AbstractNasopharyngeal carcinoma (NPC) is an Epstein-Barr virus–associated malignancy most common in East Asia
and Africa. Aberrant expression of Jab1/CSN5, a negative regulator of the cell-cycle inhibitor p27, is correlatedwith reduced p27 expression and associated with advanced tumor stage and poor prognosis in several humancancers. In this study, we examined the functional relationship between Jab1 and p27 protein expression in NPC.Immunohistochemical analysis showed an inverse association between Jab1 and p27 in NPC tissue samples, andoverexpression of Jab1 correlated with poor survival in patients with NPC. Mechanistically, Jab1 and p27 werefound to interact directly in NPC cells, with Jab1mediating p27 degradation in a proteasome-dependent manner.Knockdown of Jab1 resulted in a remarkable increase in p27 levels and inhibition of cell proliferation, indicatingthat Jab1 targets p27 for degradation, thereby controlling its stability. Jab1 depletion also enhanced the antitumoreffects of cisplatin in NPC cells. Together, our findings suggest that Jab1 overexpression plays an important role inthe pathogenesis of NPC through Jab1-mediated p27 degradation. Jab1 therefore represents a novel diagnosticmarker and therapeutic target in patients with NPC. Cancer Res; 72(7); 1890–900. �2012 AACR.
IntroductionNasopharyngeal carcinoma (NPC), originating from the
epithelial lining of the nasopharynx, is a squamous cell carci-noma (1). This neoplasm has remarkable ethnic and geograph-ic distribution, with a high prevalence in southern China,Southeast Asia, Northern Africa, and Alaska (2, 3). The annualincidence peaks at 50 cases per 100,000 people in the endemicregions but is rare in the Western world (1 per 100,000; ref. 2).Etiologic studies have indicated that Epstein-Barr virus (EBV)infection, environmental factors, and genetic susceptibility areassociated with NPC (1, 4). In China, more than 95% of NPCsare nonkeratinizing carcinoma, whereas less than 5% are
keratinizing carcinoma; thus, in our study, we use CNE1(keratinizing carcinoma) and CNE2 (nonkeratinizing carcino-ma) cells to represent the 2 main histologic types (5).
Radiotherapy and adjuvant cisplatin chemotherapy havebecome the standard treatments for NPC (6). The 5-yearsurvival rate after treatment is approximately 70% (7). Thirtyto forty percent of patients will develop distant metastasiswithin 4 years (8), and once metastasis occurs, the prognosis isvery poor.
To develop better treatment approaches, it is important tounderstand the molecular basis of the development and pro-gression of NPC. Genetic alterations have been reported inNPCs including a frequent reduction of p27 expression (9, 10).Furthermore, low p27 expression levels may contribute to theaggressive behavior of NPCs (11). Overexpression of cyclin EmRNA has been shown to predict poor prognosis in NPC andhas been correlated with an advanced stage of NPC and a lowoverall survival rate (12). Recently, it has been reported thatPIK3CA gene amplification is frequently observed in advanced-stage NPC, which emphasizes the association between PIK3CAgene amplification and poor prognosis (11). It has also beenshown that Akt promotes cell proliferation and survival inNPCs (4, 13). However, additional molecular abnormalitiesresulting in the deregulation of cell-cycle progression may alsooccur.
Jab1/CSN5 (Jab1 hereafter), as we initially identified as a c-Jun coactivator, is also known as the fifth component of theCOP9 signalosome (CSN) complex (CSN5; refs. 14, 15). Jab1promotes cell proliferation and inactivates p27 by inducingtranslocation of p27 from the nucleus to the cytoplasm, whichaccelerates p27 degradation through the ubiquitin-dependentproteasome pathway and promotes cell-cycle progression (16).
Authors' Affiliations: 1Department of Pathophysiology, ZhongshanSchool of Medicine, Sun Yat-Sen University; 2State Key Laboratory ofOncology in South China, Sun Yat-Sen University Cancer Center, Guangz-hou, Guangdong, PR China; 3Department of Systems Biology, The Uni-versity of Texas MD Anderson Cancer Center; and 4Cancer BiologyProgram and Experimental Therapeutic Program, The University of TexasGraduate School of Biomedical Sciences at Houston, Houston, Texas
