molecular chaperones as a common set of proteins that ... · molecular chaperones (19%). we...

Human Cancer Biology

Molecular Chaperones as a Common Set of Proteins That Regulatethe Invasion Phenotype of Head and Neck Cancer

Ching-Chi Chiu1, Chien-Yu Lin2, Li-Yu Lee3, Yin-Ju Chen1, Ya-Ching Lu1, Hung-Ming Wang4,Chun-Ta Liao5, Joseph Tung-Chieh Chang2, and Ann-Joy Cheng1

AbstractPurpose: The goal of this study was to establish a common set of molecules that regulate cell invasion in

head and neck cancer (HNC).

Experimental Design: Five invasive sublines derived from HNC cell lines were established using the

Matrigel selection method. Proteomic technology, MetaCore algorithm, and reverse transcriptase-PCR

methods were used to search for molecules that contribute to the invasion phenotype. Cellular functional

analyses and clinical association studies were applied to examine the significance of the molecules.

Results: Fifty-two proteins were identified in more than two of the four independent proteomic

experiments, including 10 (19%) molecular chaperones. Seven chaperones were confirmed to be differ-

entially expressed in five sublines, Hsp90a, Hsp90b, Hsp90-B1/Gp96, Hsp70-A5/Grp78, andHYOU1, that

upregulate, whereas Hsp60 and glucosidase-a neutral AB (GANAB) downregulate. Four molecules were

further investigated. In all cell lines, knockdown of Hsp60 or GANAB and silencing of Gp96 or Grp78

considerably enhanced or reduced cell migration and invasion, respectively. Clinical association studies

consistently revealed that low levels of Hsp60 or GANAB and high levels of Gp96 or Grp78 are significantly

associated with advanced cancer (P < 0.001 to P ¼ 0.047, respectively, for the four molecules) and poor

survival (P < 0.001 to P ¼ 0.025, respectively, for the four molecules).

Conclusion: Our study defined molecular chaperones as a common set of proteins that regulate the

invasionphenotype ofHNC. Loss of the tumor suppression function ofHsp60orGANABand acquisition of

the oncogenic function of Gp96 or Grp78 contribute to aggressive cancers. These molecules may serve as

prognosticmarkersandtargets forcancerdrugdevelopment.ClinCancerRes;17(14);4629–41.�2011AACR.

Introduction

Head and neck cancer (HNC) is the 6th most prevalentcancer in the worldwide, with an estimated over 500,000new cases being diagnosed annually (1, 2). HNC is chara-cterized by an aggressive growth phenotype and earlymetastasis that lead to difficult tumor control. Identifica-tion of molecular markers and effective treatment regimens

for metastasis are urgently needed. Recently, several investi-gators have conducted global expression profiling experi-ments to identify genes linked to cellular invasion.However, there was little overlap in the genes identifiedby each study, probably because of differences in the studydesigns. For example, different experimental approacheswere used to identify invasion-related genes, including thedirect comparison of 2 sets of samples with differentinvasive capabilities (3–5) and the comparison of cancercell lines with normal keratinocytes (6). However, a majordisadvantage of these approaches lies in the heterogeneityof the samples. To reduce heterogeneity and obtain acommon set of data on molecules involved in HNC inva-sion, in this study, we used an in vitro Matrigel invasionmodel to establish 5 highly invasive HNC sublines, follow-ing a global survey of the invasion-associated molecules byproteomic methods. A total of 52 proteins were identifiedwith high frequency, including a significant fraction ofmolecular chaperones (19%). We therefore further investi-gated whether this group of proteins possessed roles incellular invasion.

Molecular chaperones are required for the stability andactivity of a wide range of client proteins that are involvedinmanybiological processes, including signal transduction,cellular trafficking, chromatin remodeling, cell growth,

Authors' Affiliations: 1Department of Medical Biotechnology, ChangGung University, Taoyuan, Taiwan; Departments of 2Radiation Oncology,3Pathology, 4Medical Oncology, and 5Department of Otorhinolaryngology,Head and Neck Surgery Section, ChangGungMemorial Hospital, Taoyuan333, Taiwan

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

Ching-Chi Chiu and Chien-Yu Lin contributed equally to this work.

Corresponding Author: Ann-Joy Cheng, Department of Medical Biotech-nology, Chang Gung University, 259 Wen-Hwa 1st Road, Taoyuan 333,Taiwan. Phone: 886-3-2118800 (ext 5085); Fax: 886-3-2118247; E-mail:[email protected] and Joseph Tung-Chieh Chang, Department ofRadiation Oncology, Chang Gung Memorial Hospital, 5 Fu-Shin Road,Taoyuan 333, Taiwan. Phone: 886-3-2118800; Fax: 886-3-2118247;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-10-2107

�2011 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 4629

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Published OnlineFirst June 3, 2011; DOI: 10.1158/1078-0432.CCR-10-2107

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differentiation, and reproduction (7, 8). However, theirfunctions in regulation of cell mobility or invasion havenot been much addressed. In this study, we confirmed 7proteins differentially expressed in 5 HNC invasion sub-lines. We further validated the biological functions andclinical significance of 4 proteins, and discussed the impli-cations in the carcinogenic mechanisms.

Materials and Methods

Establishment and characterization of highly invasiveHNC cell sublines

Five HNC cell lines were used, including 2 nasopharyn-geal cancers (BM1 and BM2; ref. 9), 1 oral cancer (OECM1;ref. 10) and 2 pharyngolaryngeal cancers (Fadu andDetroit; ref. 11). All the cell culture conditions were thesame as previously reports (9–11). The Matrigel invasionmethod was used to establish HNC cell sublines with ahigh invasive capability, similar to what has been pre-viously described (12). Four generations of HNC invasivesublines were established. Although different cells showedvarying levels of invasiveness, the invasive ability of theHNC sublines increased with ascending generations. Com-pared to the parental cells, the invasive ability of the 5different cell lines increased between 13- and 21-fold after 4generations (Supplementary Fig. S1). These results indicatethat highly invasive sublines of HNC cells were successfullyestablished.

