Research Article
Dual-Specificity Phosphatase 1 Ubiquitination in Extracellular
Signal-Regulated Kinase–Mediated Control of Growth
in Human Hepatocellular Carcinoma
Diego F. Calvisi, Federico Pinna, Floriana Meloni, Sara Ladu, Rossella Pellegrino, Marcella Sini,
Lucia Daino, Maria M. Simile, Maria R. De Miglio, Patrizia Virdis, Maddalena Frau,
Maria L. Tomasi, Maria A. Seddaiu, Maria R. Muroni,
Francesco Feo, and Rosa M. Pascale
Department of Biomedical Sciences, Division of Experimental Pathology and Oncology, University of Sassari, 07100 Sassari, Italy
Abstract
Sustained activation of extracellular signal-regulated kinase
(ERK) has been detected previously in numerous tumors in the
absence of RAS-activating mutations. However, the molecular
mechanisms responsible for ERK-unrestrained activity independent of RAS mutations remain unknown. Here, we evaluated the effects of the functional interactions of ERK proteins
with dual-specificity phosphatase 1 (DUSP1), a specific inhibitor of ERK, and S-phase kinase-associated protein 2 (SKP2)/
CDC28 protein kinase 1b (CKS1) ubiquitin ligase complex in
human hepatocellular carcinoma (HCC). Levels of DUSP1, as
assessed by real-time reverse transcription–PCR and Western
blot analysis, were significantly higher in tumors with better
prognosis (as defined by the length of patients’ survival) when
compared with both normal and nontumorous surrounding
livers, whereas DUSP1 protein expression sharply declined
in all HCC with poorer prognosis. In the latter HCC subtype,
DUSP1 inactivation was due to either ERK/SKP2/CKS1dependent ubiquitination or promoter hypermethylation
associated with loss of heterozygosity at the DUSP1 locus.
Noticeably, expression levels of DUSP1 inversely correlated
with those of activated ERK, as well as with proliferation index
and microvessel density, and directly with apoptosis and
survival rate. Subsequent functional studies revealed that
DUSP1 reactivation led to suppression of ERK, CKS1, and
SKP2 activity, inhibition of proliferation and induction of
apoptosis in human hepatoma cell lines. Taken together, the
present data indicate that ERK achieves unrestrained activity
during HCC progression by triggering ubiquitin-mediated
proteolysis of its specific inhibitor DUSP1. Thus, DUSP1 may
represent a valuable prognostic marker and ERK, CKS1, or
SKP2 potential therapeutic targets for human HCC. [Cancer
Res 2008;68(11):4192–200]
Introduction
Human hepatocellular carcinoma (HCC) is one of the most
common and deadliest tumors worldwide (1, 2). HCC is endemic
in certain areas of Southeast Asia and Southern Africa, and its
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Francesco Feo, Dipartimento di Scienze Biomediche,
Sezione di Patologia Sperimentale e Oncologia, Università di Sassari, Via P. Manzella 4,
07100 Sassari, Italy. Phone: 39-079-228307; Fax: 39-079-228485; E-mail: [email protected].
I2008 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-07-6157
Cancer Res 2008; 68: (11). June 1, 2008
incidence is rapidly rising in Western countries (1, 2). Only few
patients are amenable to surgery due to the late diagnosis of HCC,
and alternative treatments do not significantly improve the prognosis of patients with unresectable HCC (1, 2). Thus, the investigation of HCC molecular pathogenesis is needed to identify new
targets for its early diagnosis, chemoprevention, and treatment.
