TWIST1 is a molecular marker for a poor prognosis in oral cancer and represents a potential...

TWIST1 Is a Molecular Marker for a Poor Prognosis in OralCancer and Represents a Potential Therapeutic Target

Sabrina Daniela da Silva, PhD1,2,3; Moulay A. Alaoui-Jamali, PhD2; Fernando Augusto Soares, MD, PhD4;

Dirce Maria Carraro, PhD5; Helena Paula Brentani, MD, PhD6; Michael Hier, MD3; Silvia Regina Rogatto, PhD7; and

Luiz Paulo Kowalski, MD, PhD1

BACKGROUND: Locoregional recurrence and distant metastases are ominous events in patients with advanced oral squamous cell

carcinoma (OSCC). The objective of this study was to identify functional biomarkers that are predictive of OSCC progression to me-

tastasis. METHODS: The expression profile of a network of epithelial-mesenchymal transition (EMT) genes was investigated in a large

cohort of patients with progressive OSCC using a complimentary DNA microarray platform coupled to quantitative reverse

transcriptase-polymerase chain reaction and immunohistochemical analyses. Therapeutic potential was investigated in vitro and in

vivo using an orthotopic mouse model of metastatic OSCC growing in the tongue microenvironment. RESULTS: Among deregulated

EMT genes, the Twist-related protein 1 (TWIST1) transcription factor and several of its regulated genes were significantly overex-

pressed across advanced stages of OSCC. This result was corroborated by the clinical observation that Twist1 up-regulation predicted

the occurrence of lymph node and lung metastases as well as poor patient survival. In support of Twist1 as a driver of OSCC progres-

sion, the up-regulation of Twist1 was observed in cells isolated from patients with metastatic OSCC. The inhibition of Twist1 in these

metastatic cells induced a potent inhibition of cell invasiveness in vitro as well as progression in vivo. CONCLUSIONS: The current

results provide evidence for the prognostic value and therapeutic potential of a network of Twist genes in patients with advanced

OSCC. Cancer 2014;120:352–62. VC 2013 American Cancer Society.

KEYWORDS: oral squamous cell carcinoma, epithelial-mesenchymal transition, Twist, metastasis, clinical outcome.

INTRODUCTIONThe development of oral squamous cell carcinoma (OSCC) metastasis poses clinical challenges because of the limitedtherapeutic options available.1,2 The time course of relapse manifestation and metastasis are unpredictable; and metasta-ses, not primary tumors, account for approximately 90% of all cancer deaths.3

Metastasis is a dynamic process in which the interplay of tumor cell, host, and tissue microenvironment factorsplay a critical role, particularly in the signaling pathways that regulate homotypic and heterotypic cell-cell and cell-matrix interactions.4 In advanced cancers in general, the final stages of tumor differentiation and progression arecharacterized by the invasion of tumor cells into the surrounding tissue and their dissemination to form metastasesin distant organs.5 Conceptually, the metastatic “cascade” is multifactorial and comprises the loss of cancer cell ad-herence at the primary site, cell motility, proteolysis of the extracellular matrix (ECM) and basement membrane,invasion of local stroma, entry of vascular and lymphatic vessels (intravasion), extravasion, recolonization, andexpansion into distant sites.4–6

During this process, epithelial-mesenchymal transition (EMT) is established as a critical mechanism by which epi-thelial cells acquire sufficient plasticity and motile properties to become invasive. In addition, increasing evidence supportsa link between EMT in the selection of cancer cell variants with stem cell properties and chemotherapy resistance.6,7 EMTevents require the coordinated expression of several sets of genes and signaling pathways, many of which have demon-strated the ability to regulate specific aspects of malignant transformation and cancer progression in preclinical models.7–9

Corresponding authors: Sabrina Daniela da Silva, PhD, and Moulay A. Alaoui-Jamali, PhD, Departments of Medicine and Oncology, Otolaryngology-Head and

Neck Surgery, McGill University, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote St. Catherine Road, Montreal, QC, H3T 1E2, Canada; Fax: (514) 340-7576;

[email protected]; [email protected]

