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Biology of Human Tumors

A Prognostic Bio-Model Based on SQSTM1 andN-Stage Identifies Nasopharyngeal CarcinomaPatients at High Risk of Metastasis for AdditionalInduction ChemotherapyQi Yang1,2, Meng-Xia Zhang1,3, Xiong Zou1,2, You-Ping Liu1,2, Rui You1,2, Tao Yu1,2,Rou Jiang1,4, Yi-Nuan Zhang1,2, Jing-Yu Cao1,2, Ming-Huang Hong1,5, Qing Liu1,4,Ling Guo1,2, Tie-Bang Kang1, Xiao-Feng Zhu1, and Ming-Yuan Chen1,2

Abstract

Purpose: Metastasis is one of the most important causes oftreatment failure in nasopharyngeal carcinoma (NPC). In T4 orN2-3 patients at high-risk of metastasis, concurrent chemora-diotherapy (CCRT) is inadequate and additional induction che-motherapy (IC) is controversial. There is a critical need to developabetter patient stratification to efficiently identify patients at high-risk of metastasis for additional IC. Recently, Sequestosome 1(SQSTM1)/p62, an autophagy adaptor protein, was identified asone of the metastasis-related proteins in NPC. However, themechanism by which SQSTM1 is involved in NPC metastasiswas not investigated.

Experimental Design: The effect of SQSTM1 on cell migrationand invasion was examined in vitro and in vivo. SQSTM1 expres-sion was analyzed in clinical NPC samples using IHC. Luciferasereporter analyses were conducted to identify the effects ofSQSTM1 on NF-kB transcriptional activity. A prediction bio-

model was constructed by Cox analysis. Retrospective and pro-spective randomized clinical data were adopted to build and testthe model, respectively.

Results: SQSTM1 mediated epithelial to mesenchymaltransition (EMT) through the NF-kB pathway to promoteNPC metastasis. Inhibiting SQSTM1 enhanced sensitivity tocisplatin in NPC cells. In NPC patients, high SQSTM1 ex-pression was associated with increased risk of distant metas-tasis. Furthermore, we propose a prognostic bio-modelbased on SQSTM1 and N-stage to predict NPC metastasis.Most importantly, our prospective randomized study sug-gested that IC is beneficial for NPC patients with high metas-tasis risk.

Conclusions: The prognostic bio-model identifies NPCpatients at high-risk of metastasis for additional IC. Clin CancerRes; 24(3); 648–58. �2017 AACR.

IntroductionPatients with nasopharyngeal carcinoma (NPC), especially

with locoregionally advanced NPCs (stage III or IVa/IVb), suc-cumb to the metastatic disease (1). According to the NCCNguidelines (version 2012), concurrent chemoradiotherapy(CCRT) is the standard regimen for locoregionally advanced NPC

(LA-NPC), which can be divided into two groups according to theTNM staging system: low-risk metastasis group (T3N0-1) andhigh-risk metastasis group (T4 or N2–3; ref. 2). In low-riskmetastasis group, CCRT might decrease local recurrence anddistant metastasis. In contrast, CCRT alone is inadequate ineliminating the potential micrometastasis in the high-risk metas-tasis group. In this group of NPC patients, additional cycles ofchemotherapy, such as the addition of induction chemotherapy(IC) to CCRT, might improve the control of distant metastasis.However, IC did not reduce distantmetastasis or prolong survivalin previous clinical trials (3–7). We previously demonstrated thatIC plus CCRT increased the distant metastasis-free survival(DMFS) in LA-NPC, but the extent of the improvement was only4%, with most patients being overtreated (8). It is possible that abetter patient stratification can efficiently identify patients at high-risk of metastasis for additional IC. Traditional TNM stagingsystem can play a role but is not very effective. In this respect,incorporation of biomarkers by further elucidating the molecularmechanisms of NPC metastasis is a promising strategy.

Sequestosome 1 (SQSTM1), also known as p62 in humans, isan adaptor protein involved in selective autophagy. However,SQSTM1, independent of its role in autophagy (9), functions inmultiple signaling pathways that govern various cellular beha-viors (10–12). Excess SQSTM1 was related to poor prognosis invarious malignancies, such as lung adenocarcinoma, breast

1Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology inSouth China, Collaborative Innovation Center for Cancer Medicine, Guangzhou,China. 2Department of Nasopharyngeal Carcinoma, Sun Yat-sen UniversityCancer Center, Guangzhou, China. 3Department of Radiation Oncology, FujianMedical University Union Hospital, Fuzhou, China. 4Department of CancerPrevention, Sun Yat-sen University Cancer Center, Guangzhou, China. 5Depart-ment of Clinical Trial Center, Sun Yat-sen University Cancer Center, Guangzhou,China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Q. Yang, M.-X. Zhang, X. Zou, and Y.-P. Liu contributed equally to this study.

CorrespondingAuthor:Ming-YuanChen, Sun Yat-senUniversity Cancer Center,651 Dongfeng Road East, Guangzhou 510060, China. Phone: 8620-8734-3361;Fax: 8620-8734-3624; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-1963

�2017 American Association for Cancer Research.