Recent studies showed c-myc, cyclin D1, cyclin A, and E2f1 upregulation, rise in cyclin D1-Cdk4 and E2f1-Dp1 complexes, and
pRb hyperphosphorylation in neoplastic liver lesions of c-Myc/
Tgf-a transgenic mice (3) and Fisher 344 rats (4, 5) subjected to the
carcinogens according to the resistant hepatocyte protocol (6),
implying a deregulation of G1 and S phases in these lesions. Deregulation of these cell cycle components also occurs in human hepatocarcinogenesis (1–5, 7). A major player favoring G1-S transition
via induction of cyclin D1, CDK4, c-Myc, and pRB hyperphosphorylation is the RAS cascade, whose activation regulates numerous
signals involved in cell growth, survival, and migration (8). The
best-characterized RAS effector promoting cell cycle progression
is the mitogen-activated protein kinase (MAPK) pathway (9). In
this cascade, RAS induction triggers activation of RAF, MAPK
kinase kinase (MEK), and extracellular signal-regulated kinase
(ERK) proteins, leading to up-regulation of c-FOS, c-JUN, c-MYC,
and ETS targets (9). Sustained ERK activity is associated with
various types of tumors, including lung, ovary, colon, pancreas, and
kidney (9). This frequently depends on up-regulation of the RAS/
MEK cascade. However, constitutive ERK overexpression may
also occur independently of the RAS/MEK signaling (10, 11). Recent studies (12, 13) indicate that prolonged activation of ERK
promotes phosphorylation at the Ser296 residue of its inhibitor,
dual-specificity phosphatase 1 (DUSP1; also known as MAPK phosphatase-1). Phosphorylation of this specific residue renders the
DUSP1 protein susceptible to proteasomal degradation by two
substrate recognition proteins belonging to a large S-phase kinaseassociated protein (SKP)–cullin–F box ubiquitin ligase: the S-phase
kinase-associated protein 2 (SKP2) and CDC28 protein kinase b1
(CKS1) complex. Thus, accelerated degradation of DUSP1 may
further reinforce ERK activity and its cooperation with SKP2/CKS1
ubiquitin ligase. An additional mechanism leading to ERK upregulation could be ERK-mediated induction of SKP2/CKS1
ubiquitin ligase, which controls DUSP1 ubiquitination. This effect
could be at least partially attributed to induction by ERK of the
FOXM1 gene (14) which, in turn, up-regulates the SKP2/CKS1 ligase
(15). In contrast, transient activation of ERK leads to catalytic
activation of DUSP1 followed by inactivation of ERK (13). This body
of evidence indicates that DUSP1 feedback inhibits its activation
by ERK and that DUSP1 might be a crucial regulator of ERK activity
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DUSP1 Deregulation in Liver Cancer
in the cell. Although much effort has focused on the molecular
interactions modulating DUSP1 activity in mammalian cell lines,
little is known about the role of DUSP1 in carcinogenesis. DUSP1
inactivation is frequent in prostate and urothelial tumors (16, 17),
and recent observations indicate that immunohistochemical
positivity for DUSP1 in human HCC is associated with longer
patients’ survival (18). However, the interactions of DUSP1 with
ERK and the SKP2/CKS1 ligase and the mechanisms responsible
for DUSP1 inactivation in the liver and other tumors have not
been analyzed to date. Here, we evaluated the effects of these
interactions, at molecular and functional levels, in human HCC
subtypes with different survival times, in the attempt to evaluate
the role of DUSP1 in hepatocarcinogenesis and correlate the effects
of its molecular interactions with tumor growth and patients’
survival and identify new potential prognostic markers and therapeutic targets.
Materials and Methods
Tissue specimens, cell lines, and treatments. Six normal livers, 42
HCCs, and corresponding surrounding nontumorous livers were used.
Tumors were divided in 21 HCCs with poor prognosis (HCCP),
characterized by <3 y survival, and 21 HCCs with better prognosis (HCCB)
with >3 y survival after liver partial resection. Liver tissues were kindly
provided by Dr. Snorri S. Thorgeirsson (National Cancer Institute).
Institutional review board approval was obtained at participating hospitals
and NIH.
In vitro growing 7703, HuH7, SNU-182, and SNU-387 human HCC cell
lines were treated with 20 mmol/L UO126 (MEK inhibitor; EMD Chemicals,
Inc.) or small interfering RNA (siRNA) against SKP2, CKS1, or ERK2; 7703
cells with 10 Amol/L 5-aza-2-deoxycytidine (5-Aza-dC; Sigma-Aldrich Corp.);
and SNU-182 cells with 5 Amol/L Ro-31-8220 (DUSP1 inhibitor; EMD
Chemicals, Inc.) or siRNA against DUSP1. The cells were grown 72 and
144 h in the presence of 5-Aza-dC and 12 and 24 h with all other inhibitors
and siRNAs. siRNA experiments were done as reported (13, 19–21).
Transient transfection with either Ha-RAS cDNA (wild type), ERK2 (wild
type) in a pUSEamp plasmid (Millipore), or SKP2 cDNA in a pCMV6-XL
vector (OriGene Technologies) and stable transfection with ERK2 cDNA
(wild type; Millipore) were done on SNU-182 cells following manufacturers’
protocols. To determine the effect of proteasome-mediated proteolysis,
the proteasome inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN; 10 Amol/L)
and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132; 25 Amol/L) were
added in the final 3 h of the transient transfection experiment (13).
Proliferation and apoptotic indices. Proliferation index was determined by counting proliferating cell nuclear antigen–positive cells, as
reported (22). Apoptotic index was calculated by counting the apoptotic
figures on tumor sections stained with the ApoTag peroxidase in situ
apoptosis detection kit (Millipore) and expressed as percentage of the total
number of cells counted. Cultured cell viability and apoptosis were
determined by the WST-1 cell proliferation reagent and the cell death
detection Elisa Plus kit (Roche Diagnostics), respectively.
