The ubiquitin-specific protease USP2A enhances tumor progression by targeting cyclin A1 in bladder cancer

Article (PDF Available)inCell cycle (Georgetown, Tex.) 11(6):1123-30 · March 2012with37 Reads
DOI: 10.4161/cc.11.6.19550 · Source: PubMed
The deubiquitinating enzyme USP2a has shown oncogenic properties in many cancer types by impairing ubiquitination of FASN, MDM2, MDMX or Aurora A. Aberrant expression of USP2a has been linked to progression of human tumors, particularly prostate cancer. However, little is known about the role of USP2a or its mechanism of action in bladder cancer. Here, we provide evidence that USP2a is an oncoprotein in bladder cancer cells. Enforced expression of USP2a caused enhanced proliferation, invasion, migration and resistance to several chemotherapeutic reagents, while USP2a loss resulted in slower proliferation, greater chemosensitivity and reduced migratory/invasive capability compared with control cells. USP2a, but not a catalytically inactive mutant, enhanced proliferation in immortalized TRT-HU1 normal human bladder epithelial cells. USP2a bound to cyclin A1 and prevented cyclin A1 ubiquitination, leading to accumulation of cyclin A1 by a block in degradation. Enforced expression of wild type USP2a, but not an inactive USP2a mutant, resulted in cyclin A1 accumulation and increased cell proliferation. We conclude that USP2a impairs ubiquitination and stabilizes an important cell cycle regulator, cyclin A1, raising the possibility of USP2a targeting as a therapeutic strategy against bladder tumors in combination with chemotherapy. Cell Cycle 1123
Cell Cycle 11:6, 1123-1130; March 15, 2012; © 2012 Landes Bioscience
*Correspondence to: Jayoung Kim; Email:
Submitted: 11/28/11; Revised: 01/30/12; Accepted: 01/31/12
Bladder cancer is the second most common genitourinary malig-
nancy in the United States and Europe, with 69,250 new cases
and 14,990 deaths from bladder cancer in the US estimated in
2011.1,2 Bladder cancers present as transitional cell carcinoma
(TCC), squamous cell carcinoma, adenocarcinoma and a few
other rare subtypes. TCC is the most predominant histological
type, which includes papillary, well- or moderately differentiated
non-muscle invasive bladder cancer (NMIBC).3 Cancer recur-
rence occurs in up to 70% of patients with NMIBC, and 20–30%
of these patients show progression to muscle invasive bladder
cancer and poor outcomes.4,5 Compared with other cancer types,
bladder cancer is characteristically sensitive to cisplatin-based
The deubiquitinating enzyme USP2a has shown oncogenic properties in many cancer types by impairing ubiquitination
of FASN, MDM2, MDMX or Aurora A. Aberrant expression of USP2a has been linked to progression of human tumors,
particularly prostate cancer. However, little is known about the role of USP2a or its mechanism of action in bladder
cancer. Here, we provide evidence that USP2a is an oncoprotein in bladder cancer cells. Enforced expression of USP2a
caused enhanced proliferation, invasion, migration and resistance to several chemotherapeutic reagents, while USP2a
loss resulted in slower proliferation, greater chemosensitivity and reduced migratory/invasive capability compared with
control cells. USP2a, but not a catalytically inactive mutant, enhanced proliferation in immortalized TRT-HU1 normal
human bladder epithelial cells. USP2a boun d to cyclin A1 and prevented cyclin A1 ubiquitination, leading to accumulation
of cyclin A1 by a block in degradation. Enforced expressio n of wild-type USP2a, but not an inactive USP2a mutant, resulted
in cyclin A1 accumulation and increased cell proliferation. We conclude that USP2a impairs ubiquitination and stabilizes
an important cell cycle regulator, cyclin A1, raising the possibility of USP2a targeting as a therapeutic strategy against
bladder tumors in combination with chemotherapy.