Subcellular protein extraction and comparativeproteomic analysis

To better identify cell proteomes, subcellular proteinswere extracted as previously described (13). Fractions con-taining 50 mg of protein were separated by electrophoresison an 8% to 16% gradient gel. These protein bands werethen scanned by a densitometer and analyzed by Image-

Master software. Protein bands with differential expres-sions were excised, extracted, and identified usingMALDI-TOF mass spectrometry as previously described(14, 15). Peptide mass fingerprinting was done using theMascot search engine (Matrix Science) and the NationalCenter for Biotechnology Information protein–proteinBLAST database.

Prediction and analysis of the network pathwaysassociated with invasion

Network analyses of differentially expressed genes weredone using the MetaCore Analytical suite (GeneGo Inc.) aspreviously described (12). MetaCore was used to calculatethe statistical significance (P value) of the probability ofassembly from a random set of nodes (genes) of the samesize as the input list. To build the network of differentiallyexpressed genes, we applied the shortest paths algorithm toestablish direct paths between selected objects.

RNA extraction and reverse transcriptase-PCRanalysis

Total RNA was extracted from cells using the TRIzolreagent (Gibco BRL) following the manufacturer’sinstructions. The cDNA synthesis and PCR reaction pri-marily followed methods that have been previouslydescribed (16). The primer sequences are listed in Sup-plementary Table S1. The PCR products were analyzed by1.5% agarose gel electrophoresis. The density of eachband was determined after normalization to an actincontrol band using the Gel Image System (Scion Corpora-tion).

Protein extraction and immunoblot analysisThe protein extraction and immunoblot analysis were

done as previously described (17). Briefly, cellular proteinswere separated by SDS-PAGE and transferred to a nitro-cellulose membrane. The membrane was hybridized withprimary antibodies and then incubated with horseradishperoxidase-conjugated secondary antibodies. The primaryantibodies used were anti-Hsp60 (clone: MAB3514, Che-micon), anti-glucosidase-a neutral AB (GANAB; clone:C-16, Santa Cruz Biotech.), anti-Gp96 (clone: 9G10.F8.2, NeoMarkers), and anti-Grp78 (clone N-20, SantaCruz Biotech.). The membranes were developed andexposed to x-ray film. Using the Gel Image System (ScionCorporation), the density of each band was determinedafter normalization to an actin control band.

Cloning and transfection of short hairpin RNAplasmids

The pTOPO-U6 vector was used to construct Hsp60-,GANAB-, Gp96-, and Grp78-short hairpin RNA (shRNA)plasmids as previously described (9). Briefly, a 15 to 18sense and antisense complementary hairpin oligonucleo-tide was generated against a specific mRNA sequence ofeach gene, following cloned into pTOPO-U6 vector. Thesequences of the specific shRNA oligonucleotides are listedin Supplementary Table S2.

Translational Relevance

In this study, we identified 52 proteins whose expres-sion patterns are commonly altered in 5 invasive headand neck cancer sublines. The proteins included 10(19%) molecular chaperones. Seven proteins had con-firmed differential expression patterns, and 4 werefurther investigated. Cellular studies showed thatGp96 and Grp78 play positive roles in the regulationof cell migration and invasion, whereas Hsp60 andGANAB play negative roles. Clinical studies have con-sistently revealed that high levels of Gp96 and Grp78and low levels of Hsp60 and GANAB are significantlyassociated with advanced cancer and poor survival.Thus, losing the tumor suppressive function of Hsp60or GANAB and acquiring the oncogenic function ofGp96 or Grp78 contribute to aggressive cancer. Theseproteins may serve as prognostic markers in the predic-tion of patient survival and as targets for cancer drugdevelopment.

Chiu et al.

Clin Cancer Res; 17(14) July 15, 2011 Clinical Cancer Research4630




For cellular transfection, the Lipofectamine 2000 reagentand Opti-MEM medium (Invitrogen) were used, as pre-vious described (10, 11).

Cell growth, migration, and invasion assaysCell growth, migration, and invasion assays were done as

previously described (10). Briefly, cell growth was mon-itored by counting cells on a daily basis. Cell migration wasevaluated using the in vitro wound-healing assay. Cellinvasion assays were done using a BioCoat Matrigel andTranswell invasion chamber. The invasive ability of thesecells was determined by 2 days of incubation and bycounting the cells that had passed through the Matrigel-coated membrane into the lower chamber.

ImmunohistochemistryImmunohistochemistry analysis was done using a strep-

tavidin–biotin complex system (LSAB2 system; Dako),similarly as previous described (16). These primary anti-bodies were anti-Hsp60 (clone: MAB3514, Chemicon),anti-GANAB (clone: C-16, Santa Cruz Biotech.), anti-Gp96 (clone: 9G10.F8.2, NeoMarkers), and anti-Grp78(clone N-20, Santa Cruz Biotech.). The color was deve-loped with AEC substrate chromogen (LSAB2 system). Thestaining reactions were determined by microscopic exa-mination. The immunoreactivity was evaluated by subjec-tive assessment of themedian staining intensity, as negative(0: no staining), weak (þ1), moderate (þ2) or strong (þ3).