Evaluation of microvessel density. HCCs were stained with mouse
monoclonal anti-CD34 antibody (Vector Laboratories). Any brown-stained
endothelial cell or endothelial cell cluster was counted as one microvessel,
irrespective of the presence of a vessel lumen (23). Rare and small necrotic
areas were excluded from the analysis. The four highest microvessel density
(MVD) areas for each tumor were photographed at high power (200),
and the size of each area was standardized using the ImageJ software. MVD
was expressed as the percentage of the total CD34 stained spots per section
area (0.94 mm2).
Quantitative reverse transcription–PCR. Primers for DUSP1 and RNR18 genes were chosen with the assistance of the ‘‘Assay-on-Demand
Products’’ (Applied Biosystems). PCR reactions were done with 75 to 300 ng
of cDNA, using an ABI Prism 7000 Sequence Detection System and TaqMan
Universal PCR Master Mix (Applied Biosystems). Cycling conditions were
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10 min of denaturation at 95jC and 40 cycles at 95jC for 15 s and at 60jC
for 1 min. Quantitative values were calculated by using the PE Biosystems
Analysis software and expressed as N target (NT). NT = 2 DCt, wherein DCt
value of each sample was calculated by subtracting the average Ct value of
the target gene from the average Ct value of the RNR-18 gene.
Methylation-specific PCR, bisulfite sequencing, and microsatellite
analysis. High molecular weight DNA from human samples was isolated as
reported (24) and modified with the EZ DNA methylation kit (Zymo
Research). The CpGenome Universal Methylated DNA and CpG Universal
Unmethylated DNA (Chemicon International) were used as positive and
negative control for each reaction, respectively. Primers specific for
methylated and unmethylated DUSP1 promoter were designed for
methylation-specific PCR with the MethPrimer software (25). Results were
confirmed by genomic bisulfite sequencing using a set of primers kindly
provided by Dr. T. Visakorpi (University of Tampere). Loss of heterozygosity
(LOH) of the DUSP1 locus was investigated with D5S677, D5S498, and
WI-22759 primer pairs, as published (26). LOH was recorded when a 50%
or greater reduction in electrophoretic band intensity was detected with
silver nitrate staining (Silver Stain Plus, Bio-Rad). The complete list,
sequence, annealing temperature, and product size of primers are shown in
Supplementary Table S1.
Mutation analyses. Mutations at Ha-RAS, Ki-RAS, and N-RAS (codons
12 and 13), A-RAF (exons 10 and 13), B-RAF (exons 11 and 15), RAF-1 (exons
10 and 14), epidermal growth factor receptor (EGFR; exons 18–21), and
DUSP1 (catalytic domain) genes in normal liver, HCC and respective
surrounding nontumor liver tissues were assessed by direct DNA
sequencing, as previously described (27–30).
Western blots and immunoprecipitation analyses. Protein extracts
from liver tissues were evaluated by separating 100 Ag of total protein lysate
by 10% SDS-PAGE, as reported (4). This protein amount allowed quantitative evaluation even in the tissues with low expression levels, although
saturating conditions were sometimes reached in high expression tissues.
As a consequence, some differences in protein levels may be underestimated. Proteins were then transferred onto nitrocellulose membranes
and reacted with the antibodies listed in Supplementary Table S2. Hypoxiainducible factor-1a (HIF-1a) activation was assessed by determining the
HIF-1a/p300 complexes through immunoprecipitation (4) with the anti–
HIF-1a antibody and probing the membranes with the goat polyclonal antip300 antibody. The complexes of DUSP1 with ERK2, SKP2, and CKS1 were
determined by immunoprecipitating DUSP1 with anti-DUSP1 antibody and
probing the membranes with antibodies against ERK2, SKP2, and CKS1.
Immunoprecipitation of SKP2, followed by immunoblotting with CKS1
antibody, and DUSP1, followed by immunoblotting with the mouse
monoclonal anti-ubiquitin antibody, was used to assess the SKP2/CKS1
complex and ubiquitinated DUSP1, respectively. Bands were quantified in
arbitrary units by Molecular Imager ChemiDoc XRS using the Quantity One
1-D Analysis Software and normalized to h-actin levels.
Statistical analysis. Student’s t test and Tukey-Kramer test were used to
evaluate statistical significance. Fisher’s exact test was used for comparative
analysis of the survival of HCC patient subgroups. Linear regression
analyses were done by GraphPad Instat 3 software.1 Values of P < 0.05 were
considered to be significant. Data are expressed as means F SD.
