1 DDX31 Regulates the P53-HDM2 Pathway and Rrna Gene

Author Manuscript Published OnlineFirst on September 27, 2012; DOI: 10.1158/0008-5472.CAN-12-1645 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Fukawa T et al.

DDX31 regulates the p53-HDM2 pathway and rRNA gene transcription

through its interaction with NPM1 in renal cell carcinomas

Tomoya Fukawa1,2, Masaya Ono3, Taisuke Matsuo1, Hisanori Uehara4,

Tsuneharu Miki5, Yusuke Nakamura6, Hiro-omi Kanayama2, and Toyomasa

Katagiri1

Authors’ Affiliations: 1Division of Genome Medicine, Institute for Genome

Research, The University of Tokushima, Tokushima; 2Department of Urology,

Institute of Health Biosciences, The University of Tokushima Graduate

School, Tokushima; 3Chemotherapy Division and Cancer Proteomics Project,

National Cancer Center Research Institute, Tokyo; 4Molecular and

Environmental Pathology, Institute of Health Biosciences, The University of

Tokushima Graduate School, Tokushima; 5Department of Urology, Kyoto

Prefectural University of Medicine, Kyoto; 6Laboratory of Molecular Medicine,

Human Genome Center, Institute of Medical Science, The University of

Tokyo, Tokyo Japan.

M. Ono and T. Matsuo are contributed equally to this work.

Corresponding Author: Toyomasa Katagiri, Division of Genome Medicine,

Institute for Genome Research, The University of Tokushima, 3-18-15,

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 27, 2012; DOI: 10.1158/0008-5472.CAN-12-1645 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Fukawa T et al.

Kuramoto-cho, Tokushima, 770-8503, Japan. Phone: 81-88-633-9477; Fax:

81-88-633-7986; E-mail: [email protected].

Running title

DDX31 regulates the p53 activity in RCC cells.

Keywords

DDX31, renal cell carcinoma, NPM1, HDM2, p53

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by future advanced research project from The

University of Tokushima (T. Katagiri).

Fukawa T et al.

Abstract

Studies of renal cell carcinoma (RCC) have led to the development of new

molecular-targeted drugs but its oncogenic origins remain poorly understood.

Here we report the identification and critical roles in renal carcinogenesis for

DDX31, a novel nucleolar protein upregulated in the vast majority of human

RCC. Immunohistochemical overexpression of DDX31 was an independent

prognostic factor for RCC patients. RNAi-mediated attenuation of DDX31 in

RCC cells significantly suppressed outgrowth, whereas ectopic DDX31

overexpression in human 293 kidney cells drove their proliferation.

Endogenous DDX31 interacted and colocalized with nucleophosmin (NPM1)

in the nucleoli of RCC cells, and attenuation of DDX31 or NPM1 expression

decreased pre-ribosomal RNA biogenesis. Notably, in DDX31-attenuated

cells, NPM1 was translocated from nucleoli to the nucleoplasm or cytoplasm

where it bound to HDM2. As a result, HDM2 binding to p53 was reduced,

causing p53 stablization with concomitant G1 phase cell cycle arrest and

apoptosis. Taken together, our findings define a mechanism through which

control of the DDX31NPM1 complex is likely to play critical roles in renal

carcinogenesis.

Fukawa T et al.

Introduction

Renal cell carcinoma (RCC) is the most common cancer of the kidney (1, 2),

and up to 30% of RCC patients present with metastatic disease (3, 4). At an

early stage, RCC can be cured by surgical resection, which is the most

effective treatment for localized RCC tumors (5). Moreover, cytokine

therapies, such as interleukin-2 or interferon-, are widely used as a first-line

treatment for metastatic disease. However, these treatments can be

associated with severe toxicity (6-9). In addition to these anti-cancer drugs,

several molecular target drugs, such as anti-VEGF antibody (Bevacizumab)

and small molecule inhibitors (Sunitinib, Sorafenib, Axitinib, Temsirolimus

and Everolimus) have been recommended as treatment options for

metastatic RCC and have been shown to prolong survival (10-13). However,

such treatments have also resulted in severe side effects (14-16). The

prognostic factors that determine the treatment outcome in patients with

metastatic RCC have been identified according to the patient’s status (17).

These markers have been widely used to determine the best treatment

approach and, as a result, may improve survival. At present there are no new

relevant molecular markers that have been identified to predict the prognosis

of RCC (18). Therefore, it is highly important to develop a new molecular

target agent(s) against RCC as well as prognostic molecular markers.

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The “omics” technologies, including transcriptomics, genomics and

proteomics, provide a detailed characterization of individual cancers that

should help improve clinical strategies for neoplastic diseases through the

development of novel drugs as well as providing the basis for personalized

treatment (19). Hence, these technologies have helped identify diagnostic

and therapeutic targets for cancer and understand the detailed molecular

mechanism of carcinogenesis. To this end, we analyzed the expression

profiling of clear cell RCC (ccRCC), which is a major histological type of RCC

(the Gene Expression Omnibus database (GEO) accession number:

GSE39364) (20), and identified and characterized DEAD (Asp-Glu-Ala-Asp)

box polypeptide 31 (DDX31), a novel member of the DEAD box protein

family.

DDX31 is the human homologue of Saccharomyces cerevisiae

DEAD box protein 7 (Dbp7), which was originally identified in a BLAST

search of the Saccharomyces cerevesiae Genome Database (Stanford) as a

new ATP-dependent RNA helicase (21). Dbp7 has been shown to play an

important role in the assembly of pre-ribosomal particles during the

biogenesis of the 60S ribosomal subunit (21). However, to date, no reports

have characterized the biological function of DDX31, the human homologue

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of Dbp7, and the significance of its transactivation in clinical RCC

progression.

Here, we identify and characterize DDX31 as a clinical prognostic

factor and a potential therapeutic target, and provide evidence that DDX31 is

involved in regulating ribosomal RNA (rRNA) gene transcription and the

p53−HDM2 pathway in renal carcinogenesis.

