ASSOCIATION BETWEEN ARECA-STIMULATED ... ARTICLE ASSOCIATION BETWEEN ARECA-STIMULATED VIMENTIN...

ORIGINAL ARTICLE

ASSOCIATION BETWEEN ARECA-STIMULATED VIMENTINEXPRESSION AND THE PROGRESSION OF HEAD AND NECKCANCERS

Yu-Hsin Tseng, PhD,1 Kuo-Wei Chang, DDS, PhD,1,2,4 Cheng-Chieh Yang, DDS, PhD,1,2

Chung-Ji Liu, DDS, MS,1,3 Shou-Yen Kao, DDS, DMSC,1,2 Tsung-Yun Liu, PhD,4

Shu-Chun Lin, PhD1,2

1 Institute of Oral Biology, School of Dentistry, National Yang–Ming University, Taipei, Taiwan. E-mail: [email protected] of Stomatology, Taipei Veterans General Hospital, Taipei, Taiwan3Oral and Maxillofacial Surgery, Taipei Mackay Memorial Hospital, Taipei, Taiwan4Department of Medical Education and Research, Taipei Veterans General Hospital, Taipei, Taiwan

Accepted 14 December 2010Published online 7 March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/hed.21726

Abstract: Background. Areca nut chewing is a common

oral habit of Asians that is closely associated with the high

incidence of head and neck carcinoma. The purpose of

this study was to investigate the impacts of areca nut

chewing on neoplastic process of head and neck

carcinoma.

Methods. Head and neck carcinoma cells were treated

with areca nut extract to perceive the phenotypic impacts. Tu-

mor tissues were analyzed with immunohistochemistry (IHC) to

understand the association between areca-associated molecu-

lar changes and clinical variables.

Results. Upon treatment with areca nut extract, carcinoma

cells showed the increase of vimentin. The activation of extrac-

ellular signal-regulated kinase (ERK)/cyclooxygenase (COX)-2/

prostaglandin (PGE)-2 cascade underlay the upregulation.

These cells also exhibited the enhancement of migration and

invasion. By knocking down COX-2 and vimentin expression,

the increase of cell mobility was reversed. Tumor exhibiting

extensive vimentin and/or COX-2 expression displayed a sig-

nificantly worse disease-associated survival than contrast

groups.

Conclusion. Areca-modulated vimentin expression

enhanced the progression of head and neck carcinoma.VVC 2011 Wiley Periodicals, Inc. Head Neck 34: 245–253, 2012

Keywords: areca nut chewing; head and neck cancer;

invasion; vimentin

Areca-associated head and neck squamous cell carci-noma (HNSCC) is 1 of the leading cancers in South andSoutheast Asia.1–3 Areca nuts contain the polyphenolsarecoline and arecaidine and other alkaloids.4 To date,there are several hundred million areca nut chewers inAsia.1,3,5–7 Although areca nut has been classified as agroup I carcinogen, there is limited evidence for the car-cinogenicity of specific areca nut ingredients.5 Our pre-vious studies showed that areca nut extract-inducedhypertetraploidity in low-grade HNSCC cells and down-regulation of the differentiation marker, involucrin.6,7

Areca nut extract can activate signalings or molecularevents, including cyclooxygenase-2 (COX-2), extracellu-lar signal-regulated kinase (ERK), nuclear factor kappaB (NF-jB), and others in HNSCC cells.2,4–9 In addition,areca nut extract also upregulates lysyl oxidase, anincrease of this enzyme within HNSCC cells mayenhance aggressiveness of HNSCC cells.1

Epithelial-mesenchymal transition is an impor-tant biological process during development and onco-genesis. It is characterized by a reduction ofepithelial polarities and the production of mesenchy-mal phenotypes. Downregulation of epithelialmarkers, like E-cadherin, and increase in mesenchy-mal markers, including vimentin, are hallmarks ofthe transition.10–12 In addition to tumor progression,the expression of these markers was also associatedwith drug resistance of gefitinib in HNSCC.13 It wasknown that NF-jB, matrix metalloproteinase (MMP),and other molecules may be involved in the regula-tion of epithelial-mesenchymal transition and thuspromote tumor progression.12,14,15 Because areca nutextract can disrupt the differentiation of head andneck keratinocytes shown by downregulation of invo-lucrin and also upregulates MMP-9 expression.7,8 Inthis study, we postulated that the influence of arecanut extract on vimentin expression may subsequentlyimpact tumor phenotypes.