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
Y. Pan and Q. Zhang contributed equally to this work and should beconsidered as co-first authors.
Corresponding Authors: Francois X. Claret, Department of SystemsBiology, The University of Texas MD Anderson Cancer Center, Houston,TX 77030. Phone: 713-563-4204; Fax: 713-563-4205; E-mail:[email protected]; and Huiling Yang, Department of Pathophysi-ology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangz-hou, Guangdong 510080, PR China. Phone: 86-20-87332268; Fax: 86-20-87331209; E-mail: [email protected]
p27 is a universal cyclin-dependent kinase (Cdk) inhibitor thatdirectly inhibits the enzymatic activity of cyclin–Cdk com-plexes, resulting in cell-cycle arrest at G1 (17). In addition, p27protein levels are increased in quiescent cells and rapidlydecrease after cells are stimulated with mitogens (18).Although transcriptional regulation is possible, the cellularabundance of p27 is primarily regulated at the posttransla-tional level by the ubiquitin–proteasome pathway (19). Jab1overexpression is correlated with a loss of p27 and a lowerrate of survival in patients with breast cancer, suggesting arole in breast cancer pathogenesis (20). This inverse associ-ation between Jab1 and p27 expression has also beenobserved in anaplastic large cell lymphoma (21), ovariancancer (22), pancreatic adenocarcinomas (23, 24), and othercancer types (25–27). However, the mechanisms leading top27 downregulation in NPCs remain undefined. Because Jab1overexpression is correlated with the loss of p27 in severalcancers and low p27 expression is associated with highertumor grades (28), we hypothesized that Jab1 functions as anegative regulator of p27 and as such may play a role in thepathogenesis of NPCs.To test our hypothesis, we assessed Jab1 and p27 expression
in a series of 45 NPC and 30 nasopharyngeal inflammationtissue specimens. We found that Jab1 overexpression wasassociated with no or low expression of p27 in these samples.To further elucidate the role of Jab1 in p27 degradation inNPCs, we infected NPC cell lines with an adenoviral vectoroverexpressing Jab1 and found that p27 levels were signi-ficantly reduced. We also detected a direct physical inter-action between Jab1 and p27 in NPC cells. Furthermore,inhibition of endogenous Jab1 expression with specificsiRNAs resulted in a substantial increase of p27 levels andinhibition of cell proliferation, indicating that Jab1 controls thestability of p27 by targeting it for degradation in NPCs. Inter-estingly, siRNA-mediated depletion of Jab1 inhibited cell pro-liferation and enhanced the antitumor effects of cisplatin inNPC cells. This may suggest that Jab1 is a potential target fortreating NPCs.
Materials and MethodsPatients and tissue samplesAll patients were from the Cancer Center of Sun Yat-Sen
University (Guangzhou, Guangdong, PR China) in 2003. Thestudy group consisted of 36 men and 9 women with NPC whounderwent radiotherapy and the control group consisted of13 men and 17 women with nasopharyngeal inflammation.Patients who had preoperative diagnosis and did not receivepreoperative chemoradiation treatment were selected forthis study on the basis of the availability of archived paraf-fin-embedded NPCs and nasopharyngitis tissue blocks forimmunohistochemical analysis. Ethical approval wasobtained from the cancer center and fully informed consentfrom all patients before sample collection. Surgical staging oftumors had been done according to the American JointCommittee on Cancer tumor-node-metastasis system andtumor grading was based on currently used histopathologiccriteria.
ReagentsCell culture medium was from Mediatech Inc. and FBS was
obtained from Gibco. The antibodies used were Jab1 (SantaCruz); p27 and PARP (BD Biosciences PharMingen); caspase-3,lamin A/C, and Myc-tag (Cell Signaling Technology; and Flagand b-actin (Sigma-Aldrich). The Lipofectamine Plus andOligofectamine reagents were from Invitrogen. NE-PER nucle-ar and cytoplasmic extraction reagents andWestern LightningChemiluminescence Plus reagent were from Thermo ScientificPierce. Annexin V/PI kit was from BD Biosciences. Cell Pro-liferation Kit was from Roche.
Human tissues and immunohistochemical analysisA total of 75 formalin-fixed, paraffin-embedded human
primary NPCs and noncancerous inflamed nasopharyngealspecimens from 45 patients with NPC and 30 patients withnasopharyngitis were analyzed. The p27 and Jab1 levels in theformalin-fixed, paraffin-embedded tissue sections were mea-sured by immunohistochemical analysis, as previouslydescribed (24). Briefly, heat-induced retrieval of Jab1 andp27 antigens was conducted, and the slides were incubatedwith the primary antibodies. An immunoreactionwas detectedwith the LSABþ kit from DakoCytomation. We used 3,30-diaminobenzidine as the chromogen and hematoxylin as thecounterstain. Expression levels of Jab1 and p27 were evaluatedby counting at least 500 tumor cells in representative high-power fields. Tumor cells were considered positive for Jab1 orp27 when nuclear or cytoplasmic staining was present. Foreach tumor, we determined aproportion score and an intensityscore. The proportion score represented the estimated fractionof positively stained cells (0, <5%; 1, 5%–35%; 2, 35%–65%; and3, >65%). The intensity score represented the estimated aver-age staining intensity of the positive cells (0, no staining; 1,weak staining; 2, intermediate staining; and 3, strong staining).The overall amount of protein present in each tumor was thenexpressed as the average of the proportion and intensity scoresfor negative and positive cells (ranges, 0 and 0.5–3, respective-ly). We defined the scores 0, 0.5 to 1.0, 1.5 to 2.0, and 2.5 to 3.0 asnegative (�), weakly positive (þ), and highly positive [(þþ),(þþþ)], respectively.