Patient characteristicsThis study was approved by the Institutional Review

Broad of the Human Investigation Committee in ourinstitution. Written informed consent was obtained fromall patients participating in this study. A total of 78 patientsvisiting the Chang Gung Memorial Hospital (Taoyuan,Taiwan) were recruited for this study. Biopsies of the tumorsample and grossly normal mucosa cells were obtainedfrom each subject before chemotherapy or radiotherapy.The patients included 74 (95%) males and 4 (5%) femaleswith an age range of 28 to 76 years (mean age: 49.8 years).All patients received radical surgery and postoperativeadjuvant treatment. Thirty-six patients (46%) receivedpostoperative radiotherapy and 11 had concurrent cispla-tin based chemotherapy. The median follow-up time was5.34 years (range: 1.2–8.5 years). At the end of study, 20patients had died of disease. The 3- and 5-year disease-specific survival rates were 81% and 76%, respectively.

Clinical association studyBiopsies of cancer and grossly normalmucosa tissue were

obtained from each subject before chemotherapy or radio-therapy. Proteins were extracted from tissues and subjectedto immunoblot analysis as described earlier to determinethe expression level of the chaperone proteins (Hsp60,GANAB, Gp96, and Grp78). To define the relative levelsof protein expression in clinical samples, thebanddensityofeach tumor samplewas normalizedwith an internal control(actin protein expression) and compared with that of nor-

mal tissue from the same patient. The cutoff points weredetermined after calculation of the receiver operating char-acteristic curve for best fit of sensitivity and specificity. ForHsp60 andGANAB, theprotein expressions in tumor tissueslower than1.2-foldof thenormal counterpartswere definedas low level. In Gp90 and Grp78, the protein expressions intumor tissues greater than 1.8-fold of the normal counter-parts were defined as high level.

The Pearson’s chi-square test was used to examine theassociation of chaperone protein expressions and clinico-pathologic features, including TNM stage. Survival curveswere calculated by the Kaplan–Meier method with a log-rank test. All P values were 2-sided, and the significancelevel was set at a P < 0.05.

Results

Comparative proteomics and network pathwayprediction

Comparative proteomic analysis was done in the pa-rental and the sublines of the HNC cell lines. Four inde-pendent experiments were done, and a representativeexample is shown in Supplementary Fig. S2(A). A totalof 420 protein bands were identified bymass spectrometry.These bands represented 184 proteins. Fifty-two proteinswere identified more than twice among different cell types,indicating the significance of these proteins in regulation ofthe invasion phenotype, as summarized in Table 1. Ofthese 52 proteins, 18 (35%) function as cytoskeleton oradhesionmolecules, 10 (19%) function as molecular chap-erones, 9 (17%) are metabolic enzymes, 8 (15%) areinvolved in transcriptional or translational regulation,and 7 (13%) are involved in cellular signaling transduction(Supplementary Fig. S2B).

The 52 proteins identified in several proteomes wereimported into MetaCore, and pathways associated withinvasiveness were analyzed. Seven regulatory pathways werefound to be significantly associated with invasiveness(P < 10�23): regulationofapoptosis, actin cytoskeletonorga-nization and biogenesis, mechanism of double-strand breakrepair, cellular response to stress, branching morphogenesisof a tube, pathway inmitochondrial ornithine transport, andcell cycle regulation (Supplementary Table S3).

Tomore specifically determine themost significant path-way in the regulation of cell invasion in HNC, the shortestpath analytical model of MetaCore was applied. A total of33 of the 52 identified genes were matched to networkpathways (Fig. 1A). To validate these predicted networks,some of the network proteins were examined differentiallyexpressed between parental and subline cells. As shown inFig. 1B, several molecules were shown to be involved inthe regulation of the invasion phenotype: the MMP2 extra-cellular protein; membrane protein Annexin-II; cytoplas-mic c-Raf, ERK1/2, IKK-a, and 14-3-3-sigma signalingproteins; and nuclear STAT1 and c-Myc transcription fac-tors. These results indicate that the invasion phenotype isexquisitely controlled by complicated mechanisms inhuman cells.

