together on the E2's and E3's and transferred
as a single block to its cognate substrate.
Polyubiquitin chain topology is determined by the varying ubiqui-
tin–ubiquitin linkages which may occur. Ubiquitin chains can occur
through specific K6, K11, K27, K29, K33, K48, or K63 linkages. Unlike
HECT domain ligases where the transfer of ubiquitin to the substrate
occurs directly from the HECTs own catalytic cysteine, the E2 ligase is
accountable for the transfer of ubiquitin to the substrate when RING
ligases are involved. Therefore, it stands to reason that the type of
ubiquitination that occurs is determined largely by the characteristics
of the E2. Many RING ligases can bind a number of E2s producing
several types of ubiquitination products. This may explain the recent

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observation that chain formation may either be homotypic (the links in
the chain all occur at a single lysine residue, for example K48) or het-
erotypic (the links in the chain may switch for example from K11 to
K27). Furthermore, some ubiquitin chains have been described to
contain SUMO moieties while others have exhibited a branched for-
mation. All of this indicates that chain topology is eventually likely to
be dependent on the three-dimensional interface between ligase and
substrate. The function of these distorted structures remains to be de-
termined but their discovery opens up a new window into the basic
biology of ubiquitin function. Lastly, linear ubiquitin chains, also
known as M1 linked chains, can be generated through the formation of
a covalent bond between the carboxy terminal glycine of ubiquitin and
the amino terminal methionine of the substrate bound ubiquitin [7].
It is well described that monoubiquitination and polyubiquitination
resulting from the various assortment of linkages described above
convey distinct structural and functional information. For the most part
polyubiquitination is still epitomized by two ubiquitin chain topologies,
K48 and K63. K48-linked chains serve to act as the prototypical de-
gradation signal targeting the protein for proteasome mediated de-
gradation while K63-linked chains perform a number of non-proteolytic
functions including cellular signalling, DNA damage repair, in-
tracellular trafficking and ribosomal biogenesis. Due to the relative
infrequency of the other chain topologies they have been termed aty-
pical linkages with their cellular functions remaining largely elusive
[8].
Similar to the way that protein phosphatases oppose the action of
protein kinases, ubiquitination is a process that is also reversible,
having a family of enzymes that counteract the role of the ligase.
Ubiquitin moieties can be removed from polypeptides by DUBs. The
human genome encodes approximately 80 DUBs which are grouped
into six distinct families: ubiquitin-specific protease (USP, 57 mem-
bers), ubiquitin C-terminal hydrolase (UCH, 4 members), otubain pro-
tease, (OTU, 14 members), motif interacting with Ub-containing novel
DUB family (MINDY, potential 4 members, only 1 is discuss in this
review), and machado-joseph disease protease (MJD, 4 members). USPs
make up by far the largest family of these thiol proteases. The sixth DUB
family, is distinct from the other 5 DUB families in that they possess a
JAMM (JAB1/MPN/Mov34 metalloenzyme, 11 members) domain to
carry out their catalytic function [9,10]. The deubiquitination ability
and substrate specificity of a particular DUB is determined by chain
topology, sub cellular localization and protein complex formation. It is
feasible that any disequilibrium in the ubiquitination-deubiquitination
balance can act as a major contributing factor to tumour formation. To
further understand the potential role of DUBS in tumourgenesis we
performed expression analysis of all the known DUBs across 20 tissue
specific datasets using cBioPortal (Fig. 1). Our analysis reveals that
across many tumour types, there appears to be a general trend towards
DUB amplification. Given that gene copy number alterations are a
common phenomenon in cancer our observations could merely be a
result of this. It is, however, conceivable that the overexpression of K48
specific DUBs, by virtue of stabilizing oncogenes, could potentially be
critical determinants in tumorigenesis. Undoubtedly, a much more in-
depth analysis will be required before this can be claimed with any
certainty. The exception to this is observed in brain and prostate cancer
where deletion of a number of DUBs facilitate tumorigenesis suggesting
that in these cancer types these DUBs operate as potential tumour
suppressors. Nevertheless, in the majority of cases DUB expression and
function (tumour suppressor or oncogene) appears to be tissue and
context specific. For example, USP9X has been shown to stabilize the
oncogenes β-catenin, MCL-1, estrogen receptor, Ets-1, among others,
promoting cancer progression [11–14]. In contrast, USP9X expression is
lost in renal cell carcinoma and pancreatic ductal adenocarcinoma with
downregulation of USP9X corresponding with increased YAP and RAS
signalling, respectively [15,16]. This apparent contradiction in function
has restricted the development of novel inhibitors targeting DUBS and
limited the progression of DUB inhibitors through clinical development.
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BBA - Reviews on Cancer 1868 (2017) 456–483
Therefore, a better understanding of the dynamic interactions of DUBs
and the pathways they regulate is essential. This review focuses on the
tumour suppressor and oncogenic specific functions of E3 ligases and
DUBS in the major pathways involved in cancer progression including
TGFβ, YAP, MAPK, PI3K, and WNT pathways. In reference to the role of
ubiquitin modifying enzymes in the NFKB pathway we refer to the
excellent review by Harhaj et al. [17].
2. The role of ubiquitin modifying enzymes in the TGFβ pathway
The Transforming Growth Factor β (TGFβ) pathway is crucial for
regulating embryonic development as well as maintaining tissue
homeostasis in adult tissues [18–20]. The majority of human cell types
are responsive to TGFβ, through which it regulates cell proliferation,
differentiation, migration, adhesion, apoptosis and various aspects of
the microenvironment [18–20].
TGFβ is a member of a large family of cytokines divided into two
distinct branches: TGFβ, activin, lefty, nodal and myostatin form one
arm, whereas the other comprises of bone morphogenetic proteins
(BMPs), anti-muellerian hormone (AMH) and other growth and differ-
entiation factors (GDFs) [19]. These classifications are simply based on
sequence homology and the distinct pathways that each activates [21].
Activation of the TGFβ pathway requires the ligand induced formation
of a tetrameric complex comprised of a pair of TGFβ receptor I (TβRI)
subunits as well as a pair of TGFβ receptor II (TβRII) subunits [22].
TβRII is a constitutively active serine/threonine kinase and upon ligand
binding it is able to phosphorylate TβRI at a distinct location preceding
the kinase domain called the GS domain [23]. This results in the acti-
vation of the TβRI serine/threonine kinase allowing for effector mole-
cules, Receptor–SMADs (R-SMADs), to be phosphorylated [18–20]. The
R-SMADs belonging to the TGFβ arm of the pathway include SMAD2
and SMAD3, whereas SMAD1, SMAD5 and SMAD8 are attributed to the
BMP pathway [18,19].
SMAD proteins are made up of two globular Mad-homology (MH)
domains, MH1 and MH2, separated by a highly regulated linker region
[24]. The MH1 domain contains the conserved DNA binding motif
capable of recognizing the 51-AGAC-31 DNA sequence, more commonly
known as the SMAD-Binding-Element (SBE). MH2 is highly conserved
and mediates complex binding with other SMADs or SMAD nuclear
complexes. The variable linker region is enriched in proline residues
and contains multiple phosphorylation residues which can be phos-
phorylated by various kinases in response to stimuli such as MAPK, or
CDK8/9 [25].
R-SMADs are phosphorylated at a precise-SSXS c-terminal motif
creating an interaction interface permitting the association with the co-
SMAD, SMAD4. Upon entry to the nucleus the R-SMAD/co-SMAD
complex binds to the SMAD binding element (SBE) sequence on DNA
permitting appropriate transcriptional responses [18,20,21].The R-
SMAD/co-SMAD complex has only a weak affinity for the SBE response
elements, thus full transcriptional activation requires the association of
additional transcription factors [18–21]. This deceptive weakness in
this family of transcription factors appears to be overcome by the
overall flexible nature of the linker region whereby it is thought that the
distinct phosphorylation patterns in the R-SMAD linker region are likely
to result in varying conformational differences which will allow the
various permutations of available transcription factors and the sub-
sequent recruitment of co-repressors or co-activators to dictate whether
a gene will be repressed or activated, respectively, by TGFβ stimulation
[18–20]. Therefore, it is the versatility of the R-SMAD/co-SMAD com-
plex in binding a variety of other transcription factors which accounts
for the pleiotropy response to TGFβ.
Given the fundamental cellular processes that TGFβ regulates one
can speculate that should this signalling pathway be deregulated,
aberrations in cellular function would therefore occur promoting tu-
morigenesis. To investigate the validity of this, one must first under-
stand how TGFβ signalling is regulated. As this review will reveal, the
N. Kumari et al. BBA - Reviews on Cancer 1868 (2017) 456–483

Fig. 2. Schematic overview of regulation of TGFβ pathway by Ubiquitin ligases and deubiquitinating enzymes. (A) Factors that turn off the TGFβ pathway: At the receptor level AIP4/
Itch, in a non-catalytic manner, brings SMAD7 and TGFβRI into close proximity preventing the association of SMAD2 with the latter and thus prevents signalling. SMAD7 itself brings
HECT E3 ligases SMURF1, SMURF2, Tuil1/WWP1 and NEDD4-2 to the receptor complex resulting in complex ubiquitination and degradation. The DUB, USP26, facilitates this event by
increasing the stability of SMAD7 by removing degradative ubiquitin chains. TGFβ pathway signal mediators are also regulated by degradative ubiquitin: SMURF2 targets SMAD2 for
degradation whereas SMAD3 is targeted by U-Box E3 ubiquitin ligase, CHIP; HECT E3 ligase, NEDD4L; and SCF E3 ligase ROC1-SCFFbw1a. Co-SMAD, SMAD4, is regulated by a number of
HECT E3 ligases, namely: SMURF1, SMURF2, Tuil1/WWP1, and NEDD4-2. These ligases associate with and ubiquitinate SMAD4 indirectly by forming a complex with SMAD7, SMAD6 or
activated SMAD2, resulting in the degradation of the former. In many cancers SMAD4 harbors point mutations. In this context these protein variants are targeted for degradation by SCF
E3 ligases, SCFTrCP1β and SCFSkp2. In addition to promoting degradation, ubiquitination is also able to disrupt protein complexes: SMURF2 multi-monoubiquitinates SMAD3 in either the
cytoplasm or the nucleus resulting in the dissociation of the R-SMAD/SMAD4 complex and hence disruption of TGFβ signalling. A similar reaction occurs in the nucleus as E3 ligase
Ectodermin/Tif1γ monoubiquitinates SMAD4 also resulting in the dissociation of the R-SMAD/SMAD4 complex. Once the R-SMAD/SMAD4 complex enters the nucleus to initiate
transcription, here SMAD2 is targeted for degradation as repressive DNA binding proteins SnoN and TGIF direct NEDD4-2 and Tiul/WWP1, respectively, to this R-SMAD, resulting in the
termination of signalling. It has also been shown that the action of an undetermined ligase is able to mono-ubiquitinate the R-SMADs in the R-SMAD/SMAD4 complex thus precluding
complex binding to the DNA. (B) Factors the turn the on the TGFβ pathway: At the receptor level, USP4 is able to interact directly with TGFβRI resulting in its relative persistence at the
plasma membrane level therefore promoting TGFβ signalling. DUBs USP11, USP15 and UCH37 all augment TGFβ signalling by associating with SMAD7, which effects a plasma
membrane localization and therefore stabilizes the receptor complex by way of degradative-ubiquitin removal. SMAD7, itself an antagonist to TGFβ signalling, is degraded by ubi-
quitination by three difference ligases: Arkadia, AIP4/Itch and RNF12. Further downstream, AIP4/Itch has also been observed to ubiquitinate SMAD2, resulting in an augmentation of
TGFβ signalling. The mechanism for this is unclear but this ubiquitination ostensibly promotes SMAD2 and TGFβRI proximity as an AIP4/Itch, SMAD2 and TGFβRI trimeric complex have
been observed in TGFβ stimulated cells (not depicted). Stability of SMAD2/3 is enhanced by the action of OTUB1, which prevents E2 ligase transfer of ubiquitin to the R-SMADs. USP9X
promotes the formation of the R-SMAD/SMAD4 complex by removing the ubiquitin moiety that is attached to SMAD4 by Ectodermin/Tif1γ, therefore relieving steric inhibition. Once in
the nucleus the R-SMAD/SMAD4 complex facilitates the transcription of TGFβ target genes by inducing the destruction of transcriptional repressor SnoN: Arkadia, SMURF2 and CDH1-
APC bind to SMAD2/3 in the nucleus bringing the ligases into close proximity to SnoN allowing for ubiquitin mediated degradation. Finally, as mono-ubiquitination of R-SMADs
precludes the R-SMAD/SMAD4 complex from associating with the DNA, USP15 therefore promotes TGFβ dependent transcription by removing this ubiquitin moiety.

dynamic ubiquitination/deubiquitinating process plays a crucial role in
regulating overall TGFβ signalling at all nodes of the pathway (Fig. 2).
It will also provide pertinent examples of how aberrations in this pro-
cess can lead to cancer formation.
2.1. E3 ligases in the TGFβ pathway
The earliest observation that TGFβ signalling is regulated by ubi-
quitination was in 1999 by Lo and Massague where they observed that
prolonged TGFβ exposure reduced levels of total SMAD2 in a protea-
some dependent manner. Their discovery has led to almost 20 years of
research in ubiquitin dependent regulation of the TGFβ pathway [26].
With regard to ubiquitin mediated regulation the TGFβ superfamily as a
whole, the first major discovery was the identification of the HECT E3
ligase SMAD-specific E3 ubiquitin-protein ligase 1 (SMURF1). This was
found to be a ubiquitin ligase for SMAD1 resulting in the degradation of
SMAD1 and loss of BMP signalling [27]. Subsequent analysis found,
however, that it did not bind to SMAD2, indicating that another E3
ligase was responsible for the negative feedback mechanism originally
identified by Lo and Massague [28]. The SMURF1 homologue, SMURF2
was subsequently shown to bind SMAD2 in a TGFβ dependent manner,
targeting SMAD2 for proteasomal degradation [29,30]. However, both
Bonni et al. and Zhang et al. contest the notion that SMURF2 can de-
grade SMAD2 with Zhang et al. conceding that SMURF2 can degrade
SMAD2 but only when ectopic expression of SMURF2 is high [31,32]. It
is important to note that it remains inconclusive if endogenous SMAD2
can be degraded by SMURF2 [32,34]. Both SMURF1 and SMURF2 are
C2-WW-HECT-domain ligases capable of binding SMADs through an
intermolecular interaction between the SMURFs WW domain and the
PPXY sequence (PY) motif in SMADs [35]. SMURF1 has also been

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shown to ubiquitinate and degrade SMAD5, while SMURF2 also ubi-
quitinates SMAD1 under steady-state conditions [32,36].
NEDD4-like or NEDD4-L is another well-studied E3 ligase, which
belongs to the same E3 ubiquitin ligase family as the SMURF proteins.
NEDD4-L specifically recognizes a threonine phosphorylation site di-
rectly prior to the PY motif in the linker region of R-SMADs allowing
protein-protein recognition and marking SMAD2/3 for proteasomal
degradation. Ostensibly, therefore certain phosphorylation residues in
the linker region enhance SMAD transcriptional action before being
marked for degradation by NEDD4L.
Controversially, recent results by Tang et al. clearly indicate that
SMURF2 does not regulate protein stability of these aforementioned
SMAD proteins. Intriguingly, they demonstrate that in engineered
Smurf2 (−/−) mouse embryonic fibroblasts turnover rates of SMAD2/
3 were equivalent compared to wild type fibroblasts. However, loss of
SMURF2 did appear to alter monoubiquitination at multiple lysine re-
sidues in the MH2 domain of SMAD3. Monoubiquitination of the K333,
K378, and K409 in the MH2 domain of SMAD3 blocks formation of both
homotrimeric SMAD3 and heterotrimeric SMAD3-SMAD4 complexes
invariably limiting these complexes in binding to SMAD motifs on the
DNA [37]. The interesting finding that SMURF2 targets SMAD3 for
monoubiquitination is in line with recent results that show that NEDD4
family members (of which SMURF1 and SMURF2 are included) pre-
dominantly assemble K63 linkages [38]. The fact that SMURF proteins
may only form K63 chains and not K48 chains as was once believed
leads to a host of questions regarding the role of SMURF2 targeting
proteins for degradation. It may be that SMURF proteins may only
prime their targets for entry into early endosomes whereby other ligases
targeted these proteins for degradation. On the other hand, SMURF
proteins might function in a dual role: priming targets through mono-
ubiquitination and later catalysing polyubiquitinated chains depending
on other regulatory proteins bound in the complexes.
Tiul1/WWP1 is another member of the HECT E3 ligase family and
has also been found to promote degradation of SMAD2 [39]. Upon
stimulation with TGFβ, Tiul1/WWP1 interacts with Transforming
growth factor beta-inducing factor 1 (TGIF) and facilitates the forma-
tion of a repressor complex eventually leading to the ubiquitination and
degradation of SMAD2 [40]. Lo and Massague have previously ob-
served that SMAD2 is degraded specifically in the nucleus. This ob-
servation coupled with the fact that Tiul1/WWP1 mediated degradation
of SMAD2 is induced by prolonged TGFβ ligand (16 h) suggests that this
ligase may be responsible for the TGFβ induced negative feedback loop
first observed by Lo and Massague [28].
Importantly, in breast cancer the chromosomal band 8q21 is often
amplified [41]. Tiul1/WWP1 gene is present within this band and it
was found that copy number gain was present in approximately 51% of
breast cancer cell lines and 41% of primary breast tumours [41]. As
already described Tiul1/WWP1 is a negative regulator of TGFβ sig-
nalling and further biochemical analysis confirmed that siRNA tar-
geting of Tiul1/WWP1 in breast cancer lines enhanced TGFβ mediated
responses such as cytostasis and apoptosis [41]. This implies that ele-
vated Tiul1/WWP1 provides an early proliferative advantage in breast
cancer and in particular, in estrogen receptor positive breast cancers
[41,42]. A similar phenomenon is observed in prostate cancer with 44%
of prostate cancer xenografts and cell lines and 31% of prostate clinical
samples possessing a gain in Tiul1/WWP1 copy number [43]. Similar to
breast cancer, siRNA targeting of Tiul1/WWP1 in a prostate cancer cell
line enhanced TGFβ induced cytostasis, again implying that over-
expression of Tiul1/WWP1 has oncogenic capacity [43]. In addition,
hyperactivity of Tiul1/WWP1 has also been noted in prostate cancer
[44].To limit the intrinsic autocatalytic activity inherent to HECT E3
ligases intramolecular interactions between the C2 and HECT domains
maintain the ligase in closed inactive confirmation [41,44,45] Inter-
estingly, prostate cancer derived point mutant WWP1- E798V was
found to be hyperactive due to the disruption of the aforementioned
auto-inhibition mechanism. This disruption lead to hyper-degradation
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BBA - Reviews on Cancer 1868 (2017) 456–483
of the TβR complex hence removing the tumour suppressor effect of
TGFβ signalling [44].
Yet another HECT E3 ligase, NEDD4-2 (neural precursor cell ex-
pressed developmentally down-regulated 4-2), has been found to ubi-
quitinate and degrade SMAD2 [46]. This ubiquitination and degrada-
tion occurs in the presence of activated TGFβ signalling and since
NEDD4-2 can bind nuclear TGFβ repressor SnoN (see later regarding
SnoN) via its association with SMAD2 this suggests that this process
again takes place in the nucleus [46]. NEDD4-2 therefore may con-
tribute along with Tiul1 to the negative feedback instigated due to
prolonged TGFβ exposure.
It has been observed that SMAD3 is also regulated by ubiquitina-
tion. This ubiquitination is mediated by the RING E3 ligase ROC1-SCF-
Fbw1a where this complex binds the MH2 domain of SMAD3 [47]. SCF
complexes are comprised of three proteins, namely, SKP1, Cullins and
F-box proteins, where F-box proteins are important for substrate spe-
cificity [48]. In the aforementioned complex, a RING finger protein
ROC1 associates with the SCF and is crucial for the ubiquitination
function of the entire complex [47]. The degradation of SMAD3 by
ROC1-SCF-Fbw1a is TGFβ dependent. Interestingly, SMAD3 is exported
out of the nucleus by ROC1-SCF-Fbw1a where it is presumed that in the
cytoplasm degradation occurs [47]. This is in contrast to what is ob-
served with SMAD2, as mentioned earlier. Of note, it has been subse-
quently found that U box E3 ligase, CHIP, is able to target SMAD3 ir-
respective of phosphorylation status [49].
In addition to targeting the R-SMADs, ubiquitin based regulatory
mechanisms also exist for co-SMAD, SMAD4. Here again we see that a
negative feedback mechanisms are built in to the regulation of this
protein [50]. When TGFβ ligand is added, the F-box protein β-TrCP1
putatively recognizes a phosphorylated “DLSGLTLQS” motif on SMAD4
resulting in the degradation of the latter [50]. Post translational mod-
ification of this motif remains unproven but it has been postulated that
Jun activation domain binding protein 1 (JAB1) may promote phos-
phorylation of SMAD4 at this motif [50]. Importantly, JAB1 has been
observed to interact with and induce degradation of SMAD4 [51].
SMAD4 is prominently inactivated at the genomic level in pancreatic
cancer and is considered a key driver for tumorigenesis in this context
[52,53]. Furthermore, SMAD4 point mutations isolated from pancreatic
tumours appear to be correlated with errant ubiquitination patterns,
and overall increased protein turnover [54]. This is most likely due to
misfolding of the mutant protein and subsequent ‘quality control’ ubi-
quitination [54]. Furthermore F-box protein SKP2 which has been de-
monstrated to preferentially bind and degrade cancer derived SMAD4
mutants [54]. It interesting to note here that unlike SMAD4, SMAD2
and SMAD3 are rarely mutated in cancer suggesting that this type of
ubiquitin mediated degradation of SMAD2/3 is unlikely to operate in
cancer [19].
Degradative poly-ubiquitination of SMAD4 can also be mediated by
various HECT E3 ligases, including SMURF1/2, WWP1 and NEDD4-2
[54]. It has been proposed that these ligases do not directly interact
with SMAD4 but rather regulate SMAD4 physiological levels through
complex formation with either inhibitory SMADs (I-SMADs) or SMAD2
[54]. As this interaction appears to occur in the cytoplasm this may
suggest a mechanism of quashing latent SMAD4 which can no longer be
recycled following re-entry into the cytoplasm from the nucleus, thus
maintaining tolerable SMAD4 levels quantities for R-SMAD/Co- SMAD
binding. Conversely, Ectodermin/Tif1γ (ECTO) was identified to be a
mono-ubiquitin ligase for SMAD4 specifically ubiquitinating at the
K519 site thus preventing the formation of SMAD4/R-SMAD4 hetero-
trimer complex and inhibiting chromatin binding [57]. With ubiquiti-
nation occurring in the nucleus the monoubiquitinated form of SMAD4
is exported to the cytoplasm. Interestingly, it has been hypothesized
that the acetylation of histones in close proximity to the chromatin
bound SMAD complexes increases the affinity for ECTO thus expelling
the SMAD complex from the promoter regions on the DNA resulting in
the loss of transcription of downstream TGF-β target genes.
N. Kumari et al.
In addition to E3 ligases acting downstream in the TGF-β pathway,
they also mediate TGF-β kinetics at the receptor level. As part of a
transcriptionally regulated negative feedback loop BMP and TGFβ sig-
nalling induces the expression of the inhibitor adaptor SMADs (I-SMAD)
SMAD6 and SMAD7, respectively. I-SMADs are distinct from the R-
SMADs in that they possess weak homology with respect to the SMAD1
MH1 domain [58–60]. The function of these SMADs is principally to
alleviate TGFβ output [58–60]. SMAD6 has been shown to associate
with type 1 receptors TβRI and BMP receptor IB (BMPRIB), mitigating
the levels of p-SMAD2 and p-SMAD1, respectively [59]. It has also been
reported that SMAD6 inhibits BMP signalling by competing with
SMAD1 for physical association with SMAD4 [61]. SMAD7 has two
distinct mechanisms by which it inhibits TGFβ signalling. Firstly,
SMAD7 binds preferentially to activated TβRI hence competing with R-
SMADs and subsequently mitigating pathway activation [58]. However,
a potentially more important role for SMAD7 appears that it is the key
node for ubiquitin mediated regulation of the TGF-β pathway. Me-
chanistically, SMAD7 serves as scaffold to recruit SMURF2 and the E2
ligase UBCH7 to the TGF-β receptor complex to facilitate receptor
polyubiquitination and complex degradation [62].
The interaction of SMAD7 to SMURF2 also has a secondary function.
As previously mentioned, SMURF2 possesses autocatalytic activity
when the protein is unfolded and therefore to maintain its stability and
constrain unwanted activity towards its substrates the C2 and HECT
domains remain in a tightly closed confirmation. The binding of SMAD7
to the HECT domain of SMURF2 abrogates these inhibitory in-
tramolecular interactions between these domains facilitating SMURF2
ubiquitin ligase activity [63]. Besides acting as a scaffold for SMURF2 it
performs a similar function for the E3 ligases NEDD4-2, SMURF1, and
its homologue WW Domain Containing E3 Ubiquitin Protein Ligase 1
(WWP1) also known as Tiul1 (TGIF interacting ubiquitin ligase 1),
which also target the type I TGF-β receptors for ubiquitin mediated
degradation [39,64–66]. The existence of this apparent redundancy in
function between the HECT E3 ligase family is unclear and may ulti-
mately be context dependent. Furthermore, CD109 also binds to the
SMURF2/SMAD7 complex and regulates receptor ubiquitination and
degradation in a ligand dependent manner [67].
It has been observed in renal cell carcinoma that TGFβ signalling is
depressed when compared with normal renal tissues, with a decrease in
signalling being correlated with a post-translational decrease of TβRII
protein levels [68]. In fact, in renal cell carcinoma tissues the levels of
SMURF2 protein are inversely correlated with TGFβRII [68]. High le-
vels of SMURF2 also correlate with poor prognosis in Esophageal
Squamous Cell Carcinoma [69]. It was found that in this cancer,
SMURF2 is overexpressed compared with normal tissues and this high
expression was correlated with depth of invasion, and lymph node
metastasis [69]. Unlike renal cell carcinoma, the main substrate of
SMURF2 in this context appeared to be pSMAD2.
Atrophin 1-interacting protein 4/ITCH(AIP4/Itch) subsequently to
be referred to as ITCH is another HECT E3 ligase that acts to inhibit
TGFβ signalling [70]. In contrast to other members of the HECT family,
in this context, ITCH operates independently of its ligase activity [70].
ITCH appears to strengthen the association between SMAD7 and acti-
vated TGFβRI (without affecting turnover of TGFβRI) and in this way
blocks R-SMAD access to the receptor kinase domain [70]. In a con-
trasting study, Bai et al. observed that ITCH enhances pSMAD2 levels
by ubiquitination of SMAD2. They observe the formation of a trimeric
complex between ITCH, SMAD2 and TGFβRI in stimulated cells and
therefore postulate that this ubiquitination promotes SMAD2 proximity
with TGFβRI thus facilitating overall signalling [71]. In addition, ITCH
has also been shown to act as a positive regulator of the TGFβ pathway
by ubiquitinating and inducing degradation of SMAD7 [72]. It is un-
clear why ITCH facilitates such opposing effects with respect to TGFβRI
activity but it is likely to be context dependent.
The ubiquitin-proteasome system can also positively regulate the
TGFβ cascade. ARKADIA, a RING-finger containing E3, targets multiple
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BBA - Reviews on Cancer 1868 (2017) 456–483
negative regulators for proteasomal degradation including, SMAD7, c-
Ski, and SnoN [73,74]. As previously mentioned HECT E3 ligases bind
to PY domain of SMAD7, in contrast, ARKADIA binds to the MH2 do-
main of SMAD7 and induces the polyubiquitination and degradation of
SMAD7 leading to enhanced TGFβ signalling [74]. This uncovers per-
haps an interesting insight to how whereabouts of binding to SMAD7
influences whether a particular ligase will degrade SMAD7 or simply
uses it as an adaptor protein.
C-Ski and SnoN are related transcriptional co-repressors which as-
sociate with the R-SMAD-SMAD4 complexes and recruit histone dea-
cetylases (HDACs) to prevent transcription of TGFβ target genes [31].
SnoN levels are partially regulated through ligand induced phospho-
SMAD2 whereby phospho-SMAD2 translocates to the nucleus forming a
complex with ARKADIA targeting SMAD2 bound SnoN for ubiquitin
mediated degradation. Interestingly, ARKADIA binds and ubiquitinates
SnoN in the absence of TGFβ signalling [76]. Nevertheless, it is only
upon TGFβ pathway activation that efficient ARKADIA induced SnoN
degradation occurs due the formation of a phospho-SMAD2 -SnoN-
ARKADIA complex [76]. This insinuates that phospho-SMAD2 is re-
quired for proteasome targeting [76]. Interestingly, under conditions
where ARKADIA expression is repressed phospho-SMAD2/3 accumu-
lates in the nucleus but appears to be non-functional. Furthermore,
reconstitution of ARKADIA in null embryonic stem (ES) cells results in a
restoration of SMAD2/3 transcriptional activity. However, counter-
intuitively, under these conditions phospho-SMADs gets degraded in
the process [77]. This coupling of activation with degradation has been
suggested to provide a mechanism to regulate appropriate transcrip-
tional responses to ensure that active transcription is transient and that
transcription factors do not perpetually remain on promoter sequences.
Thus, once transcription has been initiated utilized SMAD complexes
will be targeted for degradation or recycling, allowing new SMAD
complexes to bind to the DNA. This termination signal is then one
mechanism to ensure that the level of extracellular TGFβ ligand pro-
duces the desired physiological responses. Whether the accumulation of
nuclear SMADs in ARKADIA null ES cells is the result of increased SnoN
expression and the inhibition of binding of SMAD complexes to DNA
remains inconclusive but is likely to be part of the process.
Nagano et al. report that ARKADIA can bind and degrade SnoN, as
well as c-Ski, a SnoN related repressor, independently of TGFβ signal-
ling, contrasting previous observations [73,78]. Incidentally SMURF2
has also been demonstrated to target SnoN via SMAD2 [79]. SMAD2 is
essential for this process as the utilization of SMAD2 deleted for its PY
motif limited the ability of SMURF2 to bind SnoN [31].
Interestingly, SMAD3 also functions to degrade SnoN but in this
scenario degradation occurs via the RING E3 ligase, Anaphase
Promoting Complex (APC)-CDH1 complex. CDH1 acts as an adaptor for
specific substrate recognition [80]. It is reported that this action is also
TGFβ dependent as APC only interacts weakly with SnoN in the absence
of ligand. This can be explained by the fact that destruction box motif
(D- Box) within SnoN, which the CDH1 subunit recognizes, is not per-
fect thereby limiting CDH1-SnoN affinity [80]. SMAD3 is therefore
required for efficient association of the APC complex with SnoN [80].
Recalling the observation made by Bonni et al. that phospho-SMAD2 is
required for SMURF2 recruitment to degrade SnoN, employing a
SMAD3 mutant incapable of SMURF2 binding similarly led to reduced
degradation of SnoN [79,80]. This suggests that both SMURF2 and APC
mediated degradation of SnoN operates concurrently for maximal effect
[80]. The contextual importance of the TGFβ negative regulator
SMURF2 repressing SnoN, another negative regulator remains to be
determined.
ARKADIA may also be involved in endocytosis of TβR as it has been
shown to bind and ubiquitinate the μ2 subunit of AP-2, which is in-
volved in the formation of clathrin coated pits [81]. ARKADIA's overall
importance in terms of cancer remains inconclusive. Although the loss
of ARKADIA is rare in human cancers a number of missense mutations
have been localized in colorectal cancer patients correlating with SnoN
N. Kumari et al.
stabilization thus potentially confirming the role of ARKADIA as an
tumour suppressor [82] [83]. Furthermore, mice heterozygous for
ARKADIA possess tumours with enhanced nuclear SnoN and decreased
TGFβ signalling leading to colorectal cancer development [82]. Finally,
it has been found that in the esophageal cancer cell line (SEG1) which
exhibits low levels of ARKADIA expression, degradation of SnoN is also
subsequently impaired [76].
In addition to activation of R-SMADs through the canonical TGFβ
signalling pathway, TGFβ also the recruits tumour-necrosis factor re-
ceptor (TNFR)-associated factor TRAF4 to the TGFβ receptor complex
restraining the E3 ligase activity of SMURF2 towards the receptor
complex thereby limiting ubiquitination and degradation and thus
maintaining TGFβ activity. Furthermore, TRAF4 is also able to recruit
the deubiquitinating enzyme USP15 to aid in this process (see below).
RNF12 has also been identified as a ligase for SMAD7 degradation [84].
2.2. DUBs in the TGFβ pathway
Considering the importance of the TGFβ pathway it is not surprising
that TGFβ signalling is heavily regulated by the ubiquitination process,
which is involved in crucial negative feedback loops, quality control
mechanisms and constitutive degradation to maintain homeostasis.
Ubiquitination is a reversible process and this adds another level of
regulation to the TGFβ pathway. This reversal process mediated by
deubiquitinating enzymes (DUBs) provides further opportunities for
aberrations to arise and facilitate cancer progression.
TβR stability and turnover functions as a principle juncture for the
downregulation of the pathway. Counteracting the ubiquitination and
degradation of the TβR complex six DUBs (USP4, USP11, USP15,
USP19, and UCH37) have been identified that directly affect TβR
deubiquitination and stability [62,85–87]. Making use of genome wide
DUB libraries three independent groups identified USP15 as a critical
component of the TGF-β pathway. Interestingly, akin to many of the E3
ligases USP15 appears to target a number of different nodes in the TGFβ
pathway. At the receptor level USP15 forms a complex with SMAD7 and
SMURF2 with USP15 opposing the effects of the SMURF2 ligase on TβR
stabilization [87,88]. In this context, the scaffold protein SMAD7 in-
teracts with two enzymes harbouring contrasting activities resulting in
a constant balancing act regulating TGF-β output. Recently, it has been
shown that TGFβ upregulates the translation of USP15 suggesting a
positive feedback loop, however, the extent of the TGFβ activity also
regulates the access of USP15 to the SMAD7-SMURF2 complex [87,89].
As a result, when the TGFβ signal is low SMAD7 engages both SMURF2
and USP15 to maintain TβR stability thus retaining low levels of TGFβ
output. However, when excessive levels of TGFβ is present USP15 is
dissociated from the SMAD7-SMURF2 complex leading to enhanced
ubiquitination of the TβR and degradation of the complex. This gen-
erates an elegant rheostat whereby TGFβ regulates its own activity
preventing hyperactivation of the signal cascade.
USP15 also appears to regulate other nodes within the canonical
TGF-β pathway. As well as acting as a DUB for the TβR, USP15 has
recently been identified to deubiquitinate monoubiquitinated and
polyubiquitinated isoforms of R-SMADs [90]. Post translational mod-
ifications within the DNA binding domains of R-SMADs prevents pro-
moter recognition and aberrant TGFβ signalling. As previously dis-
cussed, Tang et al. demonstrated that SMURF2 knockout MEFs, rather
than displaying the expectant stabilization of known substrates merely
exhibited decreased mono-ubiquitinated isoforms of SMAD3 [37].
These specifically occurred at K333, K378, and K409 within the MH2
domain. In contrast, USP15 deubiquitinates mono-ubiquitinated
SMAD3 at K81 and to a lesser degree at K33 and K53 suggesting that
USP15 deubiquitinates R-SMADs in the MH1 domain at sites in-
dependent of SMURF2 function [90]. Notably, all of these ubiquitina-
tion sites disrupt the R-SMAD/SMAD4 heterodimer from binding to the
chromatin. USP15 reverses this modification permitting SMAD tran-
scription factor binding and full TGF-β transcriptional responses. This
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BBA - Reviews on Cancer 1868 (2017) 456–483
of course leaves us with the question of what is the E3 ligase that
monoubiquitinates the lysine residues within the MH1 domain.
The fact that TGFβ enhances USP15 proteins levels but at the same
time dissociates USP15 from the SMAD7/SMURF2 complex is in intri-
guing enigma. Recently, it has been demonstrated that USP15 binds to
and stabilizes the transcription factor p53 [89]. Under physiological
conditions p53 can form complex with SMAD2/3 to co-activate tran-
scription of an assortment of genes involved in tumour suppression. It
may therefore be that under certain conditions USP15 acts as a general
regulator of the TGFβ tumour suppressor function by deubiquitinating
both R-SMADs and its co-transcriptional regulator p53 enhancing TGFβ
mediated transcription of genes. However, in scenarios where p53 is
mutated or lost and the levels of USP15 are abnormally high, this may
switch the binding of SMAD transcription factors from the promoters of
tumour suppressor genes to those governing more pro- proliferative
functions. This appears to be the case in certain cancers as USP15 gene
was demonstrated to be amplified in glioblastoma, breast and ovarian
cancers Using a number of independent samples sets, Eichhorn et al.
was able to confirm that USP15 expression is highly correlated with
TGFβRI and pSMAD2 in these cancers. Furthermore, glioblastoma pa-
tients with relatively high copy numbers of USP15 have a significantly
poorer prognosis compared to those with lower copy numbers [91].
As previously mentioned a number of DUBs including USP4, 11, and
19 have been identified as potent regulators of the signalling cascade.
All of these DUBs bound to the TβRI directly resulting in decreased
levels of TβRI ubiquitination and stabilization. The existence of DUBs
with apparent overlapping functions can only attest to the level of
complexity required for internalization and compartmentalization of
receptors at the cell surface. Interestingly, the fate of TGFβR is de-
pendent upon a continuous interplay between ligases and DUBS to
regulate ubiquitination and internalization. This is not only determined
by transitory ubiquitination state of the receptor but of all components
of the receptor complex including the ligases and DUBs themselves.
Preliminary results by Ten Dijke and colleagues add weight to this
theory whereby they identified that the ability of USP15 to deubiqui-
tinate TβRI requires USP4, as USP15 was unable to perform this func-
tion in USP4 deficient cells [85]. Furthermore, they demonstrate that
AKT phosphorylation of USP4 enhances the binding of USP4 to USP15
and that overexpression of USP15 increases USP4 stability. Under these
conditions AKT hijacks USP4 to stabilize the TβRI and reinforces the
pro-tumorigenic responses of the TGFβ pathway. Notably, USP4 is
overexpressed in breast cancer and may function as an oncogene to
maintain the TGFβ breast cancer progression [92]. Regarding the cross
talk observed between enzymes regulating ubiquitination and inter-
nalization of receptors it would be of great surprise if AKT did not
regulate other components of this complex.
Not unlike USP15, two other DUBS USP11 and UCH37 are able to
bind SMAD7 and target the TGFβ receptor for deubiquitination [62,93].
In the case of USP11, SMAD7 does not appear to act exclusively as a
scaffold for as SMAD7 does not appear to mediate the interaction be-
tween USP11 and TβRI, despite the fact that SMAD7 and USP11 being
able to form a complex. In addition, the interaction between USP11 and
SMAD7does not appear to be greatly affected by TGFβ ligand. However,
besides acting as TβRI deubiquitinases both USP15 and USP11 are able
to regulate their counterpart the E3 ligase SMURF2 albeit through ap-
parently divergent mechanisms [88]. USP15 appears to target key re-
sidues within the HECT domain of SMURF2 thereby limiting the cata-
lytic activity of SMURF2 towards it's substrates, while USP11 appears to
increase overall SMURF2 ubiquitination. The effect of USP11 in this
context is as of yet undetermined but it may suggest that USP11 may
indirectly regulate SMURF2 stability.
All the DUBs mentioned thus far, bar USP4 and USP11, utilize
SMAD7 as a scaffold protein to mediate their effects. Recently, we
identified that as part of a negative feedback loop TGF-β not only en-
hances the expression of SMAD7 but also of USP26, whereby USP26
acts as a SMAD7 K48 ubiquitin chain specific DUB rescuing SMAD7
N. Kumari et al. BBA - Reviews on Cancer 1868 (2017) 456–483