Results
Clinicopathologic features and mutational status. Data in
Supplementary Table S3 show the presence of two distinct patients
subgroups in our sample collection with mean survival of 67.51 and
9.19 months, respectively, after liver partial resection (P < 0.0001).
On the basis of patients’ survival length, HCCs were previously
classified as tumors with better (HCCB) or poorer (HCCP) prognosis (22). No differences between the two subgroups occurred as
concerns sex and age of patients, etiology, presence of cirrhosis,
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Figure 1. Activation of the ERK cascade in human normal livers, HCC, and corresponding nontumorous surrounding liver tissues. Left, representative Western blots of
activated ERK1/ERK2 and downstream effectors HIF-1a, VEGF-a, and HXKII. HIF-1a activation was assessed by determining the HIF-1a/p300 complexes through
immunoprecipitation with the anti–HIF-1a antibody and probing the membranes with the goat polyclonal anti-p300 antibody. Right, chemiluminescence analysis
showing mean F SD of 6 normal livers, 21 HCCBs, 21 HCCPs, and corresponding surrounding livers with better or poorer prognosis (B ). Tukey-Kramer test: ., HCC
and surrounding subtypes versus normal liver, at least P < 0.05; *, HCC subtypes versus corresponding surrounding liver, P < 0.001; b, different from HCCB for
P < 0.001. SLB and SLP, surrounding liver with better and poorer prognosis, respectively.
tumor size, differentiation (Edmondson and Steiner grade), and
a-fetoprotein serum levels. Significant higher proliferation index
and microvessel density and lower apoptotic index occurred in
the HCCP than HCCB subgroup. As concerns the status of Ha-RAS,
Ki-RAS, N-RAS, A-RAF, B-RAF, RAF-1, and EGFR , no mutations
were detected in any of the samples analyzed.
ERK activation in human HCC. A progressive up-regulation of
active ERK1 and ERK2 proteins was detected from nonneoplastic
Figure 2. Levels of DUSP1, SKP2, and CKS1 in human normal livers, HCC and corresponding nontumorous surrounding liver tissues. A, DUSP1 mRNA levels
were determined by qRT-PCR. NT = 2 DCt, DCt = CtRNR18 Cttarget gene. Columns, means of NT of six control (normal) liver, 21 HCCB, and 16 HCCP (without promoter
hypermethyhlation) with corresponding surrounding livers. B, representative Western blots of human lesions. The complexes of DUSP1 with ERK2, SKP2, and
CKS1 were determined by immunoprecipitating DUSP1 with anti-DUSP1 antibody followed by immunoblotting with antibodies against ERK2, SKP2, and CKS1.
Immunoprecipitation of SKP2, followed by immunoblotting with CKS1 antibody, and of DUSP1, followed by immunoblotting with anti-ubiquitin antibody, was used to
assess the complex SKP2/CKS1 and ubiquitinated DUSP1, respectively. C, chemiluminescence analysis showing mean F SD of 6 normal livers, 21 HCCB, 21
HCCP (including those with promoter hypermethylation), and corresponding surrounding livers. Tukey-Kramer test: ., different from control for at least P < 0.05;
*, different from correspondent surrounding liver for P < 0.001; b, different from HCCB for at least P < 0.001; C , control liver.
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DUSP1 Deregulation in Liver Cancer
surrounding tissues to HCC, with the highest levels being detected
in HCCP, when compared with normal livers. A similar expression
pattern was found for ERK targets, including HIF-1a and HIF-1a/
p300 complexes, vascular endothelial growth factor-a (VEGF-a),
and HXKII (Fig. 1). These data indicate that induction of ERK
proteins is associated with both HCC development and progression, in accordance with a previous report (31).
Because the magnitude of ERK activity may be modulated by the
specific inhibitor DUSP1 (12, 13), we determined DUSP1 expression
at mRNA and protein levels. Quantitative reverse transcription–
PCR (qRT-PCR) analysis (Fig. 2A) showed 2.9-fold increase in
DUSP1 mRNA levels in HCCB. In contrast, no increase occurred in
surrounding livers and 16 of 21 HCCPs with respect to control liver.