Correspondence to: S.-C. LinThis study was supported by grant V96C1-075 (to K.–W. Chang) fromTaipei Veterans General Hospital, grant NSC95-2314-B-010-043 (toS.–C. Lin) and fellowship awards NSC98-2811-B-010-030 and NSC99-2811-B-010-029 (to Y.–H. Tseng) from the National Science Council,and DOH99-TD-C-111-007 (to S.–Y. Kao) from the Department ofHeath, Taiwan.Yu-Hsin Tseng and Kuo-Wei Chang contributed equally to this work.VVC 2011 Wiley Periodicals, Inc.

Vimentin and Head and Neck Squamous Cell Carcinoma Progression HEAD & NECK—DOI 10.1002/hed February 2012 245

MATERIALS AND METHODS

Cell Culture, Areca Nut Extract Treatment, and

Reagents. The preparation of areca nut extract wasperformed as previously established protocols.4–6 OECM-1 is a moderately-differentiated HNSCC cell line culti-vated in RPMI-1640 medium (Invitrogen, Carlsbad,CA).4 OC3 was established in our laboratory from anareca nut chewer. It is a well-differentiated HNSCC cellline cultivated in a combined K-SFM-Dulbecco ModifiedEagle’s Medium (2:1, v/v, Invitrogen).6,15 OECM-1 andOC3 cells were treated with areca nut extract in culturemedium containing 0.5% and 3.3% fetal bovine serum(Biological Industries, Kibbutz Beit Haemek, Israel),respectively.6,7 Prostaglandin E2 (PGE2; Cayman Chem-icals, Ann Arbor, MI), NS-398 (Cayman Chemicals)COX-2 inhibitor, and U0126 (Calbiochem, Darmstadt,Germany) MAP kinase inhibitor were dissolved indimethyl sulfoxide (Sigma Aldrich, St Louis, MO) asstocks. Cell images were photographed with anAxioscope fluorescence microscope (Zeiss, Hamburg,Germany).

Patients. Formalin-fixed paraffin-embedded speci-mens of HNSCC were obtained from 52 patients receiv-ing initial surgical therapy during 2002 to 2004. Thisstudy was approved by an institutional review board.Surgical specimens were collected after receivinginformed consent from the patients. The average age ofthe patients was 52.5 � 1.5 years. There were 45patients (87%) who were areca nut chewers, 46 patients(88%) who were tobacco smokers, and 7 patients (13%)who were alcohol drinkers. Of these tumors, 26 (50%)were located on the buccal mucosa. The histologicalgrading of the HNSCC was based on the World HealthOrganization classification. Twenty (38%) of the tumorswere well-differentiated tumors and the others weremoderately differentiated or poorly differentiatedtumors. The TNM classification was according to theAmerican Joint Committee on Cancer system. Thirty-two of the tumors (62%) were T4 tumors, 26 patients(50%) had neck nodal metastasis, and 36 patients (69%)had TNM classification IV tumors.

Immunofluorescence and Confocal Microscopy

Examination. Cells were fixed, permeabilized, andthen incubated with antibody against vimentin (BD Bio-sciences, San Jose, CA) at a dilution of 1:500. It was fol-lowed by incubation with Alexa 488-conjugatedsecondary antibody (Invitrogen) at a dilution of 1:500.Filamentous (F)-actin was labeled with Rhodamine-Phal-loidine (Invitrogen) at a dilution of 1:200. Immunofluo-rescence was photographed on a Leica TCS NT confocallaser scanning microscope (Leica, Heidelberg, Germany).

Western Blot Analysis. Cell lysate (50 lg) wasresolved by electrophoresis on a 10% denaturing poly-acrylamide gel. The primary antibodies are listed in

Table 1. The secondary antibodies were horseradish per-oxidase-conjugated (Chemicon, Billerica, MA). The sig-nals were detected using a Western LightningChemiluminescence Reagent plus kit (Perkin–Elmer,Wellesley, MA) and Chemi-Smart 3000 image acquisitionsystem (Viber Lourmat, Marne La Vallee, France). Glyc-eraldehyde-3-phosphate dehydrogenase was the internalcontrol.

Immunohistochemistry. Immunohistochemistry (IHC)was performed on paraffin sections to detect theimmunoreactivities of vimentin and COX-2 (Table1). Pre-immunized mouse immunoglobulin G was usedas a negative control. The immunoreactivities weredetermined by counting tumor cells in HNSCCtissues.1 Tumor samples containing more than 50%cells exhibiting immunoreactivity were positive cases.