Materials and Methods

Cell lines and specimens

ACHN, Caki-1, Caki-2, 769-P, HK-2, HEK293 and COS-7 cells were

obtained from the American Type Culture Collection (ATCC) between

1994-2012. OS-RC-2 was provided by the RIKEN BioResource Center

(Tsukuba, Japan) in 2005. KC-12, KMRC-1, KMRC-20, and YCR-1 cells were

kindly provided by Dr. Taro Shuin, Department of Urology, Kochi Medical

School in 2006. All cells were cultured under conditions recommended by

their respective depositors. We monitored the cell morphology of these cell

lines by microscopy, and confirmed that they had maintained their

morphological states by comparison with the original morphological images.

No mycoplasma contamination was detected in cultures of all of these cell

lines using a Mycoplasma Detection Kit (Roche) in 2011. Surgically-resected

Fukawa T et al.

RCC tissue samples and their corresponding clinical information were

acquired from The University of Tokushima Hospital after obtaining written

informed consent. This study, as well as the use of all clinical materials

described above, was approved by the Ethical Committee of The University

of Tokushima.

Plasmids

Plasmids used in this study are described in Supplementary

Materials and Methods.

Quantitative and semi-quantitative reverse transcription-PCR

The extraction of total RNA from cultured cells and RCC clinical

tissues, reverse transcription, and subsequent PCR-amplification were

performed as previously described (1). -actin was used as a quantitative

control. The sequences of each primer set are described in Supplementary

Table S1.

Generation of a specific anti-DDX31 polyclonal antibody

The recombinant DDX31 protein was expressed and purified as

previously reported (22), and subsequently inoculated into rabbits (Scrum

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Inc.). The immune sera were then purified on antigen affinity columns using

Affi-gel 10 gel (Bio-Rad).

Antibodies

Antibodies used are described as follow; the anti-DDX31 polyclonal

antibody (see above; 1:100), anti-NPM1 monoclonal antibody (FC-61991,

Invitrogen 32-5200; 1:1000), anti-HA rat monoclonal antibody (3F10, Roche

11867423001; 1:1000), anti-FLAG M2 monoclonal antibody (M2,

Sigma-Aldrich F3165; 1:5000), -actin (AC-15, Sigma-Aldrich A5441;

1:5000), anti-p53 (DO-7, Dako M7001; 1:200), anti-p53 (FL-393, Santa Cruz

sc-6243; 1:1000), anti-p21 (Abcam, ab-7860; 1:200), anti-HDM2 (2A10,

Abcam, ab16895; 1:500), or anti-PARP (Cell Signaling Technology #9542;

1:200).

Western blot analysis

SDS-PAGE and immunoblotting were performed as described

previously (23). Full description for this analysis is described in

Supplementary Materials and Methods.

Immunohistochemical staining

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Preparation of frozen sections (5 m) of RCCs and normal kidney

tissues and immunohistochemical staining were performed as described

previously (2), with anti-DDX31 antibody (1:500), and classified the DDX31

expression levels on the stained tissues based on a scoring method as

follows; The percentage of DDX31-positive cancer cells was scored on a

scale of 0−2 (0: <5%, 1: 5−49%, and 2: >50%). Moreover, the intensity of

DDX31 or NPM1 staining was semiquantitatively evaluated by three

independent experienced pathologists without prior knowledge of

clinicopathologic data based on the following scoring system: 0: no staining,

1: low, 2: moderate and 3: high. Cases were accepted as positive only if all

reviewers independently defined them as such. In addition, the product of the

DDX31 staining score was divided into the following two groups: 3 and 4.

RNA interference experiments

We performed RNAi experiments as previously described (24, 25).

The detailed Materials and Methods of this experiment are described in

Supplementary Materials and Methods.

Establishment of stable transfectants

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Mock (no insert) or pCAGGS-DDX31-HA expression vectors were

transfected into HEK293 cells as previously described (20, 26). Following

experiments is described in Supplementary Materials and Methods.

Measurement of pre-rRNA biogenesis

The amount of 45S pre-ribosomal RNA in total RNA (pre-rRNA)

extracted was analyzed as described previously (27). Full description of this

analysis is described in Supplementary Materials and Methods.

Immunocytochemical staining analysis

The methodology for this analysis is described in Supplementary

Materials and Methods.

Immunoprecipitation and mass spectrometry (2DICAL)

DDX31-9 and Mock-3 stable transfectant cells were lysed in 0.5%

NP-40 IP-lysis buffer at 4°C for 30 min. The detailed Methods are described

in Supplementary Materials and Methods. LC-MS and data acquisition were

performed as previously described (28).

Co-immunoprecipitation assay

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Co-immunoprecipitation assay was performed as described in

Supplementary Materials and Methods.

p53 in vivo ubiquitination assay

The methodology for this assay is described in Supplementary

Materials and Methods.

Cell cycle analysis

We performed flow cytometry with analysis gates to remove debris

and doubles after transfection of siDDX31 or siEGFP, as previously

described (23). The DNA content of 10,000 cells was analyzed by FACS

Calibur and CellQuest software (BD Biosciences).

Statistical analysis

Progression-free survival curves were estimated using the

Kaplan−Meier method. The statistical significance of a relationship between

the clinical outcome and DDX31 expression was assessed using the trend

log-rank test. Cox proportional hazards analysis was used to identify

significant prognostic clinical factors and to test for an independent

contribution of DDX31 expression to progression-free survival. To examine

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potential confounding factors, age was treated as a continuous variable, and

gender, nuclear grade, T stage, M stage, and the DDX31 expression score

were treated as ordinal variables. Statistical significance was calculated

using Student's t-test to evaluate cell proliferation or gene expression. A

difference of P < 0.05 was considered statistically significant.

Results

Up-regulation of DDX31 in renal cell carcinomas

We verified that DDX31 was up-regulated in seven of nine ccRCC

cases compared to the corresponding normal kidney, and in eight of nine

ccRCC cell lines compared with normal human kidney by quantitative

RT-PCR (qRT-PCR) (Fig. 1A and B). To investigate DDX31 expression at the

protein level, we generated an anti-DDX31 polyclonal antibody, and

confirmed that this antibody specifically recognized exogenously expressed

DDX31 protein in HEK293 cells as well as endogenous DDX31 in Caki-1 cells

in western blot analyses (Supplementary Fig. S1A). The western blot results

accorded well with those of the qRT-PCR results in most of the tested RCC

cell lines and a normal human kidney epithelial cell line, HK-2 (Fig. 1C).

