Cancer Research Identification of Biomarkers for ... · has not yet been elucidated. High throughput proteomics has introduced a new approach to radioresistant research that aims

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Published OnlineFirst April 20, 2010; DOI: 10.1158/0008-5472.CAN-09-4099

Integrated Systems and Technologies
Cancer
Research

Identification of Biomarkers for Predicting NasopharyngealCarcinoma Response to Radiotherapy by Proteomics

Xue-Ping Feng, Hong Yi, Mao-Yu Li, Xin-Hui Li, Bin Yi, Peng-Fei Zhang, Cui Li, Fang Peng, Can-E Tang,Jian-Ling Li, Zhu-Chu Chen, and Zhi-Qiang Xiao

Abstract

Authors' AMinistry ofProvince, C

Note: SupResearch O

X-P. Feng,

Corresponteomics ofUniversity,4327239; F

doi: 10.115

©2010 Am

Cancer R

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Radiotherapy is the primary treatment for nasopharyngeal cancer (NPC), but radioresistance remains aserious obstacle to successful treatment in many cases. To identify the proteins involved in this resistanceand to evaluate their potential for predicting NPC response to radiotherapy, we first established a radioresistantsubclone cell line (CNE2-IR) derived from NPC cell line CNE2 by treating the cells with five rounds of sublethalionizing radiation. Proteomics was then performed to compare the protein profiles of CNE2-IR and CNE2, and atotal of 34 differential proteins were identified. Among them, 14-3-3σ and Maspin were downregulated andGRP78 and Mn-SOD were upregulated in the radioresistant CNE2-IR compared with control CNE2, whichwas conformed by Western blot. Immunohistochemistry was performed to detect the expression of the fourvalidated proteins in the 39 radioresistant and 51 radiosensitive NPC tissues and their value for predicting NPCresponse to radiotherapy were evaluated by receiver operating characteristic analysis. The results showed thatthe downregulation of 14-3-3σ and Maspin and the upregulation of GRP78 and Mn-SOD were significantlycorrelated with NPC radioresistance and the combination of the four proteins achieved a sensitivity of 90%and a specificity of 88% in discriminating radiosensitive from radiaoresistant NPC. Furthermore, the resistanceto ionizing radiation can be partially reversed by the overexpression of 14-3-3σ in the CNE2-IR. The data suggestthat 14-3-3σ, Maspin, GRP78, and Mn-SOD are potential biomarkers for predicting NPC response to radiother-apy and their dysregulation may be involved in the radioresistance of NPC. Cancer Res; 70(9); 3450–62. ©2010 AACR.

Introduction

Nasopharyngeal carcinoma (NPC) is an endemic diseasewith incidence rates of 15 to 50 per 100,000 in southernChina and Southeast Asia. In addition, it poses one of themost serious public health problems in these areas (1). His-tologically, these usually belong to WHO types II and III. Inthe West, NPC occurs sporadically and, histologically, usuallybelongs to WHO type I. The disease tends to be more sensi-tive to ionizing radiation than other head and neck cancers.Additionally, types II and III are more sensitive to ionizingradiation than WHO type I. Thus, the primary treatmentfor NPC at that point is radiotherapy. More accurate tumorlocalization by computed tomography and better radiother-apy technique have contributed to the improvement in thelocal control of this disease (2). Even in relatively advanced

ffiliation: Key Laboratory of Cancer Proteomics of ChineseHealth, Xiangya Hospital, Central South University, Hunanhina

plementary data for this article are available at Cancernline (http://cancerres.aacrjournals.org/).

H. Yi, and M-Y. Li contributed equally to this work.

ding Author: Zhi-Qiang Xiao, Key Laboratory of Cancer Pro-Chinese Ministry of Health, Xiangya Hospital, Central SouthChangsha 410008, Hunan Province, China. Phone: 86-731-ax: 86-731-4327321; E-mail: [email protected].

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disease, combined radiochemotherapy may increase survival(3). Nevertheless, radioresistance remains a serious obstacleto successful treatment in many cases. Some of the NPCpatients present local recurrences and distant metastases af-ter radiotherapy due to radioresistance and the majority ofthese patients surrender recurrence and metastasis within1.5 year after treatment (4, 5). Hence, revealing the molecularmechanism of NPC radioresistance and identifying subgroupof radioresistant NPC patients are urgently needed for per-sonalized therapy.Although a fraction of genes such as elements of cell cycle

control, apoptosis/antiapoptosis, and DNA repair are be-lieved to play a key role in the ionizing radiation–induced celldamages, our understanding of radioresistance in cancer at amolecular level is limited. Microarrays have been used toassess genes involved in radioresistance in various types ofcancer (6–11). For examples, analysis for gene expressionprofiles of radioresistant NPC cell lines using a cDNA arrayfound that at least two genes, gp96 and GDF15, involved inthe radioresistance of NPC cell lines (11). However, therewas no overlap in the genes found to be involved in radio-resistance in various studies. This may be because of distincttissue specificity, but it is also possible that there is somefundamental mechanism underlying radioresistance thathas not yet been elucidated. High throughput proteomicshas introduced a new approach to radioresistant researchthat aims at identifying differential expression proteinsassociated with the development of cancer radioresistance,

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Radioresistant Proteins in Nasopharyngeal Carcinoma


providing new opportunities to reveal the molecular mecha-nism underlying NPC radioresistance.At present, histologic grading and clinical tumor-node-

metastasis (TNM) staging are still the main guidelines as towhether the NPC patients receive higher radiation dose orcombined more aggressive chemotherapy. NPC patients withthe same histologic grading and clinical TNM staging usuallyreceived the same therapeutic regimens. As cancer sensitivityto radiotherapy not only is associated with its morphologyand clinical TNM staging, it also is affected by its intrinsicmolecular characteristics. Hence, the finding of biomarkersto estimate NPC response to radiotherapy would be of clinicalimportance for the identification of subgroups of patientsthat could benefit from personalized therapeutic strategies.In this study, we established a radioresistant NPC sub-

clone cell line CNE-2-IR to search for proteins responsiblefor NPC radioresistance by a proteomic approach. Thirty-four differential proteins were identified in the radioresis-tant CNE-2-IR and control CNE-2 cells and the expressionlevels of four differential proteins (14-3-3σ, Maspin, GRP78,and Mn-SOD) were confirmed. Because 14-3-3σ, Maspin,GRP78, and Mn-SOD proteins are involved in tumor radio-sensitivity and/or chemosensitivity from other studies(12–15), we further investigated their predictive values forNPC radiosensitivity by immunohistochemical detection oftheir expressions in the radiosensitive and radioresistantNPC tissues. Furthermore, we examined the correlation of14-3-3σ expression level with NPC radioresistance by over-expressing 14-3-3σ in CNE-2-IR cells. Our findings providesubstantial evidence that 14-3-3σ, Maspin, GRP78, andMn-SOD are potential biomarkers for predicting NPCresponse to radiotherapy.