Cell culturesHuman NPC cell lines CNE1 (well-differentiated, from
Cancer Center, Sun Yat-Sen University), CNE2 (poorly differ-entiated, from Cancer Center, Sun Yat-Sen University),HONE1 (poorly differentiated, the generous gift of Prof.Ronald Glaser, The Ohio State University Medical Center,Columbus, OH), and C666.1 (undifferentiated, the generousgift of Prof. Kwok-Wai Lo, Prince of Wales Hospital, TheChinese University of Hong Kong, Shatin, N.T., Hong Kong)were cultured in RPMI medium containing 10% FBS andpenicillin–streptomycin sulfate. 293T human embryonic kid-ney cells were cultured in Dulbecco's Modified Eagle's Medi-um (Invitrogen) with 10% FBS and penicillin–streptomycin.HOK16B (normal keratinocyte cells, the generous gift of Prof.Jeffrey N. Myers, MD Anderson Cancer Center, Houston, TX)was cultured in keratinocyte serum-free media containing 30mg/mL bovine pituitary extract, 0.2 ng/mL EGF, 5% FBS, and
Jab1 Expression Is Associated with Poor Prognosis in NPC
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penicillin–streptomycin sulfate. All cell lines were incubatedat 37�C in an atmosphere of 5% CO2.
Cell extracts and immunoblottingCells in the log phase of growthwere collected, washed twice
in cold PBS, and lysed as described previously (29). Nuclearfractions of the indicated cell lysates were separated with NE-PER nuclear and cytoplasmic extraction reagents according tothe manufacturer's directions. Proteins were separated by 10%SDS-PAGE, transferred to nitrocellulose membranes, andprobed with anti-Jab1, anti-p27, anti-Myc, anti-PARP, andanti-caspase-3. b-Actin was used as the internal positive con-trol for all immunoblots. Immunoreactive bandswere detectedwith horseradish peroxidase–conjugated secondary antibodieswith theWestern Lightning Chemiluminescence Plus Reagent.The protein levels were quantified by ImageJ software (NIH,Bethesda, MD; http://rsb.info.nih.gov/ij). Activity of PARP andcaspase-3 was measured as percentage of cleavage bandintensity to the total bands and calculated as follows: % PARPor caspase-3¼ 100%� Tc/Tt, where Tc is the intensity value ofthe cleavage bands and Tt is the intensity value of the totalbands.
Adenoviral vectors and gene transductionA recombinant adenoviral vector expressing a doxycycline-
regulated (Tet-Off) form of human Jab1 (Ad-Myc-Jab1) wasconstructed, as previously described (24). NPC cells weretransduced for 48 hours with a regulatory virus (adeno-XTet-Off, Clontech) and Ad-Myc-Jab1 at a multiplicity of infec-tion of 50 in a growth medium containing 10% FBS in thepresence or absence of 1 mg/mL doxycycline, a tetracyclineanalogue.
Proteasome inhibition assaysNPC cells were treated with the proteasome inhibitors LLnL
(35 mmol/L) or MG132 (30 mmol/L), or LLM (25 mmol/L), anddimethyl sulfoxide (DMSO) as a control. After 16 hours oftreatment, the cells were harvested, whole-cell lysates wereprepared, and immunoblotting was done with antibodiesagainst p27. b-Actin antibodies were used as a loading control.NPC cells were transduced with Ad-Myc-Jab1 as describedearlier. Thirty-two hours after infection, proteasome inhibitors(LLnL orMG132 or LLM) or DMSO (control) were added to themedium for 16 hours. Forty-eight hours after infection, whole-cell lysates were prepared, and proteins were analyzed byimmunoblotting.
siRNA, DNA transfection, and coimmunoprecipitationassays
For the siRNA analysis, Jab1 siRNA and control siRNAoligonucleotides were cloned into an RNAi vector (BD Bios-ciences PharMingen) according to the manufacturer's instruc-tions and as described by Kouvaraki and colleagues (24). TheMyc-Jab1 and Flag-p27, GFP-Jab1, and Cherry-p27 plasmidswere transfected into either 293T or CNE1 cells with theLipofectamine Plus reagent, and cells were either lysed forcoimmunoprecipitation assays or examined with a fluores-cence microscope.
Coimmunoprecipitation assays were done with the whole-cell lysates from NPC cells or DNA-transfected 293T cells, asdescribed earlier (24). Briefly, cell lysates were incubated inradioimmunoprecipitation assay (RIPA) buffer for 4 hours at4�C with either anti-Myc or anti-Jab1 antibodies or nonim-mune mouse serum as a control. Proteins were separated by12% SDS-PAGE, transferred to nitrocellulose membranes, andanalyzed by immunoblotting.