Furthermore, northern blot analysis showed that the DDX31 transcript was

weakly or undetectably expressed in normal human organs (data not shown).

Fukawa T et al.

In the NCBI database, cDNA sequences corresponding to three transcript

variants, denoted DDX31V1, DDX31V2 and DDX31V3, consist of 2555,

2328, and 2337 nucleotides that encode 851, 775 and 778 amino acids,

respectively. These three transcriptional variants contain 20, 20 and 18

exons, respectively, as shown in Fig. 1D. The V2 variant has a unique exon

19, which is 58-bp longer at the 5’ end than exon 19 of V1 and V3, resulting in

a frameshift within exon 19 and an early stop codon. The V3 variant lacks

exons 17 and 18 and has a unique exon 16, which is 35-bp shorter at the 5’

end than exon 16 of V1 and V2 (Fig. 1D). Subsequent semi-quantitative

RT-PCR confirmed that the DDX31V1 variant had the highest expression in

ccRCC cells (Fig. 1E). Therefore, we focused on elucidating the biological

roles of the DDX31V1 transcript due to its predominant expression in cancer

cells.

Association of higher DDX31 expression with poorer prognosis

To investigate the detailed expression pattern of the DDX31 protein

and characterize its biological functions, we performed immunohistochemical

staining of 70 ccRCC specimens with the anti-DDX31 antibody, and classified

the DDX31 expression levels on the stained tissues based on a scoring

method (see Materials and Methods). DDX31 was observed in the nuclei of

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many RCC specimens, although the staining patterns and intensities varied

across individual cases (Fig. 2A), while no staining was detected in normal

kidney tissues (Fig. 2B). Next, to investigate the clinicopathological

significance of DDX31 expression in RCC tissues, we analyzed the

relationship between the calculated immunohistochemical DDX31 scores

and the clinicopathological variables of 70 ccRCC specimens

(Supplementary Table S2). We used a Cox proportional hazards regression

analysis to examine the association between DDX31 expression and other

clinical and pathological variables in order to predict progression-free

survival. Both univariate and multivariate analyses revealed that DDX31

expression was a significant and independent prognostic factor for RCC

patients (P=0.0062 and P=0.014, respectively; hazard ratio: 1.8 per score)

(Table 1). A Kaplan–Meier survival analysis with the log-rank significance test

also showed that high DDX31 expression (Score 4) was associated with a

significantly worse progression-free survival than those with low DDX31

protein expression (Score 3) (Fig. 2C, Log-rank, P=0.00002). These results

suggested that DDX31 over-expression was indicative of worse outcomes in

RCC.

Effects of DDX31 expression on cell growth

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To investigate the growth-promoting role of DDX31 in RCC cells, we

knocked down the expression of endogenous DDX31 in Caki-1 (Fig. 3A and

B) and KMRC-1 (Fig. 3C and D) using RNAi techniques. qRT-PCR showed

significant DDX31 knockdown in cells transfected with DDX31 shRNAs or

siRNAs (#1 and #2), but not the controls (shEGFP or siEGFP). In

concordance with the knockdown effect, MTT assays clearly revealed

significant growth suppression in the two cell lines by both #1 and #2,

compared with the controls (shEGFP or siEGFP) (Fig. 3B and D). In addition,

a colony formation assay also confirmed that introducing both shRNA-DDX31

constructs remarkably suppressed the growth of Caki-1 cells, compared with

shEGFP-transfected cells (shEGFP: sh#1 or sh#2= 100%: 56% or 18%) (Fig.

3E). To further confirm the growth-promoting effects of DDX31

over-expression, we established HEK293 cell derivatives that stably express

exogenous DDX31 (DDX31-1, -8, -9 and -10). Western blot analysis with an

anti-HA antibody confirmed the high expression of DDX31 protein in these

four clones (Fig. 3F). Subsequent MTT assays showed that all four

DDX31-stable derivative cells (DDX31-1, -8, -9 and -10) grew significantly

faster than the mock-transfected controls (Mock-1, -2, and -3; Fig. 3G:

P=0.022). Together, these findings strongly imply that DDX31 plays an

important role in cell proliferation of RCC.

Fukawa T et al.

DDX31 interacts with NPM1

Because the biological functions of DDX31 are unknown, we

searched for DDX31-interacting protein(s) using immunoprecipitation and

2-Dimensional Image Converted Analysis of Liquid chromatography and

mass spectrometry (2DICAL) analyses (28). Nucleophosmin (NPM1), a

member of the nucleoplasmin superfamily (29), was identified as a candidate

DDX31-interacting protein. We first investigated the expression levels of

NPM1 in RCCs by qRT-PCR, western blot, and immunohistochemistry

analyses and found that NPM1 was highly expressed in most of the RCC

specimens and cell lines (Supplementary Fig. S1B-D). To validate this

interaction, HA-tagged DDX31 (DDX31-HA) and FLAG-tagged NPM1

(NPM1-FLAG) were co-transfected into COS-7 cells, and then the proteins

were immunoprecipitated with an anti-FLAG or anti-HA antibodies.

Subsequent western blot analysis with the indicated antibodies showed that

exogenous NPM1 co-precipitated with DDX31 (Fig. 4A). Additionally, we

confirmed that endogenous DDX31 co-precipitated with endogenous NPM1

in KMRC-1 cells (Fig. 4B). Next, we examined the effects of knocking down

NPM1 on the growth of RCC cell line KMRC-1, and found that

NPM1-depletion resulted in a significant decrease in cell viability of KMRC-1

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cells (Fig. 4C), although this effect was not comparable to siDDX31. These

findings suggest that NPM1 also has important roles in the growth of ccRCC

cells.