In the other five HCCPs (23.8%), however, sharp DUSP1 downregulation (mean value F SD, 0.003 F 0.0017) was found (data not
shown in the figure). Subsequent methylation-specific PCR and
microsatellite analyses showed concomitant DUSP1 promoter
hypermethylation and LOH at the DUSP1 locus only in the five
samples exhibiting DUSP1 down-regulation (Supplementary
Figs. S1 and S2). In accordance with qRT-PCR results, DUSP1
protein levels (Fig. 2) increased in HCCB. However, in contrast with
DUSP1 mRNA expression patterns, marked down-regulation of
DUSP1 protein levels occurred in all HCCPs. Importantly, no
mutations were identified in the catalytic domain of DUSP1 in the
collection of samples examined in this study, suggesting that
posttranscriptional mechanisms may be responsible for DUSP1
down-regulation in HCCP in the absence of DUSP1 somatic
mutations or promoter hypermethylation. Because a recent report
indicates that cooperation between ERK and the SKP2/CKS1 ligase
complex results in phosphorylation at Ser296 and ubiquitination of
DUSP1 in lung carcinoma CL3 cell line (13), the levels of SKP2 and
CKS1 proteins were assessed in the same collection of samples.
Both SKP2 and CKS1 were up-regulated in HCC, mainly in HCCP.
Furthermore, ubiquitinated DUSP1, as well as DUSP1-SKP2,
DUSP1-CKS1, and SKP2-CKS1 complexes, were highest in HCCP,
suggesting that DUSP1 down-regulation is achieved by SKP2/CKS1dependent ubiquitination of DUSP1 in human HCC. These findings
suggest the existence of a mutual control of DUSP1 and ERK levels
in HCC. This agrees with the existence of a strong negative correlation between active ERK and DUSP1 levels in preneoplastic
and neoplastic liver, as shown by linear regression analysis
(DERK1/2/DDUSP1 = 0.8466, r = 0.9292, P < 0.0001).
DUSP1 levels inversely correlate with human HCC growth.
Due to the different behavior of DUSP1 expression in HCCs with
different survival rate, we evaluated the relationship of DUSP1 with
clinicopathologic features to explore the prognostic role of DUSP1
in human HCC. HCCB exhibited proliferation index and MVD at
2-fold lower (P < 0.0001) and apoptotic index at 1.5-fold higher
(P < 0.001) than HCCP (Supplementary Table S3). A significant
inverse correlation of DUSP1 with proliferation index (r = 0.837,
P < 0.0001) and MVD (r = 0.961, P < 0.0001) and a direct correlation with apoptosis (r = 0.626, P < 0.0001) and patients’ survival
length (r = 0.943, P < 0.0001) were found (Fig. 3). No other
clinicopathologic features, including etiology, presence of cirrhosis,
a-fetoprotein levels, and tumor grading was detected. These results
indicate that DUSP1 down-regulation is associated with HCC
progression and aggressiveness regardless of the etiologic agent.
SKP2, CKS1, and ERK cooperation suppresses DUSP1 in
human HCC cell lines. Functional consequences of DUSP1, ERK,
SKP2, and CKS1 interactions were assessed in 7703, HuH7, SNU387, and SNU-182 human HCC cell lines. High DUSP1 levels were
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Figure 3. Relationships between DUSP1 levels and proliferation, MVD,
apoptosis, and survival length of human HCCs. A total of 42 cases (21 HCCB
and 21 HCCP) were used for correlation analysis. All HCC patients were followed
until their death and used for correlation analysis.
detected by Western blot analysis only in the SNU-182 cell line
(Fig. 4). Methylation-specific PCR showed that DUSP1 downregulation depended on DUSP1 promoter methylation in 7703
cells (data not shown), whereas DUSP1 low levels were associated
with SKP2 and CKS1 overexpression in HuH7 and SNU-387 cells.
Different approaches to modulate DUSP1 and related molecule
levels in the HCC cell lines were applied (Fig. 4, with quantitative
analysis in Supplementary Figs. S3 and S4). Treatment with the
demethylating agent 5-Aza-dC caused DUSP1 up-regulation only
in 7703 (Fig. 4A and Supplementary Fig. S3A), confirming silencing
of DUSP1 by promoter hypermethylation. As expected, no effect
of 5-Aza-dC on DUSP1 levels was detected in the other cell lines
where no promoter hypermethylation occurred (not shown). In
7703 cells, DUSP1 induction by 5-Aza-dC was paralleled by downregulation of SKP2, CKS1, pERK1/2, and its targets, HIF-1a and
VEGF-a. Suppression of ERK activity, either via treatment with
UO126 (MEK-ERK inhibitor; Fig. 4B and Supplementary Fig. S3B)
or siRNA against ERK2 (Fig. 4C and Supplementary Fig. S3C)
increased DUSP1 expression, whereas diminishing DUSP1-Erk2
complex, ubiquitinated DUSP1, SKP2, and CKS1 levels, as well as
SKP2-DUSP1 and CKS1-DUSP1 complexes in the SNU-387 cell line.
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Equivalent results were obtained in the HuH7 cell line (data not
shown). Together, these results point to a role of ERK proteins in
DUSP1 down-regulation and SKP2 and CKS1 transactivation.