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cancer, colon cancer, and others (13–15). A recent study has alsoidentified SQSTM1 as one of the metastasis-related proteins inNPC using iTRAQ labeling combined with 2D-LC/MS-MS (16).However, the clinical significance of SQSTM1 in NPC and themechanismbywhich it is involved inNPCmetastasis have not yetbeen investigated.

In this study, we demonstrated that elevated expression ofSQSTM1 initiated EMT to promote metastasis in NPC, asevidenced by an EMT phenotypic switch, enhanced cell migra-tion and invasion, and differential regulation of EMT markers.The phenotypic changes associated with SQSTM1 overexpres-sion required NF-kB transcriptional activity. Our in vitro resultssuggested that SQSTM1 inhibition induced apoptosis andenhanced sensitivity to cisplatin in NPC cells. We propose aprognostic model based on SQSTM1 status and N-stage topredict the risk of NPC distant metastasis. Our prospectiverandomized study confirmed the effectiveness of the prognosticmodel and suggested that compared with CCRT alone, IC plusCCRT is a more efficient treatment method for the high metas-tasis risk NPC patients.

Materials and MethodsCell lines and cell culture

Cell lines were kindly provided by Professor Chao-Nan Qian(Sun Yat-sen University Cancer Center) and cultured as describedpreviously (17). Human NPC cell lines (S18, S26, 5-8F, 6-10B,and Hone1) were cultured in DMEM (Invitrogen) with 10% FBS(HyClone). The nasopharyngeal epithelial cell line (NP69) wasgrown in defined KSFM medium supplemented with EGF (Invi-trogen). These cell lines were incubated in a humidified chamberwith 5% CO2 at 37�C. As reported previously, the highly meta-static subclone S18 and poorly metastatic subclone S26 wereestablished using limiting dilution method and conformed forin vitro functional studies and in vivo animal experiments (18). Allthe NPC cell lines used in this study were authenticated usingshort-tandem repeat profiling, and the cells were not cultured formore than 2 months.

Patients and tissue specimensIn this study, 116 paraffin-embedded pathological specimens

from primary-diagnosed, non-disseminated NPC patients wereobtained from the Department of Pathology of Sun Yat-SenUniversity Cancer Center. The median age of the patients was48 years (range, 24–77 years) and themedian follow-up timewas50.86 months (range, 12.93–72.23 months). The pathologicaltypes were WHO III in 113 patients and WHO II in 3 patients.

A total of 134 patients from a prospective clinical trial thatcompared the effect of CCRT with IC to CCRT alone in treatinglocoregional advanced NPC were enrolled in the study. Weselected these 134 patients because paraffin-embedded patholog-ical specimens of these cases were available in our hospital. Thesepatients were defined as the validation cohort of this study.

Written informed consent was provided by the sample donors,and approval was granted by the Institute ResearchMedical EthicsCommittee of Sun Yat-Sen University. This study is reportedaccording to the Reporting Recommendations for Tumor MarkerPrognostic Studies (REMARK) criteria (19).

Western blottingCellswere lysed inRIPAbuffer (150mmol/LNaCl, 0.5%EDTA,

50 mmol/L Tris, 0.5% SN40), incubated on ice for 30 minutes,and centrifuged for 25 minutes at 12,000 rpm (4�C). Fifty micro-grams of harvested total protein were separated on 10% sodiumdodecyl sulfate-polyacrylamide gradient gels and transferred ontoPVDFmembranes followed by blocking with 5%non-fat milk for2 hours at room temperature. Membranes were incubated withprimary antibody followed by incubation with horseradish per-oxidase–conjugated secondary antibody and then detected usingthe ECL chemiluminescence system (Pierce).

Quantitative real-time PCRTotal RNA of NPC cell lines was isolated using TRizol (Invitro-

gen) reagent. First-strand cDNA was synthesized using a Prime-Script RT Reagent Kit with gDNA Eraser. Quantitative PCR wasconducted for detection of SQSTM1mRNA using SYBR Premix ExTaq II (TaKaRa). The primers used for amplifying SQSTM1,E-cadherin, Snail, N-cadherin, Vimentin, MYC, VEGFC, Bcl-XL,CCND1, TNF a, and GAPDH are listed in Supplementary TableS1. The PCR parameters were as follows: 95�C for 10 minutes,followed by 40 cycles of 95�C for 20 seconds, 60�C for 20 secondsand 70�C for 30 seconds.

Plasmids, retroviral infection, and transfectionHuman SQSTM1 cDNA was amplified by PCR and cloned

into a pSin-EF2 lentiviral vector. Cell lines stably expressingcontrol or SQSTM1 short hairpin RNA (shRNA) were estab-lished by the Sigma shRNA system according to themanufacturer'sinstructions. The target sequences of the shRNA were as follows:

50-CCGGGACACCATCCAGTATTCAAAGCTCGAGCTTTGAAT-ACTGGATGGTGTCTTTTTTG-30.