Transwell Migration and Invasion Assay. Cellswere grown on a Transwell (Corning, Acton, MA)with a porous membrane having a pore diameter of 8lm for the migration assay. A membrane having apore diameter of 12 lm and coated with 1:4 matrigelbasement membrane matrix (BD Biosciences, SanJose, CA) was used for the invasion assay. After 24hours, the cells migrating across the membrane werecounted in 5 randomly selected �100 microscopicfields for each membrane.1

Vimentin and Cyclooxygenase-2 Knockdown. Theshort hairpin RNA vectors for knocking down vimentin(shVim-1 and shVim-3; code number TRCN0000029121and TRCN0000029123, respectively) were obtainedfrom the RNA interference consortium (Academia Sin-ica, Taipei, Taiwan). The shLuc vector was used as acontrol. Lentiviral particles carrying shVim-1, shVim-3, or shLuc were generated by protocols described pre-viously.1 Cell subclones were achieved by puromycinselection for 3 days. Oligonucleotides siRNA-A (control;catalog #sc-37007) and siCOX-2 (catalog #sc-29279)were purchased from Santa Cruz Biotech (Santa Cruz,CA). Cells were transfected with oligonucleotides usingTransFectin Lipid Reagent (BioRad Laboratories, Her-cules, CA) and subjected to analysis.

Statistical Analysis. Data were expressed as themean � SE. Mann–Whitney analysis, Fisher’s exacttest, and Kaplan–Meier survival analysis were used for

Table 1. Primary antibodies used for Western blot analysis and IHC.

Antibody Provider

Dilution for

Western

blot analysis

Dilution

for IHC

COX-2 BD Biosciences, San Jose, CA 1:500 1:50

Vimentin BD Biosciences 1:1000 1:500

GAPDH Chemicon, Billerica, CA 1:20,000

Abbreviations: IHC, immunohistochemistry; COX-2, cyclooxygenase-2; GAPDH,glyceraldehyde-3-phosphate dehydrogenase.

246 Vimentin and Head and Neck Squamous Cell Carcinoma Progression HEAD & NECK—DOI 10.1002/hed February 2012

the statistical analysis. The association of nonreactivevariables with disease-associated survival was assessedby the COX multivariate proportional hazard model.Differences between values were considered significantwhen the p value was < .05. Statistical analysis wasperformed with the Prism 5 (GraphPad, San Diego, CA)or SPSS 15.0 (SPSS, Chicago, IL) software.16

RESULTS

Areca Nut Extract Treatment Increased Vimentin

Expression in Head and Neck Squamous Cell

Carcinoma Cells Mediated by Extracellular Signal-

regulated Kinase Activation. After treatment with 5lg/mL of areca nut extract for 48 hours, OECM-1 cellsshowed morphological changes. The majority of theOECM-1 cells transformed from an original parabasa-loid cell shape (Figure 1Aa) into an elongated or spin-dle shape (Figure 1Ab, c). The morphological changecoincided with the upregulation of vimentin, startingfrom 48 hours and lasting to 72 hours (Figure 1B). Thisincrease exhibited a dose-dependent manner (Figure1C). The upregulation of vimentin sustained for atleast 48 hours after the termination of areca nutextract treatment (Figure 1D). Areca nut extract treat-ment also resulted in the fibroblastoid change of OC3cells. Areca nut extract was also reported to upregulateCOX-2 expression.4,9 Areca nut extract-stimulatedCOX-2 and vimentin expression was mediated by ERKactivation because pretreatment of cells withU0126 abrogated such expression in both OECM-1(Figure 1E) and OC3 cells (Figure 1F).