DDX31 is involved in rRNA gene transcription

Dbp7 (Dead box protein 7), the yeast homologue of DDX31,

reportedly plays an important role in the assembly of pre-ribosomal particles

during the biogenesis of the 60S ribosomal subunit (21). Additionally, NPM1

is also a nucleoprotein that has been implicated in ribosome biogenesis (27,

30). Particularly, a previous report showed that NPM1-depletion reduced the

transcription rate of the ribosomal RNA (rRNA) gene (27). Therefore, to

examine the effects of DDX31 or NPM1 knockdown on the transcription rate

of rRNA, we monitored the amount of pre-rRNA by qRT-PCR. DDX31

expression did not affect NPM1-knocking down in KMRC-1 cells (Fig. 4D),

but knocking down of DDX31, NPM1 or both significantly reduced the

expression of the pre-ribosomal RNA45S subunits (Fig. 4D and E).

Conversely, we confirmed that pre-ribosomal RNA synthesis was significantly

increased in DDX31-stable transfectants (Fig. 4F). These findings suggest

that the DDX31-NPM1 complex plays a crucial role in rRNA gene

transcription.

Fukawa T et al.

DDX31 regulates the p53 stabilization through its interaction with NPM1

in the nucleoli.

It has been reported that the redistribution of NPM1 upon DNA

damage, such as UV-damage, leads to an interaction with HDM2, and that

this binding could affect p53 stabilization and activity by inhibiting HDM2−p53

complex formation, indicating that NPM1 acts as a negative regulator of

HDM2−p53 interaction (31). Moreover, we found that a reduced level of

pre-rRNA biogenesis (Fig. 4E) weakly correlated with decreased cell growth

(Fig. 3) in DDX31- or NPM1-depleted RCC cells. Therefore, we hypothesized

that DDX31 regulates p53− HDM2 pathway through its interaction with NPM1

in RCC cells. We first examined the effects of knocking down DDX31 on the

nuclear localization of NPM1 in Caki-1 cells by immunocytochemistry with

anti-DDX31 or anti-NPM1 antibodies. We observed that DDX31 and NPM1

co-localized in the nucleoli of Caki-1 cells transfected with siEGFP as a

control (Fig. 5A; siEGFP), whereas translocation of endogenous NPM1 from

the nucleoli to the nucleoplasm or cytoplasm was observed in

DDX31-depleted cells (Fig. 5A; siDDX31). On the other hand, NPM1

knockdown did not affect the nucleolar localization of DDX31 (Supplementary

Fig. S2A). Moreover, translocation of endogenous nucleolin, which is also an

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abundant nucleoprotein, from the nucleoli to the nucleoplasm or cytoplasm

was not observed in DDX31-depleted KMRC-1 cells (Supplementary Fig.

S2B). These findings suggest the possibility that DDX31 specifically interacts

with NPM1, and thereby inhibits its translocation from the nucleoli to the

nucleoplasm or cytoplasm in RCC cells.

Next, we examined the expression level of p53 protein upon

siRNA-mediated knockdown of DDX31. The expression of p53 was clearly

up-regulated at the protein level upon DDX31 knockdown in both Caki-1 and

KMRC-1 cells (Fig. 5B), but the steady-state levels of p53 mRNA remained

constant in cells treated with DDX31-siRNA (Supplementary Fig. S3A). On

the other hand, p21, a p53-downstream target, was clearly up-regulated at

the protein and transcriptional levels at 72 h after siDDX31 transfection (Fig.

5B, Supplementary Fig. S3B and C). Moreover, we confirmed that DDX31

depletion did not affect NPM1 protein expression levels (Fig. 5C).

Subsequently, we investigated whether NPM1 could bind to endogenous

HDM2 when DDX31 expression was knocked down by siRNA, and found that

endogenous NPM1 and HDM2 formed a protein complex in DDX31-depleted

cells, while the formation of p53-HDM2 complex was clearly decreased (Fig.

5D).

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Because HDM2 regulates p53 turnover via its E3 ligase activity, we

hypothesized that DDX31 deficiency inhibits p53 degradation by reduction of

its HDM2-mediated ubiquitination. We transfected KMRC-1 cells with

plasmids expressing ubiquitin and p53 proteins, and transfected these cells

with siDDX31 or siEGFP as a control. Subsequent immunoprecipitation and

immunoblotting analyses revealed that the p53 poly-ubiquitination was

clearly decreased in DDX31-depleted cells, compared with that in

siEGFP-transfected cells under the presence of MG132, which is a 26S

proteasome inhibitor (Fig. 5E). These results show that DDX31 inhibition

possibly prevents HDM2−mediated p53 poly-ubiquitination.

DDX31 deficiency induced p53-dependent G1-arrest and apoptosis.

Next, to examine the effects of DDX31 ablation on the perturbation

of cell cycle of RCC cells, we performed FACS analysis. A clear increased

population of G0/G1 cell at 96 h after siDDX31 transfection (siEGFP:

siDDX31 = 69.71%: 79.01%) was observed (Fig. 6A). We detected

upregulation of p53 at protein level, but not at mRNA, whereas, upregulation

of p21 at both protein and mRNA levels (Fig. 6B, C). We also observed an

increased sub-G1 population from 96 h (siEGFP: siDDX31 =3.45%: 16.52%)

(Supplementary Fig. S4A) to 120 h (siEGFP: siDDX31 =8.52%: 28.54%) (Fig.

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6D). In addition, we detected a cleaved PARP at 120 h (Fig. 6 E), indicating

an induction of G1-arrest and subsequent apoptosis by DDX31-depletion.

More importantly, siRNA-mediated p53 knock down reversed the effect of

DDX31-silencing on cell growth, indicating that DDX31 deficiency led to

apoptosis in a p53-depedent manner (Fig. 6F). On the other hand, in

p53-mutant KMRC-20 cells, which dose not express p53 protein due to 5bp

insertion within exon 5, DDX31 knockdown did not affect p21 expression

level and growth reduction (Supplementary Fig. S4B, C), compared with that

in p53-wildtype KMRC-1 cells (Fig. 6B, C). Thus, DDX31 levels are relevant

to the p53-mediated cellular responses in RCC cells.