Migration and invasion assaysWound-healing and Transwell assays were used to evaluate the

migration and invasion abilities of the cells. All experiments wereperformed in duplicate and repeated three times. For the wound-healing assay, cells were seeded in 6-well plates. When the cellsreached 90% confluency, a scratch was created using a sterilepipette tip. Phase-contrast imageswere taken in the samefield at 0,

Translational Relevance

Metastasis is one of the most important causes of treatmentfailure in nasopharyngeal carcinoma (NPC). In T4 or N2-3patients at high-risk of metastasis, concurrent chemora-diotherapy (CCRT) is inadequate and additional inductionchemotherapy (IC) is controversial. There is a critical need todevelop a better patient stratification to efficiently identifypatients at high-risk of metastasis for additional IC. Recently,Sequestosome 1 (SQSTM1)/p62 was identified as one of themetastasis-related proteins in NPC. Herein, we demonstratethat SQSTM1 mediated epithelial to mesenchymal transition(EMT) through the NF-kB pathway to promote NPC metas-tasis. Inhibiting SQSTM1 enhanced sensitivity to cisplatin inNPC cells. In NPC patients, high SQSTM1 expression wasassociated with increased risk of distant metastasis. Further-more, we propose a prognostic bio-model based on SQSTM1and N-stage to predict NPCmetastasis. Most importantly, ourprospective randomized study suggested that IC is beneficialfor NPC patients with high metastasis risk.

A Prognostic Bio-Model for NPC Patients

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24, and 48 hours after wounding. The Transwell assays wereperformed using a Boyden chamber containing 24-well Transwellplates with 8-mm membrane pores. For the Transwell migrationassay, 5� 104 cells in 300 mL of serum-free DMEMwere added tothe cell culture inserts without extracellular matrix coating. Sub-sequently, 500mL ofDMEMcontaining 10%FBSwas added to thebottom chamber. After about 24 hours of incubation, the cells onthe lower surface of the filter were fixed, stained, and examinedusingamicroscope.Theaveragenumberofmigratedcells fromfiverandom optical fields (�100 magnification) on triplicate filterswasdetermined.For theTranswell invasionassay, the inserts of thechambers inwhich the cellswere seededwere coatedwithMatrigel(Becton Dickinson Labware). The subsequent staining and obser-vation procedures were as described for the migration assays.

Animal experimentsAll animal work was performed in accordance with protocols

approved by the Research Animal Resource Center of Sun Yat-senUniversity. Male athymicmice between 5 and 6weeks of age wereobtained from the Shanghai Institute for Biological Sciences(Shanghai, China). All animal studies were conducted in accor-dance with the principles and procedures outlined in the guide-lines of Institutional Animal Care and Use Committee at Sun Yat-sen University Cancer Center. Briefly, a total of 1 � 106 cells in150mL PBSwere injected into the tail veins of laparotomizedmiceusing insulin syringes (Becton Dickinson). After tumor cellinoculation for 50 days, the experiment was terminated. Themetastatic nodules in each lung were counted.

Luciferase reporter assayThe reporter assay was carried out as described previously (20).

Briefly, 1.4 � 105 cells per well were seeded in 12-well plates andtransfectedwithNF-kBp65 luciferase reporter and pRL-TKRenillaluciferase construct (Promega) in each well. After 48 hours, theluciferase activity was measured using the Dual-LuciferaseAssay kit (Promega). Three independent experiments were per-formed and the calculated means and standard deviations weredetermined.

ImmunohistochemistryParaffin sections were stained for SQSTM1 (Santa Cruz Bio-

technology, sc-28359) by IHC. First, the tissue slides were bakedin a 60�C oven for 2 hours and were subsequently deparaffinizedby dimethylbenzene for 10 minutes twice and rehydrated bygraded ethanol. High-pressure pretreatment in citrate buffer(pH 6.0) was used for antigen retrieval, and 0.3%H2O2 was usedto block endogenous peroxidase activity for 30 minutes. Thesamples were then washed with PBST and incubated overnightat 4�C with primary antibodies against SQSTM1/SQSTM1 (dilut-ed 1:500). Next, the slides were incubated with biotinylated anti-goat antibodies for 30 minutes at room temperature and thesecondary antibodies conjugated with horseradish peroxidase for30 minutes at 37�C. The slides were then stained with 3, 30-diaminobenzidine tetrahydrochloride (DAB) and counterstainedwith hematoxylin.

The staining results were evaluated by intensity and positiverates as previously reported (21). For staining intensity, the scorewas classified as 0–3 (no staining, weak staining, moderatestaining, and high staining). For the positive rate, the score wasclassified as 0–4 (no staining, <10%, 10%–50%, 50%–80%, and>80%). The total scoresweredeterminedbymultiplying these two

values together and were grouped as no/low expression (�3) andhigh expression (>3). The scores were determined by two pathol-ogists blinded to the patients' statuses.

Statistical analysesComparisons of the distributions of clinicopathological para-

meters of different SQSTM1 expression groups were performedusing c2 tests. Kaplan–Meier analysis was carried out for NPC-specific survival, such as locoregional relapse-free survival(LRRFS), DMFS, progression-free survival (PFS), and overall sur-vival (OS). Statistical significance was evaluated using the log-rank test. Multivariate analysis was performed using the Coxproportional hazards model by including gender, age, T-stage,N-stage, UICC clinical stage, IC, and concurrent chemotherapy asthe confounding factors. The factors that were identified asindependent predictive prognostic factors for DMFS by multivar-iate analyses were combined for the construction of the distantmetastasis predictive model. P values of less than 0.05 wereconsidered significant in this study. The statistical analyses werecarried out using SPSS software version 18.0. The raw data of thisarticle have been uploaded onto the Research Data Deposit(RDD) with an RDD number of RDDB2017000199.