The ubiquitin-specic protease USP2a enhances
tumor progression by targeting cyclin A1
in bladder cancer
Jayoung Kim,1-3,* Wun-Jae Kim,4,5 Zhiqian Liu,6 Massimo F. Loda6-8 and Michael R. Freeman1-3
1Division of Can cer Biology and Ther apeutics; Depar tments of Surgery and B iomedical Sciences; S amuel Oschin Comprehe nsive Cancer Institute; Ced ars-Sinai Medical Center ;
Los Angeles , CA USA; 2The Urologica l Diseases Research Cente r; Children’s Hospital Bos ton; Boston, MA USA; 3Depar tments of Surger y and Biological Chemi stry and Molecula r
Pharmacolo gy; Harvard Medic al School; Boston, MA USA ; 4Department of Urol ogy; 5BK21 Chungbuk Biom edical Science Center; S chool of Medicine; Chun gbuk National
University; Cheongju, Chungbuk South Korea; 6Departmen t of Medical Oncology ; 7Center for Molecular On cologic Pathology; D ana Farber Cancer Institute;
8Depart ment of Pathology; Bri gham and Women’s Hospital; Har vard Medical Scho ol; Boston, MA USA
Key words: USP2a, cyclin A1, bladder cancer, cisplatin resistance, deubiquitination
Abbreviations: 17-AAG, 17-(allylamino)-17-demethoxygeldanamycin; CHX, cycloheximide; c-PARP, cleaved form of poly
(ADP-ribose) polymerase; DMEM, dulbecco’s modified eagle medium; DUBs, deubiquitinating enzymes; EGFR, epidermal
growth factor receptor; FASN, fatty acid synthase; FBS, fetal bovine serum; HB-EGF, heparin-binding EGF-like growth
factor; MDM2, murine double minute 2; MMPs, matrix metallopeptidase; NMIBC, non-muscle invasive bladder cancer;
RNAi, RNA interference; RTKs, receptor tyrosine kinases; siRNAs, small interfering RNAs; TCC, transitional cell carcinoma;
TPA, 12- O-tetradecanoylphorbol-13-acetate; TUNEL, deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling;
USP2a, ubiquitin-specific protease 2a; VEGF, vascular endothelial growth factor
chemotherapy, which is a standard treatment.6 Molecular mech-
anisms and clinical impact of combination therapy with other
drugs based on targeted signaling strategies (e.g., Gefitinib,
Trastuzumab, Cetuximab, Everolimus and Sorafenib) or angio-
genesis inhibition (e.g., Sunitinib and Bevacizumab) are under
investigation.7-11 Recent functional genetic screens identified sev-
eral cisplatin response modulators, suggesting molecular mecha-
nisms as well as several therapeutic candidates, including DNA
damage response-related genes.12 For example, the hsp90 inhibi-
tor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) selec-
tively sensitized bladder cancers to cisplatin in a mouse model.13
Ubiquitination and protein degradation mechanisms have
been reported to participitate in regulation of oncoproteins
and tumor suppressors, and this is currently an active area of
1124 Cell Cycle Volume 11 Issue 6
misfolding and enhances the antitumor effects
of the proteasome inhibitor bortezomib.23
De-ubiquitinating enzymes (DUBs) such as
AMSH24 and UBPY25,2 6 have been implicated
in the increased level of oncoproteins, such as
epidermal growth factor receptor (EGFR). One
of the DUBs and a ubiquitin-specific prote-
ase 2 (USP2) isoform, USP2a, has been dem-
onstrated to regulate expression levels of fatty
acid synthase (FASN), murine double minute 2
(MDM2), MDMX and Aurora A,2 7-31 all well-
known oncoproteins relevant to tumor progres-
sion in many cancer types, including bladder
cancer.32 ,33 One USP2a target, FASN, plays a
key role in lipid metabolism by synthesizing
palmitate from acetyl-CoA and malonyl-CoA.
In bladder cancer, FASN expression level was
described as a predictor of recurrence and of
tumor aggressiveness when combined with
Ki-67 expression index.34 A recently identified
USP2a substrate,30 Aurora A, is involved in reg-
ulation of centrosomal and microtubule activity
and control of chromosome segregation;35 thus,
Aurora A overexpression leads to impaired chro-
mosomal stability and aneuploidy. Aurora A, an
oncogenic serine/threonine kinase, is overex-
pressed in bladder cancer tissues from patients
as assessed by both RT-PCR and immunohisto-
chemical assays.36-40 Taken together, these data
suggest a role for USP2a as a mediator of blad-
der cancer progression.
Here, we show that USP2a associates with
and stabilizes an important cell cycle regula-
tor, cyclin A1, resulting in enhanced oncogenic
characteristics expressed by bladder cancer cells.
Our findings suggest that impaired ubiquitina-
tion and protein degradation in bladder tumor
cells can be therapeutically targeted.