1Department of Head and Neck Surgery and Otorhinolaryngology, AC Camargo Cancer Center, S~ao Paulo, Brazil; 2Lady Davis Institute for Medical Research and

Segal Cancer Center, Jewish General Hospital, Montreal, Quebec, Canada; 3Department of Otolaryngology-Head and Neck Surgery, McGill University, Montreal,

Quebec, Canada; 4Department of Anatomic Pathology, AC Camargo Cancer Center and Faculty of Dentistry, University of S~ao Paulo, S~ao Paulo, Brazil; 5Laboratory

of Molecular Biology, AC Camargo Cancer Center, S~ao Paulo, Brazil; 6Laboratory of Biotechnology, AC Camargo Cancer Center, S~ao Paulo, Brazil; 7NeoGene Labo-

ratory, Department of Urology, S~ao Paulo State University and AC Camargo Cancer Center, S~ao Paulo, Brazil

DOI: 10.1002/cncr.28404, Received: June 10, 2013; Revised: August 21, 2013; Accepted: August 27, 2013, Published online October 21, 2013 in Wiley Online

Library (wileyonlinelibrary.com)

352 Cancer February 1, 2014

Original Article

In this context, early OSCC progression to invasivestages is often associated with widespread changes in can-cer cell morphologic and differentiation features10

believed to promote cancer cell dissemination to distantorgans, particularly the lungs, through either the lym-phatic system or blood vessels.11,12 In the current study,we gained new insights into the role of a network ofTwist genes in OSCC progression as well as the correla-tion of Twist1 with invasiveness and clinicopathologicoutcome. In addition, we examined the therapeuticpotential of targeting Twist1 in both in vitro and in vivoOSCC models.

MATERIALS AND METHODS

Study Population

Ten fresh OSCC samples classified as poorly differenti-ated tumors (grade 3)13 from the tongue were matchedwith morphologically normal, surgically removed tissuesfrom patients at the AC Camargo Cancer Center (S~aoPaulo, Brazil), 70% of whom developed locoregional ordistant metastasis, and were submitted to laser-capturemicrodissection. These samples were used for complimen-tary DNA (cDNA) microarrays and technical validationexperiments.

For the independent sample set, 74 paraffin-embedded OSCC tissue specimens from 14 patients whohad lung metastasis (metastatic cases) and 60 patients whohad negative lymph node status without recurrence ormetastatic disease (good outcomes; nonmetastatic cases)and were followed for at least 157 months were evaluatedusing immunohistochemistry assays in a tissue microar-ray. The OSCC metastatic cases presented with advancedT classification (P 5 .004), lymph nodes capsular rupture(P 5 .003), and lower survival probability (P 5 .001)compared with the nonmetastatic cases (Table 1).

The eligibility criteria included previously untreatedpatients without a second primary tumor who receivedtreatment in the same institution. All patients wereadvised of the procedures and provided written informedconsent. The Brazilian Ethics Committee (CONEP)approved this study (no. 875=07).

Tumor staging was reclassified according to the2002 version of the International Union Against Cancer(TNM). All patients were followed after treatment, anddisease recurrence was confirmed histologically. Histo-logic grade was determined based on the World HealthOrganization classification.13 The medical records of allpatients were examined to obtain detailed demographic,clinicopathologic, treatment, and follow-up data(Table 1).

Laser-Capture Microdissection and Total RNAExtraction and Amplification

Non-neoplastic samples and tumor samples were laser-capture microdissected using the PixCell II System (Arc-turus Engineering, Inc., Mountain View, Calif). ThePicoPure RNA Isolation Kit (Arcturus Engineering, Inc.)was used to obtain total RNA according to the manufac-turer’s recommendations, and the RNA was submittedto 2-round amplification using the RiboAmp kit (Arctu-rus Engineering, Inc.). The quantification and qualitywere evaluated using the NanoDrop ND-1000 spectro-photometer (Thermo Fisher Scientific Inc., Waltham,Mass) and an Agilent Bioanalyzer (Agilent Technologies,Inc., Santa Clara, Calif), respectively. Only RNA samplesthat had a 28=18S ribosomal ratio >1 were processed.