Cell proliferation assayThe MTT assay was used to evaluate cell viability, as de-
scribed previously (29). Briefly, 48 hours after transfection,NPC cells were seeded in 96-well plates (500 cells per well forgrowth curves or 2,000 cells per well for cisplatin treatment)in 100 mL of RPMI-1640 medium with 8% FBS. After theindicated incubation period, the MTT labeling reagent (finalconcentration, 0.5 mg/mL) was added, and the spectropho-tometric absorbance of the samples was read with a micro-plate (ELISA) reader at 570 nm. The ratios (%) of cell survivalor colony formation were calculated as the ratio of theindicated treatment group to the control group as follows:% survival ratio ¼ 100% � Nt/Nc, and % inhibition ratio ¼100%� (1�Nt/Nc), where Nt is the optical density (OD) value(colony number) of the treatment group and Nc is the ODvalue (colony number) of control group.
Colony formation assayForty-eight hours after transfection, NPC cells (200 cells
per well) were plated in 6-well plates for growth analysis inRPMI-1640 medium with 8% FBS. The following day, the cellswere treated with 0 or 0.5 mmol/L cisplatin for 48 hours. After10 days, the cells were fixed with methanol, stained with 0.1%crystal violet, and scored by counting with an inverted
Table 1. Characteristics of 45 patients withNPC included in the study
microscope, with the standard definition that a colony consistsof 50 or more cells.
Flow cytometric analysis of the cell cycle and apoptosisForty-eight hours after transfection, the cells were collected
and fixed overnight in 75% cold ethanol at �20�C. Cells werewashed twice in cold PBS and labeled with propidium iodide(PI) and analyzed immediately after staining with a FACScanflow cytometer (BD Biosciences) and FlowJo software.Flow cytometric analysis was used to differentiate between
living, early apoptotic, late apoptotic/necrotic, and necroticcells by staining with Annexin V-FITC and PI. Briefly, aftertreating cells with 5 mmol/L cisplatin for 48 hours, the trans-fected cells were labeled with Annexin V-FITC and PI accord-
ing to the manufacturer's recommendations. Quantification ofAnnexin V-FITC and PI binding was conducted with a FACS-can flow cytometer.
Statistical analysisFisher's exact test was used to compare the expression and
localization of Jab1 and p27 with various clinicopathologicvariables. The Spearman test was used to analyze the associ-ation between cytoplasmic Jab1 and p27. Overall survival wasdefined as the time from diagnosis to death. Kaplan–Meieranalysis was used to examine the association of Jab1 and p27expression and survival. Statistical analysis for the results wasdone by Student t test when only 2 groups or one-way ANOVAwhenmore than 2 groups were examined. Differences between
Figure 1. Expression patterns ofJab1 and p27 in nonneoplastictissues and NPC tissues. A, overallJab1 immunoreactivity was low innonneoplastic tissue (left) comparedwithNPC (middle). Right, percentageof cases with Jab1 expression innasopharyngeal tissues and NPC. B,nuclear p27 was strongly expressedin nonneoplastic tissue (left). A caseof NPC, with the predominantlycytoplasmic staining is shown(middle). Right, percentage of caseswith p27 expression in nonneoplastictissues and NPC. Originalmagnification,�200; insets�400. C,tumors with cytoplasmic Jab1expression were associated withnuclear and cytoplasmic p27. Rvalues and P values from Spearmantest. D, Kaplan–Meier analysis of theassociation of Jab1 expression (left)and survival as well as association ofcytoplasmic p27 (right) and survival.
groups were stated to be statistically significant when P < 0.05.All computations were carried out with SPSS 16.0 (SPSS)
ResultsPatient characteristics and demographics
To analyze the Jab1 and p27 expression patterns, we used 45NPC tissue samples (median age, 41; range, 20–73 years) and 30nasopharyngeal inflammation tissue samples (median age, 36;range, 14–65 years). The clinicopathologic characteristics ofthe patients with NPC are summarized in Table 1.
Jab1 and p27 expression patterns in tissue samples frompatients with noncancerous inflamed and NPC
Immunohistochemical analysis of nasopharyngitis and NPCtissue samples showed that Jab1was localized to the cytoplasmand nucleus of NPC cells. About 74% had positive cytoplasmicstaining and 56% had positive nuclear staining for Jab1 inNPCs. The staining in these cases was higher than that in the
noncancerous inflamed nasopharyngeal tissue, which was 10%in cytoplasmic and 16% in nuclear, respectively, P < 0.01 (Fig.1A). In contrast, nuclear p27 level was higher in noncancerousinflamed nasopharyngeal tissues (67%) than that in the NPCtissues (58%). However, 64% cytoplasmic p27 staining wasobserved in NPC cases, which was higher than that in non-cancerous tissue (37%), p < 0.05 (Fig. 1B). Furthermore, cyto-plasmic Jab1 expression in NPC tissues was inversely associ-ated with nuclear p27 patterns (R ¼ �0.389, p ¼ 0.008) andcorrelated with cytoplasm p27 (R ¼ 0.328, P ¼ 0.028; Fig. 1C).