ResultsSQSTM1 is upregulated in NPC cell lines with high metastaticpotential

To detect SQSTM1 expression in NPC cells, we performedWestern blot and real-time PCR analyses to detect the protein aswell as mRNA levels of SQSTM1 in five NPC cell lines and theimmortalized nasopharyngeal epithelial cell line (NP69). Theresults showed that the protein and mRNA levels of SQSTM1were higher in the 5 NPC cell lines than those in the NP69 cells.Furthermore, SQSTM1 expression was much higher in the highmetastatic S18 cells than in the low metastatic S26 cells(Fig. 1A). These results indicate that SQSTM1 is up-regulatedin NPC cells and may be related to the progression andmetastasis of NPC cells.

SQSTM1 promotes NPC cell migration and invasion in vitroTo investigate the potential biologic function of SQSTM1, we

established stable SQSTM1overexpression in lowmetastatic NPCcells (S26-SQSTM1) or knockdown in high metastatic NPC cells(S18-shRNA; Fig. 1B). Thewound-healing assay revealed that cellswith higher SQSTM1 expression showed a significantly moreextensive wound closure area compared with the respective con-trol (Fig. 1C). This result was further confirmed by a Transwellmigration assay (Fig. 1D). In addition, using a Transwell Matrigelassay, a distinct difference in invasion rates was observed betweenthe negative control cells and the corresponding cells with mod-ified SQSTM1 expression (Fig. 1D). Thus, we conclude thatSQSTM1 overexpression enhances the migration and invasionof NPC cells in vitro.

SQSTM1 promotes NPC cell metastasis in vivoWe next evaluated the in vivo effect of SQSTM1 on metastasis

using a lung metastasis model. The highest and lowest metastaticpotential cell lines (S18 and S26, respectively) and their derivedcells (S18-shRNA and S26-SQSTM1, respectively) were injectedinto the tail vein of nude mice. As assessed by hematoxylin andeosin (H&E) staining and IHC (Fig. 2A and D), SQSTM1

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Clin Cancer Res; 24(3) February 1, 2018 Clinical Cancer Research650




overexpression resulted in a significant increase in lung weightand numbers of metastatic pulmonary nodules (Fig. 2B, C, E, andF). These findings were consistent with the in vitro results, indi-cating that SQSTM1 promotes NPC metastasis in vivo.

SQSTM1 modulates EMT in NPCWe noticed that cells with ectopic SQSTM1 expression had

morphological changes consistent with a mesenchymal phe-notype as compared with the negative control cells. NPC cells

Figure 1.

SQSTM1 promotes the migration and invasion of NPCcells in vitro. A, SQSTM1 mRNA and protein levels inimmortalized nasopharyngeal epithelial cell line(NP69) and five NPC cell lines were examined byWestern blotting (WB) and quantitative real-timePCR (qPCR) analyses. GAPDH was used as aninternal control. B, The SQSTM1 knockdown andoverexpression effects were confirmed by WB andqPCR. GAPDHwas used as an internal control.C, StableS26 and S18 cells were subjected to scratch wound-healing assay. The wound space was photographed at0, 24, and 48 hours. D, The migration and invasionassays showed different cell motilities in stable S26 andS18 cells. The depletion of SQSTM1 clearly inhibited themigration and invasion of S18 cells. Conversely, theectopic expression of SQSTM1 promoted the migrationand invasion of S26 cells. The data are presented as themean � s.e.m. from three independent experiments;� , P < 0.05 by Student t test.






overexpressing SQSTM1 displayed a spindle shape, increasedlamellipodia, increased cell spreading, and decreased cellpolarity as indicated by phalloidin staining (Fig. 3A). We,therefore, examined whether EMT was responsible forSQSTM1-mediated changes in cell metastasis. As shownin Fig. 3B and C, compared with the S26-vector cells, S26-

SQSTM1 cells expressed a lower level of the epithelial markerE-cadherin and a higher level of the mesenchymal markerssnail, N-cadherin, and vimentin. Conversely, knockdown ofSQSTM1 in S18 cells induced the expression of E-cadherin,which was accompanied by a concomitant reduction of snail,N-cadherin, and vimentin.

Figure 2.

SQSTM1 promotes CC cell metastasis in vivo. A and D,The indicated stable cells were injected into the lateraltail vein of nude mice. Left, center, and right panelspresent macroscopic appearances of metastatic lungtumors, H&E staining, and IHC staining for SQSTM1,respectively. B, C, E, and F, Numbers of metastases(B and E) and weights (C and F) per lung of miceinjected with the indicated stable cells.