TABLE 1. Distribution of Patients With Oral Squa-mous Cell Carcinoma

No. of Patients (%)a

Fresh

Samples

Paraffin-Embedded Samples

Variable Grade 3 Metastatic Nonmetastatic P

Sex

Men 10 (100) 11 (78.6) 42 (68.9) .471

Women 0 (0) 3 (21.4) 18 (31.1)

Race

White 10 (100) 10 (71.4) 52 (85.3) .218

Nonwhite 0 4 (28.6) 8 (14.7)

Smoking habit

No 1 (16.7) 1 (7.7) 6 (10.7) .745

Yes 5 (83.3) 12 (92.3) 50 (89.3)

Alcohol consumption

No 1 (16.7) 1 (7.7) 14 (25) .173

Yes 5 (83.3) 12 (92.3) 42 (75)

Tumor classification

T1 1 T2 4 (40) 1 (7.7) 31 (51.7) 0.004b

T3 1 T4 6 (60) 12 (92.3) 29 (48.3)

Lymph node status

N0 3 (30) 3 (21.4) 60 (100) < .001b

N1 7 (70) 11 (78.6) 0

Histologic grade

1 0 (0) 8 (61.5) 6 (12.8) ND

2 0 (0) 5 (38.5) 12 (25.5)

3 10 (100) 0 (0) 29 (61.7)

Vascular embolization

No 8 (100) 10 (83.3) 32 (65.3) .227

Yes 0 (0) 2 (16.7) 17 (34.7)

Perineural infiltration

No 3 (37.5) 6 (50.0) 29 (93.5) .371

Yes 5 (62.5) 6 (50.0) 2 (6.5)

Capsular rupture

No 4 (50) 8 (57.1) 29 (93.5) .003b

Yes 4 (50) 6 (42.9) 2 (6.5)

Status

Alive 6 (60) 5 (35.7) 47 (78.7) .001b

Dead 4 (40) 9 (64.3) 13 (21.3)

Abbreviations: ND: not determined.a Percentages are based on the patients who had complete information.b These P values indicate a statistically significant difference.

Prognostic-Therapeutic Impact of TWIST1/da Silva et al

Cancer February 1, 2014 353

Complimentary DNA Microarrays and Probes

cDNA clones from open reading frame-expressedsequence tags (ORESTES) representing human geneswere successfully amplified by polymerase chain reaction(PCR), purified using G50 resin (Amersham, Little Chal-font, United Kingdom), and spotted onto glass slidesusing a Flexsys robot (Genomic Solutions Ltd., Hunting-don, United Kingdom).14 The cDNA platform represent-ing 2352 genes included 496 negative controls, and 48positive controls (Q gene fragments from phage lambda).Positive hybridization signals from all spots were consid-ered for evaluation of hybridization quality, normaliza-tion, and statistical analysis.

Labeled cDNA was generated using 4 lg of RNA,random hexamer primer (Invitrogen Corporation, Carls-bad, Calif), cyanine 3=cyanine 5 (Cy3=Cy5)-labeleddeoxycytidine triphosphate (Amersham), and SuperScriptII (Invitrogen Corporation). Equal amounts of Cy3=Cy5-labeled cDNA derived from tumor or morphologicallynormal tissue were mixed and competitively hybridized.The hybridization experiments were performed with tu-mor versus normal tissues and included 5 cDNA samplesin each group in 4 replicates. Dye swap also was per-formed in duplicate and was used as a replicate and as acontrol for dye bias. Data acquisition and normalizationwere performed as described previously.14 The genes wereclassified based on their biologic and signaling processes(Gene Ontology and Kyoto Encyclopedia of Genes andGenomes).15 The University of California-Santa CruzCancer Genomics Browser was used to analyze cancerexpression and clinical data.16

Primers and Quantitative Reverse Transcriptase-Polymerase Chain Reaction Analysis

cDNAs were synthesized from 2 lg of antisense RNAusing Superscript II reverse transcriptase (InvitrogenCorporation) and oligo-dT primers (Invitrogen Corpora-tion). Quantitative reverse transcriptase-PCR was per-formed in the ABI Prism 7900 (Applied Biosystems,Inc., Foster City, Calif) using SYBR Green (Applied Bio-systems, Inc.) in a 20 mL total volume and quality con-trols as proposed by MIQE (Minimum Information forPublication of Quantitative Real-Time PCR Experi-ments) guidelines.17 The reactions were carried out intriplicate (Fig. 1A). Glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) was the most stable control genefrom 4 endogenous genes tested (GAPDH, ACTB,HPRT1, and BCR) using the geNorm algorithm.18 Folddifferences in the relative gene expression were calculatedusing a Pfaffl model.19