Molecular Chaperones in Head and Neck Cancer Invasion

www.aacrjournals.org Clin Cancer Res; 17(14) July 15, 2011 4631




Table 1. List of 52 proteins identified in HNC invasive sublines

NCBI number Name Scorea Mass(Da)b

pIc Sequencecoveraged

Up/downregulationa

Molecular chaperone (N ¼ 10)gi|83699649 Hsp 90, a 209 98,082 5.07 55% Upgi|46249928 Hsp 90, b 234 83,212 4.97 50% Upgi|61656607 Hsp 90-B-1/Gp96 250 92,282 4.47 47% Upgi|62089222 Hsp 70-1A variant 212 77,448 5.97 41% Upgi|34783614 Hsp 70-1B 182 70,009 5.62 56% Upgi|48257068 Hsp 70-8, isoform 1 170 70,854 5.37 60% Upgi|6900104 Hsp 70-A5/Grp78 321 72,288 5.07 52% Upgi|47938913 HYOU1 protein 126 75,297 5.55 41% Upgi|31542947 Hsp60/Chaperonin 234 60,986 5.7 61% Downgi|38197033 GANAB 108 55,401 6.26 40% DownCytoskeleton/adhesion (N ¼ 18)gi|53791219 Flamin A 340 277,332 5.70 40% Upgi|62089364 Filamin B, b 193 170,056 5.29 38% Upgi|29436380 MYH9 286 158,653 5.78 60% Upgi|50513540 Neurofibromin 2 110 64,570 9.03 61% Upgi|4506787 IQ motif-GTPase-1 398 189,134 6.08 45% Upgi|39795300 MAP6 43 77,024 8.88 69% Upgi|2606094 Cyr61 40 42,041 8.68 71% Upgi|54696696 Annexin A1 101 38,690 6.57 66% Upgi|50845388 Annexin A2 250 40,386 8.53 59% Upgi|55665463 MACF1 97 505,330 5.22 50% Upgi|14719392 Cofilin 2 76 18,725 7.66 72% Upgi|18088719 Tubulin, b 163 49,640 4.79 61% Upgi|46249758 Villin 2 (ezrin) 280 69,199 5.94 58% Upgi|1477646 Plectin 291 518,173 5.57 26% Upgi|62897681 Calreticulin precursor 105 46,890 4.3 53% Upgi|62088870 Type VII collagen-a1 96 96,749 5.66 29% Upgi|299626 EMS1 197 61,599 5.24 59% Upgi|24657579 Vinculin, isoform VCL 188 116,663 5.83 46% DownMetabolic enzyme (N ¼ 9)gi|38013966 TKT 210 58,174 6.51 69% Upgi|35505 Pyruvate kinase 230 57,841 7.58 60% Upgi|13325287 Enolase 189 47,139 7.01 74% Upgi|15012036 Glutathione transferase 130 23,327 5.43 66% Upgi|4758304 Disulfide isomerase-4 78 72,887 4.96 40% Upgi|89573929 G3PDH 204 24,605 8.68 64% Upgi|30582761 Phosphoglucomutase 1 93 1,331 6.2 64% Upgi|5803225 Tyr/Tryp-monooxygenase 157 29,155 4.63 71% Upgi|12804929 NAD 2 (mitochondrial) 147 35,537 8.92 70% UpTranscription/translation (N ¼ 8)gi|10863945 ATP-dep DNA helicase II 102 82,652 5.55 38% Upgi|29126861 TIF- 4A, isoform 2 87 46,460 5.32 50% Upgi|62896661 TEF-1a, 1 variant 110 50,110 8.98 45% Upgi|4503483 TEF-2 111 95,277 6.41 46% Upgi|62088704 HnRNP-K isoform variant 135 48,774 5.48 59% Upgi|75517570 HnRNP-A1 139 29,368 9.19 67% Upgi|14124942 Ribophorin I 118 64,542 6.1 60% Upgi|189306 Nucleolin 76 76,298 4.59 29% Up

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Molecular chaperone proteins have consistentlyaltered expression in invasive cellsIt is interesting to note that, in addition to cytoskeleton

and adhesion molecules, molecular chaperones repre-sented a significant fraction (10/52, 19%) of the proteinswe identified (Table 1). We therefore explored whether thisgroup of proteins contributes to cellular invasion in HNC.Seven of the proteins that appeared frequently in theproteomic screening were selected for confirmation studies.Reverse transcriptase (RT)-PCR analysis was done and theresults are shown in Fig. 1C. As shown, Hsp90a/Hsp90-AA,Hsp90b/Hsp90-AB, Hsp90-B1/Gp96, Hsp70-A5/Grp78,and HYOU1 were consistently upregulated in all 5 invasivesublines compared with the corresponding parental cells(P < 0.001 in all the molecules). Hsp60 and GANAB weredownregulated in all the invasive sublines (P < 0.001 inboth molecules). The gene expression levels were alsodetermined by RT-quantitative PCRmethod and the similarresultswere obtained (Supplementary Fig. S3). These resultssuggest that these proteins play common and importantroles in regulation of invasive phenotype in HNC.

Cell growth was suppressed by Hsp60 and GANAB andpromoted by Gp96 and Grp78To shed more light on the biological functions of these

chaperones, the 4 molecules (Hsp60, GANAB, Gp96, andGrp78) that exhibited the greatest change in expressionswere selected for further study. Two specific shRNA con-structs were designed against each protein, and the knock-down efficacy of each plasmid was determined byimmunoblot analysis. As shown in SupplementaryFig. 4A, there were different levels of RNA expression inthe presence of each of the 2 shRNA constructs, with 1 (Sh-2 of Hsp60, Sh-2 of GANAB, Sh-1 of Gp96, and Sh-2 of

Grp78) showing more significant suppression (over 80%for all 4 genes). Therefore, we used the more potent shRNAconstructs for further study. The specificity of these shRNAconstructs was examined by Western blot analysis for the 4proteins Gp96, Grp78, Hsp60, and GANAB, as shown inthe Supplementary Fig. 4B. Results showed that the speci-ficity for each shRNA construct was very high, with onlyminimal effects on the other chaperone proteins.

The effects of the shRNAson cell growthweredetermined.In general, treatment with either Hsp60-sh or GANAB-shenhanced cell growth, whereas treatment with both Gp96-sh and Grp78-sh inhibited cell growth. Using Fadu cells forexample, cell growth rates increased with timewhen treatedwith Hsp60-sh or GANAB-sh, whereas the cell growth rateswere significantly reduced uponGp96-sh or Grp78-sh treat-ment (Supplementary Fig. S5). This phenomenon wasconsistently observed in other HNC cells, with a 1.3- to1.6-fold increase in cells treated with either Hsp60-sh orGANAB-sh (P ¼ 0.003–0.04 in all cell lines; Fig. 2A).Similarly, consistent with Fadu cells, substantial reductionswere found in other HNC cells when treated with Gp96-sh(27%–54%, P < 0.03 in all cell lines) or Grp78-sh (18%–53%, P < 0.02 in all cell lines; Fig. 2A). These results suggestthat Hsp60 and GANAB play negative roles, whereas Gp96and Grp78 play positive roles in the regulation of cellgrowth.