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To further examine whether SQSTM1 enhances EMT in vivo,IHCwas used to assess the expression levels of SQSTM1 and EMT-related proteins in the lung sections of nude mice. The resultsdemonstrated that E-cadherin expression was significantlydecreased, and vimentin was concurrently increased in theSQSTM1 overexpression group compared with the vector group(Supplementary Fig. S1), suggesting that SQSTM1 enhances EMTin vivo. Overall, these findings demonstrate that SQSTM1 mod-ulates EMT in NPC.

SQSTM1 activates the NF-kB signaling pathway in NPC cellsTo explore the mechanism by which SQSTM1 promotes cell

metastasis and the acquisition of a mesenchymal cellular phe-notype in NPC, we analyzed the expression of SQSTM1 andother genes that are regulated by various signaling signatures

using gene set enrichment analysis (GSEA) in publicly availableNPC patients' expression profiles (GSE12452). We found thatSQSTM1 expression levels in NPC showed a positive correla-tion with the NF-kB signaling pathway (Fig. 4A). Therefore, weinvestigated whether SQSTM1 could promote cell metastasisand enhance the mesenchymal cellular phenotype by activatingthe NF-kB signaling pathway in NPC. Using the luciferasereporter assay, we found that up-regulation of SQSTM1markedly enhanced the activity of NF-kB luciferase reportergene, whereas knockdown of SQSTM1 significantly reducedreporter activity (Fig. 4B). Immunofluorescence stainingrevealed that overexpression of SQSTM1 resulted in a substan-tial nuclear accumulation of p65 (Fig. 4C and D) as indicatedby subcellular fractionation. Real-time PCR analysis of down-stream target genes of the NF-kB signaling pathway indicated

Figure 3.

SQSTM1 modulates EMT in NPC cells. A,Immunofluorescence assay for phalloidin was used tocompare the morphological appearances in differentgroups of S26 and S18 cells. Phalloidin was stained in redcolor, and the nuclei were stainedwith 40 ,6-diamidino-2-phenylindole (DAPI) in blue color. B, The mRNAexpression levels of four EMTmarkers (E-cadherin, Snail,N-cadherin and Vimentin) in different groups of S26 andS18 cells were assayed by real-time PCR. The data arepresented as the mean� s.e.m. from three independentexperiments; � , P < 0.05 by Student t test. C, The proteinlevels of four EMTmarkers in different groups of S26 andS18 cells were assayed by western blotting. GAPDH wasused as an internal control.






that SQSTM1 overexpression markedly increased the transcrip-tional activity of NF-kB in NPC cells (Fig. 4E). These findingssuggested that SQSTM1 overexpression enhances the nucleartranslocation of p65 thereby promoting the transcriptionalactivity of NF-kB.

The NF-kB signaling pathway mediates SQSTM1-induced EMTand metastasis

To further explore whether SQSTM1 promotes EMT and NPCmetastasis through the NF-kB signaling pathway, we used thespecific inhibitor of NF-kB signaling, BMS-345541, in the S26

Figure 4.

SQSTM1 activates the NF-kB signaling pathway in NPC cells. A, GSEA plot correlating SQSTM1 mRNA levels with NF-kB signaling (JAIN_NF-kB_SIGNALING) basedon publicly available NPC patient gene-expression profiles (NCBI/GEO/GSE 12452, n ¼ 41). B, The indicated cells were transfected with a luciferase reporterfor NF-kB, and then subjected to luciferase assays as described in Materials and Methods. C,Western blot of NF-kB p65 in the nuclear fractions of the indicated cells.D, Immunofluorescence of subcellular NF-kB p65 localization in the indicated cells. E, Real-time PCR analysis of downstream NF-kB pathway target genemRNA levels. Bars in B and E indicate mean � SD of three independent experiments; � , P < 0.05.

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NPC cell line. As shown in Supplementary Fig. S2A, BMS-345541downregulated the expression of NF-kB signaling targets VEGFC,BCL-XL, CCND1, and XIAP. The effect of BMS-345541 on nuclearNF-kB p65 was also investigated. Results suggested that nuclearNF-kB p65 levels were decreased by BMS-345541. It is of note thatSQSTM1 overexpression obstructed NF-kB p65 nuclear transloca-tion changes induced by BMS-345541 (Supplementary Fig. S2A).

We then examined the influence of BMS-345541 on EMTmarkers. As shown in Supplementary Fig. S2A, BMS-345541decreased the levels of Snail, Vimentin, N-cadherin but increasedthe expression of E-cadherin in S26 cell. BMS-345541 alsoopposed the activation of EMT resulting from SQSTM1 over-expression Supplementary Fig. S2A).

The wound-healing assay and Transwell invasion assay werethen performed to examine the effect of BMS-345541 onNPC cellmotility. We observed that SQSTM1 overexpression enhanced themotility of NPC cells, whereas BMS-345541 impaired this func-tion (Supplementary Fig. S2B and S2C).