Aberrant expression of USP2a enhances T24 cancer cell prolif-
eration and invasion. To determine whether the deubiquitinase
USP2a plays a role in bladder cancer progression, we attempted to
analyze the relationship between USP2a expression and oncogen-
esis. T24 human bladder cancer cells were transiently transfected
with a wild-type USP2a construct (USP2aWT) or vector plasmid
only (Vec). In experiments where transfected T24 bladder cancer
cells were stimulated with growth medium after 16 h serum star-
vation, we observed that USP2aWT bladder cancer cells grew faster
than control cells (Fig. 1A). To independently assess the impact
of USP2aWT on migration, a migration assay using gelatin-coated
chambers was performed. Enforced USP2aWT enhanced migra-
tion about 2-fold, compared with control (Fig. 1B). The total
cell mass of USP2aWT cultures reached about 2-fold of control
cells 1 d after stimulation (p < 0.05). Increased cell migration by
investigation. Much effort has been made to explore the roles
of ubiquitin protein E3 ligases in mediating ubiquitination of
cancer-relevant proteins, including receptor tyrosine kinases
(RTKs).14,15 Deubiquitinating enzymes (DUBs) de-conjugate
ubiquitin from substrates and from ubiquitin chains, thus nega-
tively regulating ubiquitination.16-20
Although studies of ubiquitination and deubiquitination of
proteins remain challenging due to the limited methods that
are currently applied,21 recently, several reports have described
chemotherapeutic strategies to avoid chemoresistance based on
mechanisms of uniqitination and deubiquitination. Expression
of the pro-survival protein, Mcl-1, which is regulated by ubiq-
uitination/deubiquitination, regulates chemoresistance of cells
deficient in the tumor suppressor Fbw7.22 Neznanov et al.
reported that proteotoxic stress-targeted therapy induces protein
Figure 1. Enforced USP2aWT expression enhanced tumorigenesis in T24 human bladder
cancer cells. (A) Proliferation rate in T24 bladder cancer cells transiently engineered to
express USP2aWT, T24-USP2aWT and Vec (vector only), T24-Vec. Cells were serum starved
for 16 h before changing into growth medium. At the indicated time points, cells were
xed and stained with crystal violet solution. (B) Cell migration capabilit y of T24-USP2aWT
and T24-Vec cells were analyzed using gelatin-coated invasion chamber kit (company) as
described in the Materials and Methods section. (C) Migration was compared in T24-US-
P2aWT and T24-Vec cells by a wound-healing assay. (D) T24-USP2aWT and T24-Vec cells were
seeded at low density and incubated for colony formation for 7 d. After xing and staining
cells, number of colonies was counted. Representative images were shown. Asterisks in this
gure indicate p < 0.05. Cell Cycle 1125
As in T24 bladder cancer cells, enforced expression of USP2aWT
substantially enhanced colony formation in the immortalized
bladder epithelial cell line TRT-HU143 (Fig. 4A). However, inac-
tive USP2a, USP2aMUT, did not show this pro-proliferative effect,
suggesting that catalytic activity is important for USP2a function
(Fig. 4B).
Association of USP2a and cyclin A1 attenuated cyclin A1
expression. The above experimental results suggest that enforced
expression of USP2a in T24 cells, probably through suppression
of ubiquitination of critical protein(s), leads to a more aggres-
sive tumor cell phenotype. Because functional analysis of USP2a
suggested that this deubiquitinase might control proliferation
through regulation of cell cycle transition, we questioned whether
enforced expression or loss of USP2a expression alters expression
of cell cycle-related proteins. Blotting of T24-USP2aWT cell lysates
with antibodies against cyclin A1 or cyclin D1 suggested that
expression level of cell cycle regulator(s) positively correlates with
USP2a level (Fig. 5A and B). USP2aWT
-overexpressing T24 cells
showed an increased expression of cell cycle regulators (Fig. 4A),
consistent with the observed enhanced proliferation (Fig. 1A).
enforced expression of USP2aWT was also observed in a wound-
healing assay (Fig. 1C). Colony formation capability as mea-
sured by in vitro clonogenic assay reports oncogenic properties.
Approximately 10-fold more colonies (diameter >100 μm) were
formed in USP2aWT bladder cancer cells, compared with control
cells (Fig. 1D).