Cell migration and invasion were inhibited by Hsp60and GANAB and augmented by Gp96 and Grp78

An in vitro wound-healing assay was done to determinethe effect that silencing these chaperones has on cell migra-tion. As shown in Fig. 2B, both Hsp60-sh and GANAB-shtransfected cells migrated much faster than control cells.After 24 hours, the Hsp60-sh cells showed a 1.49-fold faster

Table 1. List of 52 proteins identified in HNC invasive sublines (Cont'd )

NCBI number Name Scorea Mass(Da)b

pIc Sequencecoveraged

Up/downregulationa

Signal transduction/others (N ¼ 7)gi|5174447 G protein, b2-like 82 35,055 7.6 56% Upgi|32455266 Peroxiredoxin 1 127 22,096 8.67 52% Upgi|13654237 DNA-activated kinase 335 468,788 6.75 28% Upgi|40789059 KIAA0051 461 19,1298 6.14 46% Upgi|5031703 SH3- binding protein 100 52,132 5.38 53% Upgi|482321 TNF-a–induced protein 58 39,625 8.41 43% Upgi|62131678 14-3-3-e 143 26,487 4.76 65% Down

aScore, probability based scoring after Mascot peptide search with mass fingerprint (50). The scores greater than 67 are consideredsignificant (P < 0.05).bMass (Da), specific protein mass provided by Mascot peptide search with the mass spectrum data from MALDI-TOF massspectrometric analysis.cpI, isoelectric point provided byMascot peptide search with the mass spectrum data fromMALDI-TOFmass spectrometric analysis.dSequence coverage, the percentage of sequence with identified peptides over predicted target protein.eUp/downregulation, upregulation or downregulation of the specific protein in invasion subline cells.






movement and GANAB-sh cells showed a 1.78-fold fastermovement compared to the vector transfectants. After 48hours, the wounded area was completely closed in theshRNA transfected cells,whereas thewoundedarea in vector

transfected cells was not closed. The silencing of Gp96and Grp78 had different effects. Both Gp96-shand Grp78-sh transfected cells migrated much more slowlythan control cells. After 48 hours, the wounded area

Figure 1. Network analysis and expression confirmation associated with invasion phenotypes of head and neck cancer (HNC). A, molecular network analysisof the 52 proteins associated with the invasive phenotype of HNC, as predicted by the shortest path analytical model of the MetaCore algorithm. B,Western blot analysis was used to confirm the expressions of selected molecules as marked with circle in the network pathways. These molecules includeIKK-a, STAT1, c-Myc, Raf, Ras, Erk1/2, Grp78, Gp96, 14-3-3-a, Annexin-2, MMP-2, and MMP-9. Actin gene expression was used as an internalcontrol for each gene.

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remained significantly larger when compared to that invector transfected cells, with a 44% and a 57% decreasefor Gp96-sh and Grp78-sh transfectants, respectively. Theseresults suggest that Hsp60 and GANAB negatively regulatecell migration, whereas Gp96 and Grp78 possess functionsthat positively regulate cell migration.The invasive ability of the cells was determined using the

Matrigel invasion assay. Cells treated with both Hsp60-shand GANAB-sh showed a significant increase in invasiveability compared to control cells, as shown in Supplemen-tary Fig. S6 and Fig. 2C. After 2 days, all 3 HNC cell linesthat had Hsp60 silenced displayed a 1.6- to 3.9-fold(P < 0.001 to P ¼ 0.018) increased ability to invade. Theeffect of GANAB silencing was even more dramatic. In allGANAB-sh cells, a 2.2- to 24.1-fold increase in invasiveness(P < 0.001 to P ¼ 0.014) was observed. Treatment witheither Gp96-sh or Grp78-sh resulted in a substantialdecrease in cell invasion, as shown in the SupplementaryFig. S6 and Fig. 2C. After 2 days, all 3 Gp96-silenced celllines displayed a 6% to 24% suppressed invasive ability(P < 0.001 in all 3 cell lines). Similarly, the 3 Grp78-silenced cell lines displayed a 4% to 23% suppressedinvasive ability after 5 days (P < 0.001 in all 3 cell lines).These results suggest that Hsp60 and GANAB have negativeroles, whereas Gp96 and Grp78 play crucially positive rolesin the regulation of cell invasion.

Low-level expression of Hsp60 and GANAB and high-level expression of Gp96 and Grp78 are correlatedwith cancer aggressiveness and poor survivalTo understand the clinical significance of these chaper-

one proteins in cancer, paired tumor and adjacent grosslynormal tissues from 78 patients with HNC were obtained

for study. For each tissue sample, total protein wasextracted and subjected to immunoblot analysis. A repre-sentative example of the results is shown in Fig. 3A. Asshown, Hsp60 and GANAB were significantly downregu-lated in many of the cancer tissues, whereas Gp96 andGrp78 were substantially upregulated. Results of immu-nohistochemistry analysis also support these findings. Ingeneral, the lower level of Hsp60 and GANAB and thehigher level of Gp96 and Grp78 were found in advancedcancers in comparison with early staged diseases(Fig. 3B).

To determine the potential association between cancerstatus and protein expression, the Pearson’s chi-squaremethod was used for statistical analysis. Results are sum-marized in Table 2. For Hsp60 and GANAB proteins, lowexpression was correlated withmore aggressive cancer, as inpathological T stage (P < 0.001 and P¼ 0.023 in Hsp60 andGANAB, respectively), N stage (P < 0.001 for both Hsp60and GANAB), and overall stage (P < 0.001 for both Hsp60andGANAB).Conversely, forGp96andGrp78, high expres-sionwas correlatedwith advanced cancer, as in pathologicalT stage (P ¼ 0.047 and P ¼ 0.024 for Gp96 and Grp78,respectively), N stage (P ¼ 0.029 and P ¼ 0.034 for Gp96and Grp78, respectively), and overall stage (P ¼ 0.016 andP ¼ 0.007 for Gp96 and Grp78, respectively).