Materials and Methods

Patients and tissues. One hundred ninety-six NPCpatients who were treated by curative-intent radiotherapy(a total dose of 60–70 Gy) using a modified linear acceleratorin the Xiangya Hospital of Central South University, Chinafrom August 2006 to July 2008 were reviewed. Among thesepatient, 90 NPC patients without distant metastasis (M0;WHO staging II and III) at the time of diagnosis, comprising39 radioresistant and 51 radiosensitive patients, were recruitedin this study. Radioresistant NPC patients were defined as oneswith persistent disease (incomplete regression of tumor)at >6 weeks after completion of radiotherapy or ones withrecurrent disease at the nasopharynx and/or neck nodes at>2 months after completion of radiotherapy (16). Radiosensi-tive NPC patients were defined as ones without the local resid-ual lesions at >6 weeks or recurrence at >2 months aftercompletion of radiotherapy (16). Distant metastasis was ex-cluded by skeletal, thoracic, and upper abdominal imaging be-fore radiotherapy. NPC tissue biopsies from these 90 patientswere obtained at the time of diagnosis before any therapy withan informed consent andwere used for immunohistochemicalstaining. Diagnoses were established by experienced pathol-ogists based on the 1978 WHO classification (17). This studywas approved by the ethics committee of Xiangya School of

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Medicine, Central South University, China. The clinicopatho-logic parameters of NPC tissues used in the present study areshown in Supplementary Table S1.Establishment of radioresistant NPC CNE2 subclone cell

line. Poorly differentiated NPC cell line CNE-2 was seeded ata density of 1 × 105 per T25 flask in DMEM (Invitrogen) with10% FCS and 1% antibiotics, and cultured in an incubator at37°C with humidified 5% CO2. After 24 hours of culture,various doses from 0 to 12 Gy were given to determine thesublethal dose for the cell line. The radiation was delivered atroom temperature at 300 cGy/min with a linear accelerator(2100EX, Varian). To establish radioresistant subclone cellline, CNE-2 cells received the sublethal dose of irradiation(11 Gy). After treatment, the surviving cells were selectedand cultured to produce the first generation of the subclonecells. Again, the subclone cells received a sublethal dose of ir-radiation and the surviving cells were cultured to produce thenext generation of subclone cells. Up to five generations ofsubclone NPC cells were produced, and we defined the fifth-generation cells as the radioresistant subclone cell line andnamed for CNE-2-IR. CEN-2 cells, used as a control, weretreated with the same procedure, except they were sham irra-diated. Experiments were performed with the CNE-2-IR cellswithin 4 to 10 passages after the termination of irradiation.Clonogenic survival assay. Radioresistance was measured

by clonogenic survival assay following exposure to irradiation.Briefly, cells were plated in six-well culture plates and wereexposed to a range of radiation doses (2–10 Gy). After irradi-ation, the cells were cultured for 12 days and the number ofsurviving colonies (defined as a colony with >50 cells) wascounted. The survival fraction was calculated as the numbersof colonies divided by the numbers of cells seeded times plat-ing efficiency. Plating efficiencies were calculated as coloniesper 10 cells. Three independent experiments were done.Cell growth analysis in response to irradiation. Cells

were plated in a 24-well culture plates (2.5 × 104/well). Afterincubation for 24 hours, the cells were irradiated with 6 Gy.Cell growth was monitored by counting cell numbers atvarious time intervals. Three independent experimentswere done in triplicate.Flow cytometry analysis of cell cycle in response to irra-

diation. Cells were growing in the complete medium andwere harvested at 24 hours after irradiation with 6 Gy. Thecell pellets were fixed with ice-cold 70% ethanol in PBS at−20°C for 1 hour and then centrifuged at 1,500 rpm for 5 min-utes. The pellets were incubated with 0.5% Triton X-100(Sigma) and 0.05% RNase (Sigma) in 1 mL PBS at 37°C for30 min and then centrifuged at 1,500 rpm for 5 min. The cellpellets were incubated with 40 μg/mL propidium iodide(Sigma) in 1 mL PBS at room temperature for 30 minutes.Samples were immediately analyzed by a FACScan flow cyto-metry (Becton Dickinson). The distribution of cell cycle wasdetermined using CellQuest Pro and the ModiFit software.Three independent experiments were done.Two-dimensional electrophoresis. Cells were lysed in ly-

sis buffer (7 mol/L urea, 2 mol/L thiourea, 100 mmol/L DTT,4%CHAPS, 40mmol/L Tris, 2% Pharmalyte, 1mg/mLDNase I)at 37°C for 1 hour and then centrifuged at 15,000 rpm for

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30 minutes at 4°C. The supernatant was transferred andthe concentration of the total proteins was determined usingthe two-dimensional Quantification kit (Amersham Bio-sciences). Two-dimensional electrophoresis was performedto separate 600 μg of protein samples as previously describedby us (18). After SDS-PAGE, the protein spots were visualizedby staining with colloidal Coomassie (Bio-Rad Laboratories).Triplicate gels were made for each cell line.Image analysis. To minimize the contribution of experi-

mental variations, three separate two-dimensional gels wereprepared for each cell line. The stained two-dimensional gelswere scanned with the MagicScan software on Imagescanner(Amersham Biosciences) and were analyzed using PDQuestsystem (Bio-Rad Laboratories). Spot intensities were quan-tified by calculating the spot volume after normalization ofthe image using the total spot volume normalization methodmultiplied by the total area of all the spots. An average 2-DEmap for each cell line was constructed with the PDQuest im-age analysis software after spot matching and the expressionlevel of a protein spot was determined by its average inten-sity volume of three gels. The average 2-DE maps were usedto perform the different expression analysis. The differencesof the matched protein spots between the two cell lines wascalculated by the ratio of their average expression levels andproteins were classified as being differentially expressed be-tween the two cell lines when average spot intensity wasshowed a difference ≥2-fold variation. Significant differencesin protein expression levels were determined by Student'st test with a set value of P < 0.05.Mass spectrometry analysis. All the differential protein