These results suggest that human T24 cells expressing
USP2aWT acquire a more aggressive phenotype, probably by
activation of oncogenic signals. To determine the mechanism
whereby this deubiquitinase would alter biological responses to
growth stimulating factors, we assessed the activation status of
several signal transduction pathways. Erk/MAPK is activated by
the autocrine regulator of human bladder epithelial cells, heparin-
binding EGF-like growth factor (HB-EGF).41 HB-EGF has been
reported to promote cell growth and induce phosphorylation of
essential signaling pathways, including Erk/MAPK, Akt1 and
p70S6K but not p38MAPK in bladder cells.42 The extent of Erk/
MAPK phosphorylation by HB-EGF treatment in USP2aWT cells
was compared with control cells (Vec). Cells expressing USP2aWT
responded more rapidly to HB-EGF stimulation 5 min after
treatment and Erk/MAPK phosphorylation was sustained until
60 min, while control cells exhibited transient Erk/MAPK phos-
phorylation (Fig. 2A). In order to assess the effect of USP2aWT
on sensitivity to cisplatin, we used a clinically available chemo-
therapeutic drug that causes crosslinking of DNA and apoptosis
induction; USP2aWT T24 cells were incubated in 10 μM cispl-
atin-containing medium for 24 h and compared with control
cells. Treating the USP2aWT cells (and control cells) with 10 μM
12-O-tetradecanoylphorbol-13-acetate (TPA), 5 μM nocodazole
(G2/M blocker) or 10 μM cycloheximide (CHX, protein synthe-
sis inhibitor) for 24 h was followed by measurement of apoptotic
markers to determine sensitivity to apoptosis inducers. TUNEL
assay (Fig. 2B) and western blot analysis detecting the cleaved
form of PARP (c-PARP) revealed that USP2aWT cells are more
resistant to apoptosis induction by cisplatin, as well as other
apoptosis inducers (Fig. 2C).
USP2a loss attenuates proliferation and migration in T24
human bladder cancer cells. Given the finding that USP2a may
enhance oncogenic properties of bladder cancer, we performed
similar experiments with USP2a-deficient T24 cells (shUSP2a)
to determine proliferation, migration and apoptosis resistance
and compared with control cells (shCtrl). USP2a-deficient
bladder cancer cells were generated by stable transfection with
shRNA directed against USP2a. Western blot analysis confirmed
reduced expression of USP2a protein in the USP2A-silenced
cell lines, T24-shUSP2a-c1 and T24-shUSP2a-c2, compared
with T24-shCtrl cells (Fig. 3A). Cell proliferation and migra-
tion were significantly suppressed by USP2a knockdown. These
biological effects were consistent with the relative expression
level of USP2a. The T24-shUSP2a-c2 cell line exhibited slower
proliferation and migration, with a greater decrease of USP2a
expression (Fig. 3B and C). Compared with control cells, USP2a-
knockdown cells invaded more slowly through matrigel-coated
filters (Fig. 3D). We also observed that USP2a deficiency made
T24 cells more sensitive to treatment with apoptotic reagents,
including cisplatin (Fig. 3E).
Figure 2. USP2aWT altered activation of signal transduction. (A) Erk /
MAPK phosphorylation in USP2aWT expressing T24 cells treated with
recombinant HB-EGF (100 ng/ml). Protein from whole-cell lysates at
the indicated times were blotted with the Abs against p-Erk /MAPK,
Erk/MAPK and β-actin. (B) TUNEL assay showed less apoptotic cells in
USP2a-expressing cells compared with control. (C) Levels of cleaved
PARP (c-PARP) were detected in whole lysates from T24-USP2aWT and
T24-Vec cells incubated in serum free medium (SF), TPA, nocodazole,
cisplatin or CHX for 24 h.
1126 Cell Cycle Volume 11 Issue 6
with anti-cyclin A1 antibody and immunob-
lotting with anti-USP2a antibody revealed
that cyclin A1 physically interacts with
USP2a (Fig. 5C). The potential influence of
USP2aWT on cyclin A1 stability was assessed
by inhibiting protein synthesis with CHX.
In the setting of enforced USP2aWT
, cyclin
A1 levels were sustained in the presence of
CHX. Enforced expression of USP2aWT
attenuated cyclin A1 degradation (Fig. 5D).
We conclude that USP2a regulates cyclin A1
at the posttranslational level.