Fig. 3. Schematic overview of regulation of Hippo pathway by Ubiquitin ligases and deubiquitinating enzymes. Activation of the Hippo pathway results through multiple extracellular
mediums including cell-cell contact or mechanical tension leading to the downstream activation of two key core inhibitory kinase modules. The first of these comprises of MST1 and MST2
and the second module comprises of LATS1 and LATS2 (Drosophila Hippo and Warts, respectively). MST1/2 in combination with its binding partner Sav1 permits phosphorylation and
kinase activation of LATS1/2 and the LATs co-factors MOB1A and MOB1B. Downstream transcription is mediated by YAP and its paralogue TAZ. These two transcription factors shuttle
back and forth between the cytoplasm and the nucleus where they regulate transcription of a multitude of genes primarily through TEA domain family members (TEAD). LATS1/2
phosphorylation of the YAP/TAZ complex displaces YAP/TAZ to cytoplasm inhibiting YAP/TAZ mediated transcription. Phosphorylation of YAP/TAZ by LATS1/2 occurs in a unique
phosphodegron site inducing the recruitment of the E3 ligase β-TrCP resulting in polyubiquitnation and degradation of YAP/TAZ. Similar to LATS1/2, Angiomotin (AMOT) regulates
hippo pathway activation by directly binding to YAP/TAZ resulting in cytoplasmic retention of both proteins. Ubiquitination regulates numerous proteins in the hippo pathway. AMOT
itself undergoes polyubiquitination and degradation by the E3 ligases NEDD4 and ITCH inhibiting AMOT regulation of YAP/TAZ. USP9X has been shown revert this effect. NEDD4, ITCH,
its HECT family member WWP1, and SIAH2 also ubiquitinate and degrade LATS1/2, and NEDD4 has also been demonstrated to perform a similar function towards SAV1. The E3 ligase
PRAJA2 ubiquitinates and inhibits the stability of MOB1. All of which result in downregulation of hippo pathway activation and enhanced YAP/TAZ transcription. The deubiquitinating
enzyme DUB3 deubiquitinates and stabilizes multiple components of the hippo pathway including the negative regulator ITCH resulting in AMOT and LATS1/2 degradation. Remarkably,
DUB3 can also deubiquitinate AMOT and LATS enhancing hippo activity.

from degradation. This then permits SMAD7 to remain in stable con-
firmation with SMURF2, permitting SMURF2 recruitment to the TGF-β
receptor complex potentiating complex degradation [94]. Other I-
SMAD regulatory mechanisms involve two members of the deubiqui-
tinating enzyme JAMM subfamily, associated molecule with the SH3
domain of STAM (AMSH) and its homologue AMSH-2 sequester SMAD6
and SMAD7, respectively, suppressing the inhibitory action of these I-
SMADs towards their targets although it has never been conclusively
determined if their deubiquitinating enzyme activity was required for
this function [95,96]. The exact targets of these DUBs are also unknown
but it is thought that AMSH is required for the regulation of TβR
turnover by the endosomal sorting complexes required for transport
(ESCRT) formation [97].
R-SMADs are also regulated by DUBs. OTUB1 has been found to
modulate TGFβ signalling by promoting the stabilization of only the
phosphorylated form of SMAD2/3 [98]. Interestingly, it was found that
the mechanism is non-canonical and non-catalytic: instead of OTUB1
directly deubiquitinating SMAD2/3 rather it interacts with the E2 li-
gases prevents the transfer of ubiquitin from the E2 ligase to the E3.
This non-canonical functionality of OTUB1 has been observed prior on
multiple occasions [99–102]. Importantly, OTUB1's role in modulating
TGFβ output has been established as a knockdown resulted in an in-
hibition of TGFβ induced migration [98] This lends support of a po-
tential role of OTUB1 in cancer, though this is yet to be verified.
By modulating the activity of co-SMAD, SMAD4, USP9X/FAM has
also been found to modulate the TGFβ pathway [57]. To counteract the
monoubiquitination of SMAD4 by Ectodermin at K519 USP9x/FAM acts
to remove the ubiquitin moiety in the cytoplasm permitting SMAD4 to
once again form a complex with phosphorylated forms of SMAD2. The
juxtaposed activities of ECTO and USP9X function as a feedback loop to
regulate overall SMAD transcriptional output by initially permitting
SMAD2/SMAD4 complex formation and then by terminating tran-
scriptional output. Recent results have demonstrated the importance of
this as abrogation of ECTO expression impeded proper embryonic de-
velopment [103]. Loss of SMAD4 is a common event in pancreatic
cancer and colon cancer, and loss of USP9X has recently been shown to
collaborate with KRAS to induce pancreatic cancer. Although the role of
USP9X in this scenario may likely be due to the stabilization of YAP it
cannot be discounted that inhibition of the TGFβ plays an integral role
in this process.
3. The role of ubiquitin modifying enzymes in the hippo pathway
The importance of the Hippo pathway was original recognized in
Drosophila through the use of genetic mosaic screens whereby random
loss-of-function mutations in components of this pathway gave rise to
severe organ overgrowth. It was later elucidated that these observed
effects were the result of increased cellular proliferation, decreased

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N. Kumari et al.
apoptosis and enhanced progenitor stem cell renewal. The observation
that this pathway is also conserved in mammals has resulted in a flurry
of intensive research. The integrity of the Hippo pathway is absolutely
essential in early development. In some adult organs however, Hippo
activity is expendable for normal homeostasis but is indispensable for
tissue repair and regeneration upon damage. Furthermore, experi-
mental evidence has indicated a strong correlation between abnormal
Hippo pathway activation and oncogenesis. The disparity between the
requirement of Hippo signalling in cancer and its relative dispensability
in adult human tissues has led to the Hippo pathway being considered
as a very attractive therapeutic target in cancer. Several early phase
clinical trials have been initiated and it will be interesting to see if any
clinical responses will be observed in patients receiving these com-
pounds.
The canonical Hippo pathway consists of two central components,
regulatory and transcriptional, which control overall Hippo activity.
The core regulatory component is a kinase cassette comprised of the
Mammalian sterile-20-like (MST1/2, orthologue of Drosophila Hippo)
and the adaptor protein Salvador family WW domain-containing pro-
tein 1 (SAV1). Complex formation of MST1/2 and SAV1 permits
phosphorylation and activation of large tumour suppressor 1(LATS1)
and LATS2 and the LATS co-factor MOB kinase activator protein 1A
(MOB1A) or MOB1B (reviewed in [104]).
The transcriptional component of the pathway is composed of the
yes-associated protein (YAP) and its paralogue, transcriptional co-ac-
tivator with PDZ-binding motif (TAZ). These two transcription factors
shuttle back and forth between the cytoplasm and the nucleus where
they regulate transcription of a multitude of genes primarily through
TEA domain family members (TEAD). When the Hippo pathway is ac-
tivated the LATS/MOB complex directly phosphorylates and inhibits
the transcriptional co-activators YAP and TAZ through 14–3-3 mediated
cytoplasmic retention. The Hippo pathway is further regulated by nu-
merous upstream molecules, which regulate Hippo signalling through a
multitude of post-translational modifications (reviewed extensively in
[105–107]). Additionally, the mechanism of action of ubiquitination
and deubiquitination in the Hippo pathway is starting to fall into place
(Fig. 3). The most documented of these is the priming of YAP and TAZ
for proteasomal mediated degradation following phosphorylation by
LATS. LATS phosphorylation of YAP and TAZ serves to retain YAP and
TAZ in the cytoplasm while concomitantly priming them for degrada-
tion. The phosphorylation by LATS occurs in a unique phosphodegron
site permitting recognition by the β-TrCP/SCF ubiquitin ligase complex.
Recently, it has also been demonstrated that the RAS oncogene may
serve to inhibit YAP protein turnover through two independent me-
chanisms. RAS has been shown to downregulate the ubiquitin ligase
complex substrate recognition factors SOCS5/6 which serves to target
YAP to the B/C-Cullin 5 ubiquitin ligase for proteasomal degradation.
Furthermore, the RAS isoform, H-RAS, induces the formation of MST1/
MST2 heterodimers muffling the kinase activity of MST1 towards LATS
resulting in downregulation of the pathway and YAP/TAZ nuclear lo-
calization [108].
While degradation of YAP and TAZ is critical to the overall tumour
suppressor activity of the Hippo pathway, ubiquitin mediated regula-
tion of pathway has been demonstrated to act at multiple nodes along
its axis. A number of members of the Neural Precursor Cell Expressed,
Developmentally Down-Regulated 4 (NEDD4) family of E3 ubiquitin
ligases including the E3 ubiquitin-protein ligase Itchy homologue
(ITCH), WW Domain Containing E3 Ubiquitin Protein Ligase 1(WWP1),
and NEDD4 itself have been shown to regulate the stability and abun-
dance of LATS1 kinase [109–112]. Although these homologues share a
common functional architecture suggesting redundancy in their activity
recent work has begun to identify the mechanisms that underlie their
specificity [113]. As such their activity towards LATS may be likely
attributed to particular extracellular signals leading to specific complex
formation. An example of this has been shown to specifically occur with
NEDD4. NEDD4 directly binds to the previously mentioned LATS
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BBA - Reviews on Cancer 1868 (2017) 456–483
adaptor SAV1 concomitantly ubiquitinating and degrading both SAV1
and LATS. Interestingly, the upstream kinase MST1 negatively regulates
the ability of NEDD4 to target SAV1 but in a disconnected fashion is
unable to inhibit NEDD4 mediated degradation of LATS [110]. Fur-
thermore, the authors indicate that the proficiency of MST1 to inhibit
NEDD4 activity towards SAV1 is highly dependent on confluency of the
cells. In such a scenario high cell density activates the Hippo pathway
by increasing phosphorylated active MST1 releasing the negative in-
hibition of NEDD4 towards SAV1 and silencing YAP transcription. In-
cidentally, of the NEDD4 family members only NEDD4 is capable of
binding SAV1 indicating that NEDD4, ITCH, and WWP1 interact with
other adaptor proteins to regulate overall LATS1/2 levels. Similarly, in
response to hypoxia the E3 ubiquitin ligase SIAH2 ubiquitinates LATS2,
resulting in its destabilization [114]. Finally, the RING ligase PRAJA2
induces proteasome mediated degradation of MOB1 [115].
Mechanotransduction and the associated actin dynamics translate
the cells kinetic physical signals with the required downstream che-
mical cues to direct both short term and long term signalling. How actin
specifically plays a role in downstream YAP and TAZ signalling remains
to be elucidated. Angiomotin (AMOT) is a filamentous actin binding
protein that predominantly localizes to cellular junctions. AMOT and
two AMOT-like proteins, AMOTL1 and AMOTL2 regulate Hippo
pathway activation by directly binding YAP and TAZ resulting in cy-
toplasmic retention of both proteins [116]. Interestingly, the interac-
tion between AMOT and YAP or TAZ occurs independent of YAP and
TAZ phosphorylation by LATS even though LATS enhances complex
formation between these proteins by directly phosphorylating AMOT
itself [117]. This indicates that LATS inhibits the nuclear localisation of
YAP and TAZ by two independent mechanisms; enhanced AMOT
complex formation and targeting YAP and TAZ for proteasomal medi-
ated degradation. AMOT proteins themselves have been shown to be
ubiquitinated on multiple residues with opposing effects on protein
function mediated by different ubiquitin topologies. Mono-ubiquitina-
tion of K347 and K408 is required for AMOTL2 to bind to the LATS1/2
complex and may act as a scaffold to permit LATS phosphorylation and
inactivation of YAP and TAZ [118]. In contrast, polyubiquitination at
K496 of AMOT targets AMOT for degradation resulting in YAP nuclear
localization [15]. Curiously however, both of these studies identify
USP9X as the deubiquitinating enzyme regulating both the mono-
ubiquitination and polyubiquitination of AMOT. This contradictory
result would suggest that deubiquitination of monoubiquitinated
AMOTL2 by USP9X would inhibit the ability of LATS to phosphorylated
YAP and TAZ, permitting YAP and TAZ to translocate to the nucleus and
induce target gene expression. In contrast, however, Nguyen and col-
leagues demonstrate that USP9X deubiquitinates polyubiquitinated
AMOT stabilizing AMOT and sequestering YAP and TAZ in the cyto-
plasm [15]. Further work will be needed to address the differences
between these two studies and identify the function of USP9X in the
YAP pathway. Recently, several groups have shown the importance of a
functional YAP1 pathway in KRAS mediated oncogenesis
[108,119–121]. In particular two independent studies revealed that loss
of YAP1 expression prevented cancer progression in KRAS driven
mouse models of pancreatic ductal adenocarcinoma and lung cancer
[122,123]. Data by Nguyen et al. indicate that USP9X transcript levels
are significantly lower in thyroid, prostate, liver, hepatocellular carci-
noma and kidney renal clear cell carcinoma with low USP9X expression
correlating with significantly worse disease free survival in these later
patients [15]. Also, USP9X appears to be a major tumour suppressor
gene in pancreatic ductal adenocarcinoma, as genetic abalation of
USP9X co-operated with KRAS to accelerate pancreatic tumourigenesis
[16]. Furthermore, USP9X has been identified to mutated or deleted in
4% of pancreatic adenocarcinomas. Ostensibly in the above pre-clinical
contexts, loss of USP9X stabilizes YAP therefore indirectly enhancing
RAS driven oncogenesis through YAP activation. As YAP inhibitors are
currently being tested in early phase clinical trials the potential that
USP9X may be a biomarker for response for YAP inhibition must be
N. Kumari et al.
established.
The stability of AMOT does not only appear to be regulated by
USP9X, but recent data has also shown that the deubiquitinating en-
zyme DUB3 plays integral role in AMOT stability. However, like a
number of DUBs in the TGFβ pathway DUB3 appears to act at several
levels to regulate Hippo pathway activation. First, DUB3 deubiquiti-
nates and stabilizes the E3 ligase ITCH. As, ITCH mediated ubiquiti-
nation has been shown to promote the turnover of LATS and AMOT
proteins this suggests that DUB3 acts to enhance YAP activity.
Interestingly DUB3 also interacts with and deubiquitinates AMOT,
AMOTL1, and the kinases LATS1 and LATS2 to promote their stability
and in so doing decrease YAP activity [124]. The authors hypothesis the
primary function of DUB3 is to stabilize the scaffold protein AMOT
permitting an unperturbed complex formation between AMOT and the
LATS kinases to downregulate YAP. The purpose of the seemingly
contradictory function of DUB3 with respect to ITCH in this setting
remains elusive but it is feasible that DUB3 binding to the AMOT/LATS
complex may be regulated by post translational modifications whereby
in the event that loss of binding to AMOT occurs, DUB3 can then act as
a potent activator of the pathway by stabilizing ITCH subsequently
leading to degradation of AMOT and LATS.
4. The role of ubiquitin modifying enzymes in the MAPK pathway
Mitogen-activated protein kinases (MAPKs) are protein Ser/Thr ki-
nases that convert proliferative signals at the cell surface receptors into
a wide range of cellular responses. The importance of this pathway lies
in maintaining a sensor dependent dynamic communication between
cells and their surrounding environment. Many studies have empha-
sized sustained proliferative signalling due to hyperactive MAPK
pathway as one of the main hallmarks of neoplastic diseases [125]. In
normal cells the classical MAPK cascade is sequentially relayed through
RAS induced phosphorlyation of MAPKKKs A-RAF, B-RAF, and C-RAF
(also known as RAF1) following to MAPKKs MEK1 and MEK2, and
eventually MAPKs ERK1 and ERK2. The culmination of ERK signalling
results in regulation of genes involved in activation of cell cycle, pro-
liferation, survival, cell migration among others. In order to maintain a
systemic balance and to ensure that extracellular signals elicit appro-
priate downstream responses a number of mechanisms have evolved to
regulate ERK activation, including ubiquitination (Fig. 4). The fact that
ubiquitination is reversible and that ubiquitinated proteins are known
to interact with a plethora of ubiquitin interacting proteins involved in
signalling highlights the influence of ubiquitination as not only a de-
gradative signal. In no other context is this more evident than in the
crucial process of endocytosis. Initial activation of most pathways is
stimulated by ligand induced dimerization of cell surface receptors re-
sulting in autophosphorylation of Tyr residues in the intercellular do-
main creating docking sites for protein adaptors. This autopho-
sphorylation event is more often than not associated with concomitant
ubiquitination of the receptors. The addition of the ubiquitin moiety is
required for cargo internalization and acts as a pilot shuttling the in-
ternalized receptor towards active signalling nodes, recycling chutes, or
a degradative fate. As a number of excellent reviews on the role of
ubiquitination in endocytosis has been written we will focus this seg-
ment on the ubiquitination enzymes which effect canonical down-
stream MAPK signalling [126,127]. Briefly, however, it must be noted
that regulation of internalization and endosomal sorting involves the
concerted action of an assembly of ligases and DUBs including for ex-
ample USP8, USP2A, USP9X, AMSH, and Cezanne [126,128]. USP8 has
been reported as an important factor for rescuing transmembrane re-
ceptors such as EGFR, HGFR (c-MET), ERBB2 and ERBB3 receptors
from endocytosis mediated lysosomal degradation thereby further
mediating downstream MAPK activation [129,130]. Interestingly,
overexpression of constitutively active mutant SRC (SRC Y527A) sig-
nificantly increases USP8 tyrosine phosphorylation [131]. Moreover,
EGF stimulation of EGFR induces USP8 tyrosine phosphorylation while,
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in contrast, TGF-α reduces tyrosine phosphorylation [131]. This phos-
phorylation of USP8 at S680 permits complex formation with 14-3-3
proteins [131]. 14-3-3 proteins are highly important for regulation of
various pathways in cells including cell cycle progression, DNA da-
mage, apoptosis, transcriptional regulation of genes etc. It has been
postulated that 14-3-3 binding to USP8 may result in: alterations in its
enzymatic activity, changes in subcellular localisation or 14-3-3 may in
fact mediate the binding of other proteins affecting USP8 activity in an
undefined way [132]. However, some studies have shown the role of
USP8 in deubiquitinating and stabilizing a RING finger containing E3
Ubiquitin ligase called Nrdp1 which is involved in degradation of
ERBB3 and ERBB4 receptor tyrosine kinases [133]. These results are
opposite of the results seen by Niendorf et al. [130] showing that de-
letion of USP8 leads to lower level of ERBB3. Further investigation is
needed to completely characterize the role of USP8 in regulation of
ERBB receptor tyrosine kinase family. Possibly, the functional network
between USP8, Nrdp1 and their targets such as EGFR family receptors is
tissue and context specific dependent.
Similarly, it has been shown that ubiquitin specific protease 2A
(USP2A) is localized in endosomes and prevents EGFR degradation
[134]. Liu et al. showed that overexpressing wild type USP2A, but not
mutant USP2A, led to stabilization of EGFR and enhanced downstream
signalling. Furthermore, overexpressing USP2A in human bladder cells
significantly extended the duration of response to Heparin binding EGF
like growth factor (HB-EGF) [135]. USP2A overexpressed cells could
maintain high level of p-ERK from 5 to 60 min after treatment with HB-
EGF while control cells showed only a transient increase in p-ERK level.
This was probably due to the potential role of USP2A in deubiquiti-
nating and stabilizing EGFR receptor.
Insulin Growth Factor recptor-1 (IGF-1) has been shown as an im-
portant initiator of MAPK and PI3K pathway activation. Intriguingly,
mutant K1003R IGF-1R, which is not able to bind to ATP, is markedly
less ubiquitinated than wild type IGF-1R upon stimulation with IGF,
suggesting that ubiquitination of IGF-1R is dependent on its autopho-
sphorylation [136]. Also, C-terminal domain of IGF-1R is needed for
ubiquitination and IGF induced ERK activation. C-terminal truncated
IGF-1R (Δ1245) is not ubiquitinated after IGF treatment and does not
induce MAPK pathway activation while no change was reported in PI3K
pathway activation [136]. Moreover, treating 3T3-like fibroblasts with
lysosome inhibitor (Chloroquine) completely rescued IGF-1R protein
level from degradation while proteasome inhibitor (epoxomicine) was
only able to partly rescue this receptor [136]. Although, degradation of
truncated Δ1245 IGF-1R, which is not ubiquitinated, was completely
rescued by lysosome inhibitor, further studies are needed to investigate
whether ubiquitination of IGF-1R has any role in degradation of this
receptor.
Receptor Tyrosine Kinases (RTKs) such as EGFR and IGF-1R bind
growth factor receptor bound protein-2 (GRB2) following ligand in-
duced activation. GRB2 subsequently recruits the nucleotide exchange
factor son of sevenless (SOS). The activity of downstream GTPase
protein, RAS, depends on the ability of SOS to convert inactive RAS-
GDP to the activate RAS-GTP. Activated RAS leads to phosphorylation
and consequently sequential activation of the MAPKKK, MAPKK, and
MAPKs. c-CBL is a RING finger E3 ligase involved in the regulation of
RTKs and contains three different domains in its N-terminal region in-
cluding 4 helix bundle domains (4H), EF hand like domain (EF) and
SH2 like domain. Together these three domains form what is known as
a Tyrosine Kinase Binding (TKB) domain. The TKB supports the binding
of c-CBL to phosphotyrosine sites (Y1045 in EGFR) on receptor tyrosine
kinases such as EGFR, ERBB2 and ERBB4 [137,138]. c-CBL is also re-
cruited to EGFR through an adaptor protein, GRB2 which binds to
phosphorylated tyrosine sites on EGFR (mainly Y1068 and Y1086)
promoting ubiquitination and degradation of this receptor [137,138].
Upon complex formation with the RTK's c-CBL is itself phosphorylated
at tyrosine Y371 allowing the previously guarded E2 binding domain of
c-CBL to become exposed and thus permitting full activation of the E3
N. Kumari et al. BBA - Reviews on Cancer 1868 (2017) 456–483

