The International Journal of Biochemistry & Cell Biology 45 (2013) 1327–1337

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The International Journal of Biochemistry

& Cell Biology
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Review

Deregulation of the COP9 signalosome–cullin-RING ubiquitin-ligase

pathway: Mechanisms and roles in urological cancers
Linda Gummlich a,b , Anja Rabien c , Klaus Jung b,c , Wolfgang Dubiel a,∗
a
Department of General, Visceral, Vascular and Thoracic Surgery, Division of Molecular Biology, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117
Berlin, Germany
b
Berlin Institute for Urological Research, Berlin, Germany
c
Department of Urology, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany

a r t i c l e i n f o a b s t r a c t

Article history: The COP9 signalosome (CSN)–cullin-RING ubiquitin (Ub)-ligase (CRL) pathway is a prominent segment
Received 30 January 2013 of the Ub proteasome system (UPS). It specifically ubiquitinates proteins and targets them for proteolytic
Received in revised form 21 March 2013 elimination. As part of the UPS it maintains essential cellular processes including cell cycle progression,
Accepted 22 March 2013
DNA repair, antigen processing and signal transduction. The CSN–CRL pathway consists of the CSN pos-
Available online 10 April 2013
sessing eight subunits (CSN1-CSN8) and one CRL consisting of a cullin, a RING-domain protein and a
substrate recognition subunit (SRS). In human cells approximately 250 CRLs exist each of which inter-
Keywords:
acting with a specific set of substrates and the CSN. The CSN–CRL interplay determines the activity and
COP9 signalosome
Cullin-RING ubiquitin ligases
specificity of CRL ubiquitination. The removal of the Ub-like protein Nedd8 from the CRL component cullin
Skp2 by the CSN (deneddylation) reduces the ubiquitinating activity and at the same time enables reassembly
Urological cancer of CRLs in order to adapt to substrate specificity requirements. On the other hand, CRLs as well as sub-
microRNAs strates negatively influence the deneddylating activity of the CSN. In recent years evidence accumulated
Tumor therapy that deregulation of the CSN–CRL pathway can cause cancer. Here we review current knowledge on mod-
ifications of CSN and CRL components including CSN subunits, SRSs and cullins causing tumorigenesis
with emphasis on urological neoplasia. The CSN–CRL pathway is a target of tumor-viruses as well as of
a multitude of miRNAs. Recently evaluated miRNAs altered in urological cancers might have impact on
the CSN–CRL pathway which has to be analyzed in future experiments. We propose that the pathway is
a suitable target for future tumor therapy.
© 2013 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1328
2. Architecture of CSN–CRL supercomplexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1328
3. Regulation of the CSN–CRL pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1328
4. Deregulation of the CSN–CRL pathway and its role in tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1329
4.1. Deregulation of substrate recognition subunits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1329
4.2. Deregulation of cullins, CAND1 and CSN subunits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1330
4.3. Impact of tumor-viruses on the CSN–CRL pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1331
5. miRNA-mediated regulation of the CSN–CRL pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1332
5.1. Impact of the miRNome on the CSN–CRL complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1332
5.2. Evaluated miRNAs altered in urological cancers and their possible impact on components of the CSN–CRL pathway . . . . . . . . . . . . . . . . . . . . 1333

Abbreviations: ␤-TrCP, ␤-transducin repeat-containing protein; CAND1, cullin-associated NEDD8-dissociated protein 1; CSN, COP9 signalosome; CRL, cullin-RING ubi-
quitin ligase; CUL, cullin; FBP, F-box protein; Fbw7, F-box protein WD40 domain 7; Keap1, Kelch-like ECH-associated protein 1; Nedd8, neural precursor cell expressed
developmentally down-regulated 8; miR, microRNA; mTOR, mammalian target of rapamycin; MPN, Mpr1p and Pad1p N-terminal; PCI, proteasome, COP9, and Initiation
factor 3; Rbx1, RING-box protein 1; RCC, renal cell carcinoma; Skp2, S-phase kinase-associated protein 2; SRS, substrate recognition subunit; Ub, ubiquitin; UPS, ubiquitin
proteasome system; VHL, von Hippel–Lindau protein.
∗ Corresponding author. Tel.: +49 30 450522305; fax: +49 30 450522928.
E-mail address: wolfgang.dubiel@charite.de (W. Dubiel).

1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biocel.2013.03.023
1328 L. Gummlich et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 1327–1337

6. Is the CSN–CRL pathway a suitable target for cancer therapy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1333

6.1. The deregulated CSN–CRL pathway as a target in cancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1333
6.2. The CSN–CRL pathway component Skp2 might be a specific therapeutic target in urological cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1333
7. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1334
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1335
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1335