spots showing consistent difference between the two celllines in triplicate experiments were excised from stainedgels using punch, were in-gel trypsin digested, and analyzedwith a Voyager System DE-STR 4307 matrix-assisted laserdesorption/ionization–time-of-flight (MALDI-TOF) MassSpectrometer (MS; ABI) to get a peptide mass fingerprint aspreviously described by us (18). The protein spots identifiedby MALDI-TOF MS were also subjected to analysis of electro-spray ionization quadrupole-time of flight MS (Micromass,Waters). Briefly, the samples were loaded on to a precolumn(320 μm*50 mm, 5 μm C18 silica beads, Waters) at 30 μL/minflow rates for concentrations and fast desalting through aWaters CapLC autosampler and then eluted to the re-versed-phase column (75 μm*150 mm, 5 μm, 100Å, LC Pack-ing) at a flow rate of 200 nL/min after flow splitting forseparation. MS/MS spectra were performed in data-dependedmode in which up to four precursor ions above an intensitythreshold of 7 counts per second were selected for MS/MSanalysis from each survey “scan.”Database search. In peptide mass fingerprint map data-

base searching, Mascot Distiller was used to obtain themonoisotopic peak list from the raw mass spectrometryfiles. Peptide matching and protein searches against theSwiss-Prot database were performed using the Mascot searchengine (http://www.matrixscience.com/) with a mass toler-ance of ±50 ppm. Protein scores of >64 (threshold) indicateidentity or extensive homology (P < 0.05) and were con-sidered significant.

Cancer Res; 70(9) May 1, 2010


In tandem mass spectrometry data database query, theMS/MS data (PKL format file) was imported into the Mascotsearch engine with a MS/MS tolerance of ± 0.3 Da to searchthe Swiss-Prot database. For the protein matching of two ormore unique peptides, individual ion scores of >28 (thresh-old) indicate identity or extensive homology (P < 0.05) andwere considered significant. For the protein matching onlyone unique peptide, individual ion scores of >35 (threshold)indicate identity or extensive homology (P < 0.01) and wereconsidered significant.Western blot. Western blot was performed to detect the

expression of 14-3-3σ, Maspin, GRP78, and Mn-SOD in CNE-2-IR and CNE-2 cells as previously described by us (19). Briefly,40 μg of lysates were separated by 10% SDS-PAGE andtransferred to a polyvinylidene difluoride membrane. Blotswere blocked with 5% nonfat dry milk for 2 hours at roomtemperature and then incubated with 1:2,000 dilution ofmonoclonal mouse anti–14-3-3σ antibody (Santa Cruz Bio-technology), 1:800 dilution of monoclonal mouse anti-Maspinantibody (NeoMarkers), 1:2,000 dilution of polyclonal goatanti-GRP78 antibody (Santa Cruz Biotechnology), or1:3,000 dilution of polyclonal rabbit anti-Mn-SOD antibody(Santa Cruz Biotechnology) for 2 hours at room temperature,followed by incubation with 1:3,000 dilution of horseradishperoxidase–conjugated secondary antibody for 1 hour atroom temperature. The signal was visualized with an en-hanced chemiluminescence detection reagent. β-Actin wasdetected simultaneously using 1:5,000 dilution of monoclonalmouse anti–β-actin antibody (Sigma) as a loading control.Three independent experiments were done.Immunohistochemistry. Immunohistochemical analysis

of 14-3-3σ, Maspin, GRP78, and Mn-SOD in the 39 radioresis-tant and 51 radiosensitive NPC tissue samples was performedon formalin-fixed and paraffin-embedded tissue sections aspreviously described by us (19). Briefly, 4 μm of tissue sectionswere treated with an antigen retrieval solution [10 mmol/Lsodium citrate buffer (pH 6.0)]; incubated with anti–14-3-3σantibody (1:400 dilution), anti-Maspin antibody (1:100 dilu-tion), anti-GRP78 antibody (1:400 dilution), or anti-Mn-SODantibody (1:600 dilution) overnight at 4°C; and then were in-cubated with 1:1,000 dilution of biotinylated secondary anti-body followed by avidin-biotin peroxidase complex (DAKO).Finally, tissue sections were incubated with 3′,3′-diaminoben-zidine (Sigma) until a brown color developed and were coun-terstained with Harris' modified hematoxylin. In negativecontrols, primary antibodies were omitted.Evaluation of staining. Sections were blindly evaluated by

two investigators in an effort to provide a consensus onstaining patterns by light microscopy. A quantitative scorewas performed by adding the score of staining area andthe score of staining intensity for each case to assess theexpression levels of the proteinss as previously describedby us (19). At least 10 high-power fields were chosen ran-domly and >1,000 cells were counted for each section. First,a quantitative score was performed by estimating thepercentage of immunopositive cells: 0, no staining of cellsin any microscopic fields; 1+, <30% of tissue stained positive;2+, between 30% and 60% stained positive; and 3+, >60%

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stained positive. Second, the intensity of staining was scoredby evaluating the average staining intensity of the positivecells (0, no staining; 1+, mild staining; 2+, moderate staining;3+, intense staining). Finally a total score (ranging from 0–6)was obtained by adding the area score and the intensityscore for each case. A combined staining score of ≤2 was con-sidered to be negative staining (no expression); a score be-tween 3 and 4 was considered to be moderate staining(expression); and a score between 5 and 6 was consideredto be strong staining (high expression).Statistical analysis of immunohistochemical data. Sta-

tistical analyses were performed with the statistical packageSPSS 13.0. Difference of the protein expression in the radio-sensitive and radiaoresistant NPC tissues was analyzed usingMann-Whitney U test. In addition, the four proteins were in-dividually and, as a penal, assessed for its ability to discrim-inate between radiosensitive and radiaoresistant NPCpatients by evaluating its receiver operating characteristic(ROC) curve based on the immunohistochemistry scores de-scribed by Hu and colleagues (20). Briefly, we built a logisticregression model and conducted ROC curve analyses to eval-uate overall predictive power of individual and the combinedfour proteins. The optimal cut point was determined for eachprotein by identifying the value that yielded the maximumcorresponding sensitivity and specificity. ROC curves werethen plotted based on the set of optimal sensitivity and spec-ificity values. The area under the curve and other attributeswere computed through the numerical integration of theROC curves. Sensitivity, specificity, positive predictive value,and negative predictive value of the four proteins werecalculated individually and as a penal. A two-sided P < 0.05was considered significant.Overexpression of 14-3-3σ in radioresistant CNE2-IR

cells. pcDNA3-14-3-3σ plasmid and empty vector pcDNA3(21) were transfected into the radiaoresistant CNE-2-IR cellswith the Lipofectamine 2000 Reagent (Invitrogen), respec-tively. After 14 days of selection in RPMI 1640 containing10% FCS and 400 mg/mL G418 (Invitrogen), the expressionof 14-3-3σ was determined by Western blot. Clonogenic sur-vival assay was done to determine the resistant level in thetransfected cells at 24 hours after exposed a range of radia-tion doses (2–10 Gy).