Fig. 4. Schematic overview of regulation of MAPK pathway by Ubiquitin ligases and deubiquitinating enzymes. (A) Activation of the pathway through binding of ligand binding to
receptor tyrosine kinase leads to phosphorylation of tyrosine sites on intracellular components of the receptor creating docking sites for GRB2. Upon binding of GRB2 to the receptor, SOS
converts inactive GDP-RAS to active GTP-RAS. Active RAS induces RAF activation which leads to phosphorylation and activation of downstream MEK and ERK. Phosphorylated ERK
translocates to the nucleus and induces transcription. Sprouty can also bind to phosphorylated sites on receptor and competitively inhibits the binding of the negative regulator c-CBL and
rescues the receptor from degradation. Both USP2A and USP8 can also independently deubiquitinate and stabilize the receptor. Activation of the pathway is maintained by through
SMURF2 and USP15. SMURF2 ubiquitinates and degrades the RAS negative regulator β-TrCP while USP15 deubiquitinates and stabilizes RAF. (B) The MAPK pathway is negatively
regulated through a number of different ubiquitin mediated mechanisms. Upon binding of ligand and receptor phosphorylation the E3 ligases c-CBL and Nrdp1 ubiquitinate the receptor
targeting the receptor for degradation. SPROUTY blocks the interaction of SOS and RAS and SPROUTY's interaction to RAF-1 blocks its kinase activity. β-TrCP ubiquitinates and degrades
RAS. Rabex-5 monoubiquitinates H-RAS and N-RAS and prevents their endosomal recycling into the membrane. IMP is a known ubiquitin ligase which targets the scaffold protein KSR
leading to its degradation. IMP autoubiquitination is reversed by USP15 stabilizing the protein. Several ubiquitin ligases and chaperones are responsible for degradation of RAF including
RNF149, HUWE1, XIAP and HSP90. Also, MEKK1 ligase is known to add ubiquitin chains to ERK and degrade this protein in proteasome dependent manner. The overall outcome of these
events is downregulation of pathway and inhibition of ERK mediated transcription.

ligase activity of c-CBL towards its bound RTK leading to proteosomal
mediated degradation of the RTKs [139].
The adaptor protein SPROUTY has been commonly regarded as a
negative regulator of MAPK pathway though negative regulation ap-
pears contextual; dependant on the specific RTK activated ligand. Like
c-CBL SPROUTY contains a TKB binding motif with an affinity for
phosphorylated EGFR tyrosine Y55 and in the event of EGF stimulation
SPROUTY is able to bind instead of c-CBL inhibiting c-CBL ubiquiti-
nation of EGFR [140]. Therefore, SPROUTY acts as a competitive in-
hibitor preventing c-CBL interaction with EGFR hence enhancing MAPK
signalling pathway [141]. In contrast, SPROUTY tends to act as a ne-
gative regulator following exposure to fibroblast growth factor (FGF),
vascular-endothelial growth factor (VEGF), platelet- derived growth
factor (PDGF), hepatocyte growth factor (HGF), glial-derived growth
factor (GDGF), or nerve growth factor [142]. It does so by acting as a
decoy adaptor for GRB2 preventing activation of RAS by inhibiting
interaction of GRB2 with RAS [143]. Secondly, SPROUTY can bind to
the cysteine rich domain to C-RAF kinase and blocking its kinase ac-
tivity [144,145]. Interestingly, SPROUTY expression is induced by
MAPK pathway activation eluding to the existence of a negative feed-
back loop [144].
In about one third of all human cancers one of three isoforms of RAS
(H-RAS, N-RAS and K-RAS) are mutated asserting the critical im-
portance of these small GTPases in proliferation and cell survival [146].
The primary difference in three isoforms of RAS lies in their subcellular
localization which is mediated by a 25 amino acid C-terminus hy-
pervariable region (HVR) containing sites for farnesylation and palmi-
toyilation for H-RAS and N-RAS and only farnesylation site (in the case
of K-RAS) [147]. H-, N- and K-RAS, are ubiquitously expressed and have
previously been described as having both overlapping and non-re-
dundant functions. Under physiological conditions RAS proteins con-
tinuously cycle between an active GTP-bound state and inactive GDP-
bound form which allows RAS proteins to bind and activate its various
downstream substrates. The majority of RAS mutations typically affect
hotspots within codons 12 and 13 most notably G12 V for KRAS. On a
molecular level these mutations impair GTP-GDP hydrolysis which in
turn leads to permanent activation of RAS and persistent stimulation of
downstream signalling pathways. The abundance and subcellular lo-
calisation of all isoforms of RAS are regulated by ubiquitination. In light
of this, though attempts to effectively pharmacologically inhibit K-RAS
have been unsuccessful, recent data suggests that targeted inhibition of
ubiquitin modifying enzymes may lead to downregulation of K-RAS
function. The F-box protein β-transducin repeat–containing protein (β-
TrCP) mediates polyubiquitination of all RAS isoforms, leading to
proteasome-dependent degradation of RAS [148]. Interestingly, this
process is partly mediated by Wnt/β-catenin signalling pathway
whereby Axin and Adenomatous Polyposis Coli (APC) enhance the
binding of the WD40 domain of β-TrCP with H-RAS leading to down-
regulation of the transforming capability of H-RAS. These results em-
phasize regulation of the RAS proteins by the WNT pathway.
β-TrCP itself undergoes ubiquitin mediated degradation. The E3
ligase SMURF2 monoubiquitinates its cognate ubiquitin-conjugating
enzyme (E2), UBCH5/UBE2D, leading to the active E3:E2 complex
formation resulting in the polyubiquitination and degradation of β-
TrCP [149]. Thus SMURF2 activity leads to the stabilization of K-RAS.
Conversely, loss of SMURF2 expression by genetic means stabilized β-
TrCP and degraded K-RAS. Interestingly, the authors also observed that
K-RAS mediated ubiquitination occurred at a higher level in mutant

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N. Kumari et al.
forms of the protein, probably owing to its persistent GTP activation.
This data would suggest that effective targeting of SMURF2 may be a
unique strategy to degrade mutant K-RAS in cancer cells. However, one
must consider that SMURF2 acts as a negative regulator of TGF-β sig-
nalling and therefore targeting of SMURF2 may activate the pro-onco-
genic effects of TGF-β while simultaneously deregulating K-RAS.
The endosomally localized Rabex5 (also known as RabGEF1) func-
tions partly as an E3 ubiquitin ligase and promotes mono- and di-ubi-
quitination of H-RAS and N-RAS, leading to the anchoring of RAS at the
endosomes and reducing downstream signalling [150,151]. It has been
shown that Rabex-5 contains an A20 like Zing finger ubiquitin ligase
domain (ZnF) which mediates the interaction of Rabex-5 with H-RAS
and N-RAS. Interestingly, mutant H-RAS which is not able to be ubi-
quitinated is significantly more effective than wild type H-RAS in its
activation of MAPK pathway highlighting the role of non-degradative
ubiquitination of RAS proteins in downregulation of MAPK pathway
[147]. The overall fate of endosomal bound RAS remains un-
determined, most likely is it either targeted for endosomal mediated
recycling or shunted towards the proteasomal degradation pathway.
Furthermore, reversal of this ubiquitination at this location by an en-
dosomal localized deubiquitinating enzyme has not yet been described.
Individual RAS family members can also undergo monoubiquitina-
tion at K117 and K147. It has been proposed that targeted ubiquitina-
tion of K-RAS specifically at K147 impairs regulator-mediated GTP
hydrolysis leading to markedly increased activation of MAPK pathway.
In contrast, monoubiquitination of H-RAS at K117 accelerates intrinsic
nucleotide exchange promoting GTP loading [152]. Interestingly, these
hyperactivated forms of RAS demonstrate enhanced interaction binding
with downstream effectors and overall activation of the pathway
through monoubiquitination at distinct sites eliciting distinct mechan-
isms of action. Though the identity of both the E3 ligase and the
counteracting DUB that regulates this site remains elusive, it is
tempting to speculate that a balance exists between E3 ligase activity
and DUB activity to regulate overall RAS activity through stabilization
and localization of the protein.
Impedes mitogenic signal propagation (IMP) is a Ring finger E3 li-
gase which negatively regulates KSR and inhibits MAPK pathway ac-
tivation through the prevention of RAF and MEK complex formation.
Binding of extracellular ligands to RTKs and activation of RAS leads to
activation of IMP E3 ligase activity and its autoubiquitination and
subsequent degradation. Therefore, increased IMP autoubiquitination
following RAS activation leads to KSR stabilization and higher level of
phosphorylated ERK1/2 [153,154]. How activation of RAS causes in-
creased E3 ligase activity of IMP remains an unanswered question
which requires further investigation. Recently, some studies have re-
ported the role of USP15 in deubiquitinating and rescuing IMP from
degradation [155]. Although, USP15 and USP4 both are involved in
deubiquitination of IMP, depletion of only USP15, not USP4, destabi-
lizes IMP promoting its proteasomal dependent degradation [155].
Hayes et al. showed that the interaction of USP15 and IMP is mediated
through DUSP-UBL domain of USP15 and coiled coil region of IMP.
Moreover, the same study showed the role of USP15 in stabilizing C-
RAF [155]. Although, USP15 depletion reduced the level of C-RAF, it
had no effect on the level of B-RAF and p-MEK in B-RAFV600E har-
bouring melanoma cells [155].
HUWE1 a HECT family E3 ligase has recently been demonstrated to
regulate of C-RAF protein level, but not B-RAF and A-RAF levels [156].
HUWE1 binds indirectly to C-RAF through a scaffold protein called
SHOC2 and ubiquitinates both SHOC2 and C-RAF proteins. As ex-
pected, depletion of SHOC2 abrogates HUWE1 mediated C-RAF ubi-
quitination and degradation [156]. Downregulation HUWE1 expression
by shRNA stabilized C-RAF and SHOC2 and increased p-ERK levels in
different cells lines, demonstrating HUWE1 as a negative regulator of
MAPK signalling.
Some studies have shown that silencing X linked Inhibitor of
apoptosis (XIAP), cellular inhibitor of apoptosis (c-IAP) or melanoma
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BBA - Reviews on Cancer 1868 (2017) 456–483
inhibitor of apoptosis (ML-IAP) using siRNA leads to stabilization of C-
RAF and promotes migration [157,158]. Although, IAP family members
contains a RING E3 Ubiquitin ligase domain, XIAP and c-IAP depletion
stabilized C-RAF level (≈2fold) in a RING domain independent manner
[157]. Moreover, the same study showed the role of chaperons and co-
chaperons (mainly CHIP) in ubiquitination and degradation of C-RAF.
Carboxy terminal Hsc70 interacting protein, CHIP, is one of the im-
portant co-chaperons which conjugates E2 ubiquitin ligases to C-RAF.
However, interaction of C-RAF and CHIP requires XIAP as an adaptor
protein which explains the increased ubiquitination and degradation of
C-RAF by overexpression of the mutant RING domain deleted XIAP
[157].
Similar to C-RAF, A-RAF and mutated B-RAF V600E (but not wild-
type B-RAF) are stabilized through HSP90 chaperone machinery [159].
HSP90 is required for C-RAF stability and membrane localization.
Geldanamycin, a HSP90 binding antibiotic, disrupts complex formation
of C-RAF and HSP90 and induces ubiquitin dependent degradation of C-
RAF [160]. C-RAF mediated downstream signalling was inhibited by
Geldanamycine possibly through reduction of C-RAF level. However,
the signalling and C-RAF level was maintained in presence of protea-
some inhibitors, even in presence of Glendamycine [161]. Also, mutant
B-RAF V600E, which is frequently mutated in different types of cancer
including melanoma, colorectal and thyroid cancer, is a HSP90 client
[159]. Although, HSP90-cdc37 complex was involved in degradation of
different B-RAF mutants (V600E, V600D, G465V, G468A), there was no
change in level of wild type B-RAF after 17-AGG (HSP90 inhibitor)
treatment, consistently shown in different melanoma cell lines [159].
Interestingly, RNF149 was reported as an E3 ligase responsible for
degradation of wild type B-RAF, but not mutant B-RAF [162]. siRNA
mediated downregulation of RNF149 leads to stabilization of wild type
B-RAF and simvastatin treatment which induces RNF149 expression
leads to lower level of wildtype B-RAF, without altering mutant V600E
B-RAF level [162]. Interestingly, some studies have investigated the
role of RAF proteins and dysregulation of its HSP90 dependent de-
gradation in the non-cancer context of metabolic diseases such as dia-
betes [163]. It has been shown that, methylglyoxal, a physiological
metabolite of glucose which is accumulated in serum of diabetic pa-
tients, significantly reduces C-RAF level through ubiquitin dependent
degradation [163].
Extracellular signal related kinases (ERKs) are a family of proteins
which are responsible for transmitting the extracellular signal into
nucleus through phosphorylation or regulation of various transcription
factors including ETS, ELK, MNK, RSK etc. ERK family kinases are
classified into two categories depending on scaffold dependence for
activation and function. C-terminal domain containing MAPKs such as
ERK3 and ERK7/8 can exist in individual subunits independent of
scaffold binding. The second category includes the classical ERK family
members ERK1 and ERK2. The signalling axis of this pathway requires
scaffolding proteins for their proper activation. As an example KSR
(Kinase suppressor of RAS) is needed as a scaffold protein to pre-
assemble and engage the multiple components of the MAPK cascade for
efficient ERK1/2 activation [153].
ERKs have been shown to be regulated by ubiquitination/deubi-
quitination system. Upstream of ERK, MEK kinase1 (MEKK1) is able to
activate MEKs in both JNK and ERK1/2 pathways. PHD (plant home-
odomain) domain of MEKK1 which is a Ring finger like domain shows
E3 ubiquitin ligase activity and is responsible for ubiquitination and
degradation of ERK1/2 [164]. Therefore, MEKK1 activates and down-
regulates ERK1/2 through its kinase and PHD domain, respectively
[164]. The PHD domain in MEKK1 contains 7 cysteines and a histidine
which resembles the structure of other known E3 ubiquitin ligases. It
has also been reported that the PHD domain is found in many proteins
which are involved in chromatin mediated transcriptional regulation
[164]. ERK1/2 maintains homeostasis by inhibiting apoptosis. Treat-
ment with sorbitol results in cellular hyperosmotic conditions. This
increases the interaction of MEKK1 and ERK1/2 leading to
N. Kumari et al.
downregulation of ERK1/2 through proteasome mediated degradation
mechanism allowing for apoptosis to occur [164]. This demonstrates
the crucial nature of ubiquitination along the ERK1/2 axis in main-
taining homeostasis at the organism level.
5. The role of ubiquitin modifying enzymes in the PI3K pathway
P13K-AKT-mTOR (PAM) pathway is a critical signalling pathway
involved in cellular growth and survival. Genetic aberrations that lead
to constitutive activation of the pathway are frequently observed in
human cancers. However, due its aberrant behavior and irregular cross-
talk with other signalling pathways targeted therapy with single agent
PI3K pathway inhibitors has generated disappointing results clinically.
Renewed enthusiasm analyzing various combination therapies, in-
cluding strategies targeting CDK4/6 with PI3K inhibitors has revived
hope that effective downregulation of the pathway will lead to pro-
longed responses in patients. Nevertheless, a better understanding of
the PI3K pathway is essential for this effort.
PI3Ks as the major downstream effector of receptor tyrosine kinases
(RTKs) and G protein coupled receptors (GPCRs), leads to the formation
of a potent second messenger, PIP3 (Phosphatidylinositol (3,4,5)-tri-
phosphate) which transduces the signals from various growth factors
and cytokines and assists in AKT activation. The serine threonine kinase
AKT once activated plays a vital role in the regulation of cell survival in
a variety of human neoplastic diseases and polices a range of cellular
processes, including cell survival, cell cycle progression, cytoskeletal
organization, vesicle trafficking, glucose transport, and platelet func-
tion. Along with a number of activating mutations within components
of the PAM pathway a number of inhibitory mutations have also been
identified in the tumour suppressor phosphatase and tensin homologue
deleted from chromosome 10 (PTEN). PTEN is a dual specific protein
and lipid phosphatase whose primary function is to degrade the phos-
phoinositide products of PI3K hydrolyzing (3,4,5)-trisphosphate (PIP3)
to phosphatidylinositol (4,5)-bisphosphate (PIP2) [165,166]. In terms
of enzymatic activity of the various component of PI3K-AKT-mTOR
pathway including AKT and PTEN the role of phosphorylation has been
well established however, very little is known about the role ubiquitin
modifying enzymes in this process (Fig. 5).
5.1. Ubiquitin mediated regulation of PI3K and IRS
Phosphatidylinositol 3-kinase (PI3K) is a conserved hetero-dimer
intracellular lipid kinase consisting of an 85-kD regulatory subunit
bound to a 110-kD catalytic subunit. The phosphoinositol-3-kinase fa-
mily is grouped into three different classes: Class I, Class II and Class III.
In response to various growth factors class IA PI3Ks get activated either
through direct binding with the activated receptors or through adapter
molecules such as phosphotyrosyl insulin receptor substrate (IRS). The
p85 subunit of PI3K has been shown to be regulated by several E3 li-
gases including SCF-FBXL2, CHIP, and CBL. SCF-FBXL2 catalyses ubi-
quitination and hence degradation of monomeric p85 [167]. Curiously,
FBXL2 mediated degradation of p85 appears to enhance PI3K activation
rather the inhibit it. The authors rationalize that FBXL2 preferentially
targets excess free monomeric p85 thereby limiting the ability of the
free p85 to outcompete the p85-p110 heterodimer for binding to IRS1
[168]. Recently, it has been demonstrated that the ErbB3-binding
protein 1 (EBP1) couples p85 to the HSP70/CHIP complex. The cha-
perone-dependent ubiquitin ligase (CHIP) drives p85 ubiquitination
and ultimately proteasomal mediated degradation of p85 [169]. It has
been also reported that CBL family ubiquitin ligases not only mediates
polyubiquitination of p85 in T cells but under certain conditions can
regulate p85 compartmentalization and protein-protein interactions at
the cell membrane [170]. CBL-B recruits p85 to CD28 and T cell antigen
receptor and negatively regulates p85 in a proteolysis-independent
manner. The contrasting effects of ubiquitin mediated degradation and
regulation of p85 on downstream PI3K activity in these three studies
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BBA - Reviews on Cancer 1868 (2017) 456–483
highlights the complex role of ubiquitination towards its targeted
substrates and may suggest a tissue specific regulatory function of p85
towards PI3K activation.
Like p85, IRS-1 is itself a target of proteasomal mediated degrada-
tion. CUL7 E3 ligase is a component of the SCF complex including
FBW8, SKP1, and the ROC RING finger protein. As part of negative
feedback loop S6K activation downstream of mTOR phosphorylates
IRS-1 at S307 stimulating the interaction of IRS-1 with the substrate
recognition subunit FBW8 and recruiting the SCF complex consequently
resulting in IRS-1 ubiquitination and degradation. Treatment with the
mTOR inhibitor rapamycin repressed IRS-1 phosphorylation and in-
hibited the CUL7 E3 ligase mediated degradation of IRS-1 revealing the
importance of mTOR/S6 activities in ubiquitin mediated upstream
regulation of IRS-1 by the SCF complex [171]. Likewise, SOCS con-
taining E3 ligases has been reported to block insulin signalling by in-
ducing ubiquitin mediated degradation of IRS1 and IRS2 and have been
implicated in the mechanism for inflammation-induced insulin re-
sistance [172]. Several other ubiquitin ligases suppress IGF/insulin
signalling by inducing ubiquitin mediated proteasomal degradation of
IRS-1/2 [173,174] whereas mono-ubiquitination of IRS-2 at single or
multiple sites by NEDD4 has been reported to enhance IGF signalling in
a manner dependent on the ubiquitin binding protein Epsin1 [175].
Ubiquitination of IRS proteins has been shown to be reversed by deu-
biquitinating enzyme USP7. USP7 forms a complex with IRS1/2 deu-
biquitinating and stabilizing IRS1/2. Interestingly, complex formation
between IRS and USP7 is modulated by PI3K signalling as the addition
of insulin led to the dissociation between USP7 and IRS1/2 enhancing
IRS1/2 degradation [176]. This dissociation was prevented by treat-
ment with the pan PI3K inhibitor LY294002. This suggests that IRS1/2
mediated activation of the PI3K pathway following insulin exposure
induces a dual feedback mechanism targeting IRS1/2 stability. Fol-
lowing activation of the PI3K pathway S6K phosphorylates IRS1/2 re-
cruiting the SCF ligase complex targeting IRS1/2 for ubiquitination.
Concomitantly, PI3K activation dissociates USP7 from the IRS1/2
complex ensuring a rapid downregulation of activated IRS1/2.
5.2. Ubiquitin mediated regulation of AKT
Substrate ubiquitination and targeting of proteins to the proteasome
is a dynamic process and can have a great impact on progression of
oncogenesis. Recent studies suggest that ubiquitination is equally im-
portant as phosphorylation for activation of PAM pathway components
and as such it is unsurprising that pharmaceutical inhibition of a
number of ubiquitin modifying enzymes with respect to PI3K activation
is being explored, including a number of enzymes targeting AKT. AKT is
a member of the AGC family of kinases, the members of which possess a
pleckstrin homology (PH) domain at its N-terminus. As indicated above
PI3K phosphorylates the inositol ring of PI(4,5)P2 at the D-3 position to
form PI(3,4,5)P3, which is required for the binding of the PH-domain of
AKT to the plasma membrane. This permits AKT to be phosphorylated
by phosphoinositol-dependent kinase 1 (PDK1) within its catalytic do-
main at T308. Following pathway activation AKT is subsequently
phosphorylated at S473 by ribosome-associated RICTOR-mTOR com-
plex (mTORC2). Phosphorylation of both T308 and S473 is required for
maximum kinase activity of AKT. Similarly, AKT kinase activity can be
both regulated by K63-linked ubiquitination and K48 linked ubiquiti-
nation. TRAF6, TRAF4, NEDD4, and SKP2 all regulate AKT signalling
by promoting K63 linked ubiquitination. K63-linked ubiquitination has
been revealed to be critical for activation of AKT by enabling its
membrane recruitment. TRAF6 ubiquitinates AKT at two lysines (K8
and K14) within its PH domain promoting translocation of AKT to
plasma membrane and enhancing AKT T308 phosphorylation and ac-
tivation [177]. Curiously, K63 ubiquitination at these two residues is
not required for PIP3 binding even though the K14 residue resides
within the PIP3 binding pocket. It has therefore been speculated that
ubiquitination may enhance a conformational change within the
N. Kumari et al. BBA - Reviews on Cancer 1868 (2017) 456–483

Fig. 5. Schematic overview of regulation of PI3K pathway by Ubiquitin ligases and deubiquitinating enzymes. (A). Ubiquitin mediated downregulation of the PI3K pathway. PI3Ks are
downstream effectors of receptor tyrosine kinases (RTKs) and G protein coupled receptors (GPCRs). Phosphatidylinositol 3-kinase (PI3K) is a hetero-dimer conserved intracellular lipid
kinase consisting of an 85-KD regulatory subunit bound to a 110-KD catalytic subunit. CHIP and CBL regulate stability and function of p85 respectively through ubiquitination. Like p85,
the RTK interacting protein IRS undergoes ubiquitination mediated degradation through CUL7 and SOCS. The downstream serine/threonine kinase AKT has been shown to undergo K48
ubiquitinated by the ligases MULAN, TTC3, BRCA1 and CHIP. These ligases degrade AKT downregulating PI3K pathway activity. Likewise, the DUB CYLD prevents activation of AKT by
counteracting K63 linked ubiquitination and inhibiting AKT recruitment to membrane. Furthermore, the phosphatase PHLPP1 functions in complex with FKBP51 to dephosphorylate and
inhibit AKT. USP49 stabilizes FKBP1 further enhancing AKT pathway inhibition by promoting interaction of PHLPP1 to AKT. Cul4-DDB1-Fbw5 mediates TSC2 stability and inhibiting
TSC1/TSC2 complex turnover resulting in constitutive downregulation of the mTOR activator, RHEB. mTOR is the key component of mTORC1 (mTOR, mLST8, RAPTOR) and mTORC2
(mTOR, mLST8, RICTOR) whereby mTORC1 enhances phosphorylation of downstream S6K1 and eIF4E while mTORC2 acts through a positive feedback loop to phosphorylate AKT at
S473 leading to full activation of AKT. Cul1-Skp-Fbw7 and USP9X negatively regulate mTOR in ubiquitin dependent manner, however, the manner through which USP9X regulates mTOR
stability is unknown. The mTORC2 component RICTOR has been shown to be ubiquitinated and degraded by SCF-FBXL7. Interestingly mROC1 and mTORC2 complex formation is
regulated by a number of ubiquitin modifying enzymes. mLST8, another component of both mTORC complexes, undergoes K63 mediated ubiquitination by TRAF2 hindering mTORC2
formation while increasing mTORC1. A mechanism offset by the DUB OTUD7B. In a similar fashion CUL4-DDB1 ubiquitinates RAPTOR limiting mTORC1 formation but enhancing
mTORC2 formation. A mechanism reversed in this case by UCH-L1. The tumour suppressor PTEN negatively regulates PI3K pathway by inhibiting formation of PIP3 but also plays an
undefined role as a tumour suppressor in the nucleus. PTEN also undergo various post translational modification including ubiquitination. PTEN was shown to be monoubiquitinated by
NEDD4-1 enhancing its nuclear translocation. Furthermore, two DUBs USP13 and OTUD3 deubiquitinate and stabilize PTEN thereby aiding in its tumour suppressor activities. (B)
Ubiquitin mediated upregulation of the PI3K pathway. The E3 ligase FBXL2 mediates degradation of p85 but interestingly this degradation enhances PI3K activity by preferentially
targeting excess free monomeric p85 thereby limiting the ability of free p85 to outcompete the p85-p1110 heterodimer for binding to IRS1. NEDD4 has been reported to enhance IGF
signalling by monoubiquitinating IRS at single or multiple sites. Similarly, USP7 counteracts ubiquitination of and stabilizes IRS1/2. PDK1 which phosphorylates and activates AKT is
monoubiquitinated and undergoes deubiquitinated by USP4. Although the exact function of this DUB with respect to PDK1 needs to be explored. Membrane recruitment of AKT is a
critical step for its activation. Various ligases such as TRAF6, SKP2, NEDD4 and TRAF4 all mediate K63 linked ubiquitination of AKT recruiting AKT to the plasma membrane. The E3
ligase SCF-β-TrCP ubiquitinates and degrades the AKT negative regulator PHLPP1. Similarly, UCH-L1 which is a deubiquitinating enzyme suppress the level of PHLPP1 thus enhancing
AKT pathway. Deptor, thought to be a negative regulator of mTORC activity undergoes ubiquitination and degradation by the E3 ligase β-TrCP. S6K1, which is downstream of mTORC1
also undergoes ubiquitin mediated modification by ROC1. ROC1 might be responsible for turnover of S6K protein. PTEN itself is also negatively regulate by a number of ligases including
WWP1, XIAP, CHIP, NEDD4-1 targeting PTEN polyubiquitination and degrading PTEN. RFP also polyubiquitinates PTEN but rather then inducing degradation it functions by inhibiting
its activity. USP7 as a deubiquitinating enzyme counteract monoubiquitination of PTEN and causes its nuclear exclusion.

protein permitting complex formation with a ubiquitin binding factor
resulting in the relocalisation of AKT from the cytosol to the plasma
membrane. Interestingly, AKT mediated ubiquitination by TRAF6 is
enhanced following growth factor stimuli. In fact, it has been noted that
AKT K63 ubiquitination is mediated by a diverse set of E3 ligases de-
pending on the stimulus, with both TRAF6 and the neural precursor cell
expressed developmentally down-regulated protein 4 (NEDD4) having
been demonstrated to regulate AKT activity through K63 linked ubi-
quitination following IGF-1 signalling whereas SKP2 and TRAF4 func-
tion downstream of ErbB receptors [178,179]. Curiously, unlike IRS1/2
where the SCF complex targets IRS1/2 for proteosomal mediated
degradation, the SCF complex (SKP2, CUL, FBOX), mediates K63 ubi-
quitination of AKT [180]. For the most part SCF complexes promote
protein degradation through the transfer of K48 ubiquitin chains. Why
in this case SKP2-SCF complex promotes a non-proteolytic K63 linked
ubiquitination of AKT remains a bit of mystery and this issue, un-
doubtedly, will need to be probed into further. It is important to note
that all three of these ligases have been reported to promote cancer
progression. Activating mutations in AKT itself are rarely observed,
with the exception of a single point mutation E17K within the PH do-
main [181]. Interestingly, this cancer associated mutant displays hy-
perubiquitination of AKT, increased PIP3 binding and increased