1. Introduction human CSN complex were performed by electron microscopy (EM)

(Kapelari et al., 2000). Images of the CSN and the lid were compared
In eukaryotic cells the dynamic state of intracellular proteins is revealing similar architectures of the two complexes. Integrating
mostly controlled by the ubiquitin (Ub) proteasome system (UPS). the EM data with known subunit–subunit interactions yielded the
In this system polyUb chains serve as markers for proteolysis. Ub first architectural model of the CSN (Kapelari et al., 2000). It has
is first activated by the Ub activating enzyme (E1). In a second been shown that the complex contains a single copy of each sub-
step activated Ub is transferred by the family of Ub conjugating unit and is composed of two symmetrical clusters, CSN1/2/3/8 and
enzymes (E2s) to a substrate supported by E3 Ub ligases. Selectivity CSN4/5/6/7, which are connected by CSN1-CSN6 (Sharon et al.,
and substrate specificity of the UPS is conferred by E3s specifically 2009). CRL1 complexes were crystallized (Goldenberg et al., 2004;
targeting proteins for degradation via ubiquitination (Hershko and Zheng et al., 2002) and molecular models of CRL1 action and
Ciechanover, 1998). The largest family of E3s are the cullin-RING CSN–CRL interaction were described (Deshaies and Joazeiro, 2009).
Ub ligases (CRLs) including approximately 250 members (Lee et al., The formation of CSN–CRL supercomplexes (Fig. 1B) has been
2011a). CRLs are multisubunit complexes composed of a cullin demonstrated in many organisms (Huang et al., 2009; Lyapina
from the CUL family (CUL-1-7), a RING domain subunit, mostly et al., 2001; Peng et al., 2003; Schwechheimer et al., 2001). Addi-
Rbx1, an adaptor protein and a substrate recognition subunit (SRS) tional progress in the field was recently obtained by EM and single
(Fig. 1A) (Petroski and Deshaies, 2005). In case of CUL-1 the SRSs particle analysis which generated a three-dimensional molecular
are F-box proteins (FBPs) and the complexes are called CRL1FBP . model for the CSN–CRL complex (Enchev et al., 2010, 2012). In
The activity of CRLs is regulated by conjugating and deconjugating this model, the PCI subunits of the CSN form an approximately
the Ub-like protein Nedd8 to cullins (Saha and Deshaies, 2008) (see coplanar surface. The four longest subunits (CSN1, CSN2, CSN3,
Chapter 3). Nedd8 activation and conjugation to proteins (cullins) and CSN4) are docked into the central region of an arc, which
occurs in a similar manner as described above for Ub (Liakopoulos is capped at each end by the shorter CSN7 and CSN8 subunits
et al., 1998). The COP9 signalosome (CSN) controls CRLs by specif- (Fig. 1A). The MPN domain subunits, CSN5 and CSN6, are pre-
ically deconjugating Nedd8 (deneddylation) from cullins (Fig. 1B) dicted to form a protrusion on the side opposite to the PCI subunits
(Bondar et al., 2006; Deshaies and Joazeiro, 2009; Lyapina et al., (Enchev et al., 2010, 2012; Sharon et al., 2009). In their model of
2001; Schwechheimer et al., 2001). The dynamic interplay and the CSN–CRL complex the authors show that the CSN predom-
activity of the CRLs and the CSN is called here the CSN–CRL path- inantly binds to the C-terminal portion of CUL-1 (Enchev et al.,
way and represents a pivotal element of the system that governs 2012). Neddylated CRL1 revealed a tighter binding to the CSN as
intracellular protein dynamics in eukaryotic cells. compared with the unneddylated one. As predicted before (Lyapina
The CSN is a conserved protein complex containing eight core et al., 2001), the molecular model of Enchev et al. (2012) con-
subunits, named CSN1–8 (Fig. 1A) (Bech-Otschir et al., 2005; firms a major interaction of CUL-1 with subunit CSN2 of the CSN
Cope and Deshaies, 2003; Deng et al., 2000; Harari-Steinberg and (Fig. 1B). The structural analysis of CSN–CRL complexes revealed a
Chamovitz, 2004; Kato and Yoneda-Kato, 2009; Schwechheimer, mutual interference of the two complexes. CSN-mediated dened-
2004; von Arnim, 2003; Wei and Deng, 2003; Wei et al., 2008; dylation causes a conformational change that deactivates the CRLs.
Wolf et al., 2003). The complex was originally discovered in Ara- On the other hand, CRL composition has impact on CSN-mediated
bidopsis thaliana as a regulator of constitutive photomorphogenesis deneddylation.
(COP) (Chamovitz et al., 1996; Wei et al., 1994). Subsequently it was
identified in human cells (Seeger et al., 1998) and studies using 3. Regulation of the CSN–CRL pathway
various eukaryotic organisms indicated that the CSN is responsi-
ble for regulating the UPS pathway (Glickman et al., 1998; Seeger The CSN–CRL pathway catalyze the specific ubiquitination of
et al., 1998; Wei and Deng, 1998). Deregulation of the CSN–CRL selected substrates which are subsequently degraded by the 26S
pathway can have dramatic effects on diverse cellular functions proteasome (Fig. 1A). Ubiquitination is stimulated by neddylation
including DNA repair (Groisman et al., 2003; Takedachi et al., 2010; and the removal of Nedd8 carried out by CSN5 through its met-
Wei et al., 2008), cell cycle control (Kato and Yoneda-Kato, 2009; alloenzyme (JAMM) domain (Cope et al., 2002) deactivates the
Liu et al., 2003), angiogenesis (Braumann et al., 2008) and tumor CRLs (Fig. 1B). In addition to this catalytic mechanism, CSN can
development as discussed in this review. also inhibit CRL function in a noncatalytic fashion (Emberley et al.,
2012; Enchev et al., 2012). The CSN-mediated inactivation of CRLs
2. Architecture of CSN–CRL supercomplexes prevents the autocatalytic breakdown and enables the reassem-
bly of the CRLs, maintaining their optimal activity (Schmidt et al.,
The CSN complex is composed of the six subunits with PCI 2009). The complex stability depends further on the availabil-
(Proteasome, COP9, and Initiation factor 3) domains (Fig. 1A, yel- ity of substrate (Bornstein et al., 2006; Chew and Hagen, 2007;
low subunits) (Aravind and Ponting, 1998; Glickman et al., 1998; Emberley et al., 2012). The presence of phosphorylated p27 e.g.
Hofmann and Bucher, 1998), and CSN5 and CSN6 with MPN (Mpr1p stimulates the activity of CRL1Skp2 and controls its own ubiquitin-
and Pad1p N-terminal) domains (Fig. 1A, blue subunits). The PCI ation and degradation (Bornstein et al., 2006). Also other CRLs were
and MPN domains are found almost exclusively in two other large found regulated by substrates (Chew and Hagen, 2007). CAND1 is
protein complexes, the 26S proteasome lid and the eukaryotic another important modulator of CRL activity maintenance in vivo. It
translation initiation factor eIF3, suggesting a common ancestor binds to the deneddylated form of cullins and competes with SRSs
in evolution (Pick et al., 2009). Initial structural studies of the (Zheng et al., 2002). Therefore, CAND1 allows SRS exchange by a
 L. Gummlich et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 1327–1337 1329