Areca Nut Extract-Stimulated Upregulation of

Vimentin Mediated by Cyclooxygenase-2/

Prostagladin2 Signaling Cascade. To explorewhether COX-2 activity may underlie the increase ofvimentin, treatment with PGE2, a downstream endproduct of COX-2, significantly upregulated vimentinlevels in both OC3 and OECM-1 cells (Figure 2A and2D). A dose-dependent effect was observed on OC3 cells(Figure 2A). Enzyme linked immunosorbent assays ofthe supernatants of OC3 and OECM-1 at 24 to 72hours after areca nut extract treatment showed signifi-cant increase of the PGE2 level, which indicated theupregulation of COX-2 activity (Figure 2C). Pretreat-ment of OC3 and OECM-1 cells with COX-2 inhibitor,NS-398, diminished the induction of vimentin expres-sion by areca nut extract (Figure 2B and 2E). A dose ofNS-398 as low as 1.25 lM was sufficient to attenuatevimentin expression in OECM-1 cells (Figure 2E),whereas higher NS-398 doses were needed to attenuatevimentin expression in OC3 cells (Figure 2B). The vali-dated doses of areca nut extract and NS-398 for OC3treatment were higher than OECM-1 treatment. Thismight be associated with the presence of higherconcentration of serum in the OC3 cell culturemedium than OECM-1 cell culture medium, which

may interfere with the drug effects. The upregulationof vimentin concurred with a fibroblastoid change inOC3 and OECM-1 cells upon PGE2 treatment (Figure2F). These results indicated COX-2/PGE2 signalingcascade played a pivotal role in areca nut extract-stimulated vimentin expression and fibroblast-likemorphological transformation of HNSCC cells.

Areca Nut Extract Treatment Enhanced Migration

and Invasion of Head and Neck Squamous Cell

Carcinoma Cells. To study the roles of this molecu-lar mechanism in head and neck carcinogenesis, ourpreliminary analysis on growth tendency showed nodifference between control and areca nut extract-treated cells. However, a significant increase ofmigration and invasion was noted in the areca nutextract-treated OECM-1 (Figure 3A) and OC3 (Figure3B) cells. To investigate the functional roles of ele-vated vimentin in areca nut extract-induced onco-genic phenotypes, HNSCC cells with vimentin stablyknocked down by short-hairpin RNA were establishedby lentiviral infection, which carried shVim-1, shVim-3, and shLuc. The stable subclones were designatedas shVim-1, shVim-3, and shLuc. They were thentreated with areca nut extract for 48 hours andfollowed by culture in areca nut extract-free mediumfor another 48 hours. Western blot analysis and quan-tification showed that there was significant reductionof vimentin expression in shVim-1 and shVim-3 sub-clones, relative to the parental control and shLucclones (Figure 3C). The areca nut extract-stimulatedmigration was significantly attenuated in thesevimentin-knocked down subclones even under arecanut extract treatment (Figure 3D). These results sug-gested that areca nut extract-stimulated vimentinexpression contributed to migratory phenotype ofOECM-1 cells. The attempts to establish stable cellsubclones with COX-2 knockdown using short hairpinRNA vectors were not successful due to the rarity ofcell survival after drug selection. Therefore, OECM-1cells were transiently transfected with small interfer-ence oligonucleotide siCOX-2. The areca nut extract-stimulated increase of OECM-1 migration was abol-ished with the treatment of siCOX-2 relative to con-trol (Figure 3E).

Association between Cyclooxygenase-2/Vimentin

Expression and Poor Survival of Head and Neck

Squamous Cell Carcinoma Cells Patients. Theareca nut chewing duration of patients was 10.7 �1.0 years with a median value of 10 years. Pack yearwas used to quantitate cigarette smoking. The con-sumption of tobacco smoking was 16.0 � 1.4 packyear in our patients. No patient received adjuvantchemotherapy or radiotherapy before surgery. No tu-mor exhibited positive margin involvement as shownby histopathological evaluation. Perineural invasionand lymphovascular permeation was found in 10


(19%) and 6 (12%) primary tumor tissues, respec-tively. Among 26 cases exhibiting neck lymph nodemetastasis, 13 had multiplicity of positive nodes and5 had extracapsular spreading. To address the impor-tance of COX-2/vimentin signaling in areca-associatedHNSCC, IHC for vimentin and COX-2 was performed.

Tumor cells exhibiting immunoreactivity of cytosolicvimentin, which were stronger than or equal to thoseof stromal fibroblasts or vascular smooth muscle cellswere positive for vimentin immunoreactivity. Thirty-seven of 52 tumors (71%) exhibiting complete absenceof vimentin or focal distribution of positive cells were

FIGURE 1. Effect of areca nut extract (ANE) on head and neck squamous cell carcinoma (HNSCC) cells. (A) Inverted microscopy

revealed a progressive change of OECM-1 morphology from a polygonal shape (a) to a spindle shape after 5 lg/mL of areca nut

extract treatment for 2 hours (b) and 48 hours (c). Bars, 25 lM. (B–F) Western blot analysis. (B) Left, Increase of vimentin expression

was shown in OECM-1 cells after 5 lg/mL of areca nut extract treatment for 48 hours or 72 hours. Right, Bar diagram showed quantifi-

cation at 48 hours of treatment. Data shown were from 6 independent analysis. ** p < .01; Mann–Whitney analysis. (C) It indicated

the increase of vimentin stimulated by areca nut extract followed a dose-dependent manner at 72 hours. (D) The sustained effect of

vimentin expression by areca nut extract treatment in OECM-1 cells was shown at 48 hours after the removal of areca nut extract.