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membrane localization indicative of the importance of PH domain
ubiquitination and AKT activation.
Counteracting these ligases the deubiquitinating enzyme CYLD acts
as a tumour suppressor and negative regulator of AKT activation by
apparently directly deubiquitinating K63 linked ubiquitin chains within
the PH domain, terminating AKT activation [182,183]. Similar results
were observed in TRAF6 depleted cells. As it has previously been de-
monstrated that CYLD deubiquitinates TRAF6 these results suggests
that CYLD deubiquitinates and suppresses the activity of both the E3
ligase and its kindred substrate [184]. An effect which has been fre-
quently observed [88,185]. Interestingly, K14 but not K8 was the cri-
tical residue for CYLD mediated deubiquitination.
Proteasomal mediated degradation of AKT is regulated by a plethora
of ubiquitin ligases including tetratricopeptide repeat domain 3 (TTC3),
breast cancer susceptibility gene 1 (BRCA1), chaperon-associated ubi-
quitin ligase (CHIP), and mitochondrial ubiquitin ligase activator of NF-
κB (MULAN) [186–189]. In the majority of these cases phosphorylation
and activation of AKT is a prerequisite determinant for ligase binding,
AKT K48 ubiquitination, and subsequent degradation. TTC3 has been
shown to preferentially bind to phosphorylated forms of AKT. Inter-
estingly, as part of a self-regulation mechanism, AKT phosphorylates
TTC3 at S378, a site elucidated to be required for TTC3 activity as
mutation of TTC3 inhibited TTC3 mediated ubiquitination and de-
gradation of AKT.
The tumour suppressor BRCA1 is frequently mutated in breast and
ovarian cancers. BRCA1 activity regulates a number of signalling
pathways involved in genome stability including DNA damage repair,
cell cycle check point control, chromatin remodeling, transcriptional
regulation, apoptosis, and ubiquitination. Like TTC3, BRCA1 is re-
cruited to phosphorylated isoforms of AKT and targets active AKT in-
ducing K48 ubiquitin chain formation and degradation of the later
[186]. This work highlights the ubiquitin mediated tumour suppressive
function of BRCA1 in tumorigenesis. Similarly, the mitochondrial as-
sociated ubiquitin ligase MULAN and CHIP target phosphorylated AKT.
Interestingly, K48 ubiquitination of phosphorylated AKT can occur both
in the nucleus (BRCA1, TTC3) and in the cytoplasm (MULAN, CHIP)
eluding to the potential role of AKT substrates in negative feedback
loops in the recruitment of these ligases in both the nucleus and cyto-
plasm to downregulate active AKT. It also suggests that to prevent
nuclear exclusion of AKT through its degradation in the cytoplasm by
ubiquitin ligases a small proportion of phosphorylated AKT may be
specifically recruited to the nucleus to perform its nuclear functions. It
will be interesting to determine if the hyperactivated form of
AKT(E17K) functions primarily in the cytoplasm or the nucleus and
ubiquitin mediated degradation of AKT (E17K) is altered at either of
these locales.
5.3. Ubiquitin mediated regulation of PDK1 and mTOR
AKT is phosphorylated at T308 by PDK1 and subsequently at S473
by mTORC2 resulting in full activation of the protein. PDK1 (3-
phospho-inositide-dependent kinase 1) is a serine/threonine kinase
which phosphorylates and activates approximately twenty-three of the
AGC family of protein kinases including the proto-oncogene AKT,
Protein Kinase C (PKC) and the p70 S6 kinase (S6K) [190–192]. PDK1
plays critical role in cell proliferation and metabolism. Mono-ubiquiti-
nated isoforms of PDK1 have been reported in a various human cell
lines suggesting PDK1 ubiquitination is a common process. The deu-
biquitinating enzyme USP4 co-localizes with PDK1 at the plasma-
membrane and has been shown to deubiquitinate monoubiquitinated
PDK1 both in vitro and in vivo [193]. However, the role of PDK1 ubi-
quitination remains unclear. It is yet to be determined if ubiquitination
is required for recruitment of PDK1 to the membrane or if PDK1 ubi-
quitination is required for AKT activation.
The mammalian target of rapamycin (mTOR) protein is a serine-
threonine kinase that belongs to the PI3K family. mTOR is the key
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BBA - Reviews on Cancer 1868 (2017) 456–483
component in two distinct signalling complexes mTORC1 and mTORC2.
mTORC1 is comprised of mTOR, the regulatory associated protein of
mTOR (RAPTOR), mammalian lethal with Sec13 protein 8 (mLST8), the
proline rich AKT substrate 40 kDA (PRAS40), and DEP-domain-con-
taining mTOR-interacting protein (DEPTOR). mTORC2 comprises of six
different proteins including rapamycin-insensitive companion of mTOR
(RICTOR), mammalian stress-activated protein kinase interacting pro-
tein (mSIN1), protein observed with RICTOR-1 (Protor-1), mLST8, and
DEPTOR [194]. Due to the importance of mTORC1/2 in cell growth and
metabolism it is unsurprising that deregulation of mTOR signalling is
associated with various diseases some of which appear to be determined
by aberrant ubiquitination of mTOR components. Even though both
mTORC1 and mTORC2 contain a number of common components ac-
tivation of mTORC1 and mTORC2 occurs through unique stimuli in-
cluding growth factors or nutrients and PI3K signalling, respectively.
Correspondingly, mTORC1 and mTORC2 appear to be regulated
through both analogous and distinctive ubiquitination schemes.
mTORC1 primarily functions downstream of AKT to regulate pro-
tein translation and cell growth by phosphorylating S6K1 and EIF4E-
BP1 Eukaryotic translation initiation factor 4E-binding protein 1
(EIF4E-BP1) [195]. It has been reported that DEPTOR is a naturally
occurring inhibitor of both mTORC1 and mTORC2 [196]. DEPTOR is
targeted for ubiquitin mediated degradation by the SCF (Skp1-Cullin-F
box proteins)- βTrCP E3 ligase in response to serum stimulation,
thereby promoting mTOR activation and cellular proliferation
[197–199]. Similarly, RAPTOR and mLST8 interact with CUL4-DDB1, a
core component of CUL4-DDB1-WDR ubiquitin E3 ligase complex, re-
sulting in enhanced mTOR mediated signalling. Loss of CUL4B and
DDB1 diminished mTORC1-dependent phosphorylation of S6K and 4E-
BP1 [200]. Interestingly, CUL4-DDB1 function does not appear to reg-
ulate RAPTOR or mLST8 stability directly but functions to enhance
mTORC1 assembly. Counteracting this process ubiquitination of
RAPTOR by CUL4-DDB1 is offset by the deubiquitinating enzyme UCH-
L1 [201]. In either setting a direct consequence of either CUL4B-DDB1
or UCH-L1 function is the reorganization and assembly of competing
mTOR complexes. CUL4B-DDB1 supports mTORC1 complex formation
while limiting mTORC2 while UCH-LI destabilizes mTORC1 enhancing
mTORC2 assembly. Comparably, the E3 ligase TRAF2 has recently been
shown to promote K63 mediated ubiquitination of mLST8 hindering
complex formation with the mTORC2 specific component SIN1 thereby
promoting mTORC1 formation, a process reversed by the deubiquiti-
nating enzyme OTUD7B (also known as CEZANNE) [202]. As such it
would be expected that both CUL4B-DDB1 and TRAF2 enhance
mTORC1 dependent S6K and 4E-BP1 phosphorylation while UCH-L1
and OTUD7B enhance mTORC2 dependent AKT phosphorylation at
S473.
mTOR signalling can further be downregulated through a number of
ubiquitin dependent mechanisms with mTOR itself being negatively
regulated by both Cul1-Skp-Fbw7 E3 ligase and the DUB USP9X
[203,204]. The targeting of the Cul1-Skp-Fbw7 complex to mTOR re-
sults in the latter's ubiquitination and degradation while the mechanism
through which USP9X inhibits mTOR signalling remains ill defined. In
contrast, Cul4-DDB1-Fbw5 E3 ligase regulates TSC1/TSC2 complex
turnover increasing in the constitutive activation of the downstream
mTOR activator RHEB. Downstream of mTOR ribosomal protein S6
kinase has been demonstrated to be ubiquitinated and targeted for
degradation by the ligase ROC1 resulting in the inhibition of S6K ac-
tivity [205].
In addition to the key ubiquitin modifying enzymes which regulate
mTORC1 a number of specific mTORC2 ubiquitin mediated activators
and repressors have been identified. RICTOR is distinctively ubiquiti-
nated and degraded by SCF-FBXL7 highlighting one of the more inter-
esting feedback loops regulating overall PI3K activity. As observed with
a large proportion of ubiquitination reactions catalyzed by E3 ligases,
ubiquitination is mediated by prior phosphorylation of the substrate.
Likewise, phosphorylation of RICTOR is prerequisite for its interaction
N. Kumari et al.
to SCF-FBXL7 and eventual degradation. Furthermore, the phosphor-
ylation of RICTOR limits RICTOR binding to AKT. The phosphorylation
of RICTOR is mediated by GSK3β which itself is inhibited by AKT
generating a positive feedback loop when AKT is activated. Meanwhile,
GSK3β can also phosphorylate the serine/threonine phosphatase
PHLPP1 promoting the interaction of PHLPP1 with the SCF-βTrCP li-
gase complex targeting PHLPP1 for degradation. PHLPP1 depho-
sphorylates AKT limiting the activity of AKT. As such under certain
conditions GSK3β can activate AKT by degrading its negative regulator
PHLPP1. Similarly, the deubiquitinating enzyme UCHL1 has been
shown to enhance AKT pathway activation by suppressing the levels of
PHLPP1 an effect found to drive the development of lymphoma in vivo
[206]. In contrast, another DUB, USP49, inhibits AKT pathway by
promoting interaction of PHLPP1 to AKT, thus facilitating the de-
phosphorylation of AKT. USP49 does so by deubiquitinating and sta-
bilizing the scaffold protein FKBP51, which enhances PHLPP1/AKT
interaction.
5.4. Ubiquitin mediated regulation of PTEN
The lipid phosphatase PTEN plays an indispensable role in con-
trolling PI3K pathway activation by inhibiting formation of the up-
stream signalling molecule PIP3. PTEN has also recently been shown to
negatively regulate EGFR activity by affecting stabilization of the EGFR
complex with the ubiquitin ligase CBL [207]. The overall outcome of
PTEN function in the cytoplasm is the inactivation of downstream on-
cogenic AKT mediated signalling. Unsurprisingly, post translational
modifications including ubiquitination has been shown to play a huge
role in regulation of PTEN stability and activity as ubiquitinated iso-
forms of PTEN have been reported in a plethora of cancers [208].As
indicated PTEN is primarily located in the cytosol with only a small
fraction of PTEN recruited to plasma membrane to convert PIP3 to
PIP2. This recruitment of PTEN to the membrane is dependent upon on
an open accessible motif on the surface of PTEN generating a membrane
binding regulatory interface. A phosphorylated C-terminal region of
PTEN forms intra molecular interactions with this interface blocking
PTEN binding to lipid membrane. Upon dephosphorylation of C-term-
inal residues PTEN reverts to the open conformation permitting mem-
brane binding and PTEN mediated dephosphorylation of PIP3 to PIP2.
Importantly, the open conformation of PTEN also leads to nuclear
translocation of the protein where PTEN exerts a tumour suppressive
role in the regulation of DNA repair and genome stability.
Mutations at K13 and K289 of PTEN, are frequently observed in
Cowdens disease and glioblastoma and are regulatory ubiquitination
sites for PTEN function [209,210]. Interestingly, these disease asso-
ciated PTEN mutants display normal phosphatase activity. However, in
both cases the nuclear translocation of the enzyme is perturbed [210].
The E3 ligase neural precursor cell expressed developmentally down-
regulated protein 4-1 (NEDD4-1) functions in a bi-modal manner to
mediate both mono- and poly-ubiquitination of PTEN with K289 acting
as a major site for NEDD4-1 activity. K289 is located within the C2
domain of PTEN and it has been suggested that mutations at these sites
may disrupt the C2 domain from binding the plasma membrane thus
maintaining high levels of PIP3 [211]. Following the catalyses of PIP3
to PIP2 PTEN is likely polyubiquitinated by NEDD4-1 at K289 to con-
finement of PTEN in cytoplasm and its subsequent degradation via the
proteasome. In contrast, mono-ubiquitination of PTEN by NEDD4-1 at
K13 and K289 leads to a regulatory modification promoting its nuclear
translocation [212]. Thus, NEDD4-1 acts as both an oncogene and a
tumour-supressor. NEDD4-1 is amplified in a number of cancers and
expression of NEDD4-1 correlates with low PTEN levels in lung cancer
[213]. In the majority of cases PTEN associated cancer mutations are
present in the C2 domain, however, in some cases the C2 domain re-
mains intact, the latter situation potentially acting as a predictive bio-
marker for cancers that may respond to NEDD4-1 inhibition. It must be
noted that data from an independent group revealed no changes in the
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expression and localization of PTEN in a Nedd4-1-deficient mouse
model signifying that NEDD4-1 may not be a relevant E3 ligase for
PTEN ubiquitination in the murine system [214]. Counteracting the
effects of NEDD4-1 ubiquitin-specific protease Herpesvirus-associated
ubiquitin-specific protease (HAUSP), also known as USP7, removes
ubiquitin from PTEN in the nucleus resulting PTEN nuclear exclusion
[215]. Critically, absence of nuclear PTEN has been linked with tu-
morigenesis [210]. In correlation with PTEN nuclear exclusion, USP7
may therefore be regarded as a potential onco-protein a point further
strengthened as USP7 has been observed to be overexpressed in pros-
tate cancer [215].
X-linked inhibitor of apoptosis protein (XIAP), a RING domain E3
ligase has been reported to regulate PTEN stability both in vitro and in
vivo. XIAP has been shown to enhance PTEN turnover through poly-
ubiquitination but similarly ectopic expression of XIAP did not induce
mono-ubiquitination of PTEN [216]. In contrast, knockdown of XIAP
using shRNA in 293T and MCF-7 cells reduced both mono, multi-mono
and poly-ubiquitination forms of PTEN resulting in decreased PTEN
expression in the nucleus followed by a corresponding accumulation of
PTEN in cytoplasm suggesting involvement of another E3 ligase reg-
ulating PTEN mono-ubiquitination. On the other hand, both WWP2
(also known as AIP2), a member of NEDD4 family of E3 ligases and
CHIP, the chaperone-associated E3 ligase, have been proposed to
mediate PTEN degradation and demonstrated to be required for neo-
plastic formation [217,218].
Along with stability, the phosphatase activity of PTEN has also been
demonstrated to be regulated by ubiquitination. Ret Finger Protein
(RFP) or TRIM27, a member of the tripartite motif (TRIM) family,
promotes poly-ubiquitination of PTEN but rather then altering PTEN
stabilization or localization RFP significantly inhibits PTEN phospha-
tase activity leading to PI3K pathway hyperactivation [219].
Apart from ubiquitination, de-ubiquitination of PTEN has also been
determined to be a critical factor in tumorigenesis. Utilising a genome
wide library encompassing all the known deubiquitinating enzymes
Zhang et al. performed a PTEN interacting DUB screen through which
they identified USP8, USP10, USP13, and USP39 as novel interactors of
PTEN [220]. They highlight USP13 as a deubiquitinating enzyme of
PTEN whereby USP13 stabilizes PTEN by counteracting ubiquitin-
mediated degradation of PTEN. Furthermore, they demonstrate that
USP13 expression is downregulated in breast cancer correlating with
the expectant loss of PTEN expression. OTUD3, plays a similar role in
regulation of PTEN, and tumour progression in breast cancer [221].
DUBs have has been also shown to control PTEN at transcription level.
The DUB Ataxin-3, a Josephin family DUB has been reported to restrict
PTEN transcription in lung cancer cells [222].
6. The role of ubiquitin modifying enzymes in the WNT pathway
The Wnt signalling pathway plays a key role during embryogenesis/
development. It has been known to be involved in cell proliferation, cell
migration and cell fate determination. Unfortunately, aberrant activa-
tion of the Wnt pathway is frequently observed in tumorigenesis. The
canonical Wnt signalling pathway undergoes activation following Wnt
ligand binding to a seven-pass transmembrane Frizzled (Fz) family of
receptors and its co-receptors LRP5/6 at the cell surface resulting in the
recruitment of the adaptor protein Dishevelled to the receptor complex.
LRP5/6 is subsequently phosphorylated by Casein kinase 1 (CK1) and
glycogen synthase kinase 3 (GSK3) resulting in a high affinity binding
site for AXIN. This then thwarts AXIN complex mediated phosphor-
ylation of β-catenin leading to an overall increase in the cytoplasmic
levels of β-catenin and permitting β-catenin to accumulate in the nu-
cleus where it activates T-cell factor (TCF) and Lymphoid enhancer
factor (LEF) leading to the transcription of key target genes such as
Cyclin D1 and c-Myc [223,224].
The ubiquitin-proteasome system has previously been described to
tightly regulate key components of Wnt pathway (Fig. 6). Cytosolic β-
N. Kumari et al. BBA - Reviews on Cancer 1868 (2017) 456–483

Fig. 6. Schematic overview of regulation of Wnt pathway by Ubiquitin ligases and deubiquitinating enzymes. (A) Wnt off: In the absence of Wnt ligand, the Axin-GSK-APC-CK1
destruction complex targets β-catenin for phosphorylation, priming β-catenin for ubiquitination by β-TRCP and resulting in β-catenin degradation. GSK can also target the E3 ligase
RNF220 inhibiting recruitment of USP7 and deubiquitination of β-catenin. USP15 can enhance stabilization of the destruction complex by deubiquitinating APC. The pro Wnt pathway
activator Dishevelled is ubiquitinated by either the E3 ligases ITCH, NEDD4L or CUL-3 reducing its stability and thus limiting Dishevelled recruitment to LRP5/6 and Wnt activation. In
some scenarios Dishevelled can act as a negative regulator of Wnt signalling by binding ZNRF3/RNF43. ZNRF3/RNF43 functions to downregulate Wnt signalling by limiting Frizzled
recruitment to the membrane. ZNRF3/RNF43 also plays a role downstream of β-catenin by sequestering the transcription factor TCF4 to the nuclear membrane. The E3 ligases CBL and
JADE-1 ubiquitinate and destabilise nuclear β-catenin. Taken together multiple ubiquitin modifying enzymes are involved in the downregulation of Wnt signalling.
(B) Wnt on: Upon Wnt ligand binding to the Fzd receptor and the coreceptor Lpr5/6, Dishevelled is recruited to the receptor complex. LRP5/6 is subsequently phosphorylated by CK1 and
GSK resulting in a high affinity binding site for Axin. The binding of Axin to the receptor complex results in the demolition of the destruction complex resulting β-catenin stability and
translocation to the nucleus where it binds to the TCF/LEF family of transcription factors to induce transcription. A number of DUBs act to stabilize the receptor complex including USP8
which deubiquitinates Fzd and USP14 which deubiquitinates Dishevelled. In contrast, the E3 ligases RNF146 and SMURF2 both ubiquitinate and degrade Axin. Axin itself is also regulated
by the SMURF2 homologue SMURF1 however, SMURF1 mediated ubiquitination of Axin results in enhanced K29 ubiquitination of the protein and downregulation of Axin activity.
USP34 also deubiquitinates Axin leading to stabilization of Axin however, the authors also noted that downregulation of USP34 downregulated β-catenin induced transcription of Wnt
target genes. Four DUBs bind and deubiquitinate β-catenin resulting in its stability: USP4, USP9X, USP35 AND USP47. A fifth DUB, USP7, performs a similar function but does so in
complex with RNF220 to limit β-catenin ubiquitination. Also the E3 ligase RAD6B functions in the nucleus to stabilize β-catenin.

catenin and its regulation by Wnt is the essence of Wnt signalling and
along with its original discovery it was noted that β-catenin levels were
policed by various degradative mechanisms. The AXIN complex is made
up of the scaffolding protein AXIN which contains dispersed binding
sites to interact with the tumour suppressor adenomatous polyposis coli
gene product (APC), casein kinase 1 (CK1), and glycogen synthase ki-
nase 3 (GSK3). GSK3 and CK1 coordinate sequential phosphorylation of
β-catenin at S45 (CK1) and subsequently at T41, S37 and S33 (GSK3)
[225]. The primary E3 ubiquitin ligase responsible in the regulation of
β-catenin stability is β-TrCP. GSK3 phosphorylation of β-catenin at S33
and S37 generates a binding site for β-TrCP, leading to β-catenin ubi-
quitination and degradation [226]. GSK3 and CK1 can also phosphor-
ylate AXIN and APC increasing the association of these components to
β-catenin further enhancing β-catenin degradation.
Another RING finger containing E3 ubiquitin ligase, c-CBL has been
described to ubiquitinate and degrade β-catenin in the nucleus [227].
The authors demonstrate that wild-type c-CBL suppresses while a ligase
deficient mutant of c-CBL enhances Wnt signalling. Although, the au-
thors describe the UBA domain on c-CBL dimerizes with β-catenin, it
was not until recently that phosphorylation was found to be involved
[228]. In contrast, JADE-1 ubiquitylates both phosphorylated and non-
phosphorylated forms of β-catenin therefore regulating β-catenin sta-
bilization in both the Wnt-off and Wnt-on phases [229].
β-catenin can also be regulated by the E3 ligase MULE with ectopic
expression of MULE decreasing β-catenin stabilization. Interestingly,
MULE appears to function through a Wnt ligand dependent negative
feedback loop whereby MULE targets both β-catenin and its upstream
regulator Dishevelled [230]. As such under low Wnt signalling activa-
tion of MULE is restricted however, under conditions of cellular hy-
perproliferation promoting constitutive Wnt signalling MULE expres-
sion limits β-catenin stability. In Apcmin mice exhibiting enhanced β-
catenin expression MULE deficiency further accelerated adenoma de-
velopment compared to Apcmin mutation alone [231].
Of note, other E3 ubiquitin ligases targeting Dishevelled have also
been described. The Cullin-3 ubiquitin ligase with the Broad complex,
tramtrack and Bric- a- Brac (BTB) protein Kelch-like 12 (KLHL12) was
one of the first E3 ubiquitin ligases found to ubiquitinate and degrade
Dishevelled thereby antagonizing Wnt pathway [232]. Furthermore,
two of the HECT domain containing-NEDD4 family of E3 ubiquitin li-
gases have been shown to regulate levels of Dishevelled. ITCH ubiqui-
tinates phosphorylated forms of Dishevelled, thereby promoting its
proteasome mediated degradation and attenuating Wnt signalling