Association between SQSTM1 and metastasis in clinical NPCsamples

Next, we explored the expression levels of SQSTM1 and itsassociation with clinical parameters using IHC in 116 NPC

samples (Fig. 5A). There was no significant correlation betweenSQSTM1 expression and gender, age, T-stage, N-stage, UICCstages, IC, or concurrent chemotherapy (Supplementary TableS2). As shown in Fig. 5B, higher SQSTM1 expression was signif-icantly correlated with shorter DMFS, PFS, and OS (5-year DMFS86.0% versus 66.2%, P ¼ 0.010; 5-year PFS 83.7% vs. 59.2%,P ¼ 0.003; 5-year OS 94.4% vs. 60.1%, P < 0.001, respectively),whereas LRRFS showed no statistically significant difference(P ¼ 0.857). Moreover, multivariate analyses revealed thatSQSTM1 overexpression and N-stage were independent, unfavor-able prognostic indicators for DMFS (Supplementary Table S3).These results suggest that SQSTM1 overexpression is clinicallyrelevant to thedistantmetastasis ofNPCand that SQSTM1maybeused as an independent prognostic predictor in NPC patients.

Generation of a prognostic model for NPC metastasisCell functional and molecular mechanism analyses confirmed

the association of SQSTM1 and aggressive phenotype ofNPC cellsand multivariate analyses identified SQSTM1 and N-stage asindependent predictive prognosis factors for DMFS. We, there-fore, constructed an SN metastasis prediction model based on N-stag e and SQSTM1/p62 expression. Patients were stratified intothree risk groups: (i) the low-risk distant metastasis group (early

Figure 5.

Clinical association between SQSTM1 and metastasis in human NPC. A, Criteria for SQSTM1 expression intensity scoring. Representative micrographs are shown(�400). All micrographs were taken and processed at identical conditions. B, Kaplan–Meier analysis of the 5-year DMFS, PFS, and OS with regard tothe SQSTM1 expression levels (a–c). TheDMFS, PFS andOSof high-SQSTM1 patientswere significantly lower than those of the low-SQSTM1 patients.C,Kaplan–Meieranalysis of the 5-year DMFS, using the SN model of low (early N-classification and low-SQSTM1), intermediate-risk (advanced N-classification or high-SQSTM1),and high-risk (advanced N-classification and high-SQSTM1) patients with NPC. (a) The 5-year DMFS curves of low, intermediate and high-risk groups forall 116 NPC patients. (b) The 5-year DMFS curves of low- and intermediate-risk groups and the high-risk group for all 116 NPC patients.D, (a) Kaplan–Meier analysis ofthe 5-year DMFS curves using the SN model of low- and intermediate-risk groups and the high-risk group for all 134 NPC patients in the validation cohort.(b and c) Kaplan–Meier analysis of the 5-year DMFS of the high- (b) and low- plus intermediate (c)-risk metastatic NPC patients receiving induction chemotherapyplus concurrent chemoradiotherapy (ICþCCRT) or CCRT.






N-stages and low-level expression of SQSTM1) comprising 21(18.1%) of the patients, (ii) the intermediate-risk distant metas-tasis group (advanced N-stages or high expression level ofSQSTM1) comprising 53 (45.7%) of the patients, and (iii) thehigh-risk distant metastasis group (advanced N-stages and highexpression level of SQSTM1) comprising 42 (36.2%) of thepatients.

Overall, 28 patients developed distant metastasis in this cohortincluding 3 of 21 (14.3%) in the low-risk, 7 of 53 (13.2%) in theintermediate-risk and 20 of 42 (47.6%) in the high-risk group.Compared with the low-risk group, the relative HRs of distantmetastasis were 0.994 and 2.154 in the intermediate- and high-risk distant metastasis groups, respectively (P ¼ 0.993 andP ¼ 0.013). Compared with the intermediate-risk group, therelative HR of distant metastasis was 4.564 in the high-risk group(P¼ 0.001). The low-risk and intermediate-risk groups were thenintegrated for comparison with the high-risk distant metastasisgroup (DMFS: P < 0.001; Fig. 5C). This indicated that the SNmodel was a better prognostic indicator for predicting distantmetastasis than the TNM-stage only for NPC.

Validation of the predictive accuracy of the SNmodel for DMFSIn the validation cohort fromaprospective randomized clinical

trial, 63, 60, and 11 patients were allocated into the high-,intermediate-, and low-risk distant metastasis groups, respective-ly, according to our SNmodel. Patients in low- and intermediate-risk groups were associated with a better DMFS than those in thehigh-risk group (5-year DMFS 83.7% and 67.7%, respectively;P¼ 0.04, Fig. 5Da), further supporting the effectiveness of the SNmodel. More significantly, we found that adding IC with CCRT tohigh-risk patients resulted in improved control of distant metas-tasis (Fig. 5Db). In contrast, patients in the low- and intermediate-risk groups did not benefit from combined IC and CCRT. Thesurvival curves indicated that CCRT was sufficient for thesepatients (Fig. 5Dc).

SQSTM1 inhibition induces apoptosis and enhances sensitivityto cisplatin in NPC cells

To further elucidate the role of SQSTM1 onNPC cell sensitivityto chemotherapeutic drugs, anMTT assay was performed after theS18-control and S18-shRNA cells were treated with increasingdoses of cisplatin, a commonly used chemotherapeutic drug for

NPC patients. As shown in the dose-dependent curve for cisplatintreatment, knockdown of SQSTM1 decreased the survival of cells(Fig. 6A). It is generally accepted that cancer cells treated withcisplatin show reduced cell viability because of increased apo-ptosis. StainingwithAnnexin V andPI showed that knockdownofSQSTM1 promoted apoptosis in cisplatin-treated S18 cells (Fig.6B). Furthermore, we analyzed targets of caspase machinery andfound that SQSTM1 knockdown triggered cleavages of both pro-caspase 9 and PARP in a concentration-dependent manner (Fig.6C). Taken together, these data indicated that knockdown ofSQSTM1 could enhance the sensitivity of NPC cells to cisplatinin a concentration-dependent manner.