Results

Establishment and validation of radioresistant NPCsubclone cell line. The sublethal radiation dose was deter-mined by dose titration for the NPC CNE-2 cell line. Nearlyall NPC CNE-2 cells were killed after 5 days of irradiation ata dose of 12 Gy and no cells were recovered at doses higherthan 12 Gy. A dose of 11 Gy was therefore considered sublethaland used for the selection of CNE-2–radioresistant subclones.The first generation of subclone cells was generated from theculture of the surviving fraction of the parental cells irradiatedwith the sublethal dose. Similarly, the second generation ofsubclone cells was generated from the culture of the survivingfraction of the first-generation cells irradiated with the suble-



thal dose. Finally, the firth generation of CNE-radioresistantsubclones was established and designated as CNE-2-IR.To verify the radioresistant phenotypes of CNE2-IR, CNE-

2-IR and control CNE-2 cells were irradiated with a rangeof radiation doses (2–10 Gy) and were examined by clono-genic survival assay. As shown in the Fig. 1A, the survivingcolonies of the CNE-2-IR cells were significantly more andbigger than control CNE-2 cells. As shown in the Fig. 1B,CNE-2-IR showed decreased radiosensitivity compared withthe level of control CNE-2 and dose-modifying factors were2.04 and 1.56, respectively, at 10% and 1% of isosurvival levelsfor CNE-2-IR. Furthermore, CNE-2-IR and control CNE-2were subjected to 6 Gy radiation to examine the effect on cellgrowth. As shown in the Fig. 1C, cell growth delay after 6 Gyof irradiation was less for CNE-2-IR than for control CNE-2.For ∼5-fold increase in cell number, the delay was ∼32 hoursfor CNE-2-IR compared with ∼61 hours for control CNE-2.The difference in response to radiation between the two

cell lines was further studied by cell cycle analysis using flowcytometry. As showed in Supplementary Table S2, no differ-ence was induced by ionizing radiation in G0-G1 phases at 24hours after 6 Gy of irradiation, whereas compared with con-trol CNE-2 cells, more CNE-2-IR cells were found detained inS phase with less cells in G2-M phase. These results suggestthat the regulation of cell cycle induced by ionizing radiationstress is altered in the radioresistant CNE-2-IR, which is alsoconsistent with the typical radioresistant phenotype. Theabove results indicate that CNE-2–radioresistant subclonecell line (CNE-2-IR) was established.Screening for radioresistance-associated proteins by

proteomic analysis. Comparative proteomic study of CNE-2-IR and control CNE-2 was performed to identify the proteinassociated with CNE-2-IR radioresistance. Two represen-tative 2-DE maps from CNE-2-IR and control CNE-2 wereshown in Fig. 2A. After comparing the average 2-DE maps,41 differential protein spots (≥2-fold) in the two cell lineswere detected and subjected to the analysis of both MAL-DI-TOF MS and ESI-Q-TOF MS, 34 differential proteins ofwhich were successfully identified. The MS/MS results of aspot 22 are shown in Fig. 2B and C. The amino acid sequenceof a doubly charged peptide from spot 22 with m/z 951.9672was identified as LAEQAERYEDMAAFMK, which is a part of14-3-3σ sequence, and the query result indicated that theprotein spot 22 is 14-3-3σ. The detailed information of allthe identified proteins is summarized in the SupplementaryTable S3 and Table 1.Validation of the identified proteins. To confirm the ex-

pression levels of the differential proteins identified by pro-teomics, the expression of four identified proteins (14-3-3σ,Maspin, Mn-SOD, and GRP78) in the CNE-2-IR and controlCNE-2 cells was detected by Western blot. As shown inFig. 3A, the expression of Mn-SOD and GRP78 was signifi-cantly higher, whereas 14-3-3σ and Maspin was significantlylower in the CNE-2-IR than that in the control CNE-2 cells,which is consistent with the results of proteomic analysis.Expression of 14-3-3σ, Maspin, Mn-SOD, and GRP78

proteins in radioresistant and radiosensitive NPC tissues.The results of both proteomics and Western blot showed

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that the expressional levels of 14-3-3σ, Maspin, Mn-SOD, andGRP78 were associated with the radiosensitivity of NPC cells.To examine the relevance of this finding to clinical NPC ra-diosensitivity, we detected the four-protein expression in theradioresistant and radiosensitive NPC tissues. As shown inFig. 3B and Table 2, compared with the radiosensitive NPC,14-3-3σ and Maspin were significantly downregulated,whereas Mn-SOD and GRP78 were significantly upregulatedin the radioresistant NPC. The results indicate the associa-tion of the four-protein expression abnormality with the clin-ical NPC radiosensitivity.Value of 14-3-3σ, Maspin, Mn-SOD, and GRP78 as bio-

markers for predicting NPC response to radiotherapy. Theability of 14-3-3σ, Maspin, Mn-SOD, and GRP78 in distinguish-ing the radiosensitive and radioresistant NPC tissues was an-alyzed by determining the ROC curves of the four proteinsindividually and as a panel. The area under curve valuesof the four proteins are listed in Supplementary Table S4 to-gether with their individual and collective values of merit.When individual protein serves as a biomarker for predictingNPC radiosensitivity, their sensitivity and specificity are 64%to 87% and 66% to 88% in discriminating radiosensitive fromradioresistant NPC samples, respectively (SupplementaryTable S4; Fig. 4A). As a panel, the four proteins achieved a sen-sitivity of 90% and a specificity of 88% in discriminating radio-sensitive from radioresistant NPC samples (SupplementaryTable S4; Fig. 4A).14-3-3σ Overexpression decreased the radioresistance

of CNE-2-IR. To address the question whether the expression



levels of the four proteins may affect the radiosensitivity ofCNE-2-IR, we selected 14-3-3σ, one of the four proteins, tobe further studied because our previous study showed thatthe downregulation of 14-3-3σ was associated with the poorsurvival of patients with NPC as an independent prognosticindicator (19). We generated a stably transfected CNE-2-IRcell line overexpressing 14-3-3σ and measured the radio-resistant levels of 14-3-3σ–overexpressing and vector-transfected CNE-2-IR cells after irradiation with a range ofradiation doses (2–10 Gy) using a clonogenic survival assay.As shown in Fig. 4B, transfection of pcDNA3-14-3-3σ plasmidinto CNE-2-IR cells increased 14-3-3σ expression comparedwith the vector-transfected cells. As shown in Fig. 4C, the14-3-3σ-overexpressing CNE-2-IR showed increased radiosen-sitivity compared with the vector-transfected CNE-2-IR anddose-modifying factors were 0.717 and 0.744, respectively, at10% and 1% of isosurvival levels for 14-3-3σ–overexpressingCNE-2-IR. The result indicates that 14-3-3σ downregulationconfers a significant protection against ionizing radiation,and increased the resistance of NPC cells to irradiation.