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[233]. Expectedly, RNAi mediated knockdown of ITCH stabilized Di-
shevelled and upregulated Wnt signalling. This group also mapped two
regions on Dishevelled, a prototypical PPXY motif and DEP domain
through which it interacts with ITCH. Mutation within the PPXY motif
at Y568F or deletion of the DEP domain led to reduced affinity for
ITCH. NEDD4L, which also belongs to the NEDD4 family of HECT
containing E3 ubiquitin ligases has been shown to target Dishevelled
and promote its proteasome mediated degradation [234]. Interestingly,
NEDD4L mediated degradation of Dishevelled involves atypical ubi-
quitin chain formation including K6, K27 and K29 but not the canonical
K48 ubiquitin chain topology, usually associated with degradation.
Dishevelled can also be regulated through K63 polyubiquitination a
modification regulated by the deubiquitinating enzyme CYLD. CYLD
directly deubiquitinates Dishevelled but the direct function of K63
ubiquitination or the ligase involved in the process remains unknown.
Nevertheless, it remains clear that depletion of CYLD enhanced Wnt
induced β-catenin accumulation in the nucleus and activation of β-ca-
tenin induced transcription [235]. Curiously, Dishevelled has also been
demonstrated to be required for ZNRF3/RNF43 mediated ubiquitina-
tion and degradation of the FZD receptor. Depletion of either Dishev-
elled or ZNRF3/RNF43 enhanced cell surface levels of FZD and LRP5/6
indicating that Dishevelled acts as an intermediary for the recruitment
of these E3 ligase to the receptor complex [236]. This also places Di-
shevelled at a crossroads for both the activation and inhibition of the
pathway by acting as a scaffold protein required for both AXIN down-
regulation and FZD stabilization. Further experimentation will no doubt
be required to tease out the organization of Dishevelled in these two
phases of Wnt regulation. ZNRF3/RNF43 can also function downstream
of β-catenin to inhibit Wnt pathway activation by physically interacting
with the TCF4 transcription factor and tethering TCF4 to the nuclear
membrane [237]. Importantly, oncogenic mutations within RNF43 as-
sociated with human gastrointestinal tumours disrupted this inhibitory
mechanism resulting in transactivation of the Wnt pathway.
Counteracting these processes, a number of ubiquitin modifying
enzymes have been identified which activate Wnt signalling by reg-
ulating proteins along multiple nodes in the pathway. USP8/UBPY
along with USP6 were identified as a deubiquitinating enzymes for the
Frizzled receptor. Ubiquitination of receptors at the plasma membrane
induces receptor endocytosis and localization of receptors into lyso-
somes. Within these lysosomes receptors can either be recycled back to
cell membrane or targeted for degradation in a ubiquitin dependent
manner. USP6 and USP8 were found to deubiquitinate FZD stimulating
FZD recycling and increase FXD expression at the plasma membrane
[238,239]. Another deubiquitinating enzyme that has been shown to
positively regulate Wnt signalling is USP14. Interestingly, both USP14
and the aforementioned CYLD deubiquitinate K63 polyubiquitinated
isoforms of Dishevelled. However, in contrast to CYLD, which acts as a
negative regulator of the pathway, USP14 positively enhanced Di-
shevelled function as genetic and chemical suppression of USP14 im-
paired downstream Wnt signalling. This disparity maybe explained by
the targeting of specific ubiquitination sites by both enzymes. CYLD
deubiquitinates K46 and K50 within the DIX domain of Dishevelled
which mediates the dynamic polymerization of the protein enhancing
binding sites for Wnt signalling partners [240]. This suggests that K63
mediated ubiquitination at K46 and K50 is required for polymerization
to occur for the binding to Wnt signalling partners, potentially AXIN, an
effect downregulated by CYLD. In contrast, USP14 deubiquitinates
K444 and K451 within the DEP domain of Dishevelled, a motif required
membrane translocation for the protein. Thus, K63 ubiquitination at
these residues in the DEP domain appears to have a negative regulatory
role by inhibiting the recruitment of Dishevelled to the membrane.
A number of components of the AXIN-APC-GSK-CK1 destruction
complex are also regulated by ubiquitination. AXIN stability is pri-
marily regulated by the poly-ADP-ribosylating enzymes tankyrase 1 and
tankyrase 2, whereby both enzymes interact with a highly conserved
domain of AXIN targeting AXIN for degradation. In this setting the E3
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ligase RNF146 directly interacts with poly(ADP-ribose) enhancing
tankyrase dependent PARsylation of AXIN promoting its degradation
[241]. AXIN is also regulated by the E3 ligase SMURF2 [242]. SMURF2
ubiquitinates K505 of AXIN leading to protein degradation. AXIN has a
number of putative PPXY motifs however, it remains undetermined if
the WW domains of SMURF2 are required for this interaction. Similarly,
the SMURF2 homologue SMURF1 interaction with AXIN is independent
of its WW domain. Rather the C2 domain of SMURF1 is required for
K29 linked polyubiquitination of AXIN mediating AXIN SMURF1 loca-
lisation at the plasma membrane, an effect which appears to be cell
cycle regulated [243]. Overall, these three E3 ligases RNF146,
SMURF1, and SMURF2 regulate either AXIN stability or function de-
pending on the distinct ubiquitin chain topology.
Opposing the tankyrase-dependent ubiquitination and degradation
of AXIN is the deubiquitinating enzyme USP34. USP34 was found to
associate with AXIN containing protein complexes through liquid
chromatography-tandem mass spectrometry. In line with previous re-
ports depletion of USP34 led to the polyubiquitnation and degradation
of AXIN. However, the authors also suggest that loss of USP34 inhibited
β-catenin mediated transcription indicating that USP34 may function
downstream of β-catenin to regulate nuclear accumulation of AXIN
[244]. Although AXIN has been prescribed as a key component of the β-
catenin destruction complex AXIN is also known to shuttle between
both the cytoplasm and nucleus where nuclear AXIN expression has
been demonstrated to be greatly enriched in diverse cancers. Never-
theless, the precise function of how nuclear AXIN enhances β-catenin
transcription remains unknown. Reflecting on the overall function of
the known ubiquitin modifying enzymes that regulate AXIN it becomes
apparent that multiple post translational modifications and unique
chain topologies are required to tightly regulate AXIN homeostasis in
the cells.
TRABID which belongs to the OTU domain containing family of
DUBs has been described as a positive regulator of the Wnt signalling
[245]. TRABID binds APC but rather then antagonizing the proteasomal
turnover of APC TRABID inhibits APC activity through preferential
deubiquitination of K63 linked ubiquitin chains. Furthermore, the effect
of TRABID on APC appears to function downstream β-catenin stabili-
zation with the authors postulating that APC may function to decrease
the rate of TCF-β-catenin complex formation, a process seemingly de-
pendent upon K63 ubiquitination of APC and one which is reverted by
TRABID.
Recently, Ubiquitin specific protease 4 (USP4) was identified as a
DUB targeting β-catenin. Deubiquitination of β-catenin by USP4 re-
verses the ubiquitination mediated degradation of β-catenin, upregu-
lating Wnt signalling. Unsurprisingly, a positive co-relation between
levels of USP4 and β-catenin was observed in tissues from patients di-
agnosed with colorectal cancer [246]. In a separate study, USP4 was
also identified in an RNAi screen performed in the colorectal cancer cell
line SW480 using the Wnt-reporter TOPFlash reporter assay [247].
However, in contrast to the previous work RNAi depletion of USP4
caused an up-regulation of Wnt signalling. In this case, USP4 was ob-
served to interact with Wnt regulators Nemo-like kinase (NLK) and
TCF4. USP4 deubiquitinated a subpopulation of TCF4. However, it re-
mains unclear if TCF4 ubiquitination is required for TCF4/LEF medi-
ated transcription. Interestingly, NLK promoted nuclear accumulation
USP4. As NLK positively regulates Wnt signalling it appears that either
NLK regulates USP4 nuclear localization as part of negative feedback
loop to downregulate TCF or that NLK enhances USP4 mediated sta-
bilization of β-catenin further promoting TCF mediated transcription.
Even though, both the studies were performed using colorectal cancer
cell lines, caution must be taken to carefully understand the role of
USP4 in both scenarios.
USP15 and USP47 have also been identified as positive regulators of
Wnt signalling. Both USP15 and USP47 form a complex with β-catenin
leading to its deubiquitination and stabilization [248,249]. USP7, a
closely related member to USP47 has been shown to function along
N. Kumari et al.
with an E3 ubiquitin ligase RNF220 to stabilize β-catenin. Although
RNF220 is an E3 ubiquitin ligase, knockdown of USP7 renders the
former inactive. Knockdown of either RNF220 or USP7 impairs overall
Wnt signalling in colon cancer cells [250].
Like the β-catenin oppressor MULE, RAD6B is a transcriptional
target of β-catenin and functions through a feedback loop to regulate
overall β-catenin levels. However, unlike MULE, RAD6B mediated
polyubiquitnation of β-catenin renders β-catenin insensitive to protea-
somal degradation. RAD6B is a 17 kDA ubiquitin conjugating enzyme
which induces K63 linked β-catenin polyubiquitination. Depletion of
RAD6B decreases β-catenin polyubiquitnation and stability limiting β-
catenin transcription. Unsurprisingly, β-catenin and RAD6B expression
is positively correlated in breast carcinoma with RAD6B levels sig-
nificantly enhanced in breast cancer compared to normal controls.
Taken together it becomes wholly apparent that a multitude of post
translational modifications are required for effective regulation of Wnt
signalling with manipulation of ubiquitin modifying enzymes offering
an exciting opportunity for cancer therapy and regenerative medicine.
7. Therapeutic intervention of dubs
Following the approval of Bortezomib, a proteasome inhibitor, as an
anti-cancer therapeutic for the treatment of multiple myeloma, in-
hibitors of the ubiquitin-proteasome system (UPS) has received much
attention and focus as a potential and attractive drug target for cancer
therapy. However, to date no inhibitors targeting the ubiquitin pro-
teasome system other than those that inhibit proteasome function have
been approved for clinical use. Deubiquitinating enzymes of course are
an integral part of the UPS, functioning to remove ubiquitin moieties
from polyubiquitin chains or target proteins. The intrinsic isopeptidase
activity of DUBs results in the cleavage of the carboxyl terminus of
ubiquitin destabilizing ubiquitin substrate binding. Taking into account
that the majority of cancers demonstrate enhanced expression of DUBs
which ostensibly act as oncogenes in a number of cancer relevant
pathways, DUB inhibition may be a considered as a valid cancer ther-
apeutic strategy. The majority of the DUBs are cysteine proteases (the
exception being the JAMM family), wherein the catalytic cysteine re-
sidues sheltered in the active sites of DUBs are highly reactive towards
electrophiles. Most ubiquitination reactions involve the formation of
thioester bonds between the c-terminal tail of ubiquitin and its sub-
strate whereby the nucleophile cysteine containing thiol side chains can
then attack this reaction. Revolving around this property, most of the
DUB inhibitors that contain α, β-unsaturated ketones have been de-
monstrated to possess DUB inhibitory activity by trapping these thiol
side chains. Towards the development of such DUB inhibitors, a wide
range of compounds ranging from synthetic small molecules to natural
products with DUB inhibitory activity have been isolated. This section
reviews effective strategies to target DUBs. We would also like the
readers to refer to the comprehensive review by D'Arcy et al. [251].
7.1. Small molecule DUB inhibitors
As highlighted in this review, DUBs catalyze the removal of ubi-
quitin moieties from substrates. For the most part the eviction of such
ubiquitin moieties results in enhanced protein stability. However, a
number of DUBs (USP14, RPN11, UCH-L5) are also physically and
functionally associated with the 26S proteasome and act in a timely and
substrate specific manner to remove ubiquitin for proper substrate
processing, and as such targeted inhibition of proteasome associated
DUBs may mimic the effect of bortezomib [252,253]. A small molecule
inhibitor of the DUB USP14 was identified through a high through put
screen called IU1 (inhibitor of USP14) [254]. This molecule, when
exposed to cells, enhanced degradation of several proteasome sub-
strates implicated in neurodegenerative disease, as USP14 is essential
for the maintenance of synaptic ubiquitin levels and neuromuscular
junction development [255,256]. USP14 inhibition led to quicker
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degradation of oxidized proteins and enhanced resistance towards
oxidative stress [254].
UCH-L1 has been linked to Parkinson's disease and has been iden-
tified as a biomarker for aggressive multiple myeloma and enhances
HIF-1 mediated metastasis [257,258]. LDN-57444, a molecule which
belongs to a class of isatin O-acyl oximes was identified by a high
throughput screen to find inhibitors against UCH-L1. Though the mo-
lecule can inhibit UCH-L1, it was found that proliferation was increased
in H1299 lung tumour cell line though this effect was not exhibited a in
lung tumour lines that did not express UCH-L1. RNAi based approaches
also had similar proliferative effects and suggests that UCH-L1 enzy-
matic activity is antiproliferative and UCH-L1 expression may be a
adaptive response to enhanced tumour growth [259]. In a second study,
LDN-57444 treatment enhanced apoptosis by increasing the levels of
highly ubiquitinated proteins activating unfolded protein response, a
protective response that promotes apoptosis when cellular stress con-
tinues to persist [260]. Similarly, LDN91946, a moderately potent in-
hibitor of UCH-L1 was identified using an assay measuring the hydro-
lysis of the fluorogenic substrate Ub-AMC. Subsequent work analyzing
structural activity relationship studies and kinetics revealed that this
compound is a noncompetitive inhibitor of UCH-L1 but not other cy-
steine hydrolases tested including such as UCH-L3, USP5 and caspase 3
[261]. Again, downregulation of UCH-L1 by LDN91946 enhanced cell
death indicating that under certain conditions downregulation of UCH-
L1 may be used to optimize patient responses [262].
USP7 is probably the most well studied of all the known deubiqui-
tinating enzymes targeting a plethora of cancer relevant genes in-
cluding both tumour suppressors (p53, PTEN) and oncogenes (MDM2,
IRS1). Nevertheless, USP7 may be considered a promising therapeutic
target in cancer. With the help of biochemical assays and activity based
protein profiling small molecule antagonists with high specificity have
been identified against USP7. Both P02207 and P5091 target USP7 with
the added function that P5091 simultaneously targets USP47 directing
the cell fate towards apoptosis. Interestingly P5091 exhibited this effect
in multiple myeloma cells resistant to bortezomib suggesting that
P5091 may be used in bortezomib resistant myeloma patients as pre-
liminary results indicate that P5091 is well tolerated in animal models
and inhibits tumour growth and prolongs survival [263,264]. In-
cidentally, blocking HDM2 and p21 abrogated the cytotoxicity induced
by P5091. Further research has led to the development of a potent
analogue of P5091 called cdp14, against both USP7 and USP47, which
has enhanced potency, solubility and a strong metabolic reactivity
profile [264]. Finally, HBX19818 has also been identified as a USP7
inhibitor. Interestingly, HBX19818 covalently modifies the active
Cys233 residue of USP7 limiting overall activity of the enzyme [265].
Colon cancer cells treated with this molecule produced a G1 arrest
mimicking results observed in cell lines following USP7 shRNA
knockdown, showcasing its high anti tumorigenic potential [265].
The USP1/UAF complex is involved in the translesion synthesis
through the deubiquitination of FANCD2 and FANCI [266]. Two im-
portant domains in UAF1 regulate FANC pathway activation. The SLD2
region of UAF1 binds FANCI and the WD40 domain of UAF1 binds and
stimulates USP1. Disruption of either of these domains promotes
FAND2/FANCI ubiquitination and enhanced DNA repair defects [267].
Utilising a high throughput screen, pimozide, an anti-psychotic drug
and GW7647, a PPAR-α-agonist were shown to act as potent inhibitors
of USP1/UAF activity [268]. These inhibitors have been shown to
target the USP and WD40 repeat protein complex. Furthermore, pi-
mozide and GW7647 act synergistically with cisplatin in inhibiting
proliferation of non-small cell lung cancer cells resistant to cisplatin
[268].
LS1 was also recently identified as a potent inhibitor of UCH-L3.
Applying a FRET based approach the authors analyzed the synthesis of
ubiquitin bioconjugates to substrates [269]. Ubiquitinated peptides
were labelled with a pair of FRET labels and were used to screen a
library comprising about 1000 compounds against UCH-L3.
N. Kumari et al.
One of the first DUB inhibitors PR-619, is a broad spectrum DUB
inhibitor which leads to an overload of ubiquitinated proteins, protein
aggregate formation and subsequent inhibition of the UPS. PR-619 also
leads to the activation of the autophagy pathway by inducing the
transport of lysosomes to the aggregates formed [270,271]. However,
due to its broad spectrum activity PR-619 is highly toxic in vivo which
has therefore limited its effect in clinical development.
7.2. Michael acceptors
The formation of covalent bonds is an important criterion for the
design of anticancer molecules. Most antineoplastic agents bind directly
to their substrates. However, most these agents lack target selectivity.
Michael acceptors, on the other hand can be structurally modified to
react selectively with specific nucleophiles. The Michael reaction
comprises both Michael acceptors and Michael donors, and comprises
of a nucleophilic addition of a carbanion or other types of nucleophiles.
The α, β- unsaturated compounds (ketone) undergoing Michael addi-
tion is called the Michael acceptor and the highly reactive nucleophiles
(cysteines) are referred to as Michael donors and together their product
is referred to as the Michael adduct. The 1,4 conjugate reaction of α, β-
unsaturated carbonyl compounds and nucleophiles is referred to as the
Michael reaction. In case of DUB inhibitors, the addition of the Michael
acceptor traps the catalytic cysteine of the DUB active site rendering the
targeted DUBs inactive.
WP1130 (Degrasyn) is a small molecule, selective deubiquitinase
inhibitor of both USP and UCH subclasses. WP1130 is derived from a
compound with a Janus-activated kinase 2 (JAK2) inhibitor activity.
WP1130 inhibits DUBs such as USP5, USP9X, USP14, UCH-L1 and
UCH37, all of which are known to regulate survival protein stability.
WP1130 also suppresses BCR/ABL, is a JAK2 transducer and activator
of transcription factor STAT. The addition of WP1130 leads to rapid
accumulation of polyubiquitinated K48/K63-linked proteins into jux-
tanuclear aggresomes, independent of 20S proteasome activity.
Furthermore, WP1130 inhibits tumour activated DUBs and results in
the downregulation of anti-apoptotic proteins and upregulation pro-
apoptotic proteins such as MCL-1 and p53 [272–274]. Preliminary data
has also indicated that it may be clinically relevant as WP1130 in
combination with bortezomib had anti-tumour activity in mantle cell
lymphoma animal models [275].
Eeyarestatin-1(Eer1) is an inhibitor of the endoplasmic reticulum
associated protein degradation (ERAD) pathway [276]. Proteins that
are terminally misfolded, are recognized by chaperones on the en-
doplasmic reticulum and are transported to depots for ubiquitination
and proteasomal degradation through the ERAD pathway. Eeyarestatin-
1 binds directly to p97 ATPase and acts by blocking the degradation of
misfolded proteins associated with the p97-associated deubiquitinating
complex in cells preventing ataxin-3 mediated deubiquitination of
substrates [277]. Notably, eeyarestatin-1 has exhibited anti-cancer
properties similar to that of bortezomib.
7.3. Cyclopentone prostaglandins
Cyclopentone prostaglandins are a subset of prostaglandins (PGs) or
eicosanoids that induce the accumulation of polyubiquitinated proteins
in cells and correspondingly inhibit DUB activity [278]. These eicosa-
noids are characterized by their electrophilic nature and their dis-
tinctive cross conjugated α, β-unsaturated ketone which act as Michael
acceptors. Both A series and J series prostaglandins are capable of in-
activating wildtype p53 by impairing the conformation and phosphor-
ylation of p53 resulting in the downregulation of p53 transcriptional
activity [278]. However, the J series of prostaglandins contain a exo-
cyclic α, β-unsaturated ketone, a unique structural determinant that
confers a pro-apoptotic effect through the inhibition of ubiquitin iso-
peptidase activity regardless of p53 mediated transcriptional suppres-
sion [278]. Additionally, prostaglandins of the J series are more potent
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inhibitors compared to the other members of the A, B or E pros-
taglandin series. PGJ2, Δ12-PGJ2 and 15Δ-PGJ2 are sequentially me-
tabolized products of PGD2 [278–280]. PGs such as Δ12-PGJ2 and Δ7-
PGA1 methyl ester have antitumour and antiviral activities and are
easily taken up by cells and accumulate in the nuclei via covalent in-
teractions that ultimately lead to growth inhibition. α, β- unsaturated
ketones are highly susceptible to nucleophilic compounds with thiols as
they are particularly strong nucleophiles that PGs react with en-
zymatically or non-enzymatically. Specifically, these cyclopentone PGs
interact with the thiol nucleophiles at the β position within the cyclo-
pentenone ring. Aside from acting as Michael acceptors another pos-
sible mechanism through which these prostaglandins can lead to UPS
inhibition is through protein carbonylation and oxidative stress induced
by cyclopentone PGs which eventually leads to the decrease in activities
of S6 ATPase and 26 proteasome [281]. This oxidative stress arises
when reactive oxygen species (ROS) are generated and can have po-
tential damaging effects on cellular components. Interestingly, UCHL1
is one such protein which is oxidized in cells treated with 15Δ-PGJ2.
Δ12-PGJ2 and 15Δ-PGJ2 are potent cyclopentone PGs and have been
reported to inhibit UCH-L3 and UCH-L1 respectively [282,283]. Ad-
ditionally, cyclopentone PGs induced unfolding and aggregation of
UCH-L1, thus inhibiting its enzyme activity [284]. UCH-L1 is abundant
in the brain with its expression particularly localized to the Lewy bodies
or the neurofibrillary tangles. Furthermore, UCH-L1 has been docu-
mented to be involved in a ubiquitin-dependent proteolytic pathway
and has been implicated in the pathogenesis of Parkinson's disease. A
single thiol group on Cys152 of UCH-L1, irrespective of the presence of
5 other cysteine groups in UCH-L1, reacts with the α, β- unsaturated
carbonyl center in the cyclopentenone rings of PG leading to the for-
mation of a covalent adduct destroying its native structure and even-
tually its DUB activity [284]. Notably, 15Δ-PGJ2 also inactivates p53
function by its binding to Cys277 reducing significantly its binding and
transcriptional activity. As discussed the majority of the DUBs are cy-
steine proteases which are highly reactive nucleophiles and cyclo-
pentone PGs have Micheal acceptors attributing to the inhibitory effect
of this set of compounds towards DUBs.
7.4. Chalcone inhibitors
Chalcone or chalconids is an aromatic ketone and an enone that
forms the central core for a plethora of critical biological compounds.
Chalcone compounds such as G5, b-AP15 and RA-9 have been described
to inhibit intrinsic deubiquitinase activity. Chalcones contain cross
conjugated α,β-unsaturated ketones and accessible β-carbons and in-
hibit DUB activity but are unrelated to PGs. Chalcone DUB inhibitors
can be highly specific such as b-AP15 or highly broad spectrum like G5.
b-AP15 and its analogue VLX1570 are other proteasome associated
DUB inhibitors involved in the specific inhibition of USP14 and UCH-
L5. b-AP15 was identified in cell based screens for compounds enhan-
cing cellular apoptosis independent of either cathepsin D or p53 func-
tion [285,286]. Dual inhibition of UCH-L5 and USP14 using RNA in-
terference not only led to the accumulation of proteasomal substrates
but led to loss of cell viability [287,288]. Furthermore, b-AP15 has been
reported to show anti-neoplastic properties in animal models, multiple
myeloma and solid tumours [287,289]. Interestingly, using gene ex-
pression and connectivity MAP analysis showed that b-AP15 induced a
similar gene expression profile to that of the J series prostaglandin 15Δ-
PGJ2 [289].
Two small molecules namely G5 and F6 were identified in a small
chemical library screen that was used to identify alternative pathways
of caspase activation. The two molecules were capable of activating an
apoptosome-independent apoptotic pathway by targeting the UPS and
inhibiting ubiquitin isopeptidase activity [290,291]. Both inhibitors
upregulate the BH3-only protein NOXA, and the inhibitor of apoptosis
antagonist SMAC [290]. NOXA has been shown to play acritical role in
the induction of mitochondrial fragmentation and caspase activation
N. Kumari et al.
[291]. F6 in particular was reported by another group to be associated
with the inhibition of USP2 and USP7 and the inhibition of the SENP2
deSUMOylase [292].
Some other known partially selective DUB inhibitors, which happen
to fall in the chalcone family are AM146, RA-9, RA-14 and RAMB1
[293,294]. These DUB inhibitors induce the rapid accumulation of
polyubiquitinated substrates depleting the pool of free ubiquitin
without affecting the 20S proteasome. The functionality of these novel
small molecule inhibitors is based on the presence of a α,β-unsaturated
carbonyl group in the inhibitor making it susceptible to the nucleophilic
attack by the sulfhydryl moieties on the cysteines present in the active
sites of DUBs. These chalcone DUB inhibitors directly suppresses the
DUB activity of UCH-L1, UCH-L3, USP2, USP5 and USP8 which are
particularly known to play key roles in the turnover and the stability of
critical regulators of cell survival and proliferation. Additionally, these
compounds down regulate cyclin D while concomitantly upregulating
p53, p27 and p16 leading to arrest in S-G2/M phase of the cell cycle.
Furthermore, these inhibitors, abrogate anchorage independent growth
and promote the onset of apoptosis in ovarian, breast and cervical
cancer cell lines, displaying no significant alterations in primary human
cells [293,294].
7.5. Natural compounds exhibiting DUB inhibitory activity
Many compounds affect the ubiquitin proteasome system without
the direct inhibition of the proteasome activity, chiefly brought about
by compounds falling under the umbrella of DUB inhibitors.
Interestingly, a number of natural products exist exhibiting DUB in-