DiscussionOur study demonstrated that upregulation of SQSTM1

increased, whereas its downregulation decreased the invasionand metastasis of NPC cells both in vitro and in vivo. The in vitrostudies also suggested that SQSTM1 inhibition induced apoptosisand enhanced sensitivity to cisplatin in NPC cells. Furthermore,we showed that SQSTM1 could activate the NF-kB signalingpathway to promote EMT leading to an aggressive phenotypeand tumor progression to metastasis. Clinically, high SQSTM1expressionwas associatedwith increased risk of distantmetastasisand poor survival inNPCpatients.We thus proposed a prognosticmodel based on SQSTM1 status andN-stage, named SNmodel, topredict the risk of distant metastasis in NPC patients. Our pro-spective study data also confirmed the benefit of additional IC intreating patients who were at a high-risk of distant metastasis.

It is known that SQSTM1, as amultifunctional protein, serves asa signaling hub that modulates various cellular processes, includ-ing cell survival and cell death (11). The robust accumulation ofSQSTM1 has been detected in gastrointestinal, prostate, liver, andbreast cancer cases (22–25), suggesting a functional relationshipbetween SQSTM1 accumulation and cancer progression.Here, wedemonstrated, for the first time, that high expression of SQSTM1was related to subsequent distant metastases and poor survival ofNPC patients. Our results suggested that overexpression ofSQSTM1 may favor aggressive clinical behavior in NPC tumorsand accelerated the metastatic properties of NPC cells in vitro andin vivo, whereas suppression of SQSTM1 had the opposite effect.This further attested to the fact that aberrant accumulation of

Figure 6.

Knockdown of SQSTM-induced apoptosis and enhanced sensitivity to cisplatin in NPC cells. A, Dose-dependent curve of cisplatin treatment in S18-control andS18-shRNA cells. B, Flow-cytometry analysis of Annexin Vþ/PI�cells after the indicated cells treated with cisplatin (10 mg/mL) for 24 hours. Results areexpressed as percentages of total cells.C,Western blot analysis of the effect of SQSTM1 on cleavedPARP and caspase-9 levels at three different concentrations in theindicated cells.

Yang et al.





SQSTM1 was closely related to the formation of distant metas-tasis. Consistent with our study, analyses of breast cancers anddigestive system cancers showed that the risk of distantmetastasiswas higher in SQSTM1-positive cases (24, 26).

A previous study revealed that accumulation of SQSTM1 leadsto EMT in human skin squamous cell carcinoma (SCC), suggest-ing that this could be a mechanism of SQSTM1-mediated skinSCC invasion and lung metastasis (27). In the present study, weobserved that SQSTM1 expression correlated significantly withthe EMT markers in NPC cells. Both real-time PCR and Westernblotting showed that SQSTM1 upregulated the expression ofsnail, N-cadherin, and vimentin and downregulated E-cadherinin NPC cells. Furthermore, similar results were detected in thelung metastatic tumor sections of mice, suggesting that SQSTM1promotes EMT in vivo.

It has been shown that NF-kB signaling has a significant impacton EMT during cancer progression (28, 29). Translocation of p65into the nucleus may result in the loss of E-cadherin and subse-quent induction of EMT. In other studies, the autophagy adaptorsignaling protein, SQSTM1 has been shown to be an importantNF-kB mediator in tumorigenesis (12, 30, 31). It has beenreported that sustained SQSTM1 expression resulting fromautop-hagy defects was sufficient to alter NF-kB regulation and geneexpression and to promote tumorigenesis (31). Therefore, weinvestigated whether SQSTM1 mediates EMT to promote NPCmetastasis through the NF-kB Pathway. By analyzing SQSTM1andNF-kBpathway signatures usingGSEAwithpublicly availableNPC patient expression profiles, we found that the levels ofSQSTM1 in NPC were associated with the NF-kB signaling path-way. Furthermore, we demonstrated that overexpression ofNF-kBenhanced p65 nuclear translocation and the subsequent tran-scriptional activity of NF-kB resulting in an enhanced mesenchy-mal cellular phenotype. These underlying mechanisms mayexplain the relationship between SQSTM1 accumulation andNPC metastasis.

Clinically, survival rates for NPC patients with distant metas-tasis are generally poor as NPC biologically differs from classichead and neck squamous cell carcinoma (HNSCC; ref. 32). This ispartially due not only to the fact that NPCpatients usually presentat a late stage with large tumors but also to the lack of effectivepredictive biomarkers.