Discussion

Through an expression profiling analysis of ccRCC, we identified the

up-regulation of DDX31 in the majority of ccRCC cases, but its minimal

expression in normal human tissues (data not shown). RNAi-mediated

knockdown of DDX31 drastically suppressed RCC cell growth, while

introducing DDX31 into HEK293 cells enhanced cellular proliferation.

Moreover, clinicopathological evidence through immunohistochemistry using

70 ccRCC specimens demonstrated that RCC cases strongly expressing

DDX31 had shorter progression-free survival periods than those with

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negative or weak DDX31 expression. These results strongly suggest that

DDX31 over-expression is critically involved in renal carcinogenesis.

The DDX31 gene encodes a putative 851-amino-acid protein with a

predicted ATP-dependent RNA helicase domain that is conserved among the

DEAD-box protein family. It has been reported that Dbp7, the

Saccharomyces cerevesiae homologue of DDX31 (21% identity and 72%

similarity), localizes to the nucleolus and is required for optimal vegetative

growth (21). Dbp7 depletion results in reduced amounts of the 60S ribosome

subunits and the accumulation of halfmer polysomes (21). Thus, Dbp7 is

likely involved in the assembly of pre-ribosomal particles during the

biogenesis of the 60S ribosomal subunit (21, 32), but the pathophysiological

functions of human DDX31 in RCC cells are unknown. Here, we

demonstrated that DDX31 interacts and co-localizes with the

nucleophosphoprotein NPM1 in the nucleoli of RCC cells. Moreover,

siRNA-mediated knockdown of DDX31 or NPM1 expression led to a

significant reduction in cell growth, suggesting that this protein complex

plays an important role in the cell growth of RCC.

Accumulating evidence indicates that NPM1 is implicated in multiple

functions, including ribosomal biogenesis (28, 30), centrosome duplication

(33), and regulation of p53 stabilization (34-38). Therefore, we first examined

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whether depleting DDX31 or NPM1 affects pre-rRNA synthesis, and found

that there was a significant reduction in cell growth in DDX31- or

NPM1-depleted RCC cells, whereas a significant enhancement of pre-rRNA

synthesis was observed in DDX31-overexpressed cells. These findings

suggest that DDX31−NPM1 complex formation in the nucleoli plays a crucial

role in rRNA gene transcription in renal carcinogenesis.

Previous reports have indicated that cellular stress causes NPM1 to

undergo nucleoplasmic translocation and associate with HDM2, and the

formation of this complex leads to the stabilization of the p53 protein (31).

Additionally, NPM1 also regulates basal levels of the p53 protein in

unstressed cells through its interaction with HDM2 (31), indicating that NPM1

negatively regulates the p53−HDM2 interaction. Thus, the nucleoli to

nucleoplasmic translocalization of NPM1 resulted in p53 activation after

cellular or nucleolar stress, but it is unclear how NPM1 is anchored in the

nucleoli of RCC cells, which highly expresses DDX31, even after

doxorubicin-induced cellular stress (Supplementary Fig. S5A). On the other

hand, in normal epithelial kidney cell HK-2 which does not show DDX31

protein at detectable levels, NPM1 translocated to the nucleoplasm with

treatment of doxorubicin, but was localized in the nucleoli without

doxorubicin (Supplementary Fig. S5B). Nucleolar stress is often caused by

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disruption of ribosomal biogenesis, which in turn can be caused by

malfunction of nucleolar proteins (39-41) and chemotherapeutic drugs such

as doxorubicin (42). As shown in Fig. 4E, DDX31-depletion caused a

reduction in rRNA gene transcription, which is a key step of ribosome

biogenesis. Our results suggest the possibility that nuclear stress such as

malfunction of rRNA gene transcription induced by DDX31-depletion resulted

in translocation of the NPM1 from the nucleoli to the nucleoplasm or

cytoplasm in DDX31-depleted RCC cells, while nuclear stress such as

doxorubicin treatment caused the translocation of NPM1 to the nucleoplasm

of normal kidney cells.

Next, we investigated the effects of DDX31−depletion on the

p53−HDM2 pathway in RCC cells. Decreased rRNA gene transcription

caused by DDX31−depletion resulted in translocation of NPM1 from the

nucleoli to the nucleoplasm, and NPM1 bound HDM2, which prevented

HDM2 from interacting with p53 leading to p53 stabilization and p21

up-regulation, p53-dependent G1-cell cycle arrest and subsequent apoptosis

(Fig. 6F, depletion of DDX31). In addition, we also observed NPM1 in the

cytoplasm of DDX31−depleted RCC cells (Fig. 5A). Accumulating evidence

indicates that NPM1 shuttles between the nucleus and cytoplasm during the

cell cycle (38, 43, 44) and translocates to the cytosol and mitochodria in

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response to apoptotic stressors (45). Therefore, our findings suggest the

possibility that cytoplasmic NPM1 translocated from the nucleoli may

influence the cell cycle or apoptosis in DDX31−depleted RCC cells, although

further analysis of the DDX31−NPM1 functions will be necessary. Together,

we demonstrated that DDX31 captures NPM1 in the nucleoli, which prevents

NPM1−HDM2 complex formation in the nucleoplasm and subsequent

enhancement of p53 ubiquitination and degradation, resulting in p53

down-regulation in RCC cells (Fig. 6F, over-expression of DDX31). To date,

mutations in the p53 gene have only been rarely observed in RCC specimens

and cell lines (46, 47). Hence, our results may propose a new mechanism of

p53 inactivation in RCC cells.

In conclusion, our findings show that DDX31 likely plays a significant

role in renal carcinogenesis by regulating the p53−HDM2 pathway and rRNA

gene transcription via its interaction with NPM1. We believe that DDX31

could be a useful prognostic marker for RCC, and inhibiting DDX31−NPM1

complex formation is a promising therapeutic target for RCC, especially in

patients with P53 protein-intact RCC.

Acknowledgements

Fukawa T et al.

The authors thank Dr. Taro Shuin for kindly providing the RCC cell lines, Drs.

Hirofumi Arakawa and Takeshi Imamura for kindly providing expression

vectors, Drs. Tetsuro Yoshimaru and Masato Komatsu for helpful

discussions, Ms. Ikarashi Ayako, National Cancer Center Research Institute

for 2DICAL analysis, and Drs. Junko Stevens and Hirofumi Aruga, Life

technologies for development of Multiplex-quantitative Real Time-PCR

system.