hibitory activities.
Gambogic acid, a xanthanoid with cytotoxic properties, is isolated
from the resin of Garcinia hanburyi. Gambogic acid has an α,β-un-
saturated ketone moiety which directs growth inhibition and the in-
duction of apoptosis in cancer cells. Gambogic acid has been used in
traditional Chinese medicine for ages and has been shown to have cy-
totoxic activity on several cancer cells [295]. Gambogic acid displays
cytotoxic activity by inducing changes in gene expression profiles and
affects the ubiquitin proteasome system. Gambogic acid also lead to
enhanced polyubiquitination potentially through the inhibition of
chymotrypsin activity associated with the 20s proteasome leading to
the induction of apoptosis and cell death. Interestingly, the gene sig-
nature profiles of cells exposed to gambogic acid show similar profiles
as cells exposed to UPS inhibitors like PGJ2, celastrol and with aferinA
marking its potential to inhibit DUB activity [296].
Betulinic acid is a naturally occurring pentacyclic triterpenoid, that
has been isolated from many diverse plants like white birch (Betula
pubescens), ber tree (Zizyphus mauritina), carpenters herb (Prunella vul-
garis), rosemary and tropical carnivorous plants such as Triphyophyllu
peltatum and Ancistrocladus heyneanus. Betulinic acid has a variety of
biological and medicinal properties including antiviral, antibacterial,
anti-inflammatory, antihelmintic and antioxidant properties.
Additionally, betulinic acid has been reported to show significant cy-
totoxic effect on several cell lines and even human melanoma xenograft
animal models [297]. A 20% betulinic acid ointment has been used in
the treatment of atypical moles (Dysplastic nevi) [298]. Interestingly,
the anticancer property of betulinic acid is carried out by inducing
apoptotic cell death in cancer cells by triggering apoptosis through the
inhibition of transmembrane potential in isolated mitochondria [299].
Betulinic acid inhibits multiple DUBs, resulting in the overall accumu-
lation of polyubiquitinated proteins and resulting in the decreased le-
vels of oncoptoteins [299]. A similar effect was also observed in TRAMP
transgenic mice model of prostate cancer, wherein betulinic acid in-
hibited primary tumours by increasing apoptosis and decreasing an-
giogenesis through a marked decrease in androgen receptor and cyclin
D1 protein levels [299].
Curcumin, a principal component of turmeric (Curcuma longa), a
prominent spice ingredient used in most south Asian dishes has DUB
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inhibitory activity. Curcumin is a diarylheptanoid, belonging to the
family of cucuminoids, a natural phenol, bestowing the yellow colour of
turmeric. Furthermore, curcumin is a homodimer of feruloylmethane,
containing a methoxy and a hydroxyl pharmacophore. Additionally,
curcumin is an extensively studied phytochemical with anti-cancer,
anti-microbial, anti-inflammatory and anti-oxidant properties to name
a few. Studies also show that the anti-tumour property of curcumin
arises from the presence of two α,β-unsaturated ketone moieties.
Curcumin was observed to induce the accumulation of poly-
ubiquitinated proteins at high concentrations, through a shotgun pro-
teomic approach [300]. This group also showed that curcumin down-
regulated some of the proteins associated with cellular assembly,
organization, biosynthesis and glycolysis. Notably, the accumulation of
polyubiquitinated oncogenes such as NF-κB and cyclin D1 potentially
indicates the mechanism of action of this intriguing compound.
AC17, a 4-arylidene curcumin analog, has been reported to inhibit
the deubiquitinase activity of 19S ribosomal particles. Though AC17 is
a synthesized product, it acts as an inhibitory kB kinase inhibitor (IκK)
and leads to a rapid accumulation of ubiquitinated proteins without
inhibiting the proteasome proteolytic activities. It acts differently from
its parent compound curcumin which is a proteasome proteolytic in-
hibitor by serving as an irreversible deubiquitinase inhibitor of 19S
ribosomal particles, leading to the inhibition of the NF-κB pathway and
reactivation of the proapoptotic protein p53. Treatment with AC17 has
been demonstrated to suppress tumour growth in lung xenograft model
[301].
7.6. Ubiquitin variant inhibitors
As highlighted in this review, ubiquitination of substrates by E3
ligases occurs through the formation of an isopeptide bond between the
C-terminal carboxyl group of ubiquitin and the lysine of the substrate, a
process which is reversed through the catalytic properties of DUBs.
Despite their low sequence similarity, USP catalytic domains share a
common fold that includes a large structurally conserved ubiquitin
binding site for the targeted ubiquitin.
Ubiquitin binds with low affinity to these ubiquitin binding domains
(UBDs) a evolutionary consequence which permits ubiquitin to bind to
all UBDs equally. Since ubiquitin binds in general with low affinity to
USPs but through a large contact area specific alterations within the
ubiquitin molecule itself would result in potential increased binding
affinity of the altered ubiquitin to the ubiquitin binding domain of the
USPs. Furthermore, as each USP displays a relatively sequence specific
UBD any ubiquitin variant should be USP specific.
Tight binding of these ubiquitin variants should therefore act as
competitive inhibitors of catalytic activity by blocking the recognition
of endogenous ubiquitinated substrates. Using this as a backdrop, Sidhu
and colleagues generated combinatorial, phage-displayed libraries of
ubiquitin variants and effectively identified inhibitors targeting USP8,
USP21, USP2a, USP7, USP10 and OTUB1 [302,303]. In all of the cases
each of the ubiquitin variants bound to their respective ubiquitin
binding domains with a greater efficiency then wild type ubiquitin and
more importantly did not cross react with any of the other USPs tested.
Importantly, ubiquitin variants targeting OTUB1 selectively inhibited
OTUB1 mediated cleavage of K48 di-ubiquitin. Although, ubiquitin
variant inhibitors cannot be used in patients due to their relative size
8 kDa ubiquitin variant technology does allow potent inhibition of
targeted USPs and can be utilized for future target validation and drug
discovery for all UBD proteins.
8. Concluding remarks
Since its identification over 35 years ago our understanding of
ubiquitin and the enzymes that manipulate its behavior has improved
dramatically. We now know that ubiquitination plays a central role in
virtually all biological processes including a number pathways involved
N. Kumari et al.
in neoplastic disease. Following on from the overall success of borte-
zomib expectation was high to develop other specific drugs targeting
components of the ubiquitin system. However, for the most part these
expectations have not been met. In the majority of cases these dis-
appointing results are not necessarily attributed to the fact that these
inhibitors are not specific towards their respective targets but rather
because both E3s and DUBs target multiple protein complexes.
Therefore, a better understanding of ubiquitin mediated signalling is
critical to designing effective therapeutic strategies. A number of key
questions still remain which if answered may ultimately lead to the
identification novel therapeutic targets. For example, what are the
mechanisms behind the specific targeting of E3 ligases and DUBs to
substrates? In the majority of cases phosphorylation precludes ligase
recruitment but this is observed in only a proportion of cases.
Furthermore, what is the effect of post-translational modifications on
the activity of ubiquitin modifying enzymes themselves and finally,
what is the role of non-canonical ubiquitin chains in substrate signal-
ling? New and emerging technologies such as high resolution crystal-
lography, CRISPR/CAS9 screens, and affordable Next-Gen sequencing
along with highly specific ubiquitin modifying enzyme inhibitors will
undoubtedly help tease out the roles of these enzymes in cancer pro-
gression and other diseases and ultimately pave the way for better
therapies.
Acknowledgments
The authors thank all the members in the Eichhorn Lab at CSI
Singapore for scientific discussions on the topics presented here. The
authors apologize to the authors whose papers we could not cite due to
space limitations.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was in part supported by the by the National Research
Foundation Singapore and the Singapore Ministry of Education under
its Research Centers of Excellence initiative, Koninklijke Philips N·V
and the Ministry of Education Academic Research Fund Tier 1 grants
(T1-2013 Sep-10) and (T1-2014 Oct-08).
References
[1] R.C. Kane, A.T. Farrell, R. Sridhara, R. Pazdur, United States Food and Drug
Administration approval summary: bortezomib for the treatment of progressive
multiple myeloma after one prior therapy, Clinical Cancer Research: An Official
Journal of the American Association for Cancer Research 12 (2006) 2955–2960.
[2] F. Bernassola, M. Karin, A. Ciechanover, G. Melino, The HECT family of E3 ubi-
quitin ligases: multiple players in cancer development, Cancer Cell 14 (2008)
10–21.
[3] R.J. Deshaies, C.A. Joazeiro, RING domain E3 ubiquitin ligases, Annu. Rev.
Biochem. 78 (2009) 399–434.
[4] M.B. Metzger, V.A. Hristova, A.M. Weissman, HECT and RING finger families of E3
ubiquitin ligases at a glance, J. Cell Sci. 125 (2012) 531–537.
[5] V. Kirkin, I. Dikic, Ubiquitin networks in cancer, Curr. Opin. Genet. Dev. 21 (2011)
21–28.
[6] Y. Ye, M. Rape, Building ubiquitin chains: E2 enzymes at work, nature reviews,
Mol. Cell. Biol. 10 (2009) 755–764.
[7] R. Yau, M. Rape, The increasing complexity of the ubiquitin code, Nat. Cell Biol.
18 (2016) 579–586.
[8] Y. Kulathu, D. Komander, Atypical ubiquitylation — the unexplored world of
polyubiquitin beyond Lys48 and Lys63 linkages, nature reviews, Mol. Cell. Biol. 13
(2012) 508–523.
[9] S.A. Abdul Rehman, Y.A. Kristariyanto, S.Y. Choi, P.J. Nkosi, S. Weidlich, K. Labib,
K. Hofmann, Y. Kulathu, MINDY-1 is a member of an evolutionarily conserved and
structurally distinct new family of deubiquitinating enzymes, Mol. Cell 63 (2016)
146–155.
[10] S.M. Nijman, M.P. Luna-Vargas, A. Velds, T.R. Brummelkamp, A.M. Dirac,
T.K. Sixma, R. Bernards, A genomic and functional inventory of deubiquitinating
enzymes, Cell 123 (2005) 773–786.
477
BBA - Reviews on Cancer 1868 (2017) 456–483
[11] H.M. Oosterkamp, E.M. Hijmans, T.R. Brummelkamp, S. Canisius, L.F. Wessels,
W. Zwart, R. Bernards, USP9X downregulation renders breast cancer cells resistant
to tamoxifen, Cancer Res. 74 (2014) 3810–3820.
[12] H. Potu, L.F. Peterson, M. Kandarpa, A. Pal, H. Sun, A. Durham, P.W. Harms,
P.C. Hollenhorst, U. Eskiocak, M. Talpaz, N.J. Donato, Usp9x regulates Ets-1
ubiquitination and stability to control NRAS expression and tumorigenicity in
melanoma, Nat. Commun. 8 (2017) 14449.
[13] M. Schwickart, X. Huang, J.R. Lill, J. Liu, R. Ferrando, D.M. French, H. Maecker,
K. O'Rourke, F. Bazan, J. Eastham-Anderson, P. Yue, D. Dornan, D.C. Huang,
V.M. Dixit, Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell
survival, Nature 463 (2010) 103–107.
[14] W. Ouyang, S. Zhang, B. Yang, C. Yang, J. Zhang, F. Zhou, C. Xie, Beta-catenin is
regulated by USP9x and mediates resistance to TRAIL-induced apoptosis in breast
cancer, Oncol. Rep. 35 (2016) 717–724.
[15] H. Thanh Nguyen, D. Andrejeva, R. Gupta, C. Choudhary, X. Hong, P.J. Eichhorn,
A.C. Loya, S.M. Cohen, Deubiquitylating enzyme USP9x regulates hippo pathway
activity by controlling angiomotin protein turnover, Cell Discov. 2 (2016) 16001.
[16] P.A. Perez-Mancera, A.G. Rust, L. van der Weyden, G. Kristiansen, A. Li,
A.L. Sarver, K.A. Silverstein, R. Grutzmann, D. Aust, P. Rummele, T. Knosel,
C. Herd, D.L. Stemple, R. Kettleborough, J.A. Brosnan, A. Li, R. Morgan, S. Knight,
J. Yu, S. Stegeman, L.S. Collier, J.J. ten Hoeve, J. de Ridder, A.P. Klein,
M. Goggins, R.H. Hruban, D.K. Chang, A.V. Biankin, S.M. Grimmond, I. Australian
Pancreatic Cancer Genome, L.F. Wessels, S.A. Wood, C.A. Iacobuzio-Donahue,
C. Pilarsky, D.A. Largaespada, D.J. Adams, D.A. Tuveson, The deubiquitinase
USP9X suppresses pancreatic ductal adenocarcinoma, Nature 486 (2012)
266–270.
[17] E.W. Harhaj, V.M. Dixit, Deubiquitinases in the regulation of NF-kappaB signaling,
Cell Res. 21 (2011) 22–39.
[18] P.M. Siegel, J. Massagué, Cytostatic and apoptotic actions of TGF-β in homeostasis
and cancer, Nat. Rev. Cancer 3 (2003) 807–820.
[19] J. Massagué, TGFβ in cancer, Cell 134 (2008) 215–230.
[20] J. Seoane, Escaping from the TGFβ anti-proliferative control, Carcinogenesis 27
(2006) 2148–2156.
[21] Y. Shi, J. Massagué, Mechanisms of TGF-β signaling from cell membrane to the
nucleus, Cell 113 (2003) 685–700.
[22] J. Groppe, C.S. Hinck, P. Samavarchi-Tehrani, C. Zubieta, J.P. Schuermann,
A.B. Taylor, P.M. Schwarz, J.L. Wrana, A.P. Hinck, Cooperative assembly of TGF-β
superfamily signaling complexes is mediated by two disparate mechanisms and
distinct modes of receptor binding, Mol. Cell 29 (2008) 157–168.
[23] J. Massagué, TGF-β signal transduction, Annu. Rev. Biochem. 67 (1998) 753–791.
[24] J. Massague, J. Seoane, D. Wotton, Smad transcription factors, Genes Dev. 19
(2005) 2783–2810.
[25] C. Alarcon, A.I. Zaromytidou, Q. Xi, S. Gao, J. Yu, S. Fujisawa, A. Barlas,
A.N. Miller, K. Manova-Todorova, M.J. Macias, G. Sapkota, D. Pan, J. Massague,
Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-
beta pathways, Cell 139 (2009) 757–769.
[26] R.S. Lo, J. Massague, Ubiquitin-dependent degradation of TGF-[bgr]-activated
Smad2, Nat. Cell Biol. 1 (1999) 472–478.
[27] H. Zhu, P. Kavsak, S. Abdollah, J.L. Wrana, G.H. Thomsen, A SMAD ubiquitin
ligase targets the BMP pathway and affects embryonic pattern formation, Nature
400 (1999) 687–693.
[28] R.S. Lo, J. Massague, Ubiquitin-dependent degradation of TGF-beta-activated
smad2, Nat. Cell Biol. 1 (1999) 472–478.
[29] X. Lin, M. Liang, X.-H. Feng, Smurf2 is a ubiquitin E3 ligase mediating proteasome-
dependent degradation of Smad2 in transforming growth factor-β signaling, J.
Biol. Chem. 275 (2000) 36818–36822.
[30] P. Kavsak, R.K. Rasmussen, C.G. Causing, S. Bonni, H. Zhu, G.H. Thomsen,
J.L. Wrana, Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the
TGFβ receptor for degradation, Mol. Cell 6 (2000) 1365–1375.
[31] S. Bonni, H.-R. Wang, C.G. Causing, P. Kavsak, S.L. Stroschein, K. Luo, J.L. Wrana,
TGF-β induces assembly of a Smad2–Smurf2 ubiquitin ligase complex that targets
SnoN for degradation, Nat. Cell Biol. 3 (2001) 587–595.
[32] Y. Zhang, C. Chang, D.J. Gehling, A. Hemmati-Brivanlou, R. Derynck, Regulation
of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase, Proc. Natl.
Acad. Sci. 98 (2001) 974–979.
[34] X. Lin, M. Liang, X.H. Feng, Smurf2 is a ubiquitin E3 ligase mediating proteasome-
dependent degradation of Smad2 in transforming growth factor-beta signaling, J.
Biol. Chem. 275 (2000) 36818–36822.
[35] H.I. Chen, M. Sudol, The WW domain of Yes-associated protein binds a proline-
rich ligand that differs from the consensus established for Src homology 3-binding
modules, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 7819–7823.
[36] G. Murakami, T. Watabe, K. Takaoka, K. Miyazono, T. Imamura, Cooperative in-
hibition of bone morphogenetic protein signaling by Smurf1 and inhibitory Smads,
Mol. Biol. Cell 14 (2003) 2809–2817.
[37] L.Y. Tang, M. Yamashita, N.P. Coussens, Y. Tang, X. Wang, C. Li, C.X. Deng,
S.Y. Cheng, Y.E. Zhang, Ablation of Smurf2 reveals an inhibition in TGF-beta
signalling through multiple mono-ubiquitination of Smad3, EMBO J. 30 (2011)
4777–4789.
[38] E. Maspero, E. Valentini, S. Mari, V. Cecatiello, P. Soffientini, S. Pasqualato,
S. Polo, Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for
catalytic priming, Nat. Struct. Mol. Biol. 20 (2013) 696–701.
[39] S.R. Seo, F. Lallemand, N. Ferrand, M. Pessah, S. L'Hoste, J. Camonis, A. Atfi, The
novel E3 ubiquitin ligase Tiul1 associates with TGIF to target Smad2 for de-
gradation, EMBO J. 23 (2004) 3780–3792.
[40] D. Wotton, R.S. Lo, L.-A.C. Swaby, J. Massagué, Multiple modes of repression by
the Smad transcriptional corepressor TGIF, J. Biol. Chem. 274 (1999)
N. Kumari et al.
37105–37110.
[41] C. Chen, Z. Zhou, J.S. Ross, W. Zhou, J.T. Dong, The amplified WWP1 gene is a
potential molecular target in breast cancer, Int. J. Cancer 121 (2007) 80–87.
[42] C. Chen, Z. Zhou, C.E. Sheehan, E. Slodkowska, C.B. Sheehan, A. Boguniewicz,
J.S. Ross, Overexpression of WWP1 is associated with the estrogen receptor and
insulin-like growth factor receptor 1 in breast carcinoma, Int. J. Cancer 124 (2009)
2829–2836.
[43] C. Chen, X. Sun, P. Guo, X. Dong, P. Sethi, W. Zhou, Z. Zhou, J. Petros, H. Frierson,
R. Vessella, Ubiquitin E3 ligase WWP1 as an oncogenic factor in human prostate
cancer, Oncogene 26 (2007) 2386–2394.
[44] T. Courivaud, N. Ferrand, A. Elkhattouti, S. Kumar, L. Levy, O. Ferrigno, A. Atfi,
C. Prunier, Functional characterization of a WWP1/Tiul1 tumor-derived mutant
reveals a paradigm of its constitutive activation in human cancer, J. Biol. Chem.
290 (2015) 21007–21018.
[45] S. Wiesner, A.A. Ogunjimi, H.-R. Wang, D. Rotin, F. Sicheri, J.L. Wrana,
J.D. Forman-Kay, Autoinhibition of the HECT-type ubiquitin ligase Smurf2
through its C2 domain, Cell 130 (2007) 651–662.
[46] G. Kuratomi, A. Komuro, G. Kouichiro, M. Shinozaki, K. Miyazawa, K. Miyazono,
T. Imamura, NEDD4-2 (neural precursor cell expressed, developmentally down-
regulated 4-2) negatively regulates TGF-β (transforming growth factor-β) signal-
ling by inducing ubiquitin-mediated degradation of Smad2 and TGF-β type I re-
ceptor, Biochem. J. 386 (2005) 461–470.
[47] M. Fukuchi, T. Imamura, T. Chiba, T. Ebisawa, M. Kawabata, K. Tanaka,
K. Miyazono, Ligand-dependent degradation of Smad3 by a ubiquitin ligase
complex of ROC1 and associated proteins, Mol. Biol. Cell 12 (2001) 1431–1443.
[48] J.D. Laney, M. Hochstrasser, Substrate targeting in the ubiquitin system, Cell 97
(1999) 427–430.
[49] H. Xin, X. Xu, L. Li, H. Ning, Y. Rong, Y. Shang, Y. Wang, X.-Y. Fu, Z. Chang, CHIP
controls the sensitivity of transforming growth factor-β signaling by modulating
the basal level of Smad3 through ubiquitin-mediated degradation, J. Biol. Chem.
280 (2005) 20842–20850.
[50] M. Wan, Y. Tang, E.M. Tytler, C. Lu, B. Jin, S.M. Vickers, L. Yang, X. Shi, X. Cao,
Smad4 protein stability is regulated by ubiquitin ligase SCFβ-TrCP1, J. Biol. Chem.
279 (2004) 14484–14487.
[51] M. Wan, X. Cao, Y. Wu, S. Bai, L. Wu, X. Shi, N. Wang, X. Cao, Jab1 antagonizes
TGF-β signaling by inducing Smad4 degradation, EMBO Rep. 3 (2002) 171–176.
[52] M. Schutte, R.H. Hruban, L. Hedrick, K.R. Cho, G.M. Nadasdy, C.L. Weinstein,
G.S. Bova, W.B. Isaacs, P. Cairns, H. Nawroz, D. Sidransky, R.A. Casero,
P.S. Meltzer, S.A. Hahn, S.E. Kern, Gene in various tumor types, Cancer Res. 56
(1996) 2527–2530.
[53] T. Sjöblom, S. Jones, L.D. Wood, D.W. Parsons, J. Lin, T.D. Barber, D. Mandelker,
R.J. Leary, J. Ptak, N. Silliman, S. Szabo, P. Buckhaults, C. Farrell, P. Meeh,
S.D. Markowitz, J. Willis, D. Dawson, J.K.V. Willson, A.F. Gazdar, J. Hartigan,
L. Wu, C. Liu, G. Parmigiani, B.H. Park, K.E. Bachman, N. Papadopoulos,
B. Vogelstein, K.W. Kinzler, V.E. Velculescu, The consensus coding sequences of
human breast and colorectal cancers, Science 314 (2006) 268–274.
[54] A. Moren, U. Hellman, Y. Inada, T. Imamura, C.H. Heldin, A. Moustakas,
Differential ubiquitination defines the functional status of the tumor suppressor
Smad4, J. Biol. Chem. 278 (2003) 33571–33582.
[57] S. Dupont, A. Mamidi, M. Cordenonsi, M. Montagner, L. Zacchigna, M. Adorno,
G. Martello, M.J. Stinchfield, S. Soligo, L. Morsut, FAM/USP9x, a deubiquitinating
enzyme essential for TGFβ signaling, controls Smad4 monoubiquitination, Cell
136 (2009) 123–135.
[58] H. Hayashi, S. Abdollah, Y. Qiu, J. Cai, Y.-Y. Xu, B.W. Grinnell, M.A. Richardson,
J.N. Topper, M.A. Gimbrone, J.L. Wrana, The MAD-related protein Smad7 as-
sociates with the TGFβ receptor and functions as an antagonist of TGFβ signaling,
Cell 89 (1997) 1165–1173.
[59] T. Imamura, M. Takase, A. Nishihara, E. Oeda, J.-I. Hanai, M. Kawabata,
K. Miyazono, Smad6 inhibits signalling by the TGF-β superfamily, Nature 389
(1997) 622–626.
[60] A. Nakao, M. Afrakhte, A. Morn, T. Nakayama, J.L. Christian, R. Heuchel, S. Itoh,
M. Kawabata, N.-E. Heldin, C.-H. Heldin, Identification of Smad7, a TGFβ-in-
ducible antagonist of TGF-β signalling, Nature 389 (1997) 631–635.
[61] A. Hata, G. Lagna, J. Massagué, A. Hemmati-Brivanlou, Smad6 inhibits BMP/
Smad1 signaling by specifically competing with the Smad4 tumor suppressor,
Genes Dev. 12 (1998) 186–197.
[62] S.J. Wicks, K. Haros, M. Maillard, L. Song, R.E. Cohen, P.T. Dijke, A. Chantry, The
deubiquitinating enzyme UCH37 interacts with Smads and regulates TGF-beta
signalling, Oncogene 24 (2005) 8080–8084.
[63] S. Wiesner, A.A. Ogunjimi, H.R. Wang, D. Rotin, F. Sicheri, J.L. Wrana,
J.D. Forman-Kay, Autoinhibition of the HECT-type ubiquitin ligase Smurf2
through its C2 domain, Cell 130 (2007) 651–662.
[64] A. Komuro, T. Imamura, M. Saitoh, Y. Yoshida, T. Yamori, K. Miyazono,
K. Miyazawa, Negative regulation of transforming growth factor-β (TGF-β) sig-
naling by WW domain-containing protein 1 (WWP1), Oncogene 23 (2004)
6914–6923.
[65] T. Ebisawa, M. Fukuchi, G. Murakami, T. Chiba, K. Tanaka, T. Imamura,
K. Miyazono, Smurf1 interacts with transforming growth factor-beta type I re-
ceptor through Smad7 and induces receptor degradation, J. Biol. Chem. 276
(2001) 12477–12480.
[66] G. Kuratomi, A. Komuro, K. Goto, M. Shinozaki, K. Miyazawa, K. Miyazono,
T. Imamura, NEDD4–2 (neural precursor cell expressed, developmentally down-
regulated 4–2) negatively regulates TGF-beta (transforming growth factor-beta)
signalling by inducing ubiquitin-mediated degradation of Smad2 and TGF-beta
type I receptor, The Biochemical Journal 386 (2005) 461–470.
[67] K.W. Finnson, B.Y. Tam, K. Liu, A. Marcoux, P. Lepage, S. Roy, A.A. Bizet,
478
BBA - Reviews on Cancer 1868 (2017) 456–483
A. Philip, Identification of CD109 as part of the TGF-beta receptor system in
human keratinocytes, FASEB J. 20 (2006) 1525–1527.
[68] H. Fukasawa, T. Yamamoto, Y. Fujigaki, T. Misaki, N. Ohashi, T. Takayama,
S. Suzuki, S. Mugiya, T. Oda, C. Uchida, Reduction of transforming growth factor-β
type II receptor is caused by the enhanced ubiquitin-dependent degradation in
human renal cell carcinoma, Int. J. Cancer 127 (2010) 1517–1525.
[69] M. Fukuchi, Y. Fukai, N. Masuda, T. Miyazaki, M. Nakajima, M. Sohda, R. Manda,
K. Tsukada, H. Kato, H. Kuwano, High-level expression of the Smad ubiquitin li-
gase Smurf2 correlates with poor prognosis in patients with esophageal squamous
cell carcinoma, Cancer Res. 62 (2002) 7162–7165.
[70] F. Lallemand, S.R. Seo, N. Ferrand, M. Pessah, S. L'Hoste, G. Rawadi, S. Roman-
Roman, J. Camonis, A. Atfi, AIP4 restricts transforming growth factor-β signaling
through a ubiquitination-independent mechanism, J. Biol. Chem. 280 (2005)
27645–27653.
[71] Y. Bai, C. Yang, K. Hu, C. Elly, Y.C. Liu, Itch E3 ligase-mediated regulation of TGF-
beta signaling by modulating smad2 phosphorylation, Mol. Cell 15 (2004)
825–831.
[72] S.H. Park, E.H. Jung, G.Y. Kim, B.C. Kim, J.H. Lim, C.H. Woo, Itch E3 ubiquitin
ligase positively regulates TGF-beta signaling to EMT via Smad7 ubiquitination,
Mol. Cell 38 (2015) 20–25.
[73] Y. Nagano, K.J. Mavrakis, K.L. Lee, T. Fujii, D. Koinuma, H. Sase, K. Yuki,
K. Isogaya, M. Saitoh, T. Imamura, V. Episkopou, K. Miyazono, K. Miyazawa,
Arkadia induces degradation of SnoN and c-Ski to enhance transforming growth
factor-beta signaling, J. Biol. Chem. 282 (2007) 20492–20501.
[74] D. Koinuma, M. Shinozaki, A. Komuro, K. Goto, M. Saitoh, A. Hanyu, M. Ebina,
T. Nukiwa, K. Miyazawa, T. Imamura, K. Miyazono, Arkadia amplifies TGF-beta
superfamily signalling through degradation of Smad7, EMBO J. 22 (2003)
6458–6470.
[76] L. Levy, M. Howell, D. Das, S. Harkin, V. Episkopou, C.S. Hill, Arkadia activates
Smad3/Smad4-dependent transcription by triggering signal-induced SnoN de-
gradation, Mol. Cell. Biol. 27 (2007) 6068–6083.
[77] K.J. Mavrakis, R.L. Andrew, K.L. Lee, C. Petropoulou, J.E. Dixon, N. Navaratnam,
D.P. Norris, V. Episkopou, Arkadia enhances Nodal/TGF-β signaling by coupling
phospho-Smad2/3 activity and turnover, PLoS Biol. 5 (2007) e67.
[78] L. Levy, M. Howell, D. Das, S. Harkin, V. Episkopou, C.S. Hill, Arkadia activates
Smad3/Smad4-dependent transcription by triggering signal-induced SnoN de-
gradation, Mol. Cell. Biol. 27 (2007) 6068–6083.
[79] S. Bonni, H.R. Wang, C.G. Causing, P. Kavsak, S.L. Stroschein, K. Luo, J.L. Wrana,
TGF-beta induces assembly of a Smad2-Smurf2 ubiquitin ligase complex that
targets SnoN for degradation, Nat. Cell Biol. 3 (2001) 587–595.
[80] S.L. Stroschein, S. Bonni, J.L. Wrana, K. Luo, Smad3 recruits the anaphase-pro-
moting complex for ubiquitination and degradation of SnoN, Genes Dev. 15 (2001)
2822–2836.
[81] A. Mizutani, M. Saitoh, T. Imamura, K. Miyazawa, K. Miyazono, Arkadia com-
plexes with clathrin adaptor AP2 and regulates EGF signalling, J. Biochem. 148
(2010) 733–741.
[82] V. Sharma, A.G. Antonacopoulou, S. Tanaka, A.A. Panoutsopoulos, V. Bravou,
H.P. Kalofonos, V. Episkopou, Enhancement of TGF-β signaling responses by the
E3 ubiquitin ligase Arkadia provides tumor suppression in colorectal cancer,
Cancer Res. 71 (2011) 6438–6449.
[83] M.A. Briones-Orta, L. Levy, C.D. Madsen, D. Das, Y. Erker, E. Sahai, C.S. Hill,
Arkadia regulates tumor metastasis by modulation of the TGF-β pathway, Cancer
Res. 73 (2013) 1800–1810.
[84] L. Zhang, H. Huang, F. Zhou, J. Schimmel, C.G. Pardo, T. Zhang, T.S. Barakat, K.-
A. Sheppard, C. Mickanin, J.A. Porter, RNF12 controls embryonic stem cell fate
and morphogenesis in zebrafish embryos by targeting Smad7 for degradation, Mol.
Cell 46 (2012) 650–661.
[85] L. Zhang, F. Zhou, Y. Drabsch, R. Gao, B.E. Snaar-Jagalska, C. Mickanin, H. Huang,
K.A. Sheppard, J.A. Porter, C.X. Lu, P. ten Dijke, USP4 is regulated by AKT
phosphorylation and directly deubiquitylates TGF-beta type I receptor, Nat. Cell
Biol. 14 (2012) 717–726.
[86] M.A. Al-Salihi, L. Herhaus, T. Macartney, G.P. Sapkota, USP11 augments TGFbeta
signalling by deubiquitylating ALK5, Open Biology 2 (2012) 120063.
[87] P.J. Eichhorn, L. Rodon, A. Gonzalez-Junca, A. Dirac, M. Gili, E. Martinez-Saez,
C. Aura, I. Barba, V. Peg, A. Prat, I. Cuartas, J. Jimenez, D. Garcia-Dorado,
J. Sahuquillo, R. Bernards, J. Baselga, J. Seoane, USP15 stabilizes TGF-beta re-
ceptor I and promotes oncogenesis through the activation of TGF-beta signaling in
glioblastoma, Nat. Med. 18 (2012) 429–435.
[88] P.V. Iyengar, P. Jaynes, L. Rodon, D. Lama, K.P. Law, Y.P. Lim, C. Verma,
J. Seoane, P.J. Eichhorn, USP15 regulates SMURF2 kinetics through C-lobe
mediated deubiquitination, Sci Rep 5 (2015) 14733.
[89] W.T. Liu, K.Y. Huang, M.C. Lu, H.L. Huang, C.Y. Chen, Y.L. Cheng, H.C. Yu,
S.Q. Liu, N.S. Lai, H.B. Huang, TGF-beta upregulates the translation of USP15 via
the PI3K/AKT pathway to promote p53 stability, Oncogene (2016).
[90] M. Inui, A. Manfrin, A. Mamidi, G. Martello, L. Morsut, S. Soligo, E. Enzo, S. Moro,
S. Polo, S. Dupont, M. Cordenonsi, S. Piccolo, USP15 is a deubiquitylating enzyme
for receptor-activated SMADs, Nat. Cell Biol. 13 (2011) 1368–1375.
[91] P.J.A. Eichhorn, L. Rodon, A. Gonzalez-Junca, A. Dirac, M. Gili, E. Martinez-Saez,