Conversely, DUSP1 suppression in SNU-182 cells by both the
DUSP1 inhibitor RO-31-8220 (Fig. 4D and Supplementary Fig. S4A)
and siRNA against DUSP1 (Fig. 4E and Supplementary Fig. S4B)
triggered up-regulation of SKP2, CKS1, pERK1/2, and ERK targets
VEGF-a and HIF-1a, further substantiating the inhibitory role of
DUSP1 on the ERK cascade. Moreover, suppression of SKP2 by
siRNA in SNU-387 (Fig. 4F and Supplementary Fig. S3C) and HuH7
(not shown) cell lines induced DUSP1 up-regulation and downregulation of the complexes of DUSP1 with SKP2, CKS1, and ERK2,
ubiquitinated DUSP1 and pERK1/2, and HIF-1a and VEGF-a, without affecting CKS1 levels. The decline in DUSP1-CKS1 complex,
apparently contradictory to the absence of variations in CKS1
expression in cells treated with siRNA against SKP2, reflects the
decrease in at least one component of the trimeric complex formed
by DUSP1, SKP2, and CKS1 proteins. As expected, SKP2 inhibition
had no effects on DUSP1 expression in 7703 and SNU-182 HCC cell
lines (data not shown) due to the absence of posttranscriptional
regulation in 7703 cells and the elevated DUSP1 activity in SNU-182
cells. Finally, down-regulation of CKS1 by siRNA resulted in DUSP1
overexpression and decrease in ubiquitinated DUSP1, without
affecting SKP2 and pERK1/2 levels in SNU-387 (Fig. 4G and
Supplementary Fig. S3D) and HuH7 (not shown) cell lines. Taken
together, these results assign a role to DUSP1 in the modulation
of the ERK cascade and show that ERK, SKP2, and CKS1 are
involved in DUSP1 degradation in human HCC.
The effect of the modulation of DUSP1 and related molecules on
cell growth and apoptosis are shown in Fig. 5. Induction of DUSP1
expression in 7703 and SNU-387 cells treated with 5-Aza-dC and
UO126, respectively, was associated with a sharp inhibition of
growth and increase in cell death. Equivalent results were obtained
in the same cell lines treated with the siRNA against ERK2 (not
shown). The same effect on cell growth and apoptosis followed
the treatment of SNU-387 cells with SKP2 or CKS1 siRNAs. Finally,
treatment of SNU-182 cells with either siRNA against DUSP1 or
the DUSP1 inhibitor RO-31-8220 (not shown) resulted in a 50%
increase in growth rate without affecting cell death.
The above results suggest a role of ERK and SKP2/CKS1 ligase in
proteasomal degradation of DUSP1 in HCC. To further substantiate
this hypothesis, we investigated the effect of the proteasomal
inhibitors ALLN and MG132 on DUSP1 levels in SNU-182 cells
transiently transfected with Ha-RAS, ERK2, or SKP2 (Fig. 6).
Figure 4. Representative Western blot analysis of the effect of DUSP1 reactivation, and ERK1/2, DUSP1, SKP2, and CKS1 inhibition in human HCC cell lines.
Immunocomplexes were determined by immunoprecipitation (IP ) of one component followed by immunoblotting with antibodies against the second component
(IB ), as indicated. The 7703 cell line was treated for 72 and 144 h with 5-Aza-dC. Cells were treated with inhibitors or siRNAs, for 12 and 24 h, as follows:
UO126 (MEK-ERK inhibitor), Ro-31-8220 (DUSP1 inhibitor) and siRNA against ERK2, DUSP1, SKP2 siRNA, and CKS1 . Controls received solvent alone or
scramble oligonucleotides.
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DUSP1 Deregulation in Liver Cancer
Figure 5. Effect of DUSP1 reactivation and ERK1/2,
DUSP1, SKP2, and CKS1 inhibition on human HCC cell
lines proliferation and apoptosis. A and A¶, 7703 cell line
was treated for 72 and 144 h with 5-Aza-dC. B and B ¶,
SNU-387 cells were grown 12 and 24 h with SKP2 siRNA,
CKS1 siRNA, or UO126. C and C ¶, SNU-182 cells were
grown 12 and 24 h with DUSP1 siRNA. Points, mean of
five experiments; bars, SD. Tukey-Kramer test: asterisked
curves, 12 and/or 24 h or 72 and/or 144 h versus 0 time,
at least P < 0.01. Treated versus control: cell viability,
72/144 h or 12/24 h, P < 0.001; apoptosis, 72/144 h for
5-Aza-dC and 24 h for all other treatments but DUSP1
siRNA, P < 0.001. Controls received solvent alone or
scramble oligonucleotides.