In the current study, we constructed a metastasis predictionmodel based on SQSTM1 status and N-stage, named SN model.This is the first study to combine SQSTM1 status with the clinicalTNM-stage system to evaluate the risk of distant metastasis.Previously, Xia and colleagues (33) reported a prognostic modelbased on C-reactive protein and N-stage to predict the risk ofdistant metastasis and death of NPC patients which may contrib-ute to individualized treatments.However, the elevatedC-reactiveprotein level can be reflective of trauma, infection, inflammation,and other diseases, such as acutemyocardial infarction. C-reactiveprotein levels fluctuate and are easily affected by other factorswhereas SQSTM1 status may be more stable and reliable.

For the newly developed prognostic SN model, we used datafrom a prospective study and verified its effectiveness. Moreimportantly, when SQSTM1 was used for risk stratification, ICshowed promising results in high metastatic risk NPC patients(advanced N-stage and high expression of SQSTM1) with anincrease in 5-year DMFS of 27.7% (ICþCCRT vs. CCRT,P ¼ 0.024). However, IC could only increase the 5-year DMFSof all patients by only 4% (ICþCCRT vs. CCRT, 86% vs. 82%,

P ¼ 0.056), with most patients being overtreated. The high-metastatic potential NPC patients are likely to form subclinicalmicro-metastasis sites, and adding IC to CCRT may result in thegreatest eradication of micro-metastases. In addition, IC canreduce tumor load and improve blood supply before irradia-tion delivery, and thus may improve the sensitivity of tumorcells to radiotherapy. On the other hand, in intermediate- andlow-risk metastasis NPC patients, IC may result in overtreat-ment of tumor cells that are not prone to metastases andtherefore form fewer micro-metastases. A combination withIC may delay the implementation of radiotherapy and lead tochemoradiotherapy-resistant tumor cells in distant metastases.These defects can outweigh the potential micro-metastaseseradication effect and result in no better DMFS in these inter-mediate- and low-risk metastasis NPC patients.

The correlation between SQSTM1 expression and IC sheds lighton the central role of SQSTM1 in NPC cell sensitivity to chemo-therapeutic drugs. It is possible that SQSTM1 inhibition enhancesthe sensitivity of NPC cells to chemotherapeutic drugs. In thisrespect, the in vitro studies performed in this study indicated thatknockdownof SQSTM1 could enhance the sensitivity ofNPC cellsto cisplatin in a concentration-dependent manner. These resultsmight explain the fact that CCRT is sufficient for patients with lowSQSTM1 expression. By contrast, increased concentration of che-motherapeutic drugs, such as the use of additional IC, might beneeded for patients with high SQSTM1 expression to achievesatisfactory efficiency.

In summary, by using cell functional and survival analyses, wedemonstrated, for the first time, that increased levels of SQSTM1were associated with high biological aggressiveness of NPC cellsand poor survival outcome. SQSTM1 promotes EMT-mediatedNPCmetastasis through the NF-kB pathway. The proposed prog-nostic model based on SQSTM1 status and N-stage can serve as auseful tool to predict the risk of distant metastasis of NPC. Ourprospective randomized study confirmed IC plus CCRT as amoreefficient treatment method for the high-metastasis risk NPCpatients (advanced N-stage and high SQSTM1 status) comparedwith CCRT.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M.-H. Hong, M.-Y. ChenDevelopment of methodology: M.-X. Zhang, X. Zou, R. You, M.-Y. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.-X. Zhang, X. Zou, R. You, J.-Y. Cao, L. Guo,M.-Y. ChenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):Q. Yang,M.-X. Zhang, X. Zou, Y.-P. Liu, T. Yu, R. Jiang,Y.-N. Zhang, Q. Liu, T.-B. Kang, M.-Y. ChenWriting, review, and/or revision of the manuscript: Q. Yang, M.-X. Zhang,X. Zou, R. You, X.-F. Zhu, M.-Y. ChenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Zou, M.-Y. ChenStudy supervision: T.-B. Kang, M.-Y. Chen

AcknowledgmentsThis work was supported by the New Century Excellent Talents in University

(NCET-12-0562), the Sun Yat-Sen University Clinical Research 5010 Program(201310), the National Natural Science Foundation of China (grant nos.81370060, 81772895, 81572848, and 81772877), the Guangdong ProvincialNatural Science Foundation of China (S2013020012726), the Program






for Training Program Foundation for the Talents by Sun Yat-sen UniversityCancer Center (MY Chen, 2009), the National High Technology Research andDevelopment Program of China (863 Program, no. 2012AA02A501), Guang-dong Province Science and Technology Development Special Funds (Frontierand Key Technology Innovation Direction—Major Science and TechnologyProject), and Guangzhou Science and Technology Planning Project—Produc-tion and Research Collaborative Innovation Major Project.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 8, 2017; revised September 6, 2017; acceptedOctober 10, 2017;published OnlineFirst October 13, 2017.

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Yang et al.



VUMCA I Trial


2018;24:648-658. Published OnlineFirst October 13, 2017.Clin Cancer Res Qi Yang, Meng-Xia Zhang, Xiong Zou, et al. Additional Induction ChemotherapyNasopharyngeal Carcinoma Patients at High Risk of Metastasis for A Prognostic Bio-Model Based on SQSTM1 and N-Stage Identifies

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