In this study, we show that increased
cyclin A1 in human bladder cancer cells may
be regulated by activity of the USP2a deu-
biquitinase. Our data suggest that USP2a is
a pro-proliferative and pro-migratory protein
relevant to features of clinically aggressive
bladder cancer, consistent with observa-
tions in other cancer types. USP2a removes
ubiquitin from protein chains, leading to
alterations in the rate of ubiquitination-
induced protein degradation. Since ubiqui-
tination and resulting protein degradation
is a dynamic and important mechanism in
the tight control of protein expression in
cells, a balance between ubiqutination and
deubiquitination processes control protein
degradation rates.16,18,20,51 In addition to the
known function of USP2a as a deubiquit-
ing enzyme, which is critical for regulated
expression of FASN, MDM2, MDMX or
Aurora A,28 -30,32 we conclude that USP2a is
involved in cyclin A1 degradation, at least in
part by a deubiquitination mechanism.
Cyclin A1, a context-dependent regulator
of the G1/S- or G2/M-phase transitions,45, 46
binds to cyclin dependent kinases 1/2
(CDK1/2) and activates downstream targets,
including the retinoblastoma protein (Rb), E2F transcription
factors and B-MYB (a cell cycle-associated transcription factor),
leading to cell cycle transition.52-55 Cyclin A1-targeting experi-
ments in the mouse showed that male cyclin A1-/- mice exhibit
impaired spermatogenesis, and males are sterile, suggesting that
cyclin A1 plays a critical role in fertility through regulation of the
cell cycle.56 Transgenic cyclin A1-overexpressing mice develop
abnormal myelopoiesis and acute myeloid leukemia.50 Cyclin
A1 may be involved in pathogenesis of primary and metastatic
cancer, because elevated cyclin A1 expression has been observed
in various types of solid tumors, including testicular, ovarian,
breast and prostate cancers.47,49,50 Recent findings have suggested
the underlying regulatory mechanism whereby cyclin A1 con-
tributes to prostate cancer invasion. Cyclin A1 associates with
USP2a knockdown by RNA interference (RNAi) using a pool of
four siRNA oligos targeted to USP2a reversed the effect caused
by USP2a overexpression (Fig. 5B). Previously, an important cell
cycle regulator, cyclin D1, was identified as a substrate of USP2,
which consists of two splice variants, USP2a and USP2b.44 In this
study, we assessed whether USP2a regulates cyclin A1 by examin-
ing whether cyclin A1 directly binds to USP2a, thus resulting in
protein accumulation. The cell cycle-regulatory protein cyclin A1
is required for G2/M progression during meitotic division of
male germ cells45 and cell cycle transition in somatic cells.46 As
a proto-oncogene, levels of cyclin A1 expression correlate with
patient outcome in various tumor types, including prostate
cancer, breast cancer, acute myeloid leukemia and acute lym-
phoblastic leukemia.4 7-50 Further analysis using co-precipitation
Figure 3. USP2a knockdown by shRNA suppressed oncogenesis. (A) Verication of USP2a gene
silencing by stable transfec tion of T24 cells using two kinds of shUSP2a. USP2a expression level
in two engineered cells, shUSP2a-c1 and shUSP2a-c2, was analyzed by western blotting. Con-
trol shRNA (shCtrl) was used as negative control. (B) Proliferation of t wo USP2a-knockdown cell
lines was compared with control cells. (C) Wound-healing assay to assess migrator y capability
of USP2a loss shUSP2a-c2 and control cells. (D) Quantitative analysis using invasion chamber kit
of cell migration in U SP2a loss cells. (E) USP2a-knockdown cells are more susceptible to apop-
totic inducers including a chemotherapeutic drug, cisplatin. Stably engineered cell lines were
treated with the indicated reagents (see the Materials and Methods), and protein extracts were
applied for western blotting with anti-c-PARP antibody. Asterisks indicate p < 0.05. Cell Cycle 1127
Cell culture and cell transfection. T24 cells (ATCC) were
cultured in DMEM supplemented with 10% FBS, 100 units/mL
penicillin and 100 μg/mL streptomycin at 37°C in a humidified
incubator with 5% CO2. Immortalized human normal bladder
epithelial TRT-HU1 cells were recently described in reference 43.
For transient transfection, cells were grown to 80% confluence,
and transfection was performed with Lipofectamine 2000 using
1 μg plasmid DNA. Wild-type USP2a (USP2aWT) or catalytically
inactive USP2a (USP2aMUT) were cloned into pEGFP and used
for transfection. Catalytic Cys was mutated to Ala in USP2aMUT
(C276A). For silencing, cells were transfected with 80 nmol/L of
d(TT)]. OFF-TARGET siRNAs were used as control. To gen-
erate stable knockout cell lines, T24 cells were transfected with
shVec or shUSP2a, followed by selection with G418 for 6 weeks.