Fig. 1. Model of the CSN–CRL pathway. (A) The COP9 signalosome (CSN) consists of eight subunits, six subunits possess PCI (Proteasome-COP9-Initiation factor-3) domains
(yellow) and two subunits MPN (Mpr1p and Pad1p N-terminal) domains (blue). The E3 cullin-RING ubiquitin ligase complex is composed of a scaffold cullin, RING box protein
(Rbx1), E2 ligase and a specific substrate recognition subunit (SRS). Neddylated CRL complexes ubiquitinate specific substrates (S) depending on their SRSs and promote
degradation by the ubiquitin proteasome system (UPS) via the 26S proteasome. (B) Deneddylation of the CRL by complex-bound CSN5 causes conformational changes and
shifts the CRL complex in an inactive state, which ensures complex stability and enables exchange of SRSs.

mechanism not yet fully understood (Emberley et al., 2012;
Goldenberg et al., 2004; Schmidt et al., 2009).
4. Deregulation of the CSN–CRL pathway and its role in
tumorigenesis
The fine-tuned regulation of the CSN–CRL pathway points to
an immense impact in case of disturbance. Thereby multiple com-
ponents may be affected (Fig. 2A–D, Tables 1 and 2). Mutations
within substrate genes or additional post-transcriptional modifi-
cation prevent substrate recognition and promote accumulation
of oncoproteins (Fig. 2A). Since SRSs play a fundamental role in
proper functioning of CRLs, their loss or gain of function can lead
to cancerogenesis (Fig. 2C). Expression patterns of cullins, CSN sub-
units or CAND1 can be altered provoking oncogenesis. Moreover,
tumor-viruses can abuse the cellular Ub machinery to manipulate
the proteasome for their advantage (Fig. 2D). Examples mentioned
below highlight the contribution of a deregulated CSN–CRL path-
way to tumorigenesis with focus on urological neoplasia.
4.1. Deregulation of substrate recognition subunits
The prominent FBP Skp2 was found overexpressed in almost
every tumor and correlates with poor prognosis fulfilling onco-
genic functions (Frescas and Pagano, 2008). Its expression is also
increased in renal cell carcinoma (RCC), a tumor often accompanied
with resistance to chemotherapy and kinase inhibitors like mam-
malian target of rapamycin (mTOR) inhibitors (Langner et al., 2004;
Liu et al., 2008). Interestingly, a correlation between mTOR inhibitor
sensitivity and Skp2 levels was detected suggesting an impact of
the FBP on therapy outcome (Totary-Jain et al., 2012) (see Chap-
ter 6.2). The downregulation of Skp2 by von Hippel–Lindau protein
(VHL) was reported proposing an interaction between these two
SRSs (Roe et al., 2011). VHL, the SRS of the CRL2VHL complex, sta-
bilizes p27 and induces apoptosis in RCC cell lines and in vivo (Kim
et al., 2004). Considering that Skp2 promotes the degradation of
p27, Roe and coworkers revealed a VHL-mediated Skp2 polyubiqui-
tination and its degradation upon induction of DNA damage which
did not depend on the VHL E3 Ub ligase activity (Roe et al., 2011). In
bladder cancer, the Wnt pathway inhibitor WIF1 (Wnt inhibitory
factor 1) negatively regulates Skp2 by preventing the binding of
␤-catenin to the Skp2 promotor, connecting the FBP to urothe-
lial tumorigenesis (Tang et al., 2009). Furthermore, deregulation of
Skp2 was found in prostate cancer (Lin et al., 2010; Yang et al., 2002)
(see Chapter 6.2). Another well-studied FBP, ␤-TrCP, behaves onco-
genic as well as tumor suppressive depending on cellular context.
␤-TrCP targets cell cycle regulators leading to decreased genetic
instability (Guardavaccaro et al., 2003), although it was observed
upregulated in numerous tumors (Lau et al., 2012). Mutations and
overexpression of ␤-TrCP were found in prostate cancer and pro-
vided the first evidence for ␤-TrCP participation in tumorigenesis
(Gerstein et al., 2002). However, CRL1ˇ-TrCP ubiquitinates the two
prominent oncoproteins ␤-catenin (Hart et al., 1999) and p53 ligase
Mdm2 (Inuzuka et al., 2010) impeding proliferation. Alterations in
the ␤-TrCP gene (BTRC) lead to accumulation of ␤-catenin and to
development of colon cancer (Kim et al., 2007; Lau et al., 2012).
Furthermore, a mutation was observed in the F-box motif of BTRC
induced tumorigenesis in murine kidneys through overexpression
and accumulation of ␤-catenin highlighting an anti-cancerogenic
property of the FBP (Belaïdouni et al., 2005). Nevertheless, thera-
peutic targeting of ␤-TrCP might be intricate since its participation
in fundamental processes is tissue-dependent.
The FBP Fbw7 represents a classical tumor suppressor. CRL1Fbw7
promotes degradation of numerous of tumor-driving proteins, and
1330 L. Gummlich et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 1327–1337