Discussion

Radioresistance continues to be a major problem in thetreatment of NPC (4, 5). The molecular mechanisms under-lying NPC radioresistance are still unclear, and till now,there have not been effective biomarkers for predictingNPC radiosensitivity. Identification of NPC radioresistance–associated proteins will be helpful for finding biomarkers

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Figure 1. Different sensitivity toradiation in radioresistant CNE2-IRand control CNE2 cells. A and B,clonogenic survival assay.CNE2-IR and control CNE2 cellsplated in six-well culture plateswere irradiated with a range ofradiation doses (2–10 Gy) andcolonies that formed afterincubation of 12 d were counted tocalculate the survival fractions. C,growth delay of CNE2-IR inducedby irradiation. CNE2-IR and controlCNE2 cells were seeded in 24-wellculture plate 24 h beforeirradiation. Cell numbers werecounted at different times after6 Gy of irradiation. Cellmultiplication of CNE2-IR andcontrol CNE2 cells without 6 Gy ofirradiation is also showed and usedas controls. Three experimentswere done; points, mean; bars, SD.

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to estimate NPC response to radiotherapy and decipheringthe molecular mechanisms of NPC radioresistance.In this study, we established radioresistant NPC subclone

cell line CNE-2-IR by sublethal doses of radiation and usedproteomic approach to screen for differential proteins inthe CNE-2-IR cells. Consequently, 34 differential proteinswere identified and four differential proteins (14-3-3σ, Mas-pin, GRP78, and Mn-SOD) were confirmed by Western blot,suggesting that the proteins identified by proteomic ap-proach are actually differential expression proteins. Immuno-histochemistry was performed to detect the expressions of14-3-3σ, Maspin, GRP78, and Mn-SOD in the radiosensitiveand radioresistant NPC tissues, and the correlation of theirexpression levels with NPC radioresistance were evaluated.



The results showed that the downregulation of 14-3-3σ andMaspin and upregulation of Mn-SOD and GRP78 were signif-icantly correlated with NPC radioresistance. In addition, apanel of the four proteins achieved a sensitivity of 90% anda specificity of 88% in discriminating radiosensitive from ra-diaoresistant NPC. Furthermore, transfection of 14-3-3σ–expressing plasmid to upregulate the expression of 14-3-3σ,one of the four differential proteins, rendered radioresistantCNE-2-IR cells more sensitive to radiation, which stronglyindicated that the downregulation of 14-3-3σ play animportant role in the development of NPC radioresistance.The above results suggest that 14-3-3σ, Maspin, GRP78,and Mn-SOD are potential biomarkers for predicting NPCresponse to radiotherapy.

Figure 2. Comparative proteomicanalysis of radioresistant CNE2-IRand control CNE2 cells by 2-DEand MS. A, representative 2-DEmaps of CNE2-IR and controlCNE2 cells. Arrows, 34 differentialprotein spots that have beenidentified by mass spectrometry.B, the ESI-Q-TOF MS–sequencedspectrum of spot 22. The aminoacid sequence of a doubly chargedpeptide with m/z 951.9672 wasidentified as LAEQAERYED-MAAFMK from mass differences inthe y and b fragment ions series,matched with residues 12 to 27 of14-3-3σ. C, protein sequence of14-3-3σ is shown. MatchedMS/MS fragmentation isunderlined.

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Table 1. Differential expression proteins between CNE-2-IR and control CNE-2 identified by mass spectrometry

No. Swiss-ProtAC

Protein name MW (Da) pI Coverage Uniquepeptides

Scores Peptide sequenceidentified

Expression ratio(CNE2-IR/Control CNE2)

1 Q9Y4L1 Oxygen-regulated protein150K precursor

111,266 5.16 2% 2 78 LPATEKPVLLSK ↑12.07YFQHLLGK

2 P11142 Heat shock cognate71-kDa protein

88,805 5.44 3% 2 109 SQIHDIVLVGGSTR ↑2.17MVNHFIAEFK

3 P68361 Tubulin α-1B chain 50,804 4.94 17% 5 178 AVFVDLEPTVIDEVR ↑2.664 P68368 Tubulin α-4A chain 50,503 4.95 6% 3 140 DVNAAIAAIKTK ↑2.10

EDMAALEKEIIDPVLDR

5 Q16666 γ-IFN–inducibleprotein Ifi-16

46,415 4.9 2% 2 82 LISEMHSFIQIK ↑2.69GLEVINDYHFR

6 P28331 NADH-ubiquinoneoxidoreductase 75-kDasubunit, mitochondrial(precursor)

79,417 5.89 2% 1 78 NDGAAILAAVSSIAQK ↓0.18

7 P02787 Serotransferrin (Precursor) 79,280 6.81 12% 3 106 MYLGYEYVTAIR ↓0.268 P12532 Creatine kinase, ubiquitous

mitochondrial (precursor)47,406 8.6 5% 2 108 SFLIWVNEEDHTR ↓0.12

VVVDALSGLK9 P04264 Keratin, type II cytoskeletal 1 66,152 8.16 7% 4 174 SLDLDSIIAEVK ↓0.15

WELLQQVDTSTRSLNNQFASFIDKNKLNDLEDALQQAK

10 P11021 78-kDa glucose-regulatedprotein

72,187 5.03 12% 3 206 IINEPTAAAIAYGLDKR ↑17.41SDIDEIVLVGGSTRITPSYVAFTPEGER

11 P08670 Vimentin 53,545 5.06 3% 1 91 ISLPLPNFSSLNLR ↑2.0412 Q15084 Protein disulfide-isomerase

A6 (precursor)46,518 4.95 15% 4 202 TGEAIVDAALSALR ↓0.10

GSFSEQGINEFLRLAAVDATVNQVLASRALDLFSDNAPPPELLEIINEDIAKR

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Table 1. Differential expression proteins between CNE-2-IR and control CNE-2 identified by mass spectrometry (Cont'd)