C. Aura, I. Barba, V. Peg, A. Prat, I. Cuartas, J. Jimenez, D. Garcia-Dorado,
J. Sahuquillo, R. Bernards, J. Baselga, J. Seoane, USP15 stabilizes TGF-[beta] re-
ceptor I and promotes oncogenesis through the activation of TGF-[beta] signaling
in glioblastoma, Nat. Med. 18 (2012) 429–435.
[92] L. Zhang, F. Zhou, Y. Drabsch, R. Gao, B.E. Snaar-Jagalska, C. Mickanin, H. Huang,
K.-A. Sheppard, J.A. Porter, C.X. Lu, USP4 is regulated by AKT phosphorylation
and directly deubiquitylates TGF-[beta] type I receptor, Nat. Cell Biol. 14 (2012)
717–726.
N. Kumari et al.
[93] M.A. Al-Salihi, L. Herhaus, T. Macartney, G.P. Sapkota, USP11 augments TGFβ
signalling by deubiquitylating ALK5, Open Biology 2 (2012) 120063.
[94] S. Kit Leng Lui, P.V. Iyengar, P. Jaynes, Z. Isa, B. Pang, T.Z. Tan, P.J.A. Eichhorn,
USP26 regulates TGF-beta signaling by deubiquitinating and stabilizing SMAD7,
EMBO Rep. 18 (2017) 797–808.
[95] F. Itoh, H. Asao, K. Sugamura, C.H. Heldin, P. ten Dijke, S. Itoh, Promoting bone
morphogenetic protein signaling through negative regulation of inhibitory Smads,
EMBO J. 20 (2001) 4132–4142.
[96] N. Tanaka, K. Kaneko, H. Asao, H. Kasai, Y. Endo, T. Fujita, T. Takeshita,
K. Sugamura, Possible involvement of a novel STAM-associated molecule “AMSH”
in intracellular signal transduction mediated by cytokines, J. Biol. Chem. 274
(1999) 19129–19135.
[97] J.H. Hurley, Nipped in the bud: how the AMSH MIT domain helps deubiquitinate
lysosome-bound cargo, Structure 19 (2011) 1033–1035.
[98] L. Herhaus, M. Al-Salihi, T. Macartney, S. Weidlich, G.P. Sapkota, OTUB1 en-
hances TGFβ signalling by inhibiting the ubiquitylation and degradation of active
SMAD2/3, Nat. Commun. 4 (2013).
[99] S. Nakada, I. Tai, S. Panier, A. Al-Hakim, S.-i. Iemura, Y.-C. Juang, L. O'Donnell,
A. Kumakubo, M. Munro, F. Sicheri, Non-canonical inhibition of DNA damage-
dependent ubiquitination by OTUB1, Nature 466 (2010) 941–946.
[100] Y. Sato, A. Yamagata, S. Goto-Ito, K. Kubota, R. Miyamoto, S. Nakada, S. Fukai,
Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction
between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating
enzyme UBC13, J. Biol. Chem. 287 (2012) 25860–25868.
[101] Y.-C. Juang, M.-C. Landry, M. Sanches, V. Vittal, C.C.Y. Leung, Derek
F. Ceccarelli, A.-Rachele F. Mateo, Jonathan N. Pruneda, D.Y.L. Mao, Rachel
K. Szilard, S. Orlicky, M. Munro, Peter S. Brzovic, Rachel E. Klevit, F. Sicheri,
D. Durocher, OTUB1 Co-opts Lys48-linked ubiquitin recognition to suppress E2
enzyme function, Mol. Cell 45 (2012) 384–397.
[102] R. Wiener, X. Zhang, T. Wang, C. Wolberger, The mechanism of OTUB1-mediated
inhibition of ubiquitination, Nature 483 (2012) 618–622.
[103] S. Dupont, L. Zacchigna, M. Cordenonsi, S. Soligo, M. Adorno, M. Rugge,
S. Piccolo, Germ-layer specification and control of cell growth by ectodermin, a
Smad4 ubiquitin ligase, Cell 121 (2005) 87–99.
[104] Z. Meng, T. Moroishi, K.L. Guan, Mechanisms of hippo pathway regulation, Genes
Dev. 30 (2016) 1–17.
[105] F. Zanconato, M. Cordenonsi, S. Piccolo, YAP/TAZ at the roots of cancer, Cancer
Cell 29 (2016) 783–803.
[106] T. Moroishi, C.G. Hansen, K.L. Guan, The emerging roles of YAP and TAZ in
cancer, Nat. Rev. Cancer 15 (2015) 73–79.
[107] S. Piccolo, S. Dupont, M. Cordenonsi, The biology of YAP/TAZ: hippo signaling
and beyond, Physiol. Rev. 94 (2014) 1287–1312.
[108] X. Hong, H.T. Nguyen, Q. Chen, R. Zhang, Z. Hagman, P.M. Voorhoeve,
S.M. Cohen, Opposing activities of the Ras and hippo pathways converge on
regulation of YAP protein turnover, EMBO J. 33 (2014) 2447–2457.
[109] B. Yeung, K.C. Ho, X. Yang, WWP1 E3 ligase targets LATS1 for ubiquitin-mediated
degradation in breast cancer cells, PLoS One 8 (2013) e61027.
[110] S.J. Bae, M. Kim, S.H. Kim, Y.E. Kwon, J.H. Lee, J. Kim, C.H. Chung, W.J. Lee,
J.H. Seol, NEDD4 controls intestinal stem cell homeostasis by regulating the hippo
signalling pathway, Nat. Commun. 6 (2015) 6314.
[111] K.C. Ho, Z. Zhou, Y.M. She, A. Chun, T.D. Cyr, X. Yang, Itch E3 ubiquitin ligase
regulates large tumor suppressor 1 stability [corrected], Proc. Natl. Acad. Sci. U. S.
A. 108 (2011) 4870–4875.
[112] Z. Salah, G. Melino, R.I. Aqeilan, Negative regulation of the hippo pathway by E3
ubiquitin ligase ITCH is sufficient to promote tumorigenicity, Cancer Res. 71
(2011) 2010–2020.
[113] R.J. Ingham, G. Gish, T. Pawson, The Nedd4 family of E3 ubiquitin ligases:
functional diversity within a common modular architecture, Oncogene 23 (2004)
1972–1984.
[114] B. Ma, Y. Chen, L. Chen, H. Cheng, C. Mu, J. Li, R. Gao, C. Zhou, L. Cao, J. Liu,
Y. Zhu, Q. Chen, S. Wu, Hypoxia regulates hippo signalling through the SIAH2
ubiquitin E3 ligase, Nat. Cell Biol. 17 (2015) 95–103.
[115] L. Lignitto, A. Arcella, M. Sepe, L. Rinaldi, R. Delle Donne, A. Gallo, E. Stefan,
V.A. Bachmann, M.A. Oliva, C. Tiziana Storlazzi, A. L'Abbate, A. Brunetti,
S. Gargiulo, M. Gramanzini, L. Insabato, C. Garbi, M.E. Gottesman, A. Feliciello,
Proteolysis of MOB1 by the ubiquitin ligase praja2 attenuates hippo signalling and
supports glioblastoma growth, Nat. Commun. 4 (2013) 1822.
[116] B. Zhao, L. Li, Q. Lu, L.H. Wang, C.Y. Liu, Q. Lei, K.L. Guan, Angiomotin is a novel
hippo pathway component that inhibits YAP oncoprotein, Genes Dev. 25 (2011)
51–63.
[117] J.J. Adler, D.E. Johnson, B.L. Heller, L.R. Bringman, W.P. Ranahan, M.D. Conwell,
Y. Sun, A. Hudmon, C.D. Wells, Serum deprivation inhibits the transcriptional co-
activator YAP and cell growth via phosphorylation of the 130-kDa isoform of
angiomotin by the LATS1/2 protein kinases, Proc. Natl. Acad. Sci. U. S. A. 110
(2013) 17368–17373.
[118] M. Kim, M. Kim, S.J. Park, C. Lee, D.S. Lim, Role of angiomotin-like 2 mono-
ubiquitination on YAP inhibition, EMBO Rep. 17 (2016) 64–78.
[119] H.T. Nguyen, X. Hong, S. Tan, Q. Chen, L. Chan, M. Fivaz, S.M. Cohen,
P.M. Voorhoeve, Viral small T oncoproteins transform cells by alleviating hippo-
pathway-mediated inhibition of the YAP proto-oncogene, Cell Rep. 8 (2014)
707–713.
[120] D. Shao, P. Zhai, D.P. Del Re, S. Sciarretta, N. Yabuta, H. Nojima, D.S. Lim, D. Pan,
J. Sadoshima, A functional interaction between hippo-YAP signalling and FoxO1
mediates the oxidative stress response, Nat. Commun. 5 (2014) 3315.
[121] D.D. Shao, W. Xue, E.B. Krall, A. Bhutkar, F. Piccioni, X. Wang, A.C. Schinzel,
S. Sood, J. Rosenbluh, J.W. Kim, Y. Zwang, T.M. Roberts, D.E. Root, T. Jacks,
479
BBA - Reviews on Cancer 1868 (2017) 456–483
W.C. Hahn, KRAS and YAP1 converge to regulate EMT and tumor survival, Cell
158 (2014) 171–184.
[122] W. Zhang, Y. Gao, F. Li, X. Tong, Y. Ren, X. Han, S. Yao, F. Long, Z. Yang, H. Fan,
L. Zhang, H. Ji, YAP promotes malignant progression of Lkb1-deficient lung
adenocarcinoma through downstream regulation of survivin, Cancer Res. 75
(2015) 4450–4457.
[123] W. Zhang, N. Nandakumar, Y. Shi, M. Manzano, A. Smith, G. Graham, S. Gupta,
E.E. Vietsch, S.Z. Laughlin, M. Wadhwa, M. Chetram, M. Joshi, F. Wang,
B. Kallakury, J. Toretsky, A. Wellstein, C. Yi, Downstream of mutant KRAS, the
transcription regulator YAP is essential for neoplastic progression to pancreatic
ductal adenocarcinoma, Sci. Signal. 7 (2014) ra42.
[124] H.T. Nguyen, J.M. Kugler, S.M. Cohen, DUB3 deubiquitylating enzymes regulate
hippo pathway activity by regulating the stability of ITCH, LATS and AMOT
proteins, PLoS One 12 (2017) e0169587.
[125] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144
(2011) 646–674.
[126] S. Sigismund, S. Confalonieri, A. Ciliberto, S. Polo, G. Scita, P.P. Di Fiore,
Endocytosis and signaling: cell logistics shape the eukaryotic cell plan, Physiol.
Rev. 92 (2012) 273–366.
[127] K. Haglund, I. Dikic, The role of ubiquitylation in receptor endocytosis and en-
dosomal sorting, J. Cell Sci. 125 (2012) 265–275.
[128] M.G. Savio, N. Wollscheid, E. Cavallaro, V. Algisi, P.P. Di Fiore, S. Sigismund,
E. Maspero, S. Polo, USP9X controls EGFR fate by deubiquitinating the endocytic
adaptor Eps15, Current Biology: CB 26 (2016) 173–183.
[129] I.M. Meijer, J.E. van Leeuwen, ERBB2 is a target for USP8-mediated deubiquiti-
nation, Cell. Signal. 23 (2011) 458–467.
[130] S. Niendorf, A. Oksche, A. Kisser, J. Löhler, M. Prinz, H. Schorle, S. Feller,
M. Lewitzky, I. Horak, K.-P. Knobeloch, Essential role of ubiquitin-specific pro-
tease 8 for receptor tyrosine kinase stability and endocytic trafficking in vivo, Mol.
Cell. Biol. 27 (2007) 5029–5039.
[131] I.M. Meijer, J. Kerperien, A.M. Sotoca, E.J. van Zoelen, J.E. van Leeuwen, The
Usp8 deubiquitination enzyme is post-translationally modified by tyrosine and
serine phosphorylation, Cell. Signal. 25 (2013) 919–930.
[132] T. Obsil, V. Obsilova, Structural Basis of 14-3-3 Protein Functions, Seminars in
Cell & Developmental Biology, Elsevier, 2011, pp. 663–672.
[133] X. Wu, L. Yen, L. Irwin, C. Sweeney, K.L. Carraway, Stabilization of the E3 ubi-
quitin ligase Nrdp1 by the deubiquitinating enzyme USP8, Mol. Cell. Biol. 24
(2004) 7748–7757.
[134] Z. Liu, S.M. Zanata, J. Kim, M.A. Peterson, D. Di Vizio, L.R. Chirieac, S. Pyne,
M. Agostini, M.R. Freeman, M. Loda, The ubiquitin-specific protease USP2a pre-
vents endocytosis-mediated EGFR degradation, Oncogene 32 (2013) 1660–1669.
[135] J. Kim, W.-J. Kim, Z. Liu, M. Loda, M.R. Freeman, The ubiquitin-specific protease
USP2a enhances tumor progression by targeting cyclin A1 in bladder cancer, Cell
Cycle 11 (2012) 1123–1130.
[136] B. Sehat, S. Andersson, R. Vasilcanu, L. Girnita, O. Larsson, Role of ubiquitination
in IGF-1 receptor signaling and degradation, PLoS One 2 (2007) e340.
[137] G. Levkowitz, H. Waterman, S.A. Ettenberg, M. Katz, A.Y. Tsygankov, I. Alroy,
S. Lavi, K. Iwai, Y. Reiss, A. Ciechanover, Ubiquitin ligase activity and tyrosine
phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1,
Mol. Cell 4 (1999) 1029–1040.
[138] H. Waterman, M. Katz, C. Rubin, K. Shtiegman, S. Lavi, A. Elson, T. Jovin,
Y. Yarden, A mutant EGF-receptor defective in ubiquitylation and endocytosis
unveils a role for Grb2 in negative signaling, EMBO J. 21 (2002) 303–313.
[139] H. Dou, L. Buetow, A. Hock, G.J. Sibbet, K.H. Vousden, D.T. Huang, Structural
basis for autoinhibition and phosphorylation-dependent activation of c-Cbl, Nat.
Struct. Mol. Biol. 19 (2012) 184–192.
[140] G. Christofori, Split personalities: the agonistic antagonist Sprouty, Nat. Cell Biol.
5 (2003) 377–379.
[141] C. Rubin, V. Litvak, H. Medvedovsky, Y. Zwang, S. Lev, Y. Yarden, Sprouty fine-
tunes EGF signaling through interlinked positive and negative feedback loops,
Curr. Biol. 13 (2003) 297–307.
[142] G.R. Guy, R.A. Jackson, P. Yusoff, S.Y. Chow, Sprouty proteins: modified mod-
ulators, matchmakers or missing links? J. Endocrinol. 203 (2009) 191–202.
[143] H. Hanafusa, S. Torii, T. Yasunaga, E. Nishida, Sprouty1 and Sprouty2 provide a
control mechanism for the Ras/MAPK signalling pathway, Nat. Cell Biol. 4 (2002)
850–858.
[144] M. Tsang, I.B. Dawid, Promotion and attenuation of FGF signaling through the
Ras-MAPK pathway, Sci. STKE 2004 (2004) e17.
[145] A. Sasaki, T. Taketomi, R. Kato, K. Saeki, A. Nonami, M. Sasaki, M. Kuriyama,
N. Saito, M. Shibuya, A. Yoshimura, Mammalian Sprouty4 suppresses Ras-in-
dependent ERK activation by binding to Raf1, Nat. Cell Biol. 5 (2003) 427–432.
[146] A.B. Keeton, E.A. Salter, G.A. Piazza, The RAS–effector interaction as a drug target,
Cancer Res. (2017).
[147] N. Jura, E. Scotto-Lavino, A. Sobczyk, D. Bar-Sagi, Differential modification of Ras
proteins by ubiquitination, Mol. Cell 21 (2006) 679–687.
[148] S.-E. Kim, J.-Y. Yoon, W.-J. Jeong, S.-H. Jeon, Y. Park, J.-B. Yoon, Y.N. Park,
H. Kim, K.-Y. Choi, H-Ras is degraded by Wnt/β-catenin signaling via β-TrCP-
mediated polyubiquitylation, J. Cell Sci. 122 (2009) 842–848.
[149] S. Shukla, U.S. Allam, A. Ahsan, G. Chen, P.M. Krishnamurthy, K. Marsh,
M. Rumschlag, S. Shankar, C. Whitehead, M. Schipper, V. Basrur,
D.R. Southworth, A.M. Chinnaiyan, A. Rehemtulla, D.G. Beer, T.S. Lawrence,
M.K. Nyati, D. Ray, KRAS protein stability is regulated through SMURF2: UBCH5
complex-mediated beta-TrCP1 degradation, Neoplasia 16 (2014) 115–128.
[150] H. Yan, M. Jahanshahi, E.A. Horvath, H.Y. Liu, C.M. Pfleger, Rabex-5 ubiquitin
ligase activity restricts Ras signaling to establish pathway homeostasis in droso-
phila, Current Biology: CB 20 (2010) 1378–1382.
N. Kumari et al.
[151] L. Xu, V. Lubkov, L.J. Taylor, D. Bar-Sagi, Feedback regulation of Ras signaling by
Rabex-5-mediated ubiquitination, Current Biology: CB 20 (2010) 1372–1377.
[152] R. Baker, E.M. Wilkerson, K. Sumita, D.G. Isom, A.T. Sasaki, H.G. Dohlman,
S.L. Campbell, Differences in the regulation of K-Ras and H-Ras isoforms by
monoubiquitination, J. Biol. Chem. 288 (2013) 36856–36862.
[153] A. Laine, Ubiquitin chains in the ladder of MAPK signaling, Sci. Signal. 2005
(2005) re5-re5.
[154] S.A. Matheny, C. Chen, R.L. Kortum, G.L. Razidlo, R.E. Lewis, M.A. White, Ras
regulates assembly of mitogenic signalling complexes through the effector protein
IMP, Nature 427 (2004) 256–260.
[155] S.D. Hayes, H. Liu, E. MacDonald, C.M. Sanderson, J.M. Coulson, M.J. Clague,
S. Urbé, Direct and indirect control of mitogen-activated protein kinase pathway-
associated components, BRAP/IMP E3 ubiquitin ligase and CRAF/RAF1 kinase, by
the deubiquitylating enzyme USP15, J. Biol. Chem. 287 (2012) 43007–43018.
[156] E.R. Jang, P. Shi, J. Bryant, J. Chen, V. Dukhande, M.S. Gentry, H. Jang,
M. Jeoung, E. Galperin, HUWE1 is a molecular link controlling RAF-1 activity
supported by the Shoc2 scaffold, Mol. Cell. Biol. 34 (2014) 3579–3593.
[157] T. Dogan, G.S. Harms, M. Hekman, C. Karreman, T.K. Oberoi, E.S. Alnemri,
U.R. Rapp, K. Rajalingam, X-linked and cellular IAPs modulate the stability of C-
RAF kinase and cell motility, Nat. Cell Biol. 10 (2008) 1447–1455.
[158] T.K. Oberoi-Khanuja, C. Karreman, S. Larisch, U.R. Rapp, K. Rajalingam, Role of
melanoma inhibitor of apoptosis (ML-IAP) protein, a member of the baculoviral
IAP repeat (BIR) domain family, in the regulation of C-RAF kinase and cell mi-
gration, J. Biol. Chem. 287 (2012) 28445–28455.
[159] O. Grbovic, A. Basso, A. Sawai, Q. Ye, P. Friedlander, D. Solit, N. Rosen, V600E B-
Raf requires the Hsp90 chaperone for stability and is degraded in response to
Hsp90 inhibitors, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 57–62.
[160] T.W. Schulte, M.V. Blagosklonny, C. Ingui, L. Neckers, Disruption of the Raf-1-
Hsp90 molecular complex results in destabilization of Raf-1 and loss of Raf-1-Ras
association, J. Biol. Chem. 270 (1995) 24585–24588.
[161] T.W. Schulte, W.G. An, L.M. Neckers, Geldanamycin-induced destabilization of
Raf-1 involves the proteasome, Biochem. Biophys. Res. Commun. 239 (1997)
655–659.
[162] S.-W. Hong, D.-H. Jin, J.-S. Shin, J.-H. Moon, Y.-S. Na, K.-A. Jung, S.-M. Kim,
J.C. Kim, K.-p. Kim, Y.S. Hong, Ring finger protein 149 is an E3 ubiquitin ligase
active on wild-type v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), J.
Biol. Chem. 287 (2012) 24017–24025.
[163] J. Du, J. Zeng, X. Ou, X. Ren, S. Cai, Methylglyoxal downregulates Raf-1 protein
through a ubiquitination-mediated mechanism, Int. J. Biochem. Cell Biol. 38
(2006) 1084–1091.
[164] Z. Lu, S. Xu, C. Joazeiro, M.H. Cobb, T. Hunter, The PHD domain of MEKK1 acts as
an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2,
Mol. Cell 9 (2002) 945–956.
[165] L.C. Cantley, B.G. Neel, New insights into tumor suppression: PTEN suppresses
tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway, Proc.
Natl. Acad. Sci. 96 (1999) 4240–4245.
[166] M. Cully, H. You, A.J. Levine, T.W. Mak, Beyond PTEN mutations: the PI3K
pathway as an integrator of multiple inputs during tumorigenesis, Nat. Rev.
Cancer 6 (2006) 184–192.
[167] S. Kuchay, S. Duan, E. Schenkein, A. Peschiaroli, A. Saraf, L. Florens,
M.P. Washburn, M. Pagano, FBXL2-and PTPL1-mediated degradation of p110-free
p85β regulatory subunit controls the PI (3) K signalling cascade, Nat. Cell Biol. 15
(2013) 472–480.
[168] B. Geering, P. Cutillas, B. Vanhaesebroeck, Regulation of class IA PI3Ks: is there a
role for monomeric PI3K subunits? Biochem. Soc. Trans. 35 (2007) 199–203.
[169] H. Ko, C. Kim, S. Lee, J. Song, K. Lee, K. Kim, K. Park, S. Cho, J. Ahn, P42 Ebp1
regulates the proteasomal degradation of the p85 regulatory subunit of PI3K by
recruiting a chaperone-E3 ligase complex HSP70/CHIP, Cell Death Dis. 5 (2014)
e1131.
[170] D. Fang, H.-Y. Wang, N. Fang, Y. Altman, C. Elly, Y.-C. Liu, Cbl-b, a RING-type E3
ubiquitin ligase, targets phosphatidylinositol 3-kinase for ubiquitination in T cells,
J. Biol. Chem. 276 (2001) 4872–4878.
[171] X. Xu, A. Sarikas, D.C. Dias-Santagata, G. Dolios, P.J. Lafontant, S.-C. Tsai, W. Zhu,
H. Nakajima, H.O. Nakajima, L.J. Field, The CUL7 E3 ubiquitin ligase targets in-
sulin receptor substrate 1 for ubiquitin-dependent degradation, Mol. Cell 30
(2008) 403–414.
[172] L. Rui, M. Yuan, D. Frantz, S. Shoelson, M.F. White, SOCS-1 and SOCS-3 block
insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2, J. Biol.
Chem. 277 (2002) 42394–42398.
[173] R. Nakao, K. Hirasaka, J. Goto, K. Ishidoh, C. Yamada, A. Ohno, Y. Okumura,
I. Nonaka, K. Yasutomo, K.M. Baldwin, Ubiquitin ligase Cbl-b is a negative reg-
ulator for insulin-like growth factor 1 signaling during muscle atrophy caused by
unloading, Mol. Cell. Biol. 29 (2009) 4798–4811.
[174] R. Song, W. Peng, Y. Zhang, F. Lv, H.-K. Wu, J. Guo, Y. Cao, Y. Pi, X. Zhang, L. Jin,
Central role of E3 ubiquitin ligase MG53 in insulin resistance and metabolic dis-
orders, Nature 494 (2013) 375–379.
[175] T. Fukushima, H. Yoshihara, H. Furuta, H. Kamei, F. Hakuno, J. Luan, C. Duan,
Y. Saeki, K. Tanaka, S.-I. Iemura, Nedd4-induced monoubiquitination of IRS-2
enhances IGF signalling and mitogenic activity, Nat. Commun. 6 (2015).
[176] H. Yoshihara, T. Fukushima, F. Hakuno, Y. Saeki, K. Tanaka, A. Ito, M. Yoshida, S.-
I. Iemura, T. Natsume, T. Asano, Insulin/insulin-like growth factor (IGF) stimu-
lation abrogates an association between a deubiquitinating enzyme USP7 and
insulin receptor substrates (IRSs) followed by proteasomal degradation of IRSs,
Biochem. Biophys. Res. Commun. 423 (2012) 122–127.
[177] W.-L. Yang, J. Wang, C.-H. Chan, S.-W. Lee, A.D. Campos, B. Lamothe, L. Hur,
B.C. Grabiner, X. Lin, B.G. Darnay, The E3 ligase TRAF6 regulates Akt
480
BBA - Reviews on Cancer 1868 (2017) 456–483
ubiquitination and activation, Science 325 (2009) 1134–1138.
[178] C.-D. Fan, M.A. Lum, C. Xu, J.D. Black, X. Wang, Ubiquitin-dependent regulation
of phospho-AKT dynamics by the ubiquitin E3 ligase, NEDD4-1, in the insulin-like
growth factor-1 response, J. Biol. Chem. 288 (2013) 1674–1684.
[179] W. Li, C. Peng, M.H. Lee, D. Lim, F. Zhu, Y. Fu, G. Yang, Y. Sheng, L. Xiao, X. Dong,
W. Ma, A.M. Bode, Y. Cao, Z. Dong, TRAF4 is a critical molecule for Akt activation
in lung cancer, Cancer Res. 73 (2013) 6938–6950.
[180] C.-H. Chan, C.-F. Li, W.-L. Yang, Y. Gao, S.-W. Lee, Z. Feng, H.-Y. Huang, K.K. Tsai,
L.G. Flores, Y. Shao, The Skp2-SCF E3 ligase regulates Akt ubiquitination, glyco-
lysis, herceptin sensitivity, and tumorigenesis, Cell 149 (2012) 1098–1111.
[181] J.D. Carpten, A.L. Faber, C. Horn, G.P. Donoho, S.L. Briggs, C.M. Robbins,
G. Hostetter, S. Boguslawski, T.Y. Moses, S. Savage, M. Uhlik, A. Lin, J. Du,
Y.W. Qian, D.J. Zeckner, G. Tucker-Kellogg, J. Touchman, K. Patel, S. Mousses,
M. Bittner, R. Schevitz, M.H. Lai, K.L. Blanchard, J.E. Thomas, A transforming
mutation in the pleckstrin homology domain of AKT1 in cancer, Nature 448
(2007) 439–444.
[182] J.H. Lim, H. Jono, K. Komatsu, C.-H. Woo, J. Lee, M. Miyata, T. Matsuno, X. Xu,
Y. Huang, W. Zhang, CYLD negatively regulates transforming growth factor-β-
signalling via deubiquitinating Akt, Nat. Commun. 3 (2012) 771.
[183] W.-L. Yang, G. Jin, C.-F. Li, Y.S. Jeong, A. Moten, D. Xu, Z. Feng, W. Chen, Z. Cai,
B. Darnay, Cycles of ubiquitination and deubiquitination critically regulate growth
factor-mediated activation of Akt signaling, Sci. Signal. 6 (2013).
[184] E. Guven-Maiorov, O. Keskin, A. Gursoy, R. Nussinov, A structural view of nega-
tive regulation of the toll-like receptor-mediated inflammatory pathway, Biophys.
J. 109 (2015) 1214–1226.
[185] M. Li, C.L. Brooks, N. Kon, W. Gu, A dynamic role of HAUSP in the p53-Mdm2
pathway, Mol. Cell 13 (2004) 879–886.
[186] T. Xiang, A. Ohashi, Y. Huang, T.K. Pandita, T. Ludwig, S.N. Powell, Q. Yang,
Negative regulation of AKT activation by BRCA1, Cancer Res. 68 (2008)
10040–10044.
[187] S. Bae, S.Y. Kim, J.H. Jung, Y. Yoon, H.J. Cha, H. Lee, K. Kim, J. Kim, I.S. An,
J. Kim, H.D. Um, I.C. Park, S.J. Lee, S.Y. Nam, Y.W. Jin, J.H. Lee, S. An, Akt is
negatively regulated by the MULAN E3 ligase, Cell Res. 22 (2012) 873–885.
[188] C.A. Dickey, J. Koren, Y.J. Zhang, Y.F. Xu, U.K. Jinwal, M.J. Birnbaum, B. Monks,
M. Sun, J.Q. Cheng, C. Patterson, R.M. Bailey, J. Dunmore, S. Soresh, C. Leon,
D. Morgan, L. Petrucelli, Akt and CHIP coregulate tau degradation through co-
ordinated interactions, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 3622–3627.
[189] F. Suizu, Y. Hiramuki, F. Okumura, M. Matsuda, A.J. Okumura, N. Hirata,
M. Narita, T. Kohno, J. Yokota, M. Bohgaki, C. Obuse, S. Hatakeyama, T. Obata,
M. Noguchi, The E3 ligase TTC3 facilitates ubiquitination and degradation of
phosphorylated Akt, Dev. Cell 17 (2009) 800–810.
[190] A. Toker, L.C. Cantley, Signalling through the lipid products of phosphoinositide-
3-OH kinase, Nature 387 (1997) 673–676.
[191] P. Rodriguez-Viciana, P.H. Warne, A. Khwaja, B.M. Marte, D. Pappin, P. Das,
M.D. Waterfield, A. Ridley, J. Downward, Role of phosphoinositide 3-OH kinase in
cell transformation and control of the actin cytoskeleton by Ras, Cell 89 (1997)
457–467.
[192] L.R. Pearce, D. Komander, D.R. Alessi, The nuts and bolts of AGC protein kinases,
Nat. Rev. Mol. Cell Biol. 11 (2010) 9–22.
[193] I.Z. Uras, T. List, S.M. Nijman, Ubiquitin-specific protease 4 inhibits mono-ubi-
quitination of the master growth factor signaling kinase PDK1, PLoS One 7 (2012)
e31003.
[194] M. Laplante, D.M. Sabatini, mTOR signaling at a glance, J. Cell Sci. 122 (2009)
3589–3594.
[195] M. Corradetti, K. Guan, Upstream of the mammalian target of rapamycin: do all
roads pass through mTOR? Oncogene 25 (2006) 6347–6360.
[196] T.R. Peterson, M. Laplante, C.C. Thoreen, Y. Sancak, S.A. Kang, W.M. Kuehl,
N.S. Gray, D.M. Sabatini, DEPTOR is an mTOR inhibitor frequently overexpressed
in multiple myeloma cells and required for their survival, Cell 137 (2009)
873–886.
[197] Y. Zhao, X. Xiong, Y. Sun, DEPTOR, an mTOR inhibitor, is a physiological sub-
strate of SCF βTrCP E3 ubiquitin ligase and regulates survival and autophagy, Mol.
Cell 44 (2011) 304–316.
[198] D. Gao, H. Inuzuka, M.-K.M. Tan, H. Fukushima, J.W. Locasale, P. Liu, L. Wan,
B. Zhai, Y.R. Chin, S. Shaik, mTOR drives its own activation via SCF βTrCP-de-
pendent degradation of the mTOR inhibitor DEPTOR, Mol. Cell 44 (2011)
290–303.
[199] S. Duan, J.R. Skaar, S. Kuchay, A. Toschi, N. Kanarek, Y. Ben-Neriah, M. Pagano,
mTOR generates an auto-amplification loop by triggering the βTrCP-and CK1α-
dependent degradation of DEPTOR, Mol. Cell 44 (2011) 317–324.
[200] P. Ghosh, M. Wu, H. Zhang, H. Sun, mTORC1 signaling requires proteasomal
function and the involvement of CUL4-DDB1 ubiquitin E3 ligase, Cell Cycle 7
(2008) 373–381.
[201] S. Hussain, A.L. Feldman, C. Das, S.C. Ziesmer, S.M. Ansell, P.J. Galardy, Ubiquitin
hydrolase UCH-L1 destabilizes mTOR complex 1 by antagonizing DDB1-CUL4-
mediated ubiquitination of raptor, Mol. Cell. Biol. 33 (2013) 1188–1197.
[202] B. Wang, Z. Jie, D. Joo, A. Ordureau, P. Liu, W. Gan, J. Guo, J. Zhang, B.J. North,
X. Dai, X. Cheng, X. Bian, L. Zhang, J.W. Harper, S.C. Sun, W. Wei, TRAF2 and
OTUD7B govern a ubiquitin-dependent switch that regulates mTORC2 signalling,
Nature 545 (2017) 365–369.
[203] J.-H. Mao, I.-J. Kim, D. Wu, J. Climent, H.C. Kang, R. DelRosario, A. Balmain,
FBXW7 targets mTOR for degradation and cooperates with PTEN in tumor sup-
pression, Science 321 (2008) 1499–1502.
[204] P. Agrawal, Y.-T. Chen, B. Schilling, B.W. Gibson, R.E. Hughes, Ubiquitin-specific
peptidase 9, X-linked (USP9X) modulates activity of mammalian target of rapa-
mycin (mTOR), J. Biol. Chem. 287 (2012) 21164–21175.
N. Kumari et al.
[205] G. Panasyuk, I. Nemazanyy, V. Filonenko, I. Gout, Ribosomal protein S6 kinase 1
interacts with and is ubiquitinated by ubiquitin ligase ROC1, Biochem. Biophys.
Res. Commun. 369 (2008) 339–343.
[206] S. Hussain, O. Foreman, S. Perkins, T.E. Witzig, R. Miles, J. Van Deursen,
P.J. Galardy, The de-ubiquitinase UCH-L1 is an oncogene that drives the devel-
opment of lymphoma in vivo by deregulating PHLPP1 and Akt signaling,
Leukemia 24 (2010) 1641–1655.
[207] I. Vivanco, D. Rohle, M. Versele, A. Iwanami, D. Kuga, B. Oldrini, K. Tanaka,
J. Dang, S. Kubek, N. Palaskas, The phosphatase and tensin homolog regulates
epidermal growth factor receptor (EGFR) inhibitor response by targeting EGFR for
degradation, Proc. Natl. Acad. Sci. 107 (2010) 6459–6464.
[208] T. Tolkacheva, M. Boddapati, A. Sanfiz, K. Tsuchida, A.C. Kimmelman, A.M. Chan,
Regulation of PTEN binding to MAGI-2 by two putative phosphorylation sites at
threonine 382 and 383, Cancer Res. 61 (2001) 4985–4989.
[209] E.-M. Duerr, B. Rollbrocker, Y. Hayashi, N. Peters, B. Meyer-Puttlitz, D.N. Louis,
J. Schramm, O.D. Wiestler, R. Parsons, C. Eng, PTEN mutations in gliomas and
glioneuronal tumors, Oncogene 16 (1998).
[210] L.C. Trotman, X. Wang, A. Alimonti, Z. Chen, J. Teruya-Feldstein, H. Yang,
N.P. Pavletich, B.S. Carver, C. Cordon-Cardo, H. Erdjument-Bromage,
Ubiquitination regulates PTEN nuclear import and tumor suppression, Cell 128
(2007) 141–156.
[211] X. Wang, X. Jiang, Post-translational regulation of PTEN, Oncogene 27 (2008)
5454–5463.
[212] X. Wang, L.C. Trotman, T. Koppie, A. Alimonti, Z. Chen, Z. Gao, J. Wang,
H. Erdjument-Bromage, P. Tempst, C. Cordon-Cardo, NEDD4-1 is a proto-onco-
genic ubiquitin ligase for PTEN, Cell 128 (2007) 129–139.
[213] N. Amodio, M. Scrima, L. Palaia, A.N. Salman, A. Quintiero, R. Franco, G. Botti,
P. Pirozzi, G. Rocco, N. De Rosa, G. Viglietto, Oncogenic role of the E3 ubiquitin
ligase NEDD4-1, a PTEN negative regulator, in non-small-cell lung carcinomas,
Am. J. Pathol. 177 (2010) 2622–2634.
[214] F. Fouladkou, T. Landry, H. Kawabe, A. Neeb, C. Lu, N. Brose, V. Stambolic,
D. Rotin, The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN
stability and localization, Proc. Natl. Acad. Sci. 105 (2008) 8585–8590.
[215] M.S. Song, L. Salmena, A. Carracedo, A. Egia, F. Lo-Coco, J. Teruya-Feldstein,
P.P. Pandolfi, The deubiquitinylation and localization of PTEN are regulated by a
HAUSP–PML network, Nature 455 (2008) 813–817.
[216] C. Van Themsche, V. Leblanc, S. Parent, E. Asselin, X-linked inhibitor of apoptosis
protein (XIAP) regulates PTEN ubiquitination, content, and compartmentalization,
J. Biol. Chem. 284 (2009) 20462–20466.
[217] S. Maddika, S. Kavela, N. Rani, V.R. Palicharla, J.L. Pokorny, J.N. Sarkaria,
J. Chen, WWP2 is an E3 ubiquitin ligase for PTEN, Nat. Cell Biol. 13 (2011)
728–733.
[218] S.F. Ahmed, S. Deb, I. Paul, A. Chatterjee, T. Mandal, U. Chatterjee, M.K. Ghosh,
The chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP)
targets PTEN for proteasomal degradation, J. Biol. Chem. 287 (2012)
15996–16006.
[219] J.T. Lee, J. Shan, J. Zhong, M. Li, B. Zhou, A. Zhou, R. Parsons, W. Gu, RFP-
mediated ubiquitination of PTEN modulates its effect on AKT activation, Cell Res.
23 (2013) 552–564.
[220] J. Zhang, P. Zhang, Y. Wei, H.L. Piao, W. Wang, S. Maddika, M. Wang, D. Chen,