Fig. 2. The deregulated CSN–CRL pathway participates in tumorigenesis. Alterations of the CSN–CRL pathway caused by different modifications promote accumulation of
oncoproteins or increased degradation of tumor suppressors. (A) A substrate altered either through mutation or additional posttranslational modification avoids recognition
by the SRS of CRLs and therefore, accumulates in the cell. (B) Altered co-receptors impede the recognition of substrate. For example, an alteration in Cks1, the co-receptor
of Skp2 disturbs the recognition of p27 for ubiquitination (Frescas and Pagano, 2008). (C) Overexpressed SRS promotes enhanced tumor suppressor degradation whereas
downregulated or mutated SRS allows accumulation of oncoproteins. (D) Tumor-viruses are able to hijack the CRLs through SRS exchange with viral protein, interference
with the CRL core complex or deactivation by viral deneddylases.

mutation in Fbw7 substrate-binding regions or loss of heterozy-
gosity was found in 6% of all cancers, mainly lymphomas (Welcker
and Clurman, 2008). The FBW7 gene is a direct target of p53,
another well characterized tumor suppressor, indicating an anti-
cancerogenic role (Mao et al., 2004). FBW7 gene mutations were
also identified in urological cancers (Liu et al., 2012). Consider-
ing the Fbw7 substrates mTOR and Notch loss of function of this
FBP might play an important role in RCC development (Lau et al.,
2012). Moreover, loss of Fbw7 promotes resistance to anti-tubulin
chemotherapeutics like Taxol (Wertz et al., 2011), which may
explain the leaking effect of these agents in RCC patients.
CRL1Fbw4 (containing an FBP related to Fbw7) impedes tumori-
genesis by ubiquitinating cyclin D1. Mutations that inhibit
dimerization of Fbw4 required for cyclin D1 degradation was found
in esophageal carcinomas (Barbash et al., 2008). Keap1 (Kelch-
like ECH-associated protein 1), another SRS protein, targets Nrf2
(Nuclear factor (erythroid-derived 2)-like 2) to prevent its accu-
mulation and overexpression of Nrf2 downstream genes often
observed in tumors. Its anti-oncogenic role is emphasized by 19%
of lung cancer patients harboring a somatic Keap1 mutation (Singh
et al., 2006). Taken together, altered SRSs provide a platform for
drug interference. However, it still remains a challenge to iden-
tify all substrates of SRSs in order to understand their complex
regulation function in a time and tissue-specific manner.
4.2. Deregulation of cullins, CAND1 and CSN subunits
Altered expression patterns of cullins, CSN subunits and CAND1
were observed in various tumors. Berthold et al. reported a strong
downregulation of CUL-3 in kidney cancer samples (Berthold et al.,
2008), whereas CUL-1 is upregulated in human lung tumors. Inter-
estingly, the neddylated form of CUL-1 is particularly expressed
in high grade neuroendocrine lung tumors and associated with
low levels of CAND1 (Salon et al., 2007). CUL-4A also plays a role
in oncogenesis; the CUL-4A gene was found to be amplified or
overexpressed in primary breast cancer and is associated with
aggressive growth and poor prognosis (Chen et al., 1998; Schindl
et al., 2007). CUL-4A may contribute to tumorigenesis by promoting
mTOR signaling, a key pathway in RCC, through degradation of the
mTOR inhibitor REDD1 (Katiyar et al., 2009; Lee and Zhou, 2010).
 L. Gummlich et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 1327–1337 1331

Table 1
Known deregulated components of the CSN–CRL pathway involved in tumorigenesis.

Target Group Deregulation Cancer Reference

Fbw4 SRS (FBP) Mutation prevents dimerization of Fbw4 and thus cyclin D1 Esophageal carcinomas Barbash et al. (2008)
degradation
Fbw7 SRS (FBP) Tumor suppressor, mutations in Fbw7 substrate-binding regions Lymphomas, multiple cancers Mao et al. (2004), Welcker and
Clurman (2008)
␤-TrCP SRS (FBP) Oncogenic/tumor suppressor, increases genetic instability, targets Multiple cancers Kim et al. (2007), Lau et al.
Mdm2, ␤-catenin (2012)
Skp2 SRS (FBP) Overexpression in multiple tumors, promotes degradation of p21, Multiple cancers Frescas and Pagano (2008)
p27
Keap1 SRS (BTB) Keap1 gene mutation drives Nrf2 accumulation, expression of Nrf2 Lung cancer Singh et al. (2006)
target genes
VHL SRS VHL gene negatively mutated or silenced, drives expression of Clear cell RCC Kim and Kaelin (2006)
HIF1␣ target genes
CUL-1 Cullins Expression levels increased, associated with poor prognosis, drives Melanoma, breast cancer Chen and Li (2010)
p27 degradation
CUL-3 Cullins Expression levels strongly decreased RCC samples Berthold et al. (2008)
CUL-4 Cullins CUL-4A gene amplified, overexpressed, protects mTOR through Breast cancer Chen et al. (1998), Katiyar et al.
degradation of REDD1 (2009)
CUL-5 Cullins Expression levels decreased Lung cancer Singhal et al. (2003)
CSN2 CSN CSN2 gene lost in several tumors, CSN2 over-expression drives Multiple cancers Leal et al. (2008)
chromosome instability
CSN3 CSN CSN3 gene amplified Osteosacroma Yan et al. (2007)
CSN5 CSN CSN5 overexpression in multiple tumors, CN5 gene amplified in Multiple cancers Patil et al. (2005), Shackleford
hepatocellular carcinoma and Claret (2010)
CSN6 CSN CSN6 overexpressed, stabilizes Mdm2 and promotes p53 Breast cancer, multiple cancer Lee et al. (2011b), Zhao et al.
degradation (2011)
CAND1 Expression levels decreased Prostate cancer Korzeniewski et al. (2012),
Murata et al. (2010)