Correlation of Jab1 and p27 expression with clinicaloutcome
Survival analysis by the Kaplan–Meier method showed thathigh expression of Jab1 tended to correlate with a poorprognosis, P < 0.01 (Fig. 1D). Similar results were obtainedfrom the subsets of patients with nonkeratinizing carcinoma(Supplementary Fig. S1A) and keratinizing squamous
Figure 2. Jab1 is overexpressedand regulates p27 levels in NPCcells. A, whole-cell lysates wereprepared from the cells asindicated. b-Actin was usedas a control for protein loading andintegrity. The relative Jab1 intensityfrom 5 samples is shown. B,proteolysis of p27 in NPC cells issensitive to proteasome inhibitors.C, inducible expressionof Ad-Myc-Jab1 (Tet-off) downregulatesendogenous p27 levels in NPC celllines. Lysates were prepared fromAd-Myc-Jab1–infected cells in theabsence (�) or presence (þ) ofdoxycycline (Dox). D, NPC cellswere transduced with the Ad-Myc-Jab1 virus and treated as indicatedwith proteasome inhibitor. Theprotein levels were quantified byImageJ software.
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carcinoma (Supplementary Fig. S1B). The median for sur-vival time in patients with negative and weakly positive Jab1tumors was 34 months, compared with 14 months inpatients with highly positive Jab1 tumors (P < 0.01). Mean-while, cytoplasmic p27-positive expression correlated with apoor prognosis, the median for survival time was 19 monthsin positive group whereas 36 months in negative group (P <0.05).
Jab1 promotes p27 degradation through proteolysis andis sensitive to proteasome inhibitorsImmunoblotting also showed strong Jab1 expression in
NPC cells but not normal keratinocyte cells, where weak Jab1expression was detected (Fig. 2A). Treatment of NPC cellswith specific proteasome inhibitors resulted in a significantincrease in total p27 levels (Fig. 2B). To investigate whetherJab1 overexpression in NPC cells downregulates p27 levels,we transduced those 4 cell lines with Jab1 adenovirus in theabsence or presence of doxycycline and measured Myc-Jaband p27 levels 48 hours after infection. Using this inducible(Tet-Off) system, Jab1 overexpression led to a significantdecrease in p27 levels in all cell lines tested (Fig. 2C).Downregulation of p27 mediated by Jab1 overexpressionwas inhibited in cells that had been treated with protea-some inhibitors (LLnL, MG132, and LLM) but not in thosetreated with DMSO (Fig. 2D), indicating that Jab1 promotesp27 degradation through proteolysis and is sensitive toproteasome inhibitors. These data provide direct evidenceof a functional relationship between Jab1 and p27 in NPCcells.
Jab1 specifically interacts with p27To determine whether Jab1 and p27 directly interact,
we conducted coimmunoprecipitation analyses. Lysateswere immunoprecipitated with anti-Jab1 antibodies andimmunoblotted with anti-p27 antibodies (Fig. 3A). Wefound that endogenous Jab1 indeed interacts with p27(Fig. 3A). This interaction was specific because the Jab1-containing lysates were not immunoprecipitated withnonimmune rabbit serum. To confirm these results, weexamined the ectopic Jab1 and p27 interactions by trans-fecting Myc-Jab1 and Flag-p27 plasmids into 293T cells.As expected, Myc-Jab1 binds to Flag-p27 (Fig. 3B). More-over, data showed most p27 in a predominantly nuclearlocation when Cherry-p27 was transfected into CNE1 cells,but both nuclear and cytoplasmic staining were seen whenGFP-Jab1 was induced (Fig. 3C and Supplementary Fig. S3).These data strongly indicate that Jab1 associates with p27in NPC cells.
Depletion of Jab1 by siRNA increases accumulation ofp27, induces cell-cycle arrest, and inhibits cellproliferation in NPC cell linesTo assess the effect of silencing Jab1 in human NPC cells,
we transfected NPC cells with Jab1 siRNA oligonucleotidesor control siRNA oligonucleotides cloned into the RNAi-vector system. Forty-eight hours after transfection, Jab1 pro-tein levels were substantially decreased (>97%) in all Jab1
siRNA–transfected cell lines compared with cells transfectedwith the control siRNAoligonucleotides (Fig. 4A). The decreasein endogenous Jab1 protein expression was associated with asignificant increase in p27 levels and nuclear accumulation ofp27 in nucleus (Fig. 4A), suggesting the biologic importance ofJab1 in regulating p27 proteolysis in NPCs.
Next, to determine whether knockdown of Jab1 would leadto an increase in p27 activity, as indicated by inhibition of theG1–S-phase progression, we used PI staining to analyze the
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Figure 3. Jab1 specifically interacts with p27. A, cell lysates wereimmunoprecipitated (IP) with either nonimmune mouse serum or Jab1and immunoblotted with anti-p27. Immunoglobulin G heavy chain (HC)and light chain (LC) are indicated. B, 293T cells were transfected witheither Myc-Jab1 or Flag-p27 or both for 48 hours, and then celllysates were immunoprecipitated with Myc and immunoblotted withFlag. Cell lysates immunoblotted with the indicated antibodies areshown at the bottom. C, CNE1 cells were transfected with a GFPvector and Cherry-p27 or GFP-Jab1 and Cherry-p27 for 48 hours andthen examined under a fluorescence microscope. Originalmagnification, �200.