Tissue Microarray

Core biopsies were extracted from previously definedareas using a Tissue Microarrayer (Beecher Instruments,Inc., Sun Prairie, Wis). Tissue cores that measured 1.0mm from each specimen were punched and arrayed induplicate on a recipient paraffin block. Normal oral tissuesamples from surgical margins were included as controls.

Immunohistochemistry

Immunohistochemical reactions were performed as pre-viously described.20 Incubation with the primary anti-bodies diluted in phosphate-buffered saline (PBS) wereconducted overnight at 4�C for anti-Twist1 (1:100 dilu-tion; Abnova, Jhongli City, Taiwan), anti-E-cadherin(1:400 dilution; Dako, Carpenteria, Calif), anti-b-catenin (1:1500 dilution; Thermo Fisher Scientific Inc.),antivimentin (1:50 dilution; Dako), antivascular endo-thelial growth factor A (anti-VEGFA) (1:200 dilution;Dako), and anti-Ki-67 antigen (1:300 dilution; Dako).The sections were washed and incubated with secondaryantibodies (Dako) for 30 minutes, then the polymerdetection system (Dako) was used for 30 minutes atroom temperature. Positive controls were included in allreactions in accordance with the manufacturer’s proto-cols. Negative controls consisted of omitting the primaryantibody, incubating the slides with PBS, and replacingthe primary antibody with normal serum.

The slides were analyzed by 2 certified pathologistswho were blinded to clinical information. The reactionswere analyzed according to staining intensity, with 0 indi-cating no visible reaction; 1, weak expression; and 2, strongpositivity. Each core was scanned in a low-power field tochoose the most stained area predominant in at least 10%of tumor cells.20 The immunohistochemical analyses wereperformed in duplicate on different tissue microarray levels,representing 4-fold redundancy for each case. The secondslides were 25 sections deeper than the first, resulting in adistance of at least 250 lm between the 2 sections, withdifferent cell samples from each tumor. For statistical analy-sis, samples were categorized into 2 groups: negative andweakly positive cases versus strongly positive cases.21

Statistical Analysis

For frequency analysis in contingency tables, statisticalanalyses of associations between variables were per-formed using the Fisher exact test (with significance setat P< .05) and, for continuous variables, the nonpara-metric Mann-Whitney U test. Overall survival was cal-culated from the beginning of treatment (surgery) to thedate of either death or the last information (for censored

Original Article


observations). Survival probabilities were estimatedusing the Kaplan-Meier method, and the log-rank testwas applied to assess the significance of differencesamong actuarial survival curves with 95% confidenceintervals.

Cell Culture

Normal oral epithelial (NOE) cell (kindly provided by DrAla-Eddin Al Moustafa) were isolated from normalhuman tongue tissue and maintained in culture in kerati-nocyte serum-free medium supplemented with 5 mg=100mL bovine pituitary extract, as described previously.22

The OSCC1.2 cell line was established by our group froma metastatic OSCC and was maintained in culture as pre-viously described.23,24

Small Interfering RNA Expression

We used 2 target sequences for knockdown of TWIST1(available from GenBank under accession no.NM_000474): 50-UUGAGGGUCUGAAUCUUGCUCAGCU-30 and 50-AGCUGAGCAAGAUUCAGACCCUCAA-30. Transfections were carried out using 100 nMof small interfering RNA (siRNA) oligonucleotides incu-bated with DharmaFECT1 (Thermo Fisher Scientific

Inc.) in Opti-MEM I reduced serum medium (InvitrogenCorporation) according to the manufacturer’s instruc-tions. For in vivo studies, the TWIST1-target siRNAsequence was cloned as inverted repeats into pSR puromy-cin vector according to the manufacturer’s instructions(Oligoengine, Seattle, Wash). PSR was used as a control.These vectors were expressed in target cells using a retrovi-ral system as previously described.25