Furthermore, CUL-5 is slightly upregulated in lung cancer shown
by expression profiling as it was also demonstrated for additional
cell cycle genes (Singhal et al., 2003).
CAND1 expression levels are decreased in prostate cancer
(Korzeniewski et al., 2012; Murata et al., 2010). Since it is respon-
sible for proper CRL function by promoting FBP exchange, CAND1
defects should also be considered important for prostate neoplasia
development.
The deregulation of pleiotropic functions of the CSN can also
participate in tumor development (Richardson and Zundel, 2005).
The CSN2 gene is lost in several types of cancer (Leal et al., 2008).
Amplification of the CSN3 gene in osteosarcomas indicates an onco-
genic role of the CSN subunit (Yan et al., 2007). CSN3 gene alteration
is linked to the non-cancerous cerebric malignancy Smith–Magenis
syndrome (Elsea et al., 1999). The CSN5 gene was found to be ampli-
fied in hepatocellular carcinomas elucidating the high expression
levels in these tumors (Patil et al., 2005). CSN5 overexpression
correlated with tumor growth and negative clinical outcome was
detected in numerous cancers (Shackleford and Claret, 2010). CSN6
gene amplification was observed in breast cancer followed by CSN6
overexpression and Mdm2 stabilization (Zhao et al., 2011). How-
ever, it should be stressed out that most studies reporting elevated
CSN5 or CSN6 expression levels failed to give further informa-
tion on the amounts of other CSN subunit. Therefore it cannot be
excluded that the whole CSN complex rather than a single sub-
unit is upregulated since convincing evidence for the independent
function of single CSN subunits is still lacking. A high cellular CSN
level can induce cancer, because it accelerates the degradation of
the tumor suppressors p53 (Bech-Otschir et al., 2001) and p27
(Huang et al., 2006). In addition, upregulated CSN causes deregu-
lation of the CSN–CRL pathway. Unfortunately, only little is known
about the function of the CSN in urological cancers so far. Since
CRL-bound FBPs are protected by the CSN-mediated deneddylation
(Cope and Deshaies, 2006; Schmidt et al., 2009) it is likely that pri-
mary modifications of the CSN cause alterations of FBPs resulting
in tumorigenesis.
4.3. Impact of tumor-viruses on the CSN–CRL pathway
Tumor-viruses are able to hijack cellular Ub ligases (Fig. 2D,
Table 2) (Barry and Früh, 2006; Lee and Zhou, 2010). The HPV
(human papilloma virus) E7 oncoprotein associates with the CUL-2
complex to CRL2E7 and redirects it to target the tumor suppressor
retinoblastoma protein contributing to the development of cervi-
cal carcinoma (Huh et al., 2007). Moreover, the adenovirus-derived
oncoprotein E1A inhibits CRL1Fbw7 by direct interaction with Rbx1
and CUL-1 and promotes proliferation (Isobe et al., 2009). A nega-
tive regulation of CRL activity through a deneddylase encoded by
Epstein–Barr virus, a tumor-virus, was found in lymphomas and
stomach cancer (Gastaldello et al., 2010). The same group revealed
a further viral modulation of CRL activity by preventing the recruit-
ment of CAND1, which can be rescued by CAND1 overexpression
(Gastaldello et al., 2012). In addition, the Kaposi’s sarcoma (KSH)-
virus protein latency-associated nuclear antigen (LANA) serves as
a SRS in CRL5LANA and mediates the degradation of VHL and p53 to
promote KSH development, one of the first recognized HIV-related

Table 2
Tumor-viruses attack the CSN–CRL pathway and promote tumorigenesis.