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proportion of cells that were in the G1 and S-phases of the cellcycle. In CNE1 cells, 24% of the control siRNA-transfectedcells were in the S-phase compared with 37% of cells inthe G1 phase, and 14% of the Jab1 siRNA–transfected cellswere in the S-phase compared with 47% of cells in the G1 phase(Fig. 4B). Similar results were obtained from CNE2 cells (25%in S-phase and 37% in G1 phase for control cells vs. 18% inS-phase and 42% in G1 phase for Jab1 siRNA–transfected cells)and HONE1 cells (33% in S-phase and 28% in G1 phase forcontrol cells vs. 22% in S-phase and 42% in G1 phase for Jab1siRNA–transfected cells). To confirm the above conclusion,we next conducted a coimmunoprecipitation experimentto measure the amount of p27–cyclin E–Cdk2 inhibitorycomplex formation. As shown in Fig. 4C, p27 levels in Jab1knockdown cells were higher when immunoprecipitated withCdk2, indicating more complex formation. These data suggestthat downregulation of Jab1 induces stabilization of p27,therefore increasing p27 levels and activity.
To investigate whether downregulation of Jab1 was involvedin growth suppression, we first analyzed whether the siRNA-mediated Jab1 inhibition could recapitulate the tumor sup-pressor effect in NPC cell lines. The expression levels of Jab1protein in Jab1 siRNA–transfected NPC cells were significantlyless than those in the control siRNA-transfected cells (Fig. 4A).Moreover, the siRNA-mediated Jab1 knockdown also signifi-cantly inhibited the in vitro growth and colony formation ofNPC cells (Fig. 5A andB). Notably, the growth-inhibitory effectsof the Jab1 siRNA knockdown suggest that Jab1 targeting is amechanism of tumor suppression in NPC cells. In addition, wetested the effect of knocking down Jab1 on apoptosis. Forty-eight hours after the siRNAwas transfected, we usedAnnexin Vand PI staining (Fig. 5C) or analysis of cleaved PARP andcaspase-3 (Fig. 5D). However, there was no significant differ-ence between the control and Jab1 siRNA–treated groups; thus,we cannot conclude that Jab1 knockdown initiates apoptosisunder these conditions.
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Figure 4. Downregulation of Jab1increases p27 levels and inducescell-cycle arrest in NPCs. A, cellswere transfected with either ansiRNA targeting Jab1 or ascrambled sequence (control);48 hours later, the total cellularproteins and nuclear proteins wereisolated and immunoblotted withanti-Jab1 and anti-p27 antibodies.Anti-b-actin or Lamin A/Cantibodies were used as loadingcontrols. B, NPC cells weretransfected as described in A; 48hours later, cells were stained withPI and then analyzed by flowcytometry. Left, representativeresults of cell-cycle assays withNPC cells. Right, the percentage ofG1 and S-phase cells are shown.Data represent 3 independentexperiments, mean � SD�, P < 0.05; ��, P < 0.01. C, cellswere transfected with either ansiRNA targeting Jab1 or ascrambled sequence (control); 48hours later, cell lysates wereimmunoprecipitated (IP) with Cdk2and immunoblotted (IB) withanti-p27. Cont-si, control siRNA;HC, heavy chain; Jab1-si, Jab1siRNA; LC, light chain.
Pan et al.
Cancer Res; 72(7) April 1, 2012 Cancer Research1896
Jab1 depletion enhances the antitumor effects ofcisplatin in NPCBecause cisplatin is themain treatment ofNPC,we proposed
to investigate that Jab1 may be involved in the antitumoreffects of cisplatin. We first tested whether the inhibition ofJab1 enhances the antitumor effects of cisplatin by MTT assay.Data showed that 21%, 18%, and 7% of CNE1, CNE2, andHONE1 cells treated with control siRNA and cisplatin wereinhibited, whereas, 38%, 38%, and 26% cells were inhibited byJab1 siRNA and cisplatin, suggesting that Jab1-knockdownNPC cells were more sensitive to cisplatin than cells treatedwith control siRNA (Fig. 6A). To test whether Jab1 plays a rolein anchorage-independent growth in response to cisplatin,
which may reflect in vivo tumorigenicity, we conducted acolony formation assay in NPC cells treated with Jab1 siRNAand cisplatin. We found that the Jab1-knockdown cells hadsignificant inhibition of colony formation (73% in CNE1, 90% inCNE2, and 66% in HONE1) compared with the control siRNA-treated cells (22% in CNE1, 60% in CNE2, and 14% inHONE1) inresponse to cisplatin (Fig. 6B).