Y. Sun, M.C. Hung, J. Chen, L. Ma, Deubiquitylation and stabilization of PTEN by
USP13, Nat. Cell Biol. 15 (2013) 1486–1494.
[221] L. Yuan, Y. Lv, H. Li, H. Gao, S. Song, Y. Zhang, G. Xing, X. Kong, L. Wang, Y. Li,
T. Zhou, D. Gao, Z.X. Xiao, Y. Yin, W. Wei, F. He, L. Zhang, Deubiquitylase OTUD3
regulates PTEN stability and suppresses tumorigenesis, Nat. Cell Biol. 17 (2015)
1169–1181.
[222] L. Yuan, Y. Lv, H. Li, H. Gao, S. Song, Y. Zhang, G. Xing, X. Kong, L. Wang, Y. Li,
Deubiquitylase OTUD3 regulates PTEN stability and suppresses tumorigenesis,
Nat. Cell Biol. (2015).
[223] O. Tetsu, F. McCormick, Beta-catenin regulates expression of cyclin D1 in colon
carcinoma cells, Nature 398 (1999) 422–426.
[224] T.C. He, A.B. Sparks, C. Rago, H. Hermeking, L. Zawel, L.T. da Costa, P.J. Morin,
B. Vogelstein, K.W. Kinzler, Identification of c-MYC as a target of the APC
pathway, Science 281 (1998) 1509–1512.
[225] D. Kimelman, W. Xu, Beta-catenin destruction complex: insights and questions
from a structural perspective, Oncogene 25 (2006) 7482–7491.
[226] M. Hart, J.P. Concordet, I. Lassot, I. Albert, R. del los Santos, H. Durand, C. Perret,
B. Rubinfeld, F. Margottin, R. Benarous, P. Polakis, The F-box protein beta-TrCP
associates with phosphorylated beta-catenin and regulates its activity in the cell,
Curr. Biol. 9 (1999) 207–210.
[227] V. Chitalia, S. Shivanna, J. Martorell, R. Meyer, E. Edelman, N. Rahimi, c-Cbl, a
ubiquitin E3 ligase that targets active beta-catenin: a novel layer of Wnt signaling
regulation, J. Biol. Chem. 288 (2013) 23505–23517.
[228] S. Shivanna, I. Harrold, M. Shashar, R. Meyer, C. Kiang, J. Francis, Q. Zhao,
H. Feng, E.R. Edelman, N. Rahimi, V.C. Chitalia, The c-Cbl ubiquitin ligase reg-
ulates nuclear beta-catenin and angiogenesis by its tyrosine phosphorylation
mediated through the Wnt signaling pathway, J. Biol. Chem. 290 (2015)
12537–12546.
[229] V.C. Chitalia, R.L. Foy, M.M. Bachschmid, L. Zeng, M.V. Panchenko, M.I. Zhou,
A. Bharti, D.C. Seldin, S.H. Lecker, I. Dominguez, H.T. Cohen, Jade-1 inhibits Wnt
signalling by ubiquitylating beta-catenin and mediates Wnt pathway inhibition by
pVHL, Nat. Cell Biol. 10 (2008) 1208–1216.
[230] R.E. de Groot, R.S. Ganji, O. Bernatik, B. Lloyd-Lewis, K. Seipel, K. Sedova,
Z. Zdrahal, V.M. Dhople, T.C. Dale, H.C. Korswagen, V. Bryja, Huwe1-mediated
ubiquitylation of dishevelled defines a negative feedback loop in the Wnt signaling
pathway, Sci. Signal. 7 (2014) ra26.
481
BBA - Reviews on Cancer 1868 (2017) 456–483
[231] C. Dominguez-Brauer, R. Khatun, A.J. Elia, K.L. Thu, P. Ramachandran,
S.P. Baniasadi, Z. Hao, L.D. Jones, J. Haight, Y. Sheng, T.W. Mak, E3 ubiquitin
ligase Mule targets beta-catenin under conditions of hyperactive Wnt signaling,
Proc. Natl. Acad. Sci. U. S. A. 114 (2017) E1148–E1157.
[232] S. Angers, C.J. Thorpe, T.L. Biechele, S.J. Goldenberg, N. Zheng, M.J. MacCoss,
R.T. Moon, The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-
beta-catenin pathway by targeting dishevelled for degradation, Nat. Cell Biol. 8
(2006) 348–357.
[233] W. Wei, M. Li, J. Wang, F. Nie, L. Li, The E3 ubiquitin ligase ITCH negatively
regulates canonical Wnt signaling by targeting dishevelled protein, Mol. Cell. Biol.
32 (2012) 3903–3912.
[234] Y. Ding, Y. Zhang, C. Xu, Q.H. Tao, Y.G. Chen, HECT domain-containing E3 ubi-
quitin ligase NEDD4L negatively regulates Wnt signaling by targeting dishevelled
for proteasomal degradation, J. Biol. Chem. 288 (2013) 8289–8298.
[235] D.V. Tauriello, A. Haegebarth, I. Kuper, M.J. Edelmann, M. Henraat,
M.R. Canninga-van Dijk, B.M. Kessler, H. Clevers, M.M. Maurice, Loss of the tumor
suppressor CYLD enhances Wnt/beta-catenin signaling through K63-linked ubi-
quitination of Dvl, Mol. Cell 37 (2010) 607–619.
[236] X. Jiang, O. Charlat, R. Zamponi, Y. Yang, F. Cong, Dishevelled promotes Wnt
receptor degradation through recruitment of ZNRF3/RNF43 E3 ubiquitin ligases,
Mol. Cell 58 (2015) 522–533.
[237] A. Loregger, M. Grandl, R. Mejias-Luque, M. Allgauer, K. Degenhart,
V. Haselmann, C. Oikonomou, P. Hatzis, K.P. Janssen, U. Nitsche, D. Gradl, O. van
den Broek, O. Destree, K. Ulm, M. Neumaier, B. Kalali, A. Jung, I. Varela,
R.M. Schmid, R. Rad, D.H. Busch, M. Gerhard, The E3 ligase RNF43 inhibits Wnt
signaling downstream of mutated beta-catenin by sequestering TCF4 to the nu-
clear membrane, Sci. Signal. 8 (2015) ra90.
[238] A. Mukai, M. Yamamoto-Hino, W. Awano, W. Watanabe, M. Komada, S. Goto,
Balanced ubiquitylation and deubiquitylation of frizzled regulate cellular re-
sponsiveness to Wg/Wnt, EMBO J. 29 (2010) 2114–2125.
[239] B. Madan, M.P. Walker, R. Young, L. Quick, K.A. Orgel, M. Ryan, P. Gupta,
I.C. Henrich, M. Ferrer, S. Marine, B.S. Roberts, W.T. Arthur, J.D. Berndt,
A.M. Oliveira, R.T. Moon, D.M. Virshup, M.M. Chou, M.B. Major, USP6 oncogene
promotes Wnt signaling by deubiquitylating Frizzleds, Proc. Natl. Acad. Sci. U. S.
A. 113 (2016) E2945–2954.
[240] T. Schwarz-Romond, M. Fiedler, N. Shibata, P.J. Butler, A. Kikuchi, Y. Higuchi,
M. Bienz, The DIX domain of dishevelled confers Wnt signaling by dynamic
polymerization, Nat. Struct. Mol. Biol. 14 (2007) 484–492.
[241] Y. Zhang, S. Liu, C. Mickanin, Y. Feng, O. Charlat, G.A. Michaud, M. Schirle, X. Shi,
M. Hild, A. Bauer, V.E. Myer, P.M. Finan, J.A. Porter, S.M. Huang, F. Cong,
RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation
and Wnt signalling, Nat. Cell Biol. 13 (2011) 623–629.
[242] S. Kim, E.H. Jho, The protein stability of Axin, a negative regulator of Wnt sig-
naling, is regulated by Smad ubiquitination regulatory factor 2 (Smurf2), J. Biol.
Chem. 285 (2010) 36420–36426.
[243] C. Fei, X. He, S. Xie, H. Miao, Z. Zhou, L. Li, Smurf1-mediated axin ubiquitination
requires Smurf1 C2 domain and is cell cycle-dependent, J. Biol. Chem. 289 (2014)
14170–14177.
[244] T.T. Lui, C. Lacroix, S.M. Ahmed, S.J. Goldenberg, C.A. Leach, A.M. Daulat,
S. Angers, The ubiquitin-specific protease USP34 regulates axin stability and Wnt/
beta-catenin signaling, Mol. Cell. Biol. 31 (2011) 2053–2065.
[245] H. Tran, F. Hamada, T. Schwarz-Romond, M. Bienz, Trabid, a new positive reg-
ulator of Wnt-induced transcription with preference for binding and cleaving K63-
linked ubiquitin chains, Genes Dev. 22 (2008) 528–542.
[246] S.I. Yun, H.H. Kim, J.H. Yoon, W.S. Park, M.J. Hahn, H.C. Kim, C.H. Chung,
K.K. Kim, Ubiquitin specific protease 4 positively regulates the WNT/beta-catenin
signaling in colorectal cancer, Mol. Oncol. 9 (2015) 1834–1851.
[247] B. Zhao, C. Schlesiger, M.G. Masucci, K. Lindsten, The ubiquitin specific protease 4
(USP4) is a new player in the Wnt signalling pathway, J. Cell. Mol. Med. 13 (2009)
1886–1895.
[248] J. Shi, Y. Liu, X. Xu, W. Zhang, T. Yu, J. Jia, C. Liu, Deubiquitinase USP47/UBP64E
regulates beta-catenin ubiquitination and degradation and plays a positive role in
Wnt signaling, Mol. Cell. Biol. 35 (2015) 3301–3311.
[249] M.B. Greenblatt, D.Y. Shin, H. Oh, K.Y. Lee, B. Zhai, S.P. Gygi, S. Lotinun,
R. Baron, D. Liu, B. Su, L.H. Glimcher, J.H. Shim, MEKK2 mediates an alternative
beta-catenin pathway that promotes bone formation, Proc. Natl. Acad. Sci. U. S. A.
113 (2016) E1226–1235.
[250] P. Ma, X. Yang, Q. Kong, C. Li, S. Yang, Y. Li, B. Mao, The ubiquitin ligase RNF220
enhances canonical Wnt signaling through USP7-mediated deubiquitination of
beta-catenin, Mol. Cell. Biol. 34 (2014) 4355–4366.
[251] P. D'Arcy, X. Wang, S. Linder, Deubiquitinase inhibition as a cancer therapeutic
strategy, Pharmacol. Ther. 147 (2015) 32–54.
[252] E. Koulich, X. Li, G.N. DeMartino, Relative structural and functional roles of
multiple deubiquitylating proteins associated with mammalian 26S proteasome,
Mol. Biol. Cell 19 (2008) 1072–1082.
[253] B.H. Lee, Y. Lu, M.A. Prado, Y. Shi, G. Tian, S. Sun, S. Elsasser, S.P. Gygi,
R.W. King, D. Finley, USP14 deubiquitinates proteasome-bound substrates that are
ubiquitinated at multiple sites, Nature 532 (2016) 398–401.
[254] B.H. Lee, M.J. Lee, S. Park, D.C. Oh, S. Elsasser, P.C. Chen, C. Gartner, N. Dimova,
J. Hanna, S.P. Gygi, S.M. Wilson, R.W. King, D. Finley, Enhancement of protea-
some activity by a small-molecule inhibitor of USP14, Nature 467 (2010)
179–184.
[255] P.C. Chen, L.N. Qin, X.M. Li, B.J. Walters, J.A. Wilson, L. Mei, S.M. Wilson, The
proteasome-associated deubiquitinating enzyme Usp14 is essential for the main-
tenance of synaptic ubiquitin levels and the development of neuromuscular
junctions, The Journal of Neuroscience: The Official Journal of the Society for
N. Kumari et al.
Neuroscience 29 (2009) 10909–10919.
[256] S.M. Wilson, B. Bhattacharyya, R.A. Rachel, V. Coppola, L. Tessarollo,
D.B. Householder, C.F. Fletcher, R.J. Miller, N.G. Copeland, N.A. Jenkins, Synaptic
defects in ataxia mice result from a mutation in Usp14, encoding a ubiquitin-
specific protease, Nat. Genet. 32 (2002) 420–425.
[257] S. Hussain, T. Bedekovics, M. Chesi, P.L. Bergsagel, P.J. Galardy, UCHL1 is a
biomarker of aggressive multiple myeloma required for disease progression,
Oncotarget 6 (2015) 40704–40718.
[258] Y. Goto, L. Zeng, C.J. Yeom, Y. Zhu, A. Morinibu, K. Shinomiya, M. Kobayashi,
K. Hirota, S. Itasaka, M. Yoshimura, K. Tanimoto, M. Torii, T. Sowa, T. Menju,
M. Sonobe, H. Kakeya, M. Toi, H. Date, E.M. Hammond, M. Hiraoka, H. Harada,
UCHL1 provides diagnostic and antimetastatic strategies due to its deubiquiti-
nating effect on HIF-1alpha, Nat. Commun. 6 (2015) 6153.
[259] Y. Liu, H.A. Lashuel, S. Choi, X. Xing, A. Case, J. Ni, L.A. Yeh, G.D. Cuny,
R.L. Stein, P.T. Lansbury Jr., Discovery of inhibitors that elucidate the role of UCH-
L1 activity in the H1299 lung cancer cell line, Chem. Biol. 10 (2003) 837–846.
[260] Y.Y. Tan, H.Y. Zhou, Z.Q. Wang, S.D. Chen, Endoplasmic reticulum stress con-
tributes to the cell death induced by UCH-L1 inhibitor, Mol. Cell. Biochem. 318
(2008) 109–115.
[261] A.H. Mermerian, A. Case, R.L. Stein, G.D. Cuny, Structure-activity relationship,
kinetic mechanism, and selectivity for a new class of ubiquitin C-terminal hy-
drolase-L1 (UCH-L1) inhibitors, Bioorg. Med. Chem. Lett. 17 (2007) 3729–3732.
[262] J. Sosna, S. Philipp, J. Fuchslocher Chico, C. Saggau, J. Fritsch, A. Foll, J. Plenge,
C. Arenz, T. Pinkert, H. Kalthoff, A. Trauzold, I. Schmitz, S. Schutze, D. Adam,
Differences and similarities in TRAIL- and tumor necrosis factor-mediated ne-
croptotic signaling in cancer cells, Mol. Cell. Biol. 36 (2016) 2626–2644.
[263] X. Tian, N.S. Isamiddinova, R.J. Peroutka, S.J. Goldenberg, M.R. Mattern,
B. Nicholson, C. Leach, Characterization of selective ubiquitin and ubiquitin-like
protease inhibitors using a fluorescence-based multiplex assay format, Assay Drug
Dev. Technol. 9 (2011) 165–173.
[264] J. Weinstock, J. Wu, P. Cao, W.D. Kingsbury, J.L. McDermott, M.P. Kodrasov,
D.M. McKelvey, K.G. Suresh Kumar, S.J. Goldenberg, M.R. Mattern, B. Nicholson,
Selective dual inhibitors of the cancer-related deubiquitylating proteases USP7
and USP47, ACS Med. Chem. Lett. 3 (2012) 789–792.
[265] C. Reverdy, S. Conrath, R. Lopez, C. Planquette, C. Atmanene, V. Collura,
J. Harpon, V. Battaglia, V. Vivat, W. Sippl, F. Colland, Discovery of specific in-
hibitors of human USP7/HAUSP deubiquitinating enzyme, Chem. Biol. 19 (2012)
467–477.
[266] S.M. Nijman, T.T. Huang, A.M. Dirac, T.R. Brummelkamp, R.M. Kerkhoven,
A.D. D'Andrea, R. Bernards, The deubiquitinating enzyme USP1 regulates the
Fanconi anemia pathway, Mol. Cell 17 (2005) 331–339.
[267] K. Yang, G.L. Moldovan, P. Vinciguerra, J. Murai, S. Takeda, A.D. D'Andrea,
Regulation of the Fanconi anemia pathway by a SUMO-like delivery network,
Genes Dev. 25 (2011) 1847–1858.
[268] J. Chen, T.S. Dexheimer, Y. Ai, Q. Liang, M.A. Villamil, J. Inglese, D.J. Maloney,
A. Jadhav, A. Simeonov, Z. Zhuang, Selective and cell-active inhibitors of the
USP1/UAF1 deubiquitinase complex reverse cisplatin resistance in non-small cell
lung cancer cells, Chem. Biol. 18 (2011) 1390–1400.
[269] S. Ohayon, L. Spasser, A. Aharoni, A. Brik, Targeting deubiquitinases enabled by
chemical synthesis of proteins, J. Am. Chem. Soc. 134 (2012) 3281–3289.
[270] V. Seiberlich, O. Goldbaum, V. Zhukareva, C. Richter-Landsberg, The small mo-
lecule inhibitor PR-619 of deubiquitinating enzymes affects the microtubule net-
work and causes protein aggregate formation in neural cells: implications for
neurodegenerative diseases, Biochim. Biophys. Acta 1823 (2012) 2057–2068.
[271] V. Seiberlich, J. Borchert, V. Zhukareva, C. Richter-Landsberg, Inhibition of pro-
tein deubiquitination by PR-619 activates the autophagic pathway in OLN-t40
oligodendroglial cells, Cell Biochem. Biophys. 67 (2013) 149–160.
[272] V. Kapuria, L.F. Peterson, D. Fang, W.G. Bornmann, M. Talpaz, N.J. Donato,
Deubiquitinase inhibition by small-molecule WP1130 triggers aggresome forma-
tion and tumor cell apoptosis, Cancer Res. 70 (2010) 9265–9276.
[273] L.F. Peterson, H. Sun, Y. Liu, H. Potu, M. Kandarpa, M. Ermann, S.M. Courtney,
M. Young, H.D. Showalter, D. Sun, A. Jakubowiak, S.N. Malek, M. Talpaz,
N.J. Donato, Targeting deubiquitinase activity with a novel small-molecule in-
hibitor as therapy for B-cell malignancies, Blood 125 (2015) 3588–3597.
[274] J.W. Perry, M. Ahmed, K.O. Chang, N.J. Donato, H.D. Showalter, C.E. Wobus,
Antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction
of the unfolded protein response, PLoS Pathog. 8 (2012) e1002783.
[275] L.V. Pham, A.T. Tamayo, C. Li, W. Bornmann, W. Priebe, R.J. Ford, Degrasyn
potentiates the antitumor effects of bortezomib in mantle cell lymphoma cells in
vitro and in vivo: therapeutic implications, Mol. Cancer Ther. 9 (2010)
2026–2036.
[276] E. Fiebiger, C. Hirsch, J.M. Vyas, E. Gordon, H.L. Ploegh, D. Tortorella, Dissection
of the dislocation pathway for type I membrane proteins with a new small mo-
lecule inhibitor, eeyarestatin, Mol. Biol. Cell 15 (2004) 1635–1646.
[277] Q. Wang, L. Li, Y. Ye, Inhibition of p97-dependent protein degradation by
eeyarestatin I, J. Biol. Chem. 283 (2008) 7445–7454.
[278] J.E. Mullally, P.J. Moos, K. Edes, F.A. Fitzpatrick, Cyclopentenone prostaglandins
of the J series inhibit the ubiquitin isopeptidase activity of the proteasome
pathway, J. Biol. Chem. 276 (2001) 30366–30373.
[279] F.A. Fitzpatrick, M.A. Wynalda, Albumin-catalyzed metabolism of prostaglandin
D2. Identification of products formed in vitro, J. Biol. Chem. 258 (1983)
11713–11718.
[280] J.E. Mullally, F.A. Fitzpatrick, Pharmacophore model for novel inhibitors of ubi-
quitin isopeptidases that induce p53-independent cell death, Mol. Pharmacol. 62
(2002) 351–358.
[281] T. Ishii, T. Sakurai, H. Usami, K. Uchida, Oxidative modification of proteasome:
482
BBA - Reviews on Cancer 1868 (2017) 456–483
identification of an oxidation-sensitive subunit in 26 S proteasome, Biochemistry
44 (2005) 13893–13901.
[282] H. Liu, W. Li, M. Ahmad, T.M. Miller, M.E. Rose, S.M. Poloyac, G. Uechi,
M. Balasubramani, R.W. Hickey, S.H. Graham, Modification of ubiquitin-C-term-
inal hydrolase-L1 by cyclopentenone prostaglandins exacerbates hypoxic injury,
Neurobiol. Dis. 41 (2011) 318–328.
[283] Z. Li, F. Melandri, I. Berdo, M. Jansen, L. Hunter, S. Wright, D. Valbrun,
M.E. Figueiredo-Pereira, Delta12-prostaglandin J2 inhibits the ubiquitin hydrolase
UCH-L1 and elicits ubiquitin-protein aggregation without proteasome inhibition,
Biochem. Biophys. Res. Commun. 319 (2004) 1171–1180.
[284] L.M. Koharudin, H. Liu, R. Di Maio, R.B. Kodali, S.H. Graham, A.M. Gronenborn,
Cyclopentenone prostaglandin-induced unfolding and aggregation of the
Parkinson disease-associated UCH-L1, Proc. Natl. Acad. Sci. U. S. A. 107 (2010)
6835–6840.
[285] H. Erdal, M. Berndtsson, J. Castro, U. Brunk, M.C. Shoshan, S. Linder, Induction of
lysosomal membrane permeabilization by compounds that activate p53-in-
dependent apoptosis, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 192–197.
[286] M. Berndtsson, M. Beaujouin, L. Rickardson, A.M. Havelka, R. Larsson,
J. Westman, E. Liaudet-Coopman, S. Linder, Induction of the lysosomal apoptosis
pathway by inhibitors of the ubiquitin-proteasome system, Int. J. Cancer 124
(2009) 1463–1469.
[287] Z. Tian, P. D'Arcy, X. Wang, A. Ray, Y.T. Tai, Y. Hu, R.D. Carrasco, P. Richardson,
S. Linder, D. Chauhan, K.C. Anderson, A novel small molecule inhibitor of deu-
biquitylating enzyme USP14 and UCHL5 induces apoptosis in multiple myeloma
and overcomes bortezomib resistance, Blood 123 (2014) 706–716.
[288] X. Wang, M. Mazurkiewicz, E.K. Hillert, M.H. Olofsson, S. Pierrou, P. Hillertz,
J. Gullbo, K. Selvaraju, A. Paulus, S. Akhtar, F. Bossler, A.C. Khan, S. Linder,
P. D'Arcy, The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for
ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells,
Sci Rep 6 (2016) 26979.
[289] P. D'Arcy, S. Brnjic, M.H. Olofsson, M. Fryknas, K. Lindsten, M. De Cesare,
P. Perego, B. Sadeghi, M. Hassan, R. Larsson, S. Linder, Inhibition of proteasome
deubiquitinating activity as a new cancer therapy, Nat. Med. 17 (2011)
1636–1640.
[290] E. Aleo, C.J. Henderson, A. Fontanini, B. Solazzo, C. Brancolini, Identification of
new compounds that trigger apoptosome-independent caspase activation and
apoptosis, Cancer Res. 66 (2006) 9235–9244.
[291] A. Fontanini, C. Foti, H. Potu, E. Crivellato, R. Maestro, P. Bernardi, F. Demarchi,
C. Brancolini, The isopeptidase inhibitor G5 triggers a caspase-independent ne-
crotic death in cells resistant to apoptosis: a comparative study with the protea-
some inhibitor bortezomib, J. Biol. Chem. 284 (2009) 8369–8381.
[292] B. Nicholson, C.A. Leach, S.J. Goldenberg, D.M. Francis, M.P. Kodrasov, X. Tian,
J. Shanks, D.E. Sterner, A. Bernal, M.R. Mattern, K.D. Wilkinson, T.R. Butt,
Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities,
Protein Sci. 17 (2008) 1035–1043.
[293] R.K. Anchoori, S.R. Khan, T. Sueblinvong, A. Felthauser, Y. Iizuka, R. Gavioli,
F. Destro, R. Isaksson Vogel, S. Peng, R.B. Roden, M. Bazzaro, Stressing the ubi-
quitin-proteasome system without 20S proteolytic inhibition selectively kills cer-
vical cancer cells, PLoS One 6 (2011) e23888.
[294] O.A. Issaenko, A.Y. Amerik, Chalcone-based small-molecule inhibitors attenuate
malignant phenotype via targeting deubiquitinating enzymes, Cell Cycle 11
(2012) 1804–1817.
[295] H.Z. Zhang, S. Kasibhatla, Y. Wang, J. Herich, J. Guastella, B. Tseng, J. Drewe,
S.X. Cai, Discovery, characterization and SAR of gambogic acid as a potent
apoptosis inducer by a HTS assay, Bioorg. Med. Chem. 12 (2004) 309–317.
[296] J. Felth, K. Lesiak-Mieczkowska, P. D'Arcy, C. Haglund, J. Gullbo, R. Larsson,
S. Linder, L. Bohlin, M. Fryknas, L. Rickardson, Gambogic acid is cytotoxic to
cancer cells through inhibition of the ubiquitin-proteasome system, Investig. New
Drugs 31 (2013) 587–598.
[297] E. Pisha, H. Chai, I.S. Lee, T.E. Chagwedera, N.R. Farnsworth, G.A. Cordell,
C.W. Beecher, H.H. Fong, A.D. Kinghorn, D.M. Brown, et al., Discovery of betulinic
acid as a selective inhibitor of human melanoma that functions by induction of
apoptosis, Nat. Med. 1 (1995) 1046–1051.
[298] F.B. Mullauer, J.H. Kessler, J.P. Medema, Betulinic acid, a natural compound with
potent anticancer effects, Anti-Cancer Drugs 21 (2010) 215–227.
[299] T. Reiner, R. Parrondo, A. de Las Pozas, D. Palenzuela, C. Perez-Stable, Betulinic
acid selectively increases protein degradation and enhances prostate cancer-spe-
cific apoptosis: possible role for inhibition of deubiquitinase activity, PLoS One 8
(2013) e56234.
[300] S. D'Aguanno, I. D'Agnano, M. De Canio, C. Rossi, S. Bernardini, G. Federici,
A. Urbani, Shotgun proteomics and network analysis of neuroblastoma cell lines
treated with curcumin, Mol. BioSyst. 8 (2012) 1068–1077.
[301] B. Zhou, Y. Zuo, B. Li, H. Wang, H. Liu, X. Wang, X. Qiu, Y. Hu, S. Wen, J. Du,
X. Bu, Deubiquitinase inhibition of 19S regulatory particles by 4-arylidene cur-
cumin analog AC17 causes NF-kappaB inhibition and p53 reactivation in human
lung cancer cells, Mol. Cancer Ther. 12 (2013) 1381–1392.
[302] A. Ernst, G. Avvakumov, J. Tong, Y. Fan, Y. Zhao, P. Alberts, A. Persaud,
J.R. Walker, A.M. Neculai, D. Neculai, A. Vorobyov, P. Garg, L. Beatty, P.K. Chan,
Y.C. Juang, M.C. Landry, C. Yeh, E. Zeqiraj, K. Karamboulas, A. Allali-Hassani,
M. Vedadi, M. Tyers, J. Moffat, F. Sicheri, L. Pelletier, D. Durocher, B. Raught,
D. Rotin, J. Yang, M.F. Moran, S. Dhe-Paganon, S.S. Sidhu, A strategy for mod-
ulation of enzymes in the ubiquitin system, Science 339 (2013) 590–595.
[303] W. Zhang, M.A. Sartori, T. Makhnevych, K.E. Federowicz, X. Dong, L. Liu, S. Nim,
A. Dong, J. Yang, Y. Li, D. Haddad, A. Ernst, D. Heerding, Y. Tong, J. Moffat,
S.S. Sidhu, Generation and validation of intracellular ubiquitin variant inhibitors
for USP7 and USP10, J. Mol. Biol. (2017).
N. Kumari et al.
[304] N. Cancer Genome Atlas Research, Comprehensive molecular characterization of
urothelial bladder carcinoma, Nature 507 (2014) 315–322.
[305] B. Pereira, S.F. Chin, O.M. Rueda, H.K. Vollan, E. Provenzano, H.A. Bardwell,
M. Pugh, L. Jones, R. Russell, S.J. Sammut, D.W. Tsui, B. Liu, S.J. Dawson,
J. Abraham, H. Northen, J.F. Peden, A. Mukherjee, G. Turashvili, A.R. Green,
S. McKinney, A. Oloumi, S. Shah, N. Rosenfeld, L. Murphy, D.R. Bentley, I.O. Ellis,
A. Purushotham, S.E. Pinder, A.L. Borresen-Dale, H.M. Earl, P.D. Pharoah,
M.T. Ross, S. Aparicio, C. Caldas, The somatic mutation profiles of 2,433 breast
cancers refines their genomic and transcriptomic landscapes, Nat. Commun. 7
(2016) 11479.
[306] N. Cancer Genome Atlas, Comprehensive molecular portraits of human breast
tumours, Nature 490 (2012) 61–70.
[307] M. Ceccarelli, F.P. Barthel, T.M. Malta, T.S. Sabedot, S.R. Salama, B.A. Murray,
O. Morozova, Y. Newton, A. Radenbaugh, S.M. Pagnotta, S. Anjum, J. Wang,
G. Manyam, P. Zoppoli, S. Ling, A.A. Rao, M. Grifford, A.D. Cherniack, H. Zhang,
L. Poisson, C.G. Carlotti Jr., D.P. Tirapelli, A. Rao, T. Mikkelsen, C.C. Lau,
W.K. Yung, R. Rabadan, J. Huse, D.J. Brat, N.L. Lehman, J.S. Barnholtz-Sloan,
S. Zheng, K. Hess, G. Rao, M. Meyerson, R. Beroukhim, L. Cooper, R. Akbani,
M. Wrensch, D. Haussler, K.D. Aldape, P.W. Laird, D.H. Gutmann, T.R. Network,
H. Noushmehr, A. Iavarone, R.G. Verhaak, Molecular profiling reveals biologically
discrete subsets and pathways of progression in diffuse glioma, Cell 164 (2016)
550–563.
[308] C.W. Brennan, R.G. Verhaak, A. McKenna, B. Campos, H. Noushmehr, S.R. Salama,
S. Zheng, D. Chakravarty, J.Z. Sanborn, S.H. Berman, R. Beroukhim, B. Bernard,
C.J. Wu, G. Genovese, I. Shmulevich, J. Barnholtz-Sloan, L. Zou, R. Vegesna,
S.A. Shukla, G. Ciriello, W.K. Yung, W. Zhang, C. Sougnez, T. Mikkelsen,
K. Aldape, D.D. Bigner, E.G. Van Meir, M. Prados, A. Sloan, K.L. Black,
J. Eschbacher, G. Finocchiaro, W. Friedman, D.W. Andrews, A. Guha, M. Iacocca,
B.P. O'Neill, G. Foltz, J. Myers, D.J. Weisenberger, R. Penny, R. Kucherlapati,
C.M. Perou, D.N. Hayes, R. Gibbs, M. Marra, G.B. Mills, E. Lander, P. Spellman,
R. Wilson, C. Sander, J. Weinstein, M. Meyerson, S. Gabriel, P.W. Laird,
D. Haussler, G. Getz, L. Chin, T.R. Network, The somatic genomic landscape of
glioblastoma, Cell 155 (2013) 462–477.
[309] N. Cancer Genome Atlas Research, U. Analysis Working Group, Asan, B.C.C.
Agency, Brigham, H. Women's, I. Broad, U. Brown, U. Case Western Reserve, I.
Dana-Farber Cancer, U. Duke, C. Greater Poland Cancer, S. Harvard Medical, B.
Institute for Systems, K.U. Leuven, C. Mayo, C. Memorial Sloan Kettering Cancer, I.
National Cancer, H. Nationwide Children's, U. Stanford, A. University of, M.
University of, C. University of North, P. University of, R. University of, C.
University of Southern, M.D.A.C.C. University of Texas, W. University of, I. Van
Andel Research, U. Vanderbilt, U. Washington, I. Genome Sequencing Center:
Broad, L. Washington University in St, B.C.C.A. Genome Characterization Centers,
I. Broad, S. Harvard Medical, U. Sidney Kimmel Comprehensive Cancer Center at
Johns Hopkins, C. University of North, C. University of Southern California
Epigenome, M.D.A.C.C. University of Texas, I. Van Andel Research, I. Genome
Data Analysis Centers: Broad, U. Brown, S. Harvard Medical, B. Institute for
483
BBA - Reviews on Cancer 1868 (2017) 456–483
Systems, C. Memorial Sloan Kettering Cancer, C. University of California Santa,
M.D.A.C.C. University of Texas, C. Biospecimen Core Resource: International
Genomics, H. Research Institute at Nationwide Children's, S. Tissue Source Sites:
Analytic Biologic, C. Asan Medical, B. Asterand, H. Barretos Cancer,
BioreclamationIvt, C. Botkin Municipal, S. Chonnam National University Medical,
S. Christiana Care Health, Cureline, U. Duke, U. Emory, U. Erasmus, M. Indiana
University School of, M. Institute of Oncology of, C. International Genomics,
Invidumed, H. Israelitisches Krankenhaus, M. Keimyung University School of, C.
Memorial Sloan Kettering Cancer, G. National Cancer Center, B. Ontario Tumour,
C. Peter MacCallum Cancer, S. Pusan National University Medical, S. Ribeirao
Preto Medical, H. St. Joseph's, C. Medical, U. St. Petersburg Academic, B. Tayside
Tissue, D. University of, C. University of Kansas Medical, M. University of, H.
University of North Carolina at Chapel, M. University of Pittsburgh School of,
M.D.A.C.C. University of Texas, U. Disease Working Group: Duke, C. Memorial
Sloan Kettering Cancer, I. National Cancer, M.D.A.C.C. University of Texas, M.
Yonsei University College of, C.I. Data Coordination Center, H. Project Team:
National Institutes of, Integrated genomic characterization of oesophageal carci-
noma, Nature 541 (2017) 169–175.
[310] N. Cancer Genome Atlas Research, Comprehensive molecular characterization of
gastric adenocarcinoma, Nature 513 (2014) 202–209.
[311] N. Cancer Genome Atlas Research, Comprehensive molecular characterization of
clear cell renal cell carcinoma, Nature 499 (2013) 43–49.
[312] N. Cancer Genome Atlas Research, Comprehensive molecular profiling of lung
adenocarcinoma, Nature 511 (2014) 543–550.
[313] N. Cancer Genome Atlas Research, The molecular taxonomy of primary prostate
cancer, Cell 163 (2015) 1011–1025.
[314] J. Barretina, B.S. Taylor, S. Banerji, A.H. Ramos, M. Lagos-Quintana,
P.L. Decarolis, K. Shah, N.D. Socci, B.A. Weir, A. Ho, D.Y. Chiang, B. Reva,
C.H. Mermel, G. Getz, Y. Antipin, R. Beroukhim, J.E. Major, C. Hatton, R. Nicoletti,
M. Hanna, T. Sharpe, T.J. Fennell, K. Cibulskis, R.C. Onofrio, T. Saito, N. Shukla,
C. Lau, S. Nelander, S.J. Silver, C. Sougnez, A. Viale, W. Winckler, R.G. Maki,
L.A. Garraway, A. Lash, H. Greulich, D.E. Root, W.R. Sellers, G.K. Schwartz,
C.R. Antonescu, E.S. Lander, H.E. Varmus, M. Ladanyi, C. Sander, M. Meyerson,
S. Singer, Subtype-specific genomic alterations define new targets for soft-tissue
sarcoma therapy, Nat. Genet. 42 (2010) 715–721.
[315] N. Cancer Genome Atlas, Comprehensive genomic characterization of head and
neck squamous cell carcinomas, Nature 517 (2015) 576–582.
[316] A.K. Witkiewicz, E.A. McMillan, U. Balaji, G. Baek, W.C. Lin, J. Mansour,
M. Mollaee, K.U. Wagner, P. Koduru, A. Yopp, M.A. Choti, C.J. Yeo, P. McCue,
M.A. White, E.S. Knudsen, Whole-exome sequencing of pancreatic cancer defines
genetic diversity and therapeutic targets, Nat. Commun. 6 (2015) 6744.
[317] N. Cancer Genome Atlas Research, C. Kandoth, N. Schultz, A.D. Cherniack,
R. Akbani, Y. Liu, H. Shen, A.G. Robertson, I. Pashtan, R. Shen, C.C. Benz, C. Yau,
P.W. Laird, L. Ding, W. Zhang, G.B. Mills, R. Kucherlapati, E.R. Mardis,
D.A. Levine, Integrated genomic characterization of endometrial carcinoma,
Nature 497 (2013) 67–73.