Tumor-virus protein Function Cancer Reference

Human papilloma virus E7 Associates with CRL2 core complex, drives retino-blastoma degradation Cervix carcinoma Huh et al. (2007), White et al.
(2012)
KSH-virus LANA Associates with CRL5 core complex, drives VHL, p53 degradation Kaposi’s sarcoma Cai et al. (2006)
Epstein–Barr-virus BPLF1 Viral deneddylase hydrolyses Nedd8 and stabilizes CRL substrates Lymphoma, stomach cancer Gastaldello et al. (2010)
Epstein–Barr-virus BZLF1 Associates with CRL2 or CRL5 core complex, promotes p53 degradation Lymphoma, stomach cancer Sato et al. (2009)
Hepatitis B virus Hbx Binds to Skp2 F-box region and protects c-Myc from degradation Hepatocellular carcinoma Kalra and Kumar (2006)
SV40 Large T antigen Interacts with Fbw7 and protects cyclin E from degradation Multiple tumors Welcker and Clurman (2005)
1332 L. Gummlich et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 1327–1337
Fig. 3. Regulation of the CSN–CRL pathway by published miRNAs. Different angles of the CSN–CRL pathway are affected by deregulated miRNAs. They either reduce (block
arrow) or indirectly induce the protein expression (arrow). The particular function of each miRNA is listed in Table 3.
illnesses (Cai et al., 2006). These examples illustrate that tumor-
viruses target the CSN–CRL pathway to use it for their own purpose,
providing another example for the significance of the pathway in
cancer development.
5. miRNA-mediated regulation of the CSN–CRL pathway
5.1. Impact of the miRNome on the CSN–CRL complex
The enormous influx of novel miRNA studies in public databases
point out the significance of miRNAs in tumor development, in
monitoring tumor progression and as possible therapeutic tar-
gets. Deregulation within the miRNA collectivity (miRNome) has
a major impact on the equilibrium of the proteome in cells on a
post-transcriptional level often leading to pathological conditions.
On the other hand, post-translational modification e.g. ubiquitin-
ation can lead to fine-tuned protein degradation and physiological
maintenance. Both fundamental processes play an important role
in tumorigenesis. But how does the miRNome interact with the
protein degradation machinery? Here we examine whether dereg-
ulated miRNAs could also affect the UPS with focus on the CSN–CRL
pathway. Fig. 3 displays an overview on known miRNAs affecting
the ubiquitination mechanism. Furthermore, the function of these
particular miRNAs and their deregulation in cancer is provided in
Table 3.
The regulation of CSN subunit expression by the let-7 miRNA
family was first discovered in the context of CSN assembly in 2011
(Leppert et al., 2011). In addition, the subunit CSN8 is targeted by
miR-146a in gastric cancer (Crone et al., 2012). Like CSN subunits,
also E2 ligase Cdc34 mRNA is regulated by the let-7 family. Low
Cdc34 levels due to let-7b overexpression resulted in decreased
CRL1 activity, stabilizing Wee1 and G2 /M accumulation (Legesse-
Miller et al., 2009). Furthermore, CAND1 was spotted as a target of
androgen-responsive miR-148a in prostate cancer (Murata et al.,
2010).
Besides CSN and CAND1 also cullins and SRSs are negatively reg-
ulated by miRNAs. The hypoxia-sensitive miR-424 decreases CUL-2
levels and promotes angiogenesis, a phenotype that often occurs in
RCC (Ghosh et al., 2010). Targeting of CUL-5 by miR-19a/19b was
reported in cervical carcinoma cells (Xu et al., 2012). The miR-19a
and miR-19b belong to the miR-17-92 cluster and were demon-
strated to be strongly oncogenic by targeting E2Fs and by promoting
overexpression of the MYC gene in many tumors. Paradoxically,
besides the oncogenic characteristics, this cluster also shows tumor
suppressor features which need to be further inspected (Zhang
et al., 2009). The strongest targeted SRS remained the tumor sup-
pressor protein Fbw7. Its mRNA is negatively regulated by miR-25,
-27a and -223 in different contexts (Lerner et al., 2011). Increased
genome instability triggered by miR-223 targeting Fbw7 provided
the first evidence that CRL1s are affected by the miRNome (Xu et al.,
2010). In addition, FBP ␤-TrCP is targeted by miRNAs. The miR-183
interacts with ␤-TrCP mRNA, a mechanism that can be prevented
by binding of specific proteins (Elcheva et al., 2009). The inves-
tigation of the correlation of miRNAs and cisplatin resistance in
ovarian cancers revealed a direct regulation of Keap1 by miR-141
(van Jaarsveld et al., 2012). The miR-141 also plays a critical role
 L. Gummlich et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 1327–1337 1333

Table 3
Regulation of the CSN–CRL pathway by miRNAs.

Target miRNA Function Tissue References

CSN miR-Let-7 Targets CSN subunit mRNAs, inhibits assembly Cervical cancer Leppert et al. (2011)
CSN8 miR-146a Targets CSN8, inhibits NF␬B activation Gastric cancer (mouse model) Crone et al. (2012)
CAND1 miR-148a Negative regulation of CAND1 expression Prostate cancer Murata et al. (2010)
Cdc34 miR-Let-7 Targets Cdc34 expression, promotes cell cycle arrest Primary human fibroblasts Legesse-Miller et al. (2009)
CUL-2 miR-424 Targets CUL-2, promotes angiogenesis Cervical cancer Ghosh et al. (2010)
CUL-5 miR-19a, 19b Negative regulation of CUL-5 expression Cervical cancer Xu et al. (2012)
Fbw7 miR-223 Targets Fbxw7, promotes genomic instability Embryonic fibroblasts Li et al. (2012), Xu et al. (2010)
miR-27a Targets Fbxw7, promotes cell cycle progression Various cancer cell lines Lerner et al. (2011)
miR-25 Targets Fbxw7 Embryonic fibroblasts Lu et al. (2012)
␤-TrCP miR-183 Negative regulation of ␤-TrCP expression Colorectal cancer cell lines Elcheva et al. (2009)
VHL miR-92a Negative regulation of VHL expression RCC samples Valera et al. (2011)
Keap1 miR-141 Targets Keap1, induces cisplatin resistance Ovarian cancer van Jaarsveld et al. (2012)
p27 miR-221 miR-222 Negative regulation of p27 expression Cardiomyocytes Wang et al. (2012b)
miR-181 Negative regulation of p27 expression Hepatic stellate cells Wang et al. (2012a)
p53 miR-214 Indirect upregulation of p53 Myeloma cell lines Misiewicz-Krzeminska et al. (2012)
c-Myc miR-34a Targets c-Myc, inhibits invasion Prostate cancer Yamamura et al. (2012)
FOXO1 miR-27a, miR-96,-182 Negative regulation of FOXO1 expression Breast cancer Guttilla and White (2009)
c-Jun miR-125b Negative regulation of c-Jun expression Melanoma cell lines, samples Kappelmann et al. (2012)
miR-155 Negative regulation of c-Jun expression Human dermal fibroblasts Song et al. (2012)
Cyclin D1 miR-520b Negative regulation of cyclin D1 and MEKK2 expression Hepatoma cell lines Zhang et al. (2012)