Before removal, the cells had been treated with 5 lg/mL of areca nut extract for 48 hours. (E, F) Pretreatment with 10 lM of

U0126 for 1 hour significantly attenuated the vimentin induction by 5 lg/mL of areca nut extract in OECM-1 cells (E) and 20 lg/mL of

areca nut extract in OC3 cells (F). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide; COX-2, cyclooxy-

genase 2.


FIGURE 2. Areca nut extract (ANE)-stimulated cyclooxygenase (COX)-2 activation mediated upregulation of vimentin in head and

neck squamous cell carcinoma (HNSCC) cells. (A, B, D, E) Western blot analysis. (A) Treatment of OC3 cells with 1.25 to 5 lM of

prostaglandin-2 (PGE2) for 48 hours increased vimentin expression in a dose-dependent manner. (B) Pretreated OC3 cells with 2.5 or

5 lM of COX-2 inhibitor, NS-398, remarkably attenuated vimentin expression stimulated by areca nut extract. (C) Enzyme linked immu-

nosorbent assays revealed that areca nut extract induced PGE2 production in the supernatant of both OC3 and OECM-1 cells from 24

to 72 hours. (D) Left, Treatment of OECM-1 cells with 5 lM of PGE2 for 48 hours increased vimentin expression. Right, Quantification

showed vimentin expression increased by more than three-fold after PGE2 treatment. (E) Left, Pretreated OECM-1 cells with 1.25 and

2.5 lM of NS-398 largely reduced vimentin expression stimulated by areca nut extract. Right, Quantification showed the reduction of

vimentin by NS-398 treatment was dose dependent. (F) Compared with control groups (upper panels), when OC3 and OECM-1 cells

were treated with 10 lM of PGE2 for 72 hours and 5 lM of PGE2 for 48 hours, respectively, morphological transformation from polyg-

onal to spindle shape was observed by inverted light microscopy. Using confocal microscopy, the increase of cytosolic vimentin of

OECM-1 cells was demonstrated. Bars, 25 lM. Data shown in (C, D, E) were from triplicate analysis. * p < .05; ** p < .01; *** p <

.001; Mann–Whitney analysis. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide. [Color figure can be

viewed in the online issue, which is available at wileyonlinelibrary.com.]

scored as vimentin (�) (Figure 4A). Fifteen HNSCCsamples (29%) were scored as vimentin (þ), whichcontained more than 50% of cells positive for vimen-tin immunoreactivity (Figure 4B). There were 45tumors (87%) exhibiting sporadic distribution or anabsence of COX-2 immunoreactivity were scored asCOX-2 (�) (Figure 4C). Seven HNSCC samples (13%)were scored as COX-2 (þ) (Figure 4D).

Vimentin immunoreactivity seemed stronger inpatients with longer duration of areca nut chewing.However, the statistical difference was marginally sig-nificant (Table 2). Vimentin immunoreactivity was sig-nificantly associated with the presence of perineuralinvasion in primary tumor tissues (Table 2). In addition,

vimentin immunoreactivity was significantly associatedwith the patient’s nodal status, and the multiplicity ofnodal involvement (Table 2). Vimentin immunoreactivitywas not associated with other clinicopathological varia-bles including site of the primary tumor, dose of tobaccosmoking, size of primary tumor, TNM classification, lym-phovascular permeation, tumor differentiation, andextracapsular spreading. COX-2 immunoreactivity wasnot associated with any clinicopathological variable.