Western Blot Analysis

Total cell extracts were used for Western blot analysis aspreviously described.25 Blots were detected using the anti-bodies for anti-E-cadherin (1:1000 dilution; Cell Signal-ing Technology, Inc., Beverly, Mass), anti-Twist1 (1:500dilution; Abnova), anti-b-catenin (1:1000 dilution;Abcam plc., Cambridge, United Kingdom), and anti-GAPDH (1:10,000 dilution; Cedarlane, Burlington, On-tario, Canada), and signals were detected with peroxidase-conjugated secondary antibodies and an enhanced chemi-luminescence detection system.

Invasion and Migration Assays

Cell invasion assays were performed using 8-lm porouschambers coated with Matrigel (BD Biosciences, East

Figure 1. (A,B) Primer characteristics and relative messenger RNA (mRNA) expression levels of Twist-related protein 1 (TWIST1);cadherin 1 type 1 (CDH1); catenin (cadherin-associated protein), b1 (CTNNB1); and vimentin (VIM) were analyzed using quantita-tive reverse transcriptase-polymerase chain reaction (PCR) in samples of oral cell squamous cell carcinoma (OSCC) and normalepithelium after glyceraldehyde-3-phosphate dehydrogenase (GAPDH) normalization (an asterisk indicates P<.05; t test).(C) Box plots illustrate the relation between mRNA levels and immunohistochemical labeling in the OSCC samples.



Rutherford, NJ) according to the manufacturer’s recom-mendations. Cell migration was assayed using a qualita-tive wound-healing assay. Each experiment wasperformed at least 3 times, and the results are expressed asthe average 6 standard deviation. Statistical significancewas determined using the Student t test.

In Vivo Preclinical Studies

In vivo studies were conducted in compliance with institu-tional and federal Canadian guidelines and were approvedby McGill University. Exponentially growing cells were sus-pended in PBS (106 cells per 0.1 mL) and injected ortho-topically into the tongues of male severe combined-immunodeficiency (SCID) mice; or, alternatively, cells wereimplanted into the mice subcutaneously to address distincttumor microenvironment, as previously described.23,24 Tu-mor length (L) and width (W) were monitored every sec-ond day using a caliper that was accurate to 0.5 mm.Tumor volume (V) was calculated using the following for-mula: V 5 L3 W3 1=2 W. Once the tumor reached themaximal size allowed by institutional guidelines, the micewere killed, and the tumors were dissected and weightedusing a precision balance. The invasive phenotype of thesetumors was investigated macroscopically and by histologicexamination. The results are expressed as the average 6 -standard deviation (n 5 8 mice per condition), and statisti-cal analysis was done using the Student t test.

RESULTS

Twist-Regulated Genes Are MolecularSignatures for Invasive Oral Squamous CellCarcinoma

We conducted a microarray expression analysis usingRNA extracted from matched normal tissue and invasiveOSCC samples (Table 1). The results revealed differen-tial expression between normal tissues and advancedOSCC samples for several genes that significantlyaffected primary functions in cell-cell and cell-matrixinteractions and in the signal transduction pathwaysinvolved in motility and invasion processes. Amongthem, Twist1 was strongly correlated with invasiveOSCC and was predictive of a poor clinical outcome(Tables 2, 3). In addition, the expression of severalTwist-regulated genes (Fig. 1A) was altered, includingdown-regulation of E-cadherin and up-regulation ofvimentin and VEGFA. In agreement with the cDNAmicroarray results, quantitative reverse transcriptase-PCR analysis confirmed TWIST1 overexpression(P 5 .0001) and down-expression of cadherin 1 type 1(CDH1) (P 5 .050) in progressive OSCC compared

with normal epithelium (Fig. 1B). A significant correla-tion was observed between transcription levels and pro-tein expression in the same set of OSCC samples, asrepresented in Figure 1C by box-and-whisker plots. Insupport of our results, analysis of a publically availablegenomic database revealed an association of TWIST1overexpression with OSCC and TWIST1 down-regulation in normal tissues (Fig. 2).