Two clones showing maximum knockdown, shUSP2a-c1 and
shUSP2a-c2, were chosen and used for the experiments described.
Western blot analysis. Whole-cell lysates were prepared with
lysis buffer (1% Nonidet P-40, 50 mM Tris pH 7.4, 10 mM NaCl,
1 mM NaF, 5 mM MgCl2, 0.1 mM EDTA, 1 mM PMSF, prote-
ase inhibitor cocktail) and centrifuged at 12,000x g for 15 min.
Protein concentrations were determined by Micro BCA assay
according to the manufacturer’s protocol. Proteins were separated
by SDS-PAGE and transferred onto nitrocellulose membranes for
immunoblotting analysis. To standardize protein levels, the blots
were reprobed with antibodies against β-actin.
Cell proliferation assay. T24 human bladder cancer cells
were plated onto 6-well culture plates at a density of 1 x 103 cells
per well in standard DMEM growth medium with 10% FBS.
When cells grew to ~80% confluence, they were transfected with
androgen receptor (AR) and regulates
the expression of matrix metalloproteases
(MMPs) and vascular endothelial growth
factor (VEGF), leading to a more invasive
and metastatic phenotype.4 7,5 7 However,
pathoclinical studies or functional analy-
ses to understand the expression status or
the precise role of cyclin A1 in primary and
metastatic bladder cancers are limited. Our
results suggest that further investigation
into the mechanisms whereby cyclin A1
contributes to bladder cancer development
and progression to advanced disease are
Our findings show that aberrant expres-
sion of USP2a significantly upregulated
the proliferation and migration capability
of T24 cells (Fig. 1), while USP2a knock-
down suppressed those effects compared
with controls (Fig. 3). These findings sug-
gest the possibility of USP2a-suppressing
reagents as an approach to cancer therapy.
A recently reported synthetic compound
with antioxidant activity, HO-3867,
exhibited anticancer effects on many can-
cer types, including breast, colon, head and
neck, liver, lung, ovarian and prostate cancers,58 by significant
downregulation of USP2a, causing apoptosis. Knockdown of
USP2a enhanced the cisplatin sensitivity in testicular embryonal
carcinoma cells.29 This finding is suggestive of the possibility that
clinical application of USP2a inhibitors to the cisplatin-resistant
patient population may result in clinical benefit.
In summary, the present study provides evidence suggesting
a role for the USP2a deubiquitinase in proliferation and migra-
tion of human bladder cancer cells. We showed that USP2a tar-
gets a cell cycle regulator, cyclin A1, thereby regulating cyclin
A1 expression level, cell growth and apoptosis (Fig. 6). These
findings provide a potential strategy in which the USP2a-cyclin
A1 pathway might be pharmacologically manipulated as a means
of bladder cancer therapy.
Experimental Procedures
Reagents. Dialyzed fetal bovine serum (FBS), Dulbecco’s
Modified Ea gle Medium (DMEM) and Lipofectamine 2000 were
from Invitrogen. Micro BCA protein assay kit was obtained from
Pierce. Coomassie Blue R-250 staining solution and destaining
solution were from Bio-Rad. Small interfering RNAs (siRNAs)
aga inst USP2a a nd OFF-TARGET controls were from Dha rmacon.
Protease inhibitor cocktail tablets were from Roche Diagnostics.
The following antibodies were used: antibodies to USP2a
(Millennium Pharmaceuticals, Inc., and/or ABGENT), cyclin
D1 (Biosource), cyclin A1 (Abcam Inc.), ubiquitin (Calbiochem),
FASN (Santa Cruz Biotechnology), EGFR (Invitrogen),
β-actin (Sigma-Aldrich). All other reagents were highest quality
and were obtained from Sigma-Aldrich or Promega.
Figure 4. USP2a enhanced the proliferation rate of immortalized human bladder epithelial cells
(TRT-HU1) via catalytic activity. (A) A greater colonization by USP2a overexpression in TRT-HU1
cells. Cells were seeded at low density (102/dish) and incubated for 14 d. Colonies were counted
after crystal violet staining. (B) Eect of a mutant form of USP2a on proliferation in TRT-HU1 cells.
Cell proliferation was evaluated using crystal violet.
1128 Cell Cycle Volume 11 Issue 6
expression constructs or siRNAs. Three days after transfection,
cell proliferation rate was determined by crystal violet staining.59
Absorbance was measured at 570 nm.