The association of patient survival and protein expres-sion was examined using the Kaplan–Meier method with alog-rank test. As shown in Fig. 3C, lower levels of Hsp60 orGANAB were associated with adverse treatment outcomes(5-year disease-specific survival was 93% vs. 56%, P < 0.001for Hsp60, and 90% vs. 62%, P ¼ 0.006 for GANAB).Conversely, higher levels of Gp96 or Grp78 were associatedwith a poor treatment outcome (5-year disease-specific

Figure 1. (Continued) C,examination of differentialexpressed genes in highly invasiveHNC cells as determined byRT-PCRmethod. The expressionsof 7 genes in 5 parental (Pt) andinvasive (Iv) subline cells wereanalyzed. Actin gene expressionwas used as an internal control foreach gene. The relative level ofeach gene was determined afternormalization to the actin level ineach individual sample. Theaverage fold change of each genewas calculated with the meanvalue after comparing theexpressions between invasion andparental cells.






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Effect of Hsp60-sh on cell invasion


Effect of GANAB-sh on cell invasion

Figure 2. Alteration of cellphenotypes (growth, migration,and invasion) after knockdown ofgene expressions by specificshRNAs. A, effects of cell growthin HNC cells after specificknockdowns of Hsp60, GANAB,Gp96, or Grp78. The cell linesused in the study were indicated atthe bottom of each panel figure. Ineach experiment, 5 � 105 cellswere seeded in a 10-mm plate andtransfected with either the shRNA(Hsp60-sh, GANA-sh, Gp96-sh, orGrp78-sh) or the vector plasmid.Cell numbers were calculated after5 days. In each cell line, the cellnumber of shRNA transfectionwas normalized by that of vectortransfection to assess relativeinvasion ability. Each experimentwas done in triplicate. B, effects ofcell migration in HNC cells afterspecific knockdowns of Hsp60,GANAB, Gp96, or Grp78. In eachexperiment, 5 � 105 cells wereseeded in a 10-mm plate andtransfected with either the shRNAor the vector plasmid. Cells(2� 106) were then seeded in eachwell of a 6-well fibronectin-coatedplate and further incubated for 6hours in the present of 1% FBS inmedium to allow monolayerformation. Cells were woundedwith a micropipette tip andcontinuously incubated for 48hours in a tissue culture incubator.Cell migration into the woundedarea was observed every 24 hoursand photographed. C, effects ofcell invasion in HNC cells afterspecific knockdowns of Hsp60,GANAB, Gp96, or Grp78. The celllines used in the study wereindicated at the bottom of eachpanel figure. In each experiment,5 � 105 cells were seeded in a10-mm plate and transfected witheither the shRNA or the vectorplasmid. Cells were then seededinto the upper chamber of theTranswell in 1% FBSmedium. Thelower chamber containedcomplete culture medium, whichincluded 10% FBS to trap cellinvade through Matrigel-coatedmembrane. Cell numbers in thelower chambers were calculatedafter 2 days. In each cell line, thecell number of shRNA transfectionwas normalized by that of vectortransfection to assess relativeinvasion ability. Each experimentwas done 2 times with triplicate.The P values were generated fromthe mean values of the 2experiments.

Chiu et al.





survival was 55% vs. 85%, P¼ 0.025 for Gp96, and 63% vs.87%, P ¼ 0.005 for Grp78).

Discussion

In HNC, tumor invasion and lymph node metastasis arecommon causes of treatment failure. To investigate theinvasion phenotype of HNC, we established 5 sublinesof highly invasive HNC cells. We used these cells to identifya common set of proteins that regulate the invasion phe-notype. Proteomic analysis identified 52 proteins whoseexpression patters were frequently altered in the invasivesublines (Table 1). Network pathway prediction revealedthat several cellular processes may respond to invasion,suggesting the involvement of complex circuits in theregulation of the invasion phenotype. Furthermore, speci-fic proteins in the predicted pathways were identified(Fig. 1B), suggesting a high probability that the algorithmicanalysis in this study is valid.The biological functions of the identified proteins in the

invasive sublines were examined. In addition to cytoskel-eton adhesion molecules, molecular chaperone proteinsrepresented a considerable fraction (19%) of the identifiedproteins. These molecular chaperone proteins included 3proteins that belong to the Hsp90 family and 4 proteinsthat belong to the Hsp70 family (Table 1). We furtherconfirmed that 7 proteins were differentially expressed inthe 5 invasive HNC sublines. Of these, 5 (Hsp90a/Hsp90-AA, Hsp90b/Hsp90-AB, Hsp90-B1/Gp96, Hsp70-A5/Grp78, and HYOU1) were consistently upregulated, and2 (Hsp60 and GANAB) were consistently downregulated(Fig. 1C). The 4 proteins with the greatest changes in theexpression levels were selected for functional validation.Although Hsp60 and GANAB had only a marginal effect oncell growth, the knockdown of these 2 genes considerablyenhanced cell migration and invasion. This suggests thatthese 2 proteins possess tumor suppressive functions, espe-cially on the regulation of cell mobility (Fig. 2). In contrast,the knockdown of Gp96 or Grp78 drastically reduced cellgrowth, migration, and invasion, suggesting that these 2proteins possess oncogenic functions (Fig. 2). Although theunderlined mechanism between these chaperone proteinsand invasion is still unclear, because there is no crossinteractions between these 4 chaperones (SupplementaryFig. S4B), the effects of these molecules leading to cellinvasions may be through mutual independent pathways.A number of molecular chaperones, including Hsp60,