No. Swiss-ProtAC




13 Q04695 Keratin, type Icytoskeletal 17

40,524 4.9 18% 5 321 TMQALEIELQSQLSMK ↓0.15

14 P23381 Tryptophanyl-tRNAsynthetase

53,132 5.83 7% 1 49 DRTDIQCLIPCAIDQDPYFR ↓0.17

15 P36952 Serpin B5 (precursor;Maspin)

42,568 5.72 5% 2 74 ELETVDFKDK ↓0.43IIELPFQNK

16 P35998 Proteasome 26S subunitMSS1-human

49,009 5.71 5% 2 91 LREVVETPLLHPER ↓0.46ALDEGDIALLK

17 Q00987 E3 ubiquitin-proteinligase Mdm2

55,198 4.6 7% 2 96 NLVVVNQQESSDSGTSVSENR ↑7.42RAISETEENSDELSGER

18 P17980 26S protease regulatorysubunit 6A

45,512 5.39 3% 1 101 QTYFLPVIGLVDAEK ↑2.50

19 P08727 Keratin 19, type I,cytoskeletal-human

44,065 5.04 13% 3 86 VLDELTLAR ↑2.20DYSHYYTTIQDLRDKFVSSSSSGGYGGGYGGVLTASDGLLAGNEK

20 Q04760 Lactoylglutathione lyase 21,381 5.12 10% 2 87 FEELGVKFVK,DFLLQQTMLR ↑3.4821 P13693 Translationally controlled

tumor protein21,512 5.34 8% 1 94 DLISHDEMFSDIYK ↓0.26

22 P31947 14-3-3 protein σ 28,325 4.8 6% 1 59 LAEQAERYEDMAAFMK ↓0.2423 Q01105 Template-activating factor-I 33,469 4.23 8% 2 45 IDFYFDENPYFENK ↑3.25

VEVTEFEDIK24 Q13162 Peroxiredoxin-4 30,749 5.86 4% 1 55 IPLLSDLTHQISK ↓0.4025 Q3T0X5 Proteasome subunit

α type-129,822 6.15 9% 2 74 IHQIEYAMEAVK,ETLPAEQDLTTK ↑3.64

26 P21976 Voltage-dependentanion-selectivechannel protein 1

30,868 8.62 20% 3 176 VNNSSLIGLGYTQTLKPGIK ↑2.27KLETAVNLAWTAGNSNTRSENGLEFTSSGSANTETTK

(Continued on the following page)

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Table 1. Differential expression proteins between CNE-2-IR and control CNE-2 identified by mass spectrometry (Cont'd)

No. Swiss-ProtAC




27 P04792 Heat shock protein 27 22,768 5.98 13% 2 128 LATQSNEITIPVTFESR ↑2.22LFDQAFGLPR

28 Q06830 Peroxiredoxin-1 22,324 8.27 32% 4 300 GLFIIDDKGILR ↑3.07TIAQDYGVLKADEGISFRQITVNDLPVGRLNCQVIGASVDSHFCHLAWVNTPK

29 Q99479 Protein DJ-1 20,050 6.33 54% 7 451 EGPYDVVVLPGGNLGAQNLSESAAVK ↓0.31GAEEMETVIPVDVMRGLIAAICAGPTALLAHEIGFGSKVTVAGLAGKDPVQCSRDVVICPDASLEDAKDVVICPDASLEDAKKKGLIAAICAGPTALLAHEIGFGSK

30 P03755 Annexin A2 38,822 7.57 33% 5 191 AEDGSVIDYELIDQDAR ↑8.63GVDEVTIVNILTNRGLGTDEDSLIEIICSRSALSGHLETVILGLLKLSLEGDHSTPPSAYGSVK

31 Q9Y277 Voltage-dependentanion-selectivechannel protein 3

32,060 7.49 4% 1 67 LTLDTIFVPNTGK ↑3.75

32 P31943 Heterogeneous nuclearribonucleoprotein H

49,198 5.89 4% 2 135 SNNVEMDWVLK ↑8.39VHIEIGPDGR

33 P04179 Superoxide dismutase(Mn), mitochondrial

(precursor)

24,906 8.35 23% 3 155 HHAAYVNNLNVTEEKYQEALAK ↑5.62GDVTAQIALQPALKAIWNVINWENVTER

34 O15235 Ribosomal protein S12 14,905 6.81 18% 1 49 EAVCFIPGEGHTLQEHQIVLVEGGR ↓0.08

Abbreviations: AC, accession code; MW, molecular weight; pI, isoelectric point.

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14-3-3σ, a potential tumor suppressor protein, can nega-tively regulate cell cycle progression by inducing G2-M phasearrest (22, 23). It has been shown that 14-3-3σ is transacti-vated by p53 in response to DNA damage and, in turn, inter-acts with p53 and positively regulates p53 activity (24). It is



known that p53 is involved in the complex response to ion-izing radiation, such as inducing irreversible growth arrestand apoptosis, and sensitizes cells to radiochemotherapy(25). As a negative regulator of cell cycle and mediator ofp53 function in response to DNA, 14-3-3σ may also play a

Table 2. The difference of 14-3-3σ, Maspin, Mn-SOD, and GRP78 expression in the radioresistant andradiosensitive NPC tissues

n
Score
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P

Low (0–2)
Moderate (3–4) High (5–6)
14-3-3σ
Radioresistance 39 18 9 12 0.0001 Radiosensitivity 51 9 17 25
Maspin
GRP78
Mn-SOD
NOTE: P < 0.01 by Mann-Whitney U test, radioresistant versus radiosensitive NPC tissues.

Figure 3. Expression of 14-3-3σ,Maspin, Mn-SOD, and GRP78in the radioresistant andradiosensitive NPC cell line andtissues. A, a representative resultof Western blot analysis showsthe downregulation of 14-3-3σand Maspin, and the upregulationof Mn-SOD and GRP78 inradioresistant CNE-2-IR cellscompared with control CNE-2cells. β-Actin was used as aninternal control for loading.B, a representative result ofimmunohistochemistry shows thedownregulation of 14-3-3σ andMaspin, and the upregulationof Mn-SOD and GRP78 in theradioresistant NPC tissuescompared with the radiosensitiveNPC tissues. IR-NPC,radioresistant NPC tissues.IS-NPC, radiosensitive NPCtissues. Original magnification,×200.