During the median follow-up for 5.2 years afterinitial therapy, 22 patients (42%) showed regionalrecurrence and 7 patients (13%) developed distantmetastasis. Twenty-four patients (46%) died of dis-eases. In vimentin (þ) group, 12 of 15 tumors (80%)

FIGURE 3. Areca nut extract (ANE) treatment enhanced the migration and invasion of head and neck squamous cell carcinoma

(HNSCC) cells. (A, B) OECM-1 cells and OC3 cells, respectively. Cells treated with 5 lg/mL (for OECM-1 cells) and 20 lg/mL (for

OC3 cells) areca nut extract for 48 hours were followed by cultivation in areca nut extract free medium for another 48 hours. Transwell

assays were then carried out. The results indicated areca nut extract treatment increased both migration and invasion. (C) Western

blot analysis and (D) Transwell migration assays on parental OECM-1 cells and shLuc, shVim-1, and shVim-3 subclones with or with-

out areca nut extract treatment, followed the conditions identical to (A). Vimentin levels were efficiently downregulated by introducing

specific short-hairpin RNA. Cyclooxygenase (COX)-2 levels were not influenced by knockdown of vimentin (C). Areca nut extract-stimu-

lated cell mobility was significantly attenuated by knocking down vimentin expression (D). (E) Transwell migration assays on OECM-1

cells transfected with siRNA-A and siCOX-2 oligonucleotides with or without areca nut extract treatment, followed the conditions identi-

cal to (A). Areca nut extract-stimulated cell mobility was significantly attenuated by siCOX-2 treatment. Data shown in (A, B, D, F)

were from triplicate analysis. * p < .05; ** p < .01; Mann–Whitney analysis. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


developed local recurrence or metastasis, whereas46% of vimentin (�) cases (17 of 37) exhibited thesame aggressive clinical behaviors during follow-up (p¼ .033; Fisher’s exact test). Patients with tumorsexhibiting either vimentin (þ) or COX-2 (þ) had sig-nificantly worse disease-associated survival than thecontrasting group (Figure 4E). If we combined bothvimentin and COX-2, patients carrying vimentin (þ)and/or COX-2 (þ) tumors had even worse disease-associated survival than the contrasting patients(Figure 4F). Multivariate analysis also indicated thateither vimentin (þ) or the combination of vimentin(þ) and COX-2 (þ) were poor survival factors of

patients with HNSCC (Table 3). In consistence withother studies, our analysis also indicated that nodalmetastasis of primary tumors is an independent pre-dictor for disease-associated survival in patients withHNSCC. However, variables including the duration ofareca nut chewing, perineural invasion, multiplicityof positive nodes, and COX-2 immunoreactivity wereunable to predict survival independently.

DISCUSSION

Although there is a strong link between areca nutchewing and HNSCC genesis that has been proposed

FIGURE 4. Vimentin expression was correlated with the survival of patients with head and neck squamous cell carcinoma (HNSCC). (A, B)

The immunoreactivity of vimentin was shown in representative HNSCC tissues, scored as vimentin (�) and vimentin (þ), respectively (origi-

nal magnification � 200). (C, D) The immunoreactivity of cyclooxygenase (COX)-2 was revealed in representative HNSCC tissues, scored

as COX-2 (�) and COX-2 (þ), respectively (original magnification � 100). Arrows in (A, C) indicate scattered immunoreactive tumor cells.

(E, F) Kaplan–Meier analysis of disease-associated survival. (E) It indicated that HNSCC tissues exhibiting vimentin (þ) or COX-2 (þ) pre-

sented a worse survival than the opposite group. (F) It indicated that HNSCC tissues exhibiting vimentin (þ) and/or COX-2 (þ) had a signifi-

cantly worse survival than the opposite group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


in Asians, the molecular mechanisms for head andneck epithelial oncogenesis associated with areca nutchewing are largely unclear.3 In this study, we pre-sented evidence that areca nut extract treatmentcould convert HNSCC cells into fibroblast-like cellswith simultaneous upregulation of COX-2, vimentin,and enhancement of invasion. Our previous studieshave shown that areca nut extract also induced thefibroblastoid transition of normal head and neck kera-tinocytes.7,8 Based on the above findings, the molecu-lar and phenotypic changes induced by areca nutextract may contribute to HNSCC pathogenesis inboth early and late stages. ERK activation was shownto mediate areca nut extract-stimulated COX-2 upreg-ulation.4,9 PGE2 production in HNSCC cells wasCOX-2-dependent. Inhibition of COX-2 suppressedthe invasion of HNSCC cells.17 The association ofERK/COX-2/PGE2 cascade with increased vimentinexpression in this study indicated the involvement ofthis signaling axis in the invasion of areca-associatedHNSCC. Both NF-jB and MMP could induce the epi-thelial-mesenchymal transition and invasion oftumors.12,14 Our previous study also showed thatareca nut extract-stimulated NF-jB may underlie theupregulation of MMP-9 in head and neck keratino-cytes.8 Whether the NF-jB activity also contributesto the increase of vimentin expression in areca-associ-ated HNSCCs needs further investigation in thefuture.