are typically located in mitochondria and are presumed tofunction mainly within this organelle. However, there isnow accumulating evidence that these chaperones are alsolocated in a variety of cellular compartments where they doimportant functions (17–19). For example, Hsp60 in thecytosol can interact with procaspase-3 or p53 to orchestratesurvival, whereas disruption of these complexes can exertan apoptotic effect (20, 21). All of these reports indicatethat Hsp60 is involved in multiple cellular functionsrelated to maintaining homeostasis. Recently, aberrantexpression of Hsp60 was found in many clinical cancer

tissues; however, this protein has been reported to be bothpositively and negatively correlated with cancer status. Forexample, elevated expression of Hsp60 was found in cer-vical, prostate, and breast cancers (22–24). On the otherhand, decreased expression of this protein was reported inbronchial and bladder cancers (25, 26). Our results are inagreement with the latter findings that Hsp60 expressionwas lost during cancer progression (Fig. 3A and B). Further-more, lower levels of Hsp60 expression were associatedwith clinicopathological stage and poor survival (Table 2,Fig. 3C), indicating that loss of the Hsp60 tumor suppres-sion function may lead to advanced malignancy.

GANAB, also named the glucosidase II-a subunit, is aneutral glucosidase involved in the transaction and foldingof newly synthesized glycoproteins in the endoplasmicreticulum (ER; refs. 27, 28). Disruption of GANAB led tothe accumulation of misfolded glycoproteins and theinduction of the unfolded protein response (28). GANABis also a key regulator of glycosylation. Deletion of GANABled to a novel N-glycosylation mechanism in the biosynth-esis of variant cell surface glycoproteins (29). The loss ofglycosidase II is associated with polycystic liver disease, inwhich hepatocystin fails to assemble with GANAB duringcarbohydrate processing, leading to altered cellular prolif-eration and differentiation (30, 31). Aside from polycysticliver disease, to the best of our knowledge, GANAB has notbeen reported to be associated with other diseases. In thisstudy, we first showed that GANAB is associated withcancer development, through negative regulation of cellmigration and invasion abilities (Fig. 2). This downregula-tion further supports a correlation between GANAB expres-sion andmore aggressive cancer and poor survival (Table 2,Fig. 3). Because N-glycosylation of several cellular proteinsmay change tumor invasion andmetastatic ability (32, 33),GANAB may participate in carcinogenesis throughthe regulation of the N-glycosylation of specific clientproteins.

There are 3 types of Hsp90s in mammalian cells: (a)cytosolic Hsp90, which includes Hsp90a/Hsp90-AA andHsp90b/Hsp90-AB; (b) ER Hsp90, Hsp90-B1/Gp96/Grp94/endoplasmin; and (c) mitochondrial Hsp90, Trap-1 (7, 34). Because several oncoproteins are client proteins ofcytosolic Hsp90, many Hsp90 inhibitors have been devel-oped as anticancer therapeutic agents in preclinical andclinical evaluations (35, 36). The ER Hsp90, Gp96, wasfirst identified to be associated with malignancy by screen-ing a human teratocarcinoma cDNA library with mouseGp96, which encodes the TRA1 protein (37). As knowledgeincreased, Gp96 was found to play an important role in thecell-mediated immunity of the antitumor response by form-ing stable complexes with tumor-derived antigenic peptidesthat in turn present these peptides toMHC class I complexes(38). Recently, Gp96 overexpression has been observed inseveral cancers, suggesting a direct link between this proteinand malignant diseases (39–42). In this study, we foundthat the levels of both cytosolic (Hsp90a and Hsp90b) andendoplasmic reticulum (Gp96) Hsp90s are elevated in theinvasive subline cells, and the level of Gp96was higher than






that of other Hsp90s (Fig. 1C).We further showed that highlevels of Gp96 are associated with more aggressive pheno-types and poor survival of HNC patients (Table 2, Fig. 3). Inaddition, silencing Gp96 significantly suppressed cellgrowth and invasion (Fig. 2). Therefore, Gp96 may activelyregulate multiple cancer behaviors and contribute to malig-nant transformation.

Grp78 (also named Hsp70-5, HspA5, or Bip) is a well-characterized ER chaperone that is part of the Hsp70 family(43). Previously, the study of Grp78 emphasized thecytoprotective and antiapoptotic function of Grp78 inresponse to ER stress, which leads to the modulation of

chemosensitivity (44). Recently, it has been shown thatGrp78 plays more roles than originally appreciated. Studieshave shown that this protein is also expressed on the cellsurface and may actively regulate multiple malignant phe-notypes (45–48). Silencing of Grp78 expression may resultin the loss of PTEN tumor suppression and oncogenic AKTactivation (46). Grp78 may also form a complex with thecripto protein at the cell surface, and knockdown of thiscomplex may eventually lead to suppression of cell pro-liferation and invasion (47, 48). In agreement with thesereports, Grp78 has been found to be overexpressed inmanycancers, including lung, colon, esophageal, gastric, and oral

Figure 3. Clinical associationstudies of Hsp60, GANAB, Gp96,and Grp78 with disease status ofHNC. A, differential expression ofHsp60, GANAB, Gp96, and Grp78in tumor (T) tissues compared withmatched grossly normal mucosa(N) from patients with HNC. Theprotein expression levels weredetermined by Western blotanalysis. Expression of actinprotein served as an internalcontrol. Relative expression levelof each paired sample wasindicated at the bottom of thepanel figure after normalizationwith the actin expression. B,immunohistochemistry analysis ofHsp60, GANAB, Gp96, and Grp78in the tumor tissues from HNCpatients with early stage (overallstage I or II) or advanced stage(overall stage III or IV). Theintensity of the immunoreactivitywas documented in the left-handcorner of the correspondingfigure.