References

1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics.

CA Cancer J Clin 2008;58:71–96.

2. American Cancer Society: Cancer facts and figures 2010. Atlanta, Ga:

American Cancer Society, 2011.

3. Lam JS, Leppert JT, Belldegrun AS, Figlin RA. Novel approaches in the

therapy of metastatic renal cell carcinoma. World J Urol 2005;23:202–12.

4. Motzer RJ, Bander NH, Nanus DM. Renal-cell carcinoma. N Engl J Med

1996;335:865–75.

5. Cohen HT, McGovern FJ. Renal-cell carcinoma. N Engl J Med 2005;353:

2477–90.

Fukawa T et al.

6. Stadler WM, Huo D, George C, Yang X, Ryan CW, Karrison T, et al.

Prognostic factors for survival with gemcitabine plus 5-fluorouracil based

regimens for metastatic renal cancer. J Urol 2003;170:1141–5.

7. Fyfe G, Fisher RI, Rosenberg SA, Sznol M, Parkinson DR, Louie AC.

Results of treatment of 255 patients with metastatic renal cell carcinoma

who received highdose recombinant interleukin-2 therapy. J Clin Oncol

1995;13:688–96.

8. Negrier S, Escudier B, Lasset C, Douillard JY, Savary J, Chevreau C, et al.

Recombinant human interleukin-2, recombinant human interferon alfa-2a,

or both in metastatic renal-cell carcinoma. N Engl J Med

1998;338:1272–8.

9. Negrier S, Maral J, Drevon M, Vinke J, Escudier B, Phillip T. Long-term

follow-up of patients with metastatic renal cell carcinoma treated with

intravenous recombinant interleukin-2 in Europe. Cancer J Sci Am

2000;6:93–8.

10. Lane BR, Rini BI, Novick AC, Campbell SC. Targeted molecular therapy

for renal cell carcinoma. Urology 2007;69:3–10.

11. Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Siebels M, et al.

Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med

2007;356:125–34.

Fukawa T et al.

12. Hudes G, Carducci M, Tomczak P, Dutcher J, Figlin R, Kapoor A, et al.

Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma.

N Engl J Med 2007;356:2271–81.

13. Motzer RJ, Hutson TE, Tomczak PP, Michaelson MD, Bukowski RM, Rixe

O, et al. Sunitinib versus interferon alfa in metastatic renal-cell carcinoma.

N Engl J Med 2007;356:115–24.

14. Wu S, Chen JJ, Kudelka A, Lu J, Zhu X. Incidence and risk of

hypertension with sorafenib in patients with cancer: a systematic review

and meta-analysis. Lancet Oncol 2008;9:117–23.

15. Bhojani N, Jeldres C, Patard JJ, Perrotte P, Suardi N, Hutterer G, et al.

Toxicities associated with the administration of sorafenib, sunitinib, and

temsirolimus and their management in patients with metastatic renal cell

carcinoma. Euro Urol 2008;53:917–30.

16. Bellmunt J, Szczylik C, Feingold J, Strahas A, Berkenblit A. Temsirolimus

safety profile and management of toxic effects in patients with advanced

renal cell carcinoma and poor prognostic features. Ann Oncol

2008;19:1287–392.

17. Motzer RJ, Bacik J, Murphy BA, Russo P, Mazumdar M. Interferon-alfa as

a comparative treatment for clinical trials of new therapies against

advanced renal cell carcinoma. J Clin Oncol 2002;20:289–96.

Fukawa T et al.

18. Zhao H, Ljungberg B, Grankvist K, Rasmuson T, Tibshirani R, Brooks JD.

Gene expression profiling predicts survival in conventional renal cell

carcinoma. PLoS Med 2006;3:115–24.

19. Petricoin EF 3rd, Hackett JL, Lesko LJ, Puri RK, Gutman SI, Chumakov K,

et al. Medical applications of microarray technologies: a regulatory

science perspective. Nat Genet 2002;32:474–9.

20. Hirota E, Yan L, Tsunoda T, Ashida S, Fujime M, Shuin T, et al.

Genome-wide gene expression profiles of clear cell renal cell carcinoma:

Identification of molecular targets for treatment of renal cell carcinoma. Int

J Oncol 2006;29:799–827.

21. Daugeron MC, Linder P. Dbp7p, a putative ATP-dependent RNA helicase

from Saccharomyces cerevisiae, is required for 60S ribosomal subunit.

RNA 1998;4:566–81.

22. Kanehira M, Harada Y, Takata R, Shuin T, Miki T, Fujioka T, et al.

Involvement of upregulation of DEPDC1 (DEP domain containing 1) in

bladder carcinogenesis. Oncogene 2007;26:6448–55.

23. Park JH, Lin ML, Nishidate T, Nakamura Y, Katagiri T. PDZ-bindingkinase

T-LAK cell-originated protein kinase, a putative cancer testisantigen with

an oncogenic activity in breast cancer. Cancer Res 2006;66:9186–95.

Fukawa T et al.

24. Dobashi S, Katagiri T, Hirota E, Ashida S, Daigo Y, Shuin T, et al.

Involvement of TMEM22 overexpression in the growth of renal cell

carcinoma cells. Oncol Rep 2009;21:305–12.

25. Endo A, Kitamura N, Komada M. Nucleophosmin/B23 regulates ubiquitin

dynamics in Nucleoli by recruiting deubiquitylating enzyme USP36. J Bio

Chem 2009;284:27918–23.

26. Togashi A, Katagiri T, Ashida S, Fujioka T, Maruyama O, Wakumoto Y, et

al. Hypoxiainducible protein 2 (HIG2), a novel diagnostic marker for renal

cell carcinoma and potential target for molecular therapy. Cancer Res

2005;65:4817–26.

27. Murano K, Okuwaki M, Hisaoka M, Nagata K. Transcription regulation of

the rRNA gene by a multifunctional nucleolar protein,

B23/Nucleophosmin, through Its histone chaperone activity. Mol Cell Biol

2008;28:3114–26

28. Ono M, Shitashige M, Honda K, Isobe T, Kuwabara H, Matsuzuki H, et al.

Label-free quantitative proteomics using large peptide data sets

generated by nanoflow liquid chromatography and mass spectrometry.