Migration and wound-healing assay. Matrigel-coated inserts
(Millipore Corp.) were rehydrated before seeding T24 cells at
3 x 105 cells/ml on the upper surface of inserts. Sixteen h after
seeding, non-migratory cells in interior surface of inserts were
removed. Invaded cells on the bottom surface of the inserts were
stained with the cell staining solution and counted. Eluates with
10% acetic acid based extraction solution were transferred to
96-well microtiter plates and absorbance read at 560 nm using a
FLUOstar plate reader. Wound-healing assays were performed in
6-well plates after cell density reached confluence. After scratch-
ing cells with a sharp tip, the plates were returned to the tissue
culture incubator for 16 h before fixation and imaging.
Colony formation assay. Transfected cells were seeded in
150 mm plate at 1 x 102/well. Cells were fixed and stained with
crystal violet reagents for counting at 7 d.
Figure 5. USP2a stabilized cyclin A1. (A) USP2aWT expressing T24
cells expressed higher level of cyclin D1 and cyclin A1. (B) USP2a-
targeted oligos, siUSP2a, reversed an increase of cyclin D1 and cyclin
A1. (C)Physical interaction of USP2a and cyclin A1. Whole-cell lysates
(200 μg) from T24 cells were applied to immunoprecipitation (IP) with
anti-cyclin A1 antibody, and IP product was blotted with anti-USP2a an-
tibody (~70kDa). The reciprocal co-immunoprecipitation assay was not
applied due to no dierence between cyclin A1 and IgG (50–55 kDa),
although the IP itself was working and we could detect EGFR and FASN
co-immunoprecipitated with USP2a. (D) Cyclin A1 degradation was
measured in absence or presence of 10 μg/ml CHX in the T24-USP2aWT
and T24-Vec cells.
Apoptosis analysis. Apoptosis was determined by two inde-
pendent methods, the terminal deoxynucleotidyltransferase-
mediated dUTP-biotin nick end labeling (TUNEL) assay and
western blotting with anti-c-PARP antibody. For TUNEL assay
(Roche Applied Science), reaction mixture or negative con-
trol solution was applied to each slide after cell fixation using
4% paraformaldehyde. Detection of apoptotic cells and counter-
staining with propidium iodide (PI) were performed according to
the instructions provided by the manufacturer. TUNEL positiv-
ity was counted and analyzed under an Axioplan 2 microscope
(Carl Zeiss MicroImaging, Inc).
Co-immunoprecipitation. Whole-cell lysates from T24 cells
were prepared with lysis buffer containing 50 mM HEPES pH 7.3,
10% glycerol, 1% Triton X-100, 0.5 mM Na3VO4, protease inhib-
itor tablet (Roche), 10 mM NEM. Lysate was then incubated with
protein A/G-Sepharose beads pre-loaded with antibodies against
cyclin A1 or USP2a for 4 h in a cold room. Immunoprecipitates
were washed twice with lysis buffer and twice with lysis buffer
containing 500 mM NaCl. The precipitates were resuspended in
2x SDS loading buffer for western blot analysis.
Cyclin A1 degradation. T24 cells transiently transfected with
an USP2aWT (or Vec) were incubated with or without 10 μg/ml
cycloheximide (CHX). After treatment for 15, 30 or 45 min,
whole-cell lysates were prepared and applied for western blot with
anti-cyclin A1 antibody.
Statistical analysis. Statistical analysis was performed using
data from at least 3 independent experiments and at least 3
samples per experiment. Data are shown as mean ± standard
deviation (SD) and statistical significance of differences between
means was assessed by two-tailed Student t-test, and p < 0.05 was
considered statistically significant.
Author Contributions
J. K. and M.R.F. conceived and designed experiments and wrote
the paper. W.K., Z.L. and M.F.L. contributed reagents, materi-
als, expertise related to bladder cancer and USP2a and partici-
pated data analysis. J.K., W.K., M.F.L. and M.R.F. interpreted
data as well as helped to prepare and revise the manuscript.
Figure 6. Diagram showing potential USP2a signal network during
bladder cancer progression. Cell Cycle 1129
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
We thank Dr. Leland W. Chung (Cedars-Sinai Medical Center)
for critical review of the manuscript and helpful advice. This
work was supported by the following funding sources: NIH
grants R01 DK087806, R01 CA143777 and P50 DK65298
(to M.R.F.); the Fishbein Family IC Research Foundation/
Interstitial Cystitis Association (ICA), Pilot Research Program/
ICA, New York Academy of Medicine and Children’s Hospital
Boston Faculty Development (to J.K.). J.K. is an American
Urological Association Foundation Research Scholar and a
Harvard Medical School Eleanor and Miles Shore Scholar.