in urological cancers. The VHL protein is under the control of miR-
92a leading to stabilization of pro-angiogenic factors (Valera et al.,
2011). It should be mentioned that substrates of CRLs are regulated
by miRNAs as well (Table 3).
This data gives an insight in the regulation of the CSN–CRL path-
way by the miRNome. Different angles of the Ub machinery are
affected by deregulated miRNAs in various tumors (Fig. 3, Table 3).
Interestingly, most of these miRNAs are also altered in urological
cancers providing a new regulation cross-talk and possible com-
mon targets for these tumors.
5.2. Evaluated miRNAs altered in urological cancers and their
possible impact on components of the CSN–CRL pathway
anti-neddylation agent MLN4924 strikes by inhibiting complete
CRL activity (Soucy et al., 2010) and is only useful when an over-
expressed cullin or FBP is the main key player in a specific cancer
type. Nevertheless, radio sensitization of cancer cells was found
after inhibition of CRLs, which could provide an advantage for
tumor patients (Wei et al., 2012). In order to reduce adverse reac-
tion, more specific drugs are needed which interfere with substrate
recognition or required kinases like the cyclin-dependent kinase
regulatory subunit (Cks1) for Skp2-mediated p27 recognition. The
small molecule enhancer of rapamycin (SMER3) is able to inhibit
the binding of FBP Met30 to the CRL1 core complex (Aghajan et al.,
2010) and is an example for such a unique interference with the
CSN–CRL pathway.

Insufficient data were published so far concerning urological
cancer-associated miRNAs and the impact of the CSN–CRL complex.
To identify putative miRNA binding sites in the mRNAs of members
of the CSN–CRL complex, an in silico target prediction using Tar-
getScan 6.2 algorithm was performed, a stringent tool to identify
miRNA binding sites (http://www.targetscan.org/). Table 4 summa-
rizes predicted targeting of components of the CSN–CRL pathway
by selected miRNAs found to be deregulated in urological neoplasia
(Schaefer et al., 2010; Wotschofsky et al., 2012). The CSN, CAND1,
cullins and FBPs were spotted as predicted targets of these partic-
ular deregulated miRNAs. Especially CSN2, CSN7b, CUL-3, CUL-4,
␤-TrCP and FBPs containing a WD40 domain (FBXWs) provide puta-
tive miRNA binding sites, which need to be verified in experimental
context. Based on this prediction, a urological cancer-promoting
miRNome seems to play a role in the regulation of the CSN–CRL
pathway (Table 4) and should be considered in future investigations
of tumor therapeutics.
6. Is the CSN–CRL pathway a suitable target for cancer
therapy?
6.1. The deregulated CSN–CRL pathway as a target in cancer
therapy
The deregulated CSN–CRL pathway is an attractive target for
cancer therapy. This approach will improve and complement ther-
apies already developed in the past decade. Proteasome inhibitors
effectively downregulate protein degradation but cause a vari-
ety of unwanted side effects and are only approved for specific
types of myeloma and lymphoma (D’Arcy and Linder, 2012). The
6.2. The CSN–CRL pathway component Skp2 might be a specific
therapeutic target in urological cancers
Urological cancers are accompanied by increasing incidence and
poor prognosis longing for a better understanding of molecular
mechanisms and new therapeutic targets. In RCC, the therapeutic
opportunities are limited since RCC patients are mostly resistant to
chemo- and radiation therapy (Corn, 2007) and they only partly
respond to currently available agents. The proteasome inhibitor
bortezomib was found in a phase II clinical trial with only par-
tial response in 10% of RCC patients (Kondagunta et al., 2004).
The toxicity caused by the agent recommend inhibition of E3
Ub ligases as a more specific treatment (Corn, 2007). Totary-Jain
et al. revealed an interesting interplay between the mTOR inhibitor
rapamycin and Skp2. Rapamycin down-regulates Skp2 by impair-
ing its phosphorylation through AKT (Protein kinase B) and RNA
interference-mediated silencing of Skp2 increased sensitivity of
tumor cells to rapamycin in vitro and in vivo (Totary-Jain et al.,
2012). Therefore, the Skp2 expression level can serve as a pre-
dictor for mTOR inhibitor-sensitivity. For RCC tumors resistant to
mTOR inhibitors, additional targeting of Skp2 might be a ther-
apeutic option. In recent years, Skp2 emerged as an attractive
target in prostate cancer therapy. Evidence of Skp2 overexpress-
ion in neoplastic prostate tissue accumulates (Lin et al., 2010;
Shim et al., 2003; Yang et al., 2002; Zheng et al., 2004), correlating
with tumor grade and poor prognosis like in most types of cancer
(Frescas and Pagano, 2008). Using high-throughput screening, the
small molecule inhibitor CpdA (Compound A) was identified which
blocks the recruitment of Skp2 to the CRL core complex (Chen et al.,
2008). CpdA was able to overcome the resistance to chemotherapy
1334 L. Gummlich et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 1327–1337

Table 4
Predicted targets of deregulated miRNAs in prostate and renal cell cancer (Schaefer et al., 2010; Wotschofsky et al., 2012) with focus on the CSN–CRL pathway.

Target Gene Protein Kidney cancer Prostate cancer

CSN COPS2 CSN2 miR-16,-31,-32,-92a,-92b, miR-107,-181b

COPS7A CSN7a miR-16
COPS7B CSN7b miR-10 miR-16,-34a, -34a,c,-125, miR-486-5p
COPS8 CSN8 miR-200c
CAND1 CAND1 CAND1 miR-148
Cullins CUL2 CUL-2 miR-16,-574-3p
CUL3 CUL-3 miR-130a miR-20a,-23b,-32,-34b, miR-92a,-92b,-101,-106a,
miR-106b,-141,-181b,-218, miR-373, -485-3p,-636
CUL4A CUL-4a miR-514 miR-96,-107,-181b
CUL4B CUL-4b miR-101,-141,-194
CUL5 CUL-5 miR-19a,-19b miR-31,-96,-101,-145,-182, miR-181b
F-box proteins BTRC ␤-TrCP miR-10b, -195, miR-224, -514 miR-16,-34b,135b,-107
SKP1 Skp1 miR-221,-145
FBXWs FBXW2 Fbw2 miR-29a,-141,-146a, -194, miR-197,-331-3p
FBXW4 Fbw4 miR-125b,-149
FBXW5 Fbw5 miR-296
FBXW7 Fbw7 miR-223 miR-1,-16,-29a,-32,-92a, miR-92b,-101,-107,-194, miR-197
FBW9 Fbw9 miR-29b miR-29a
FBXW11 Fbw11 miR-20a,-101, miR-200c miR-20a,-23b,-96,-101, miR-181b,-182