Patient A B C D E

Tissue

Hsp60

Hsp60

Actin1 0.05 1 0.07 1 0.02 1 0.40 1 0.52

1 0.65 1 0.02 1 0.65 1 0.40 1 0.52

1 2.93 1 2.59 1 2.05 1 2.19 1 2.13

1 3.37

Early staged Cancers

PatientA

A

BPatientB PatientC PatientD

Advanced staged Cancers

1 6.07 1 12.95 1 30.79 1 18.06

Actin

GANAB

GANAB

Actin

Gp96

Gp96

Actin

Grp78

Grp78

N T N T N T N T N T

Chiu et al.





Table 2 . Association of HSP60, GANAB, Gp96, and Grp78 expression with clinicopathological status

Clinical status Na HSP60 (%) GANAB (%) Gp96 (%) Grp78 (%)

Low High Low High Low High Low High

Pathological T stageT1–T2 60 19 (32) 41 (68) 25 (42) 35 (58) 36 (60) 24 (40) 41 (68) 19 (32)T3–T4 18 16 (89) 2 (11) 13 (72) 5 (28) 6 (33) 12 (67) 7 (39) 11 (61)P value <0.001 0.023 0.047 0.024Pathological N stageN ¼ 0 55 16 (29) 39 (71) 19 (34) 36 (66) 34 (62) 21 (38) 38 (69) 17 (31)N > 0 23 19 (83) 4 (17) 19 (83) 4 (17) 8 (35) 15 (65) 10 (43) 13 (57)P value <0.001 <0.001 0.029 0.034Overall stageI-II 46 9 (20) 37 (80) 14 (30) 32 (70) 30 (65) 16 (35) 34 (74) 12 (26)III-IV 32 26 (81) 6 (19) 24 (75) 8 (25) 12 (37) 20 (63) 14 (44) 18 (56)P value <0.001 <0.001 0.016 0.007DifferentiationWell 30 12 (40) 18 (60) 13 (43) 17 (57) 17 (57) 13 (43) 22 (73) 8 (27)2M-P 48 23 (48) 25 (52) 25 (52) 23 (48) 25 (52) 23 (48) 26 (54) 22 (46)P value 0.494 0.795 0.693 0.091Total 78 35 (45) 43 (55) 38 (49) 40 (51) 42 (54) 36 (46) 48 (61) 30 (39)

NOTE: Total of 78 head and neck cancer patients were recruited. In each cancer tissue, the expression levels of the 4 proteins (HSP60,GANAB, Gp96, and Grp78) were determined as low or high levels by Western blot analysis, as described in Materials and Methods.The Pearson's chi-square test was used to examine the association of these protein expressions with clinicopathologic features,including pathological T stage (T1–T2 vs. T3–T4), pathological N stage (N¼ 0 vs.N > 0), overall stage (stages I–II vs. stages III–IV), andthe differentiation status (well differentiation vs. moderate to poor differentiation). P < 0.05 was considered statistical significance.aN, number of patients.bM–P, moderate to poor differentiation.

Figure 3. (Continued) C, clinicalassociation between disease-freesurvival of patients and the proteinexpression status of Hsp60,GANAB, Gp96, or Grp78. Survivalcurves were calculated using theKaplan–Meier method with a log-rank test.

(I) HSP60C

Hsp60 expressionLow P < 0.0001 P < 0.006

P = 0.025 P = 0.005

High

Gp96 expressionLowHigh

Grp78 expressionLowHigh

0 2 4

Time (y)

6 8 10 0 2 4

Time (y)

6 8 10

0 2 4

Time (y)

6 8 10 0 2 4

Time (y)

6 8 10

GANAB expressionLowHigh

Dis

eas

e-s

pec

ific

su

rviv

al

1.0

0.8

0.6

0.4

0.2

0.0D

ise

as

e-s

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ific

su

rviv

al

1.0

0.8

0.6

0.4

0.2

0.0

Dis

ea

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-sp

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ific

su

rviv

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1.0

0.8

0.6

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ific

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rviv

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1.0

0.8

0.6

0.4

0.2

0.0

(II) GANAB

(III) Gp96 (IV) Grp78






carcinomas (39–42, 49). In this study, we found high levelsof Grp78 in HNC tissues, and these levels were associatedwith more aggressive status and poor prognosis (Table 2,Fig. 2). In addition, silencing of Grp78 significantly sup-pressed cell growth and invasion (Fig. 2). Thus, Grp78 hasbeen shown to be an important oncogenic protein in manyaspects.

Understanding the mechanisms underlying the regula-tion of invasion provides insight into the management ofbulky or metastatic cancers. Our study shows the greatpotential of several molecular chaperones, especiallyHsp60, GANAB, Gp96, and Grp78, in clinical applicationsinvolving HNC prognosis and treatment.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This study is supported by Grants from Chang Gung Memorial Hospital(CMRPG390421, J.T. Chang) and National Science Counsel of Taiwan(NSC 98-2314-B-182-039-MY3).

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 August 4, 2010; revised April 29, 2011; accepted May 9, 2011;published OnlineFirst June 3, 2011.

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2011;17:4629-4641. Published OnlineFirst June 3, 2011.Clin Cancer Res Ching-Chi Chiu, Chien-Yu Lin, Li-Yu Lee, et al. the Invasion Phenotype of Head and Neck CancerMolecular Chaperones as a Common Set of Proteins That Regulate

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