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    • "USP2a, as mentioned above, has been linked to different types of cancer. In bladder cancer cells, USP2a was found to deubiquitylate and to stabilize the cell cycle regulator, Cyclin A1, controlling proliferation of these cells (Kim et al., 2012). Taken together, ∼15 (out of ∼90) DUBs have been directly linked to molecular processes of the cell cycle (Figure 1). "
    [Show abstract] [Hide abstract] ABSTRACT: Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.
    Full-text · Article · Jul 2016
    • "The function of Cyclin A1 in normal and malignant somatic cells is only partially understood and might depend on the expression level, differentiation grade, and tissue of origin. Cyclin A1 expression enhances G1/S transition in somatic cells and is associated with enhanced proliferation and invasiveness in cancers of the breast, prostate, urothelium, and thyroid2829303132. At the same time, the induction of apoptosis by both intrinsic and extrinsic pathways increases the Cyclin A1 protein level by both p53-mediated transcription and posttranslational modification. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Cyclin A1 is essential for male gametopoiesis. In acute myeloid leukemia, it acts as a leukemia-associated antigen. Cyclin A1 expression has been reported in several epithelial malignancies, including testicular, endometrial, and epithelial ovarian cancer (EOC). We analyzed Cyclin A1 expression in EOC and its correlation with clinical features to evaluate Cyclin A1 as a T-cell target in EOC. Methods: Cyclin A1 mRNA expression in EOC and healthy tissues was quantified by microarray analysis and quantitative real-time PCR (qRT-PCR). Protein expression in clinical samples was assessed by immunohistochemistry (IHC) and was correlated to clinical features. Results: Cyclin A1 protein was homogeneously expressed in 43 of 62 grade 3 tumor samples and in 1 of 10 grade 2 specimens (p < 0.001). Survival analysis showed longer time to progression (TTP) among patients with at least moderate Cyclin A1 expression (univariate: p = 0.018, multivariate: p = 0.035). FIGO stage, grading, age, macroscopic residual tumor after debulking, and peritoneal carcinomatosis / distant metastasis had no impact on TTP or overall survival (OS). Conclusion: Cyclin A1 is highly expressed in most EOCs. The mechanism behind the prolonged TTP in patients with high Cyclin A1 expression warrants further investigation. The frequent, selectively high expression of Cyclin A1 in EOC makes it a promising target for T-cell therapies.
    Full-text · Article · Oct 2015
    • "These proteins are deregulated in many human cancers and exert their oncogenic activity predominantly by inhibiting the p53 tumor suppressor. Other USP2 targets involved in tumorigenesis are Aurora-A [5], Cyclin D1 [6], Cyclin A1 [7] and EGFR (epidermal growth factor receptor) [8] . These findings are in line with the result that USP2-1 is overexpressed in 44% of all prostate carcinomas [9] and contributes to resistance against treatment with Cisplatin in tumor cells [3, 10, 11] . "
    [Show abstract] [Hide abstract] ABSTRACT: The human deubiquitinating enzyme ubiquitin-specific protease 2 (USP2) regulates multiple cellular pathways, including cell proliferation and apoptosis. As a result of alternative splicing four USP2 isoenzymes are expressed in human cells of which all contain a weak peroxisome targeting signal of type 1 (PTS1) at their C-termini. Here, we systematically analyzed apoptotic effects induced by overexpression and intracellular localization for each isoform. All isoforms exhibit proapoptotic activity and are post-translationally imported into the matrix of peroxisomes in a PEX5-dependent manner. However, a significant fraction of the USP2 pool resides in the cytosol due to a weaker PTS1 and thus low affinity to the PTS receptor PEX5. Blocking of peroxisomal import did not interfere with the proapoptotic activity of USP2, suggesting that the enzyme performs its critical function outside of this compartment. Instead, increase of the efficiency of USP2 import into peroxisomes either by optimization of its peroxisomal targeting signal or by overexpression of the PTS1 receptor did result in a reduction of the apoptotic rate of transfected cells. Our studies suggest that peroxisomal import of USP2 provides additional control over the proapoptotic activity of cytosolic USP2 by spatial separation of the deubiquitinating enzymes from their interaction partners in the cytosol and nucleus.
    Full-text · Article · Oct 2015
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