We further examined whether Jab1 knockdown couldenhance cisplatin-induced apoptosis in NPC cells. We foundthat cisplatin inducedmore apoptosis in Jab1-knockdown cellsthan that in control siRNA-treated cells, which is 1.4-fold inCNE1 and 4.7-fold in CNE2, respectively (Fig. 6C), when usingAnnexin V and PI staining, and 1.6-fold in CNE1 and 3.2-fold in
Figure 5. Knockdown of Jab1inhibits cell proliferation. A,NPCcellswere transfectedwith Jab1 siRNA for48 hours, and then cell growth wasdetermined by an MTT assay. B,representative results of colonyformation assays with NPC cellstreated with control siRNA or 1 nmol/L (þ) or 5 nmol/L (þþ) of Jab1 siRNA.Bottom, quantification of the relativenumber of colonies is shown. Datarepresent 3 independentexperiments, mean� SD. �, P < 0.05;��,P < 0.01. C, cells were treatedwithcontrol siRNA or Jab1 siRNA for 48hours and then stained with AnnexinV and PI and measured by flowcytometry. Bottom, quantification ofthe percentage of living cells. Datarepresent 3 independentexperiments, mean � SD. D, cellswere transfected with siRNA for 48hours, and then cell lysates wereimmunoblotted with the indicatedantibodies for apoptotic analysis. C,cleaved; Cont-si, control siRNA;Jab1-si, Jab1 siRNA.
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2.0
1.6
1.2
0.8
0.4
0.0
1.0
0.8
0.6
0.4
0.2
0.0
Cont-si
Jab1-si:
Cont-si:
Jab1-si: + +Cont-si:
Jab1-si+
Jab1-si++
Cont-si* *
**
****
Jab1-si:
PARP
C-PARP
Caspase-3
C-caspase-3
β-Actin
+ +
Cont-si: + +
+ +
–
+
+
–
++
–
Jab1-siCont-si
Jab1-siCont-si
CNE1
Absorb
ance (
570 n
m)
Absorb
ance (
570 n
m)
Absorb
ance (
570 n
m)
**** **
**
****
**
**
*
CNE2
CNE1 CNE2
CNE1 CNE2
HONE1
CNE1 CNE2 HONE1
CN
E1 C
NE
1
PI
Via
ble
cells
%
Rela
tive
colo
nie
s (
%)
CN
E2 C
NE
2
HO
NE
1
Jab1 Expression Is Associated with Poor Prognosis in NPC
www.aacrjournals.org Cancer Res; 72(7) April 1, 2012 1897
CNE2, when using Hoechst 33342 staining (Supplementary Fig.S2). However, therewere no significant differences in apoptosisinduced by control and Jab1 siRNA. Proteolytic cleavage ofPARP and cleaved caspase-3 is the hallmark of apoptosis. Thus,we also examined the effect of Jab1 siRNA on the proteolytic
cleavage of PARP and cleaved caspase-3 in response to cis-platin. Compared with the control siRNA-treated cells, cisplat-in consistently induced more proteolytic cleavage of PARP(12% change in CNE1, 30% change in CNE2, and 6% change inHONE1) and cleaved caspase-3 (11% change in CNE1, 50%
Figure 6. Jab1 depletion enhancesthe antitumor effects of cisplatin inNPCcells. A, forty-eight hours afterthe cells were treated with control(Cont-si) or Jab1 siRNA (Jab1-si),NPC cells were treated with2 mmol/L of cisplatin (CP) foranother 48 hours, and the growth-inhibitory effects of cisplatin werequantified by an MTT assay. Theinhibition ratio (%) is marked onthe graphs. Data represent3 independent experiments,mean � SD. ��, P < 0.01. B,1 nmol/L (þ) or 5 nmol/L (þþ) ofJab1 siRNA–treated NPC cellswere seeded into 6-well plates andexposed to cisplatin for 48 hoursand, 10 days later, the number ofcolonies formed were counted.Left, representative results ofcolony formation assays. Right,quantification of the relativenumber of colonies. The inhibitionratios (%) are marked on thegraphs. Data represent 3independent experiments, mean �SD. �, P < 0.05; ��, P < 0.01. C,siRNA-transfected NPC cells werestained with Annexin V and PI aftercisplatin treatment for 48 hours.Left, representative results ofapoptotic assays in NPC cells.Right, quantification of thepercentage of apoptotic cells. Datarepresent 3 independentexperiments, mean � SD.�, P < 0.05; ��, P < 0.01. D, siRNA-transfectedNPCcell were exposedto 10 mmol/l of CP for 48 hours;apoptotic cells were measured bythe Western blot analysis ofcleaved (C) PARP and cleavedcaspase-3. Protein levels werequantified by ImageJ software.
Pan et al.
Cancer Res; 72(7) April 1, 2012 Cancer Research1898
change in CNE2, and 31% change in HONE1) in Jab1-knock-down cells (Fig. 6D).