Transfection of pUSEamp/Ha-RAS, pUSEamp/ERK2, and pCMV6XL/SKP2 constructs led to equivalent results, determining a
remarkable increase in Ha-RAS, ERK2, and SKP2 expression with
respect to cells receiving the plasmid alone (Fig. 6A–C). These
changes were not affected by ALLN, whereas MG132 induced
further increase in ERK2, Ha-RAS, and SKP2 levels (Fig. 6A, B, and
D). Both Ha-RAS and ERK2 transfection resulted in DUSP1 downregulation and increase in ubiquitinated DUSP1, but these effects
were prevented by ALLN and MG132 (Fig. 6A and B). Moreover,
increase in SKP2 and CKS1 levels occurred in ERK2-transfected
cells, which was not/poorly affected by proteasomal inhibitors
(Fig. 6B). SKP2 transfection led to sharp decrease in DUSP1 levels,
associated with increase in ubiquitinated DUSP1, ERK2, DUSP1ERK2 complex, and CKS1 and DUSP1 complexes with CKS1 and
SKP2 (Fig. 6C). Similar to that described for Ha-RAS–transfected
and ERK2-transfected cells, DUSP1 down-regulation was prevented
by proteasomal inhibitors in SKP2-transfected cells (Fig. 6D).
Finally, overexpression of ERK2 was reversed by siRNA against
either SKP2 or CKS1 in ERK2-transfected cells (Fig. 6E). In the
latter cells, reduction of ERK2 levels via SKP2 or CKS1 siRNA was
paralleled by DUSP1 up-regulation and decrease in ubiquitinated
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DUSP1. No complexes of ERK with SKP2 and/or CKS1 were found
(data not shown).
Discussion
A number of observations support a prominent role of the RASMAPK cascade in hepatocarcinogenesis (31–34). This signaling
pathway leads to ERK activation, which may favor HCC progression
by promoting tumor growth and angiogenesis (Supplementary
Fig. S4). Recent researches on the molecular interactions between
ERK and DUSP1 have shown a reciprocal regulation between these
two proteins in mammalian cell lines. The physical interaction of
transiently activated ERK2 with DUSP1 induces the catalytic activation of the latter and the subsequent inactivation of MAPKs
(12, 13). In contrast, sustained ERK2 activation triggers DUSP1
degradation via the ubiquitination proteasomal pathway (13).
The present results show a progressive unrestrained activation
of ERK proteins from nontumorous surrounding liver to HCC,
with the highest increase characteristic of HCCP, suggesting a role
for DUSP1, whose expression is significantly higher in HCCB than
HCCP, as a putative tumor suppressor which negatively regulates
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ERK in the progression stage of hepatocarcinogenesis. Mutations of
the RAS, RAF, and EGFR genes, supporting active RAS-MAPK
pathway, are rare in HCC (refs. 31–35 and present study). RAS
mutations and silencing of RAS inhibitors are mutually exclusive
events in pancreatic (36), colorectal (37), and non–small cell lung
cancer (38). Our results suggest a prominent role of ERK upregulation in reducing DUSP1 expression in HCCP. Indeed,
suppression of active ERK1, ERK2, SKP2, or CKS1 expression in
cultured HCC cells resulted in DUSP1 up-regulation. Furthermore,
ERK proteins were active in surrounding liver and HCCB (although
at a significantly lower extent than in HCCP), where DUSP1
expression did not change (surrounding liver) or even increased
(HCCB). These findings indicate that although DUSP1 inhibition
theoretically could contribute to ERK overactivity, this is presumably
not the main causative event responsible for pERK1/2 up-regulation
in human HCC. This agrees with the notion that DUSP1 acts as a
feedback inhibitor by limiting the duration or magnitude of pERK1/2
activity rather than preventing pERK1/2 expression (13). DUSP1
suppression by ERK proteins may instead further strengthen their
effects on HCC growth by prolonging the half-life of active ERK.