Mol Cell Proteomics 2006;5:1338–47.

Fukawa T et al.

29. Frehlick LJ, Eirin-Lopez JM, Ausio J. New insights into the

nucleophosmin/nucleoplasmin family of nuclear chaperones. Bioessays

2007;29:49–59.

30. Savkur RS, Olson MO. Preferential cleavage in pre-ribosomal RNA by

protein B23 endoribonuclease. Nucleic Acids Res1998;26:4508–15.

31. Kurki S, Peltonen K, Latonen L, Kiviharju TM, Ojala PM, Meek D, et al.

Nucleolar protein NPM interacts with HDM2 and protects tumor

suppressor protein p53 from HDM2-mediated degradation. Cancer Cell

2004;5:465–75

32. Bernstein KA, Granneman S, Lee AV, Manickam S, Baserga SJ.

Comprehensive mutational analysis of Yeast DEXD/H Box RNA helicases

involved in large ribosomal subunit biogenesis. Mol Cell Biol

2006;26:1195–208.

33. Okuda M, Horn HF, Tarapore P, Tokuyama Y, Smulian AG, Chan PK, et al.

Nucleophosmin/ B23 is a target of CDK2/cyclin E in centrosome

duplication. Cell 2000;103:127–40.

34. Wu MH, Chang JH, Chou CC, Yung BY. Involvement of

nucleophosmin/B23 in the response of HeLa cells to UV irradiation. Int J

Cancer 2002;97:297–305.

Fukawa T et al.

35. Yao Z, Duan S, Hou D, Wang W, Wang G, Liu Y, et al. B23 acts as a

nucleolar stress sensor and promotes cell survival through its dynamic

interaction with hnRNPU and hnRNPA1. Oncogene 2010;29:1821–34.

36. Li J, Zhang X, Sejas DP, Bagby GC, Pang Q. Hypoxia-induced

nucleophosmin protects cell death through inhibition of p53. J Biol Chem

2004;279:41275–9.

37. Li J, Sejas DP, Burma S, Chen DJ, Pang Q. Nucleophosmin suppresses

oncogene-induced apoptosis and senescence and enhances oncogenic

cooperation in cells with genomic instability. Carcinogenesis

2007;28:1163–70.

38. Grisendi S, Mecucci C, Falini B, Pandolfi PP. Nucleophosmin and cancer.

Nat Rev Cancer 2006;6:493–505.

39. Fumagalli S, Di Cara A, Neb-Gulati A, Natt F, Schwemberger S, Hall J, et

al. al. Absence of nucleolar disruption after impairment of 40S ribosome

biogenesis reveals an rpL11-translation-dependent mechanism of p53

induction. Nat Cell Biol 2009;11:501–8.

40. Zhang Y, Lu H. Signaling to p53: ribosomal proteins find their way.

Cancer Cell 2009;16:369-77.

41. Warner JR, McIntosh KB. How common are extraribosomal functions of

ribosomal proteins? Mol Cell 2009; 34:3-11.

Fukawa T et al.

42. Burger K, Muhl B, Harasim T, Rohrmoser M, Malamoussi A, Orban M, et

al. Chemotherapeutic drugs inhibit ribosome biogenesis at various levels.

J Biol Chem 2010;285:12416-25.

43. Borer RA, Lehner CF, Eppenberger HM, Nigg E. Major nucleolarproteins

shuttle between nucleus and cytoplasm. Cell 1989;56:379–90.

44. Szebeni A, Herrera JE, Olson MO. Interaction of nucleolar protein B23

with peptides related to nuclear localization signals. Biochemistry

1995;34:8037–42.

45. Lindenboim L, Blacher E, Borner C, Stein R. Regulation of stress-induced

nuclear protein redistribution: a new function of Bax and Bak uncoupled

from Bcl-x(L). Cell Death Differ 2010;17:346–59.

46. Stickle NH, Cheng LS, Watson IR, Alon N, Malkin D, Irwin MS, et al.

Expression of p53 in renal carcinoma cells is independent of pVHL.

Mutant Res 2005:578:23–32.

47. Gurova KV, Hill JE, Razorenova OV, Chumakov PM, Gudkov AV. p53

Pathway in Renal Cell Carcinoma Is Repressed by a Dominant

Mechanism. Cancer Res 2004;64:1951–8.

Fukawa T et al.

Table 1. The association of DDX31 expression with other clinical and

pathologic variables.

Univariate Multivariate Variables P-value Hazard ratio (95% CI) P-value Hazard ratio (95% CI)

Age 0.61 1.02 (0.95-1.10)

Gender 0.89 1.13 (0.218-5.83)

Nuclear grade 0.036 3.51 (1.09-11.36) 0.13 3.53 (0.71-17.69)

Score 0.0062 1.72 (1.17-2.52) 0.014 1.8 (1.13-2.87)

T stage 0.041 2.45 (1.04-5.79) 0.16 1.98 (0.76-5.15)

N stage Not calculated

M stage 0.0008 16.13 (3.16-82.30) 0.029 7.40 (1.23-44.3)

Cox proportional hazard regression analysis was used to test for prediction

of the progression-free survival. Univariate analysis showed that nuclear

grade, T stage, M stage and DDX31 score were significant predictors of

progression (P=0.036, 0.041,0.0008 and 0.0062, respectively).

Fukawa T et al.

Figure legends

Figure 1. DDX31 expression in RCCs. A and B, expression of the DDX31

gene in nine RCC tissues (T), the adjacent normal kidney (N), and nine RCC

cell lines and a normal kidney, as determined by quantitative RT-PCR

(qRT-PCR). -actin served as a quantitative internal control. C, DDX31

protein expression in RCC cell lines and HK-2 cell line, as determined by

western blot. W: p53 wild-type, M: p53 mutant. D, genomic structure of three

DDX31 splicing variants (V1, V2 and V3). White and black triangles indicate

the initiation and stop codons, respectively. Arrows indicate the primer set

that detects each of the three variants. The number above each box indicates

the exon number. E, expression pattern of each of the three DDX31 splicing

variants in three RCC cell lines and a normal human kidney examined by

semi-quantitative RT-PCR.