DiscussionThe molecular basis of NPC pathogenesis remains poorly
defined, and this has hindered the development of new treat-ments. Therefore, identification of novel mechanisms involvedin NPC oncogenesis is needed.In this study, we identified the role of Jab1-mediated p27
degradation in NPC oncogenesis. First, we found that Jab1 wasaberrantly expressed in either cytoplasm or nucleus NPCtissues compared with noncancerous inflamed nasopharyn-geal tissue. All (14 of 14) strong Jab1-positive patients diedwithin 41 months whereas 11 of 31 weak or Jab1-negativepatients were alive after 60 months. We also showed that Jab1protein is overexpressed in NPC cell lines but not in pairednormal keratinocyte cells. Our findings on p27 expression alsoconfirm those of previous reports. For instance, Hwang andcolleagues (11) reported that in patients with NPC, 47 of 69cases expressed low levels of p27. In our study, 29 of 45 NPCcases (64%) expressed low or no nuclear p27, and 28 cases(62%) expressed low or no cytoplasmic p27. In addition, thenuclear cytoplasmic translocation of p27 has been observed inhuman tumors and is associated with poor survival (30–33), itwas in agreement with our studies that patients with NPCwithcytoplasmic-positive p27 had a poor survival.Furthermore, cytoplasmic Jab1 expression in NPC tissues
was inversely associated with nuclear p27 patterns (R ¼�0.389, p ¼ 0.008) and correlated with cytoplasm p27 (R ¼0.328, P¼ 0.028). Our finding of an inverse correlation betweenJab1 and p27 expression levels in NPC is in agreement with ourpreviously published results on breast cancer and pancreaticcarcinomas (20, 24) and similar associations reported in otherepithelial or lymphoidmalignancies (21, 22). This suggests thatJab1 has a physiologic role in controlling p27 levels in NPCs. Toour knowledge, this is the first study on the expression andfunction of Jab1 in NPCs to be reported.Jab1 promotes cell proliferation by interacting directly
with p27 and induces nuclear export and subsequent p27degradation (34). p27 is a critical component of the cell-cyclemachinery (16). As an inhibitor of cyclin E–Cdk2, p27 plays apivotal role in controlling cell proliferation and therefore thecell's entry into S-phase and exit from G1 phase duringdevelopment and tumorigenesis (18, 35). We infected NPCcell lines with an inducible Jab1-expressing adenoviral vectorand found that p27 levels were significantly reduced. Theseresults are in agreement with those we previously obtained inbreast cancer and pancreatic cell lines (20, 24). Furthermore,knockdown of endogenous Jab1 expression with siRNA in
NPC cell lines led to a significant increase in total p27 levelsand nuclear accumulation of p27 in the nucleus and p27–cyclin E–Cdk2 complex, substantial decreases in the numberof S-phase cells, and increases in the number of G1 phasecells. In addition, a direct p27–Jab1 interaction was con-firmed by coimmunoprecipitation assays and fluorescentmicroscopy. These results provide additional evidence thatJab1 controls p27 levels posttranslationally by acceleratingp27 degradation in NPCs.
We also showed that loss of Jab1 resulted in growth inhi-bition in NPC cell lines. Similarly, a study by Supriatno andcolleagues (36) showed that suppression of Jab1 inhibited thegrowth of human head and neck cancer cells (HNt and HSYcells) and delayed tumor growth in murine xenografts. Itsuggests that strong expression of Jab1 not only representsa prognostic marker for malignant transformation but alsocontributes to cancer cell proliferation and survival. Further-more, we found that knockdown of Jab1 sensitized NPC cells tocisplatin chemotherapy. Jab1 siRNA–treated cells were moresusceptible to apoptotic stimuli than the control siRNA-trea-ted cells. Taken together, the data implicate Jab1 as a potentialbiomarker and further suggest exploration of whether decreas-ing Jab1 levels or function would overcome resistance tocisplatin chemotherapy in NPCs. The significance of thesefunctions of Jab1 in NPCs needs to be explored in futuremechanistic studies.
Overall, we found Jab1 to be overexpressed and inverselycorrelated with p27 expression levels in NPC. Jab1 overexpres-sion associated with low p27 levels or other factors maycontribute to uncontrolled cell-cycle progression and tumor-igenesis. Depletion of Jab1 by siRNA, which leads to inhibitionof proliferation, enhanced cisplatin activity in NPC. Therefore,modulation of the Jab1 gene product is a novel target fortherapies in NPCs.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
AcknowledgmentsThe authors thank Kate J. Newberry for editing the manuscript.
Grant SupportThis work was supported by a fellowship from China Scholarship Council
(2010638087 to Y. Pan), a grant from National Cancer Institute (RO1-CA90853 toF.X. Claret); National Natural Science Foundation of China (81071837; 30670627);Natural Science Foundation of Guangdong Province, China (9251008901000005;06021210); and Scientific and Technological Project of Guangdong, China(2008A030201009; 2010B050700016 to H. Yang).
The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received October 22, 2011; revised January 25, 2012; accepted January 31, 2012;published OnlineFirst February 20, 2012.
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2012;72:1890-1900. Published OnlineFirst February 20, 2012.Cancer Res Yunbao Pan, Qingxiu Zhang, Ling Tian, et al. Pathogenesis of Nasopharyngeal CarcinomaJab1/CSN5 Negatively Regulates p27 and Plays a Role in the
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