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critical role in sensitizing tumor cells to radiochemother-apy. Indeed, decreased expression of 14-3-3σ has been ob-served in drug-resistant human breast cancer cells (12).Our previous study showed that 14-3-3σ was decreased inNPC and that its downregulation was associated with thepoor survival of NPC patients as an independent prognosticfactor which indicates that 14-3-3σ downregulation maycause radioresistance in NPC (19). It is also a reason for se-lecting 14-3-3σ to examine the effects of its expression inCNE-2-IR cells. Our results showed that 14-3-3σ was down-regulated in the radioresistant NPC cells and tissues; itsdownregulation was associated with NPC radioresistance;and upregulating 14-4-3σ expression lead to the partial radio-sensitization of CNE-2-IR cells. The data strongly suggest that14-3-3σ might be a biomarker for predicting NPC response toradiotherapy. However, we found that the overexpression of14-4-3σ did not completely reverse the radioresistant pheno-type of CNE-2-IR, indicating that there are other factors con-tributing to radioresistance other than 14-4-3σ in NPC.Maspin is a serpin with tumor suppressive properties; can

inhibit tumor growth, angiogenesis, invasion, and metastasis;



and promote apoptosis (26). Maspin is also an importantapoptosis-sensitizing factor, making tumor sensitive todrug-induced apoptosis (27, 28). It has been shown thatMaspin expression was directly correlated with radioche-motherapy response in head and neck squamous cell carci-noma (13, 29) and the use of Maspin as a therapeutic targetagainst cancer through the reexpression of Maspin by phar-macologic intervention have been explored (30). In this study,Maspin was downregulated in the radioresistant NPC cellsand tissues and its downregulation was associated withNPC radioresistance, which together with reporters suggeststhat the decrease of Maspin expression in NPC increasedits radiation resistance and Maspin might be a biomarkerfor predicting NPC response to radiotherapy.The glucose-regulated protein GRP78, an endoplasmic re-

ticulum stress–induced molecular chaperone, plays a criticalrole in the endoplasmic reticulum stress and in maintainingcellular homeostasis as an important stress response survivalprotein (31, 32). GRP78 is induced in cancers due to endo-plasmic reticulum stress, including glucose deprivation,acidosis, and severe hypoxia (33), and is also induced by

h. 6, 2020. © 2010 Am

Figure 4. Efficacy of 14-3-3σ, Maspin,Mn-SOD, and GRP78 in discriminatingradiosensitive from radioresistant NPC tissues,as well as the effect of 14-3-3 σ overexpressionon the CNE2-IR radiosensitivity. A, ROC curvesof 14-3-3σ, Maspin, Mn-SOD, and GRP78 indiscriminating radiosensitive from radioresistantNPC tissues, individually and as a panel.B, a representative result of Western blotshows the levels of 14-3-3σ expression inCNE2-IR, the vector transfected–, andpcDNA3-14-3-3σ–transfected CNE2-IR cells.β-Actin was used as an internal control forloading. C, clonogenic survival assay. CNE2-IRcells and its transfectants plated in six-wellculture plates were irradiated with a range ofradiation doses (2–10 Gy) and colonies thatformed after incubation of 12 d were countedto calculate the survival fractions. Threeexperiments were done; points, mean; bars, SD.

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chemotherapeutic drugs and radiation, conferring tumorresistance to chemotherapeutic agents and radiation (14,34). Knockdown of GRP78 sensitizes tumor cells to drugtreatment in multiple types of cancer (35). Given the impor-tance of GRP78 in cancer cell survival, it represents a primetarget for anticancer treatment. Furthermore, GRP78 canserve as a biomarker for predicting treatment response inbreast cancer patients (14). In the present study, GRP78 isupregulated in the radioresistant NPC cells and tissues, andits upregulation was associated with NPC radioresistance,which together with reporters suggests that overexpressionof GRP78 increased NPC radioresistance and GRP78 mightbe a biomarker for predicting NPC response to radiotherapy.Mn-SOD, one of the mitochondrial antioxidant enzymes,

plays a central role in protecting cells against reactive oxygenspecies injury (36). Exposure of cells to ionizing radiationleads to the formation of reactive oxygen species that candamage DNA, RNA, protein, and lipid components, and is as-sociated with radiation-induced injury (36). Mn-SOD canscavenge reactive oxygen species produced by ionizing radi-ation and protect cancer cells from radiation-induced oxida-tive damage (37). Both in vitro and in vivo studies showedthat the expression level of Mn-SOD negatively correlateswith tumor radiosensitivity (15, 38–40). In the present study,Mn-SOD was upregulated in the radioresistant NPC cells andtissues, and its upregulation was associated with NPC radio-resistance, which together with reporters suggests that the



overexpression of Mn-SOD increased NPC radiation resis-tance and Mn-SOD might be a biomarker for predictingNPC response to radiotherapy.In summary, we used proteomic approach identifying

34 differential proteins in the radioresistant NPC cell lineCNE2-IR. We further showed that the expression abnormalityof 14-3-3σ, Maspin, GRP78, and Mn-SOD might contribute toNPC radioresistance and be potential biomarkers for predict-ing NPC response to radiotherapy. The findings reportedhere could have clinical value in distinguishing radiosensitivefrom radiaoresistant NPC and in identifying subgroups ofNPC patients that could benefit from personalized therapeu-tic strategies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

National Nature Science Foundation of China (30973290), OutstandingScholars of New Era from Ministry of Education of China (2002-48), LotusScholars Program of Hunan Province, China (2007-362), and Key research pro-gram from Science and Technology Committee of Hunan, China (06SK2004).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/07/2009; revised 01/23/2010; accepted 02/12/2010; publishedOnlineFirst 04/20/2010.

References
1. Ho JH. An epidemiologic and clinical study of nasopharyngeal
carcinoma. Int J Radiat Oncol Biol Phys 1978;4:182–98.2. Teo P, Yu P, Lee WY, et al. Significant prognosticators after primary

radiotherapy in 903 nondisseminated nasopharyngeal carcinomaevaluated by computer tomography. Int J Radiat Oncol Biol Phys1996;36:291–304.

3. DeNittis AS, Liu L, Rosenthal DI, Machtay M. Nasopharyngealcarcinoma treated with external radiotherapy, brachytherapy, andconcurrent/adjuvant chemotherapy. Am J Clin Oncol 2002;25:93–5.

4. Lee AW, Poon YF, Foo W, et al. Retrospective analysis of 5037patients with nasopharyngeal carcinoma treated during 1976-1985:overall survival and patterns of failure. Int J Radiat Oncol Biol Phys1992;23:261–70.

5. Leung SF, Teo PM, Shiu WW, Tsao SY, Leung TW. Clinical fea-tures and management of distant metastases of nasopharyngealcarcinoma. J Otolaryngol 1991;20:27–9.

6. Wong YF, Sahota DS, Cheung TH, et al. Gene expression patternassociated with radiotherapy sensitivity in cervical cancer. CancerJ 2006;12:189–93.

7. Ogawa K, Utsunomiya T, Mimori K, et al. Differential gene expressionprofiles of radioresistant pancreatic cancer cell lines established byfractionated irradiation. Int J Oncol 2006;28:705–13.

8. Higo M, Uzawa K, Kouzu Y, et al. Identification of candidate radio-resistant genes in human squamous cell carcinoma cells throughgene expression analysis using DNA microarrays. Oncol Rep 2005;14:1293–8.

9. Guo WF, Lin RX, Huang J, et al. Identification of differentiallyexpressed genes contributing to radioresistance in lung cancer cellsusing microarray analysis. Radiat Res 2005;164:27–35.