Figure 2. Expression of DDX31 in RCC and normal kidney tissue sections. A,

B, Immunohistochemistry analysis of DDX31 in ccRCC and normal kidney

tissue sections. Representative images of RCC tissues evaluated as score 2

(1109), score 4 (1077) and score 6 (993) as well as normal kidney tissues

(Sample No. 993 and 1109) are shown in A and B, respectively. Enlarged

images of the rectangular region of each RCC tissue in A. Scale bar shows

Fukawa T et al.

50 m. C, Kaplan–Meier survival curves of progression-free survival in RCC

patients according to DDX31 expression. High DDX31 expression was

significantly associated with poor progression-free survival (log rank test,

P=0.00002).

Figure 3. Growth-promoting effects of DDX31 in Caki-1 (A and B) and

KMRC-1 (C and D). A, C, DDX31 knockdown by both vector-based shRNA

(sh#1 and sh#2) and DDX31-specific siRNA oligonucleotides (si#1 and si#2)

was validated by qRT-PCR. siRNA targeting EGFP (siEGFP) was used as a

negative control. -actin was used as a quantitative control. B, D, MTT assay

demonstrating a decrease in cell number upon DDX31 knockdown in Caki-1

(B) and KMRC-1 (D) cells. E, colony formation assay demonstrating

decreased colony numbers in DDX31-depleted Caki-1 cells. F, western blot

analysis of cells expressing exogenous DDX31 (DDX31) or those transfected

with a Mock vector. Endogenous (left) and exogenous DDX31 (right)

expression was verified with the indicated antibodies. G, In vitro growth of

HEK293–DDX31 and –Mock stable transfectant cells.

Figure 4. Interaction of DDX31 with NPM1. A, co-immunoprecipitation of

DDX31 and NPM1 proteins. Cell lysates from COS-7 cells that were

Fukawa T et al.

transfected with DDX31-HA and NPM1-FLAG were immunoprecipitated (IP)

and subsequently immunoblotted (IB) with indicated tag-antibodies. B,

confirmation of an endogenous interaction between DDX31 and NPM1 in

KMRC-1 cells. C, growth-inhibitory effects of NPM1 knockdown on RCC

cells. qRT-PCR showing the suppression of endogenous NPM1 expression

by NPM1 siRNA (left). MTT assay demonstrating a decrease in cell number

upon NPM1 knockdown in KMRC-1 cells (right). D, effects of DDX31, NPM1

or both knockdown on pre-ribosomal RNA synthesis in KMRC-1 cells.

qRT-PCR showing suppression of endogenous DDX31 (left) and NPM1

expression (right) by DDX31, NPM1 and both siRNA, respectively. E, effects

of DDX31, NPM1 or both knockdown on pre-rRNA expression in KMRC-1

cells. F, enhancement of pre-ribosomal RNA synthesis in HEK293 cells

over-expressing DDX31.

Figure 5. DDX31 regulates p53 stabilization through its interaction with

NPM1. A, effects of DDX31 knockdown on the subcellular-localization of

NPM1. Forty-eight hours after transfection with siDDX31 or siEGFP,

immunocytochemical staining was performed to assess the localization of

NPM1 in Caki-1 cells. B, effects of DDX31 knockdown on p53 and p21

protein levels in Caki-1 and KMRC-1. C, effects of DDX31 or NPM1

Fukawa T et al.

knockdown on HDM2, p53 and NPM1 expression. D, HDM2−NPM1 and

HDM2−p53 interactions in DDX31-depleted KMRC-1 cells. E, KMRC-1 cells

were transfected with the p53 expression vector. After 24 h, cells were

transfected with siDDX31 or siEGFP for 48 h, followed by treatment with

MG132 (20 M) for 3 h before lysis. Lysates were immunoprecipitated with

anti-p53 antibody (FL-393) and then, immunoblotted with antibodies to p53

(DO-7) and ubiquitin (Ub). Relative poly-ubiquitination level was estimated by

the intensity ratio of poly-ubiquitinated p53/total p53 after

immunoprecipitation with anti-p53 antibody. Representative results are

shown from one of three independent experiments.

Figure 6. DDX31 silencing induces p53-dependent G1-arrest and

subsequent apoptosis. A, FACS profiles of KMRC-1 cells that were treated

with siDDX31 or siEGFP, and then stained with propidium iodide (PI) and

analyzed by FACS annalysis at 96 h after transfection. B, western blot

analysis of p53 and p21 protein expression in DDX31-depleted KMRC-1

cells. C, qRT-PCR analysis of p53 (left panel) and p21 (right panel)

expression at the transcriptional level in DDX31-depleted KMRC-1 cells. ns;

not significant. D, FACS profiles of KMRC-1 cells that were treated with

siDDX31 or siEGFP. Sub-G1 cells were measured by FACS analysis at 120 h

Fukawa T et al.

after transfection. E, western blot analysis of caspase-specific PARP

cleavage in DDX31-depleted KMRC-1 cells. F, KMRC-1 cells were

co-transfected with siEGFP, siDDX31 and/or sip53, followed by MTT assay.

G, Schematic model of the regulation of the p53−HDM2 pathway and rRNA

gene transcription by DDX31−NPM1. DDX31-depletion causes the failure of

rRNA gene transcription, resulting in NPM1 translocation to nucleoplasm or

cytoplasm. Subsequently, NPM1 binds to HDM2 and results in TP53

accumulation (Depletion of DDX31). On the other hand, DDX31 plays an

important role in rRNA gene transcription, and regulates the p53−HDM2

pathway through its interaction with NPM1 in the nucleoli of RCC cells

(Overexpression of DDX31).

DDX31 regulates the p53-HDM2 pathway and rRNA gene transcription through its interaction with NPM1 in renal cell carcinomas

Tomoya Fukawa, Masaya Ono, Taisuke Matsuo, et al.

Cancer Res Published OnlineFirst September 27, 2012.

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