Vimentin is a mechanical transducer connectingintegrins on cell surface to cellular organelles and,thus, potentially controls cell migration through theregulation of cell adhesion.18 We found that areca nutextract-treated HNSCC cells had higher competenceof migration and invasion. Knocking down vimentinby shRNA inhibited such cellular migration. The find-ing of the important roles of vimentin in mobility ofHNSCC cells is in agreement with those observed inprostate cancer cells.19 A previous study of normal

breast tissue showed that vimentin was expressedonly in myoepithelial cells and that its high expres-sion in breast cancers was associated with a higherhistological grade and worse prognosis.20 Moreover,vimentin-positive colon cancer and breast cancer cellsshowed higher potential of invasion.21 In addition,the appearances of mesenchymal markers have beenlinked to the resistance of gefitinib in HNSCC cells.13

Our immunohistochemical analysis demonstratedstrong vimentin expression in 29% of HNSCC sam-ples and the expression of this protein predicted apoor prognosis. The relevance being found betweenvimentin expression and the perineural spreading,the more advance in nodal metastasis and the multi-plicity of nodal involvement could underlie theadvancement of tumors. Furthermore, because vimen-tin (þ) tumors exhibited stronger recurrent or meta-static tendencies, it suggested a significant role ofvimentin in the poor prognosis of patients withHNSCC. Our clinical findings were consistent withthe aggressiveness of HNSCC cells mediated byvimentin. The results in this study together with ourprevious findings, which indicated that areca nutextract disrupted keratinocyte differentiation andactivated lysyl oxidase during tumor progression,highlight the pathogenic roles of the areca nut inhead and neck carcinogenesis.1,7

This study provides evidence that areca nutextract was able to upregulate vimentin expressionand facilitate invasion of HNSCC by activating intra-cellular signaling cascades. Due to the complexity inareca nut chewing habits,3 the daily amount of arecaconsumption in patients was difficult to assess. How-ever, our preliminary analysis showed a trend ofvimentin upregulation in line with the duration ofareca exposure in HNSCC tissues. Future studiesusing a larger number of HNSCC samples and precisequantification of areca consumption is required to val-idate tissue evidences. This study also showed thattumors having higher vimentin and/or COX-2 expres-sion possessed worse prognosis and the knockdown ofvimentin or COX-2 reduced the mobility of HNSCCcells. Application of COX-2 inhibitors may benefit thetherapies of patients with HNSCC.17 Downregulationof vimentin expression has been shown to impairmigration in various cancer cells.19,21,22 Therefore,the combined use of COX-2 inhibitors and functionalknockdown of vimentin could potentially be validatedas a regimen against areca-associated HNSCC.

Table 3. Multivariate analysis using COX proportional hazard model.

Variables Comparison HR 95% CI p value

Nodal metastasis N2 or N3 vs N0

or N1

5.064 1.960–13.083 .001

Vimentin þ vs � 3.071 1.344–7.021 .008

Vimentin and

COX-2

þ and/or þ vs

others

3.780 1.221–11.702 .021

Abbreviations: HR, hazard ratio; CI, confidence interval; COX-2, cyclooxygenase-2.

Table 2. Vimentin immunoreactivity related to clinocopathological

variables.

Positive, no.

of samples (%)

Negative,

no. of

samples (%) p value

Areca nut chewing duration

�10 y 11 (73) 15 (41)

<10 y 4 (27) 22 (59) .064

Perineural invasion

Presence 6 (40) 4 (11)

Absence 9 (60) 33(89) .024

Nodal metastasis

N ¼ 2 or 3 8 (53) 7 (19)

N ¼ 0 or 1 7 (47) 30 (81) .020

Multiplicity of positive nodes*

Presence 7 (50) 6 (17)

Absence 7 (50) 29 (83) .031

Fisher’s exact test.*Forty-nine cases received the dissection of multiple levels of neck lymph nodeswere analyzed.


Acknowledgment. We acknowledge Dr.His–Feng Tu for his assistance in statisticalanalysis.

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