and proteasome inhibitors in multiple myeloma, highlighting the
potential role of targeting Skp2 in the battle against tumor progres-
sion (Chen et al., 2008). Moreover, natural occurring compounds
like curcumin, vitamin D and others promoted the downregulation
of Skp2 inducing p27 stabilization (Huang et al., 2011; Yang and
Burnstein, 2003). The novel energy restriction-mimic agent CG-
12 also downregulates Skp2 and induces ␤-TrCP upregulation by
preventing the Skp2-␤-TrCP-Sp1-feedback loop, a novel cross-talk
found between these two important FBPs (Wei et al., 2012). Never-
theless, the development of Skp2 therapeutics is still accompanied
by obstructions due to a lack of appropriate delivery systems. The
Skp2 substrate recognition of both tumor suppressors and onco-
proteins might also complicate their usage. However, Skp2 forms a
structurally active site in the presence of co-receptor kinase Cks1
(Fig. 2B) resulting in specific ubiquitination of tumor suppressor
substrates like p27 (Huang et al., 2012). Drugs interfering with the
Skp2-Cks1 binding site or Skp2-p27 interface might be attractive
therapeutic agents which exclusively prevent nuclear p27 degra-
dation associated with tumor development (Cardozo and Pagano,
2007).
7. Future prospects
The CSN–CRL pathway is dependent on the elaborate interplay
between enzymatic protein complexes. Whereas the CSN influ-
ences CRL activity mostly by deneddylation, CRL components and
substrates modulate CSN-mediated deneddylation. The pathway
confers substrate specificity to the UPS and is virtually responsible
for the degradation of a specific protein, at a certain time in a par-
ticular compartment. Because oncoproteins and tumor suppressors
are among the substrates of the CSN–CRL complex, deregulation
of its parts impairs intracellular protein dynamics with the preva-
lent consequence of tumor development as outlined in this review.
Because modifications of the CSN–CRL pathway are increasingly
found in cancers, the pathway becomes an interesting field for
pharmacological intervention in cancer.
What are relevant open questions in our understanding of
molecular mechanisms of the CSN–CRL pathway?
Future investigations should focus on concrete mechanisms and
molecular structures of the pathway. At the moment the struc-
tural model of the CSN–CRL complex is a prediction and has to be
verified by cryo-electron microscopy and crystallography. A com-
prehensive characterization of SRSs and their substrates and their
deregulation in cancer is a prerequisite for the development of
specific compounds targeting SRS/substrate interactions. Little is
known about the regulation of the CSN–CRL pathway by miRNAs
and by signaling pathways, and research in this field might open a
new area of pharmacological intervention.
Which therapeutics is desirable and applicable in cancer
therapy?
In the past decade progress has been made in the develop-
ment of drugs interfering with the UPS. Proteasome inhibitors
prevent degradation of essential tumor suppressors. However,
overall blocking of the proteasome leads to severe side effects,
because of the pleiotropic role of the enzyme complex. Similar
outcome can be expected with the use of neddylation as well as
CSN-mediated deneddylation inhibitors, since the function of all
CRLs will be impeded. To reduce adverse reactions more specific
drugs are needed which interfere with particular SRSs or prevent
tumor suppressor recognition and elimination. RNAi-based agents
are imaginable to stop the biosynthesis of a particular cullin or
SRS. Moreover, engineering specific SRS that target oncoproteins
for degradation are up for debate. The potential involvement of
deregulated miRNAs should also be considered. The determination
of miRNA levels emerged as powerful diagnostic and prognostic
tool in urological cancers. miRNAs are stable, and non-invasive
detection is possible. Here, we provide evidence that an altered
miRNome influences the CSN–CRL pathway and, therefore, could
be an attractive target for further examinations.
What is the role of the CSN–CRL pathway in urological
tumorigenesis?
New applicable targets are needed for the treatment of uro-
logical cancer. Indeed, altered expression of important FBPs in
urological cancers influences the presence of key tumor pro-
moting proteins in urological tissues as reported in this review.
The exact mechanism of how deregulated SRSs are integrated
in urological tumorigenesis is currently unknown. Unfortunately,
clinical data of CSN subunits in urological cancer samples are still
lacking. Therefore, for the further understanding and therapy of
urological tumors, the CSN–CRL pathway would be an exciting
subject.
How can gained knowledge be transferred to cancer therapy
development?
Pre-clinical results reviewed here clearly recommend the
CSN–CRL pathway as a major target for future tumor therapy.
Compounds with clear anti-tumor evidence in cell culture and in
animals should be advanced in clinical trials. In the near future,
well designed clinical studies should to be carried out to assess the
clinical properties and application of possible new drugs that target
the CSN–CRL pathway.
 L. Gummlich et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 1327–1337
Acknowledgments
This work was funded by the Foundation of Urological Research,
Berlin and the Charité-Universitätsmedizin, Berlin. Due to space
limitations we could not consider each article of the field in this
review. We apologize for that incompleteness.
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