DUSP1 down-regulation was significantly correlated to increased
tumor aggressiveness and reduced patient’s survival, strongly
suggesting its prognostic role in HCC regardless of the etiologic
agent, in accordance with previous reports in urothelial, prostate,
and liver cancer (16–18). In contrast, DUSP1 is overexpressed in
breast, gastric, and lung cancer (39–41), and its inhibition reduces
pancreatic tumor development in nude mice (42). The means by
which DUSP1 exerts such opposing effect on growth of different
cancer types remain elusive. It has been shown that elevated
DUSP1 protects cancer cells against Cisplatin-induced apoptosis by
suppressing c-Jun-NH2-kinase (JNK) activity (43). However, the lack
of JNK activation after DUSP1 inhibition by siRNA in SNU-182 HCC
Figure 6. Effect of proteasomal inhibitors on DUSP1 levels in SNU-182 cells transfected with Ha-RAS (A), ERK2 (B), or SKP2 (C and D ). Transient transfection
of SNU-182 cells was done with ERK2 or SKP2 cDNA carried by pCMV6-XL plasmid or with Ha-RAS (wild type) in pUSEamp plasmid. E, ERK2 stably transfected
SNU-182 cells treated with siRNA against either SKP2 or CKS1. Cells were grown at 37jC for 48 h. When indicated, the proteasomal inhibitors ALLN or MG132
were added in the last 3 h of the incubation, at the concentrations of 10 and 25 Amol/L, respectively. Cells were harvested 48 h after transfection. Controls are
nontransfected cells and cells transfected with plasmids alone.
Cancer Res 2008; 68: (11). June 1, 2008
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DUSP1 Deregulation in Liver Cancer
cell line (data not shown) argues against JNK regulation by DUSP1
in liver cancer. It has been shown that the protein IEX-1, an ERK
substrate, plays a role in prolonging ERK activation (44), thus
contributing to DUSP1 inactivation (13). IEX-1, upon phosphorylation by ERK, prevents cell death and favors cell proliferation (44).
Therefore, relative amounts of IEX-1 protein and duration of ERK
activation in different tumors should modulate DUSP1 effect.
Interestingly, preliminary results indicate the presence of elevated
amounts of IEX-1 protein in HCCP (data not presented),
substantiating the role of low DUSP1 levels in these tumors.
A previous report suggested epigenetic regulation of DUSP1
expression in prostate cancer (16). We show here that DUSP1
down-regulation, similarly to that of RASSF1A oncosuppressor
gene (34, 45), may occur through two mutually exclusive
mechanisms, namely promoter methylation associated with LOH
at DUSP1 locus or, most frequently, posttranscriptional events. The
latter results from the combined activity of ERK, SKP2, and CKS1,
leading to proteasomal degradation of DUSP1. This is shown as
follows: (a) DUSP1 induction by 5-Aza-dC is paralleled by downregulation of SKP2, CKS1, pERK1/2, and its targets (VEGF-A and
HIF-1a); (b) SKP2 or CKS1 suppression by specific siRNAs is
associated with DUSP1 up-regulation and down-regulation of
ubiquitinated DUSP1, as well as of pERK1/2 and its targets; (c)
siRNAs targeting SKP2 affects the formation of the SKP2/CKS1
complex (this does not occur by siRNA silencing of CKS1,
suggesting that SKP2 is a rate-limiting step in ERK-mediated
control of DUSP1 ubiquitination); (d) ERK inhibition by UO126 or
siRNA rescues DUSP1 expression and induces a decrease in
ubiquitinated DUSP1; (e) suppression of either SKP2 or CKS1 by
specific siRNA markedly decreases ERK-dependent proteolysis of
DUSP1 in ERK-overexpressing cells; ( f ) inhibition of DUSP1 by RO31-8220 or DUSP1 siRNA is associated with up-regulation of SKP2,
CKS1, pERK1/2, and its targets. Furthermore, the role of ERK and
SKP2 in DUSP1 proteolysis is substantiated by the inhibition of
DUSP1 degradation in SNU-182 cells overexpressing SKP2, ERK2, or
Ha-RAS, in which proteasomal inhibitors were added to the culture
medium. These findings confirm recent observations in embryonic
kidney cells, where down-regulation of DUSP1 was achieved by
ERK/SKP2/CKS1 cooperation, with sustained ERK activity promoting phosphorylation at the Ser296 residue of DUSP1, followed by
SKP2/CKS1-dependent degradation of DUSP1 (13). Moreover, our
results show a positive regulatory role of ERK on SKP2 and CKS1
expression in ERK-transfected cells. The mechanism underlying
this effect is not clarified by the present data. However, it may be
envisaged the possibility that ERK-induced up-regulation of
FOXM1 gene (14) is followed by up-regulation of SKP2/CKS1 ligase
(15). The induction of SKP1/CKS1 ligase by inducing DUSP1
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 11/8/2007; revised 1/24/2008; accepted 2/26/2008.
Grant support: Associazione Italiana Ricerche sul Cancro, Ministero dell’Istruzione, Università e Ricerca, and Assessorato Igiene e Sanità RAS.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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Dual-Specificity Phosphatase 1 Ubiquitination in
Extracellular Signal-Regulated Kinase−Mediated Control of
Growth in Human Hepatocellular Carcinoma
Diego F. Calvisi, Federico Pinna, Floriana Meloni, et al.
Cancer Res 2008;68:4192-4200.
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