10. Fukuda K, Sakakura C, Miyagawa K, et al. Differential gene ex-pression profiles of radioresistant oesophageal cancer cell linesestablished by continuous fractionated irradiation. Br J Cancer2004;91:1543–50.

11. Chang JT, Chan SH, Lin CY, et al. Differentially expressed genes

in radioresistant nasopharyngeal cancer cells:gp96 and GDF15.Mol Cancer Ther 2007;6:2271–9.

12. Sang M, Li Y, Ozaki T, et al. p73-dependent induction of 14-3-3σincreases the chemo-sensitivity of drug-resistant human breastcancers. Biochem Biophys Res Commun 2006;347:327–33.

13. Marioni G, Koussis H, Gaio E, et al. MASPIN's prognostic role inpatients with advanced head and neck carcinoma treated with pri-mary chemotherapy (carboplatin plus vinorelbine) and radiotherapy:preliminary evidence. Acta Otolaryngol 2009;129:786–92.

14. Lee E, Nichols P, Spicer D, Groshen S, Yu MC, Lee AS. GRP78 as anovel predictor of responsiveness to chemotherapy in breast cancer.Cancer Res 2006;66:7849–53.

15. Suresh A, Tung F, Moreb J, et al. Role of manganese superoxidedismutase in radioprotection using gene transfer studies. CancerGene Ther 1994;1:85–90.

16. To EW, Chan KC, Leung SF, et al. Rapid clearance of plasmaEpstein-Barr virus DNA after surgical treatment of nasopharyngealcarcinoma. Clin Cancer Res 2003;9:3254–9.

17. Shanmugaratnam K, Sobin LH. The World Health Organizationhistological classification of tumours of the upper respiratorytract and ear. A commentary on the second edition. Cancer 1993;71:2689–97.

18. Chen Y, Ouyang GL, Yi H, et al. Identification of RKIP as an invasionsuppressor protein in nasopharyngeal carcinoma by proteomicanalysis. J Proteome Res 2008;7:5254–62.

19. Cheng AL, Huang WG, Chen ZC, et al. Identification of novel naso-pharyngeal carcinoma biomarkers by laser capture microdissectionand proteomic analysis. Clin Cancer Res 2008;14:435–45.

20. Hu S, Arellano M, Boontheung P, et al. Salivary proteomics for oralcancer biomarker discovery. Clin Cancer Res 2008;14:6246–52.

21. Liu Y, Liu H, Han B, Zhang JT. Identification of 14-3-3σ as acontributor to drug resistance in human breast cancer cells usingfunctional proteomic analysis. Cancer Res 2006;66:3248–55.

22. Mhawech P. 14-3-3 proteins-an update. Cell Res 2005;15:228–36.

Cancer Res; 70(9) May 1, 2010 3461



Feng et al.

3462


23. Laronga C, Yang HY, Neal C, Lee MH. Association of the cyclin-dependent kinases and 14-3-3 σ negatively regulates cell cycleprogression. J Biol Chem 2000;275:23106–12.

24. Yang HY, Wen YY, Chen CH, Lozano G, Lee MH. 14-3-3 σ positivelyregulates p53 and suppresses tumor growth. Mol Cell Biol 2003;23:7096–107.

25. Gudkov AV, Komarova EA. The role of p53 in determining sensitivityto radiotherapy. Nat Rev Cancer 2003;3:117–29.

26. Sager R, Sheng S, Pemberton P, Hendrix MJ. Maspin. A tumorsuppressing serpin. Adv Exp Med Biol 1997;425:77–88.

27. Sheng S. The promise and challenge toward the clinical applicationof maspin in cancer. Front Biosci 2004;9:2733–45.

28. Schaefer JS, Zhang M. Targeting maspin in endothelial cells toinduce cell apoptosis. Expert Opin Ther Targets 2006;10:401–8.

29. Mhawech-Fauceglia P, Dulguerov P, Beck A, Bonet M, Allal AS.Value of ezrin, maspin and nm23-1 protein expressions in predictingoutcome of patients with head and neck squamous-cell carcinomatreated with radical radiotherapy. J Clin Pathol 2007;60:185–9.

30. Sheng S. A role of novel serpin maspin in tumor progression:thedivergence revealed through efforts to converge. J Cell Physiol2006;209:631–5.

31. Lee AS. The glucose-regulated proteins: stress induction and clinicalapplications. Trends Biochem Sci 2001;26:504–10.

32. Hendershot LM. The ER function BiP is a master regulator of ERfunction. Mt Sinai J Med 2004;71:289–97.

33. Dong D, Dubeau L, Bading J, et al. Spontaneous and controllable



activation of suicide gene expression driven by the stress-induciblegrp78 promoter resulting in eradication of sizable human tumors.Hum Gene Ther 2004;15:553–61.

34. Zhai L, Kita K, Wano C, Wu Y, Sugaya S, Suzuki N. Decreased cellsurvival and DNA repair capacity after UVC irradiation in associationwith down-regulation of GRP78/BiP in human RSa cells. Exp CellRes 2005;305:244–52.

35. Li J, Lee AS. Stress induction of GRP78/BiP and its role in cancer.Curr Mol Med 2006;6:45–54.

36. Sun J, Chen Y, Li M, Ge Z. Role of antioxidant enzymes on ionizingradiation resistance. Free Radic Biol Med 1998;24:586–93.

37. Rhee SG, Kim KH, Chae HZ, et al. Antioxidant defense mechanisms:a new thiol-specific antioxidant enzyme. Ann N Y Acad Sci 1994;738:86–92.

38. Epperly MW, Epstein CJ, Travis EL, Greenberger JS. Decreasedpulmonary radiation resistance of manganese superoxide dismutase(MnSOD)-deficient mice is corrected by human manganese super-oxide dismutase-Plasmid/Liposome (SOD2-PL) intratracheal genetherapy. Radiat Res 2000;154:365–74.

39. Ozeki M, Tamae D, Hou DX, et al. Response of cyclin B1 to ionizingradiation: regulation by NF-κB and mitochondrial antioxidant enzymeMnSOD. Anticancer Res 2004;24:2657–63.

40. Kalen AL, Sarsour EH, Venkataraman S, Goswami PC. Mn-superoxide dismutase overexpression enhances G2 accumulationand radioresistance in human oral squamous carcinoma cells.Antioxid Redox Signal 2006;8:1273–81.

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2010;70:3450-3462. Published OnlineFirst April 20, 2010.Cancer Res Xue-Ping Feng, Hong Yi, Mao-Yu Li, et al. Carcinoma Response to Radiotherapy by ProteomicsIdentification of Biomarkers for Predicting Nasopharyngeal

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