Proteins Encoded by Twist-Regulated GenesAre Differentially Expressed in Invasive OralSquamous Cell Carcinoma

To confirm the genomic results, protein expression levelswere determined for Twist1 and selected targets, includ-ing E-cadherin, b-catenin, vimentin, and VEGFA(Fig. 3A-F). A significant association was observedbetween lymph node metastasis and increased protein lev-els of Twist1 (P 5 .035) and Ki-67 (P 5 .007) (Table 3).OSCC that presented with vascular embolization had anincreased cell proliferation index based on positive Ki-67status (P 5 .029). The tumors with increased Ki-67 hadup-regulation of both Twist1 (P 5 .051) and VEGFA(P 5 .031) protein expression (results not shown). Posi-tivity for E-cadherin was more intense, but not statisticallysignificant, in well differentiated tumors compared withmoderately or poorly differentiated tumors. However, theabsence or reduced expression of E-cadherin was

TABLE 2. Protein Expression in Patients with Meta-static and Nonmetastatic Oral Squamous CellCarcinoma

No. of Patients (%)

Variable Metastatic OSCC Nonmetastatic OSCC Pa

Twist1

Negative 4 (30.8) 31 (68.9) .013

Positive 9 (69.2) 14 (31.1)

E-cadherin

Negative 7 (70) 10 (19.6) .001

Positive 3 (30) 41 (80.4)

Beta-cadherin

Negative 10 (83.3) 10 (19.61) < .001

Positive 2 (16.7) 41 (80.4)

Vimentin

Negative 5 (38.5) 41 (85.4) .001

Positive 8 (61.5) 7 (14.6)

VEGFA

Negative 6 (50) 43 (86) .006

Positive 6 (50) 7 (14)

Ki-67

�25% 3 (27.3) 35 (63.6) .026

>25% 8 (72.7) 20 (36.4)

Abbreviations: OSCC, oral squamous cell carcinoma; Twist1, Twist-related

protein 1; VEGFA, vascular endothelial growth factor A.a All P values indicate a statistically significant difference.

Original Article


associated with advanced clinical stage (P 5 .005) andlymph node involvement (P< .001) (Table 3).

Of significant relevance to OSCC progression, weobserved higher expression levels of Twist1 (P 5 .013) indistant metastasis compared with nonmetastasized tumors(Table 2). In Twist1-overexpessing tumors, negativeexpression of E-cadherin was associated with high expres-sion levels of vimentin (P< .001); and the highest expres-

sion was observed in tumors in advanced stages(P 5 .005) (Table 3).

Elevated Twist Expression Correlates With aPoor Prognosis

To determine whether Twist1 could have a prognosticvalue, survival probability was compared between patientswho had lung metastasis and patients who had good

TABLE 3. Correlations Among Protein Expression and Clinical Stage, Lymph Node Status, and HistologicGrade in Patients With Oral Squamous Cell Carcinoma

Tumor Classification No. (%)a Lymph Node Status No. (%)a Histologic Grade No. (%)a

Variable T1 1 T2 T3 1 T4 P N0 N1 P 1 2 3 P

Twist1

Negative 15 (44.1) 19 (55.9) .962 31 (91.2) 3 (8.8) .035b 7 (23.3) 7 (23.3) 16 (53.4) .356

Positive 10 (43.5) 13 (56.5) 16 (69.6) 7 (30.4) 5 (22.7) 9 (40.9) 8 (36.4)

E-cadherin

Negative 2 (12.5) 14 (87.5) .005b 10 (58.8) 7 (41.2) < .001b 4 (26.7) 5 (33.3) 6 (40) .651

Positive 23 (53.5) 20 (46.5) 40 (93.0) 3 (6.7) 7 (20) 9 (25.7) 19 (54.3)

Beta-catenin

Negative 5 (26.3) 14 (73.7) .047b 10 (50.0) 10 (50) < .001b 6 (33.3) 7 (38.9) 5 (27.8) .074

Positive 23 (53.5) 20 (46.5) 42 (97.7) 1 (1.3) 5 (15.2) 8 (24.2) 20 (60.6)

Vimentin

Negative 24 (54.5) 20 (45.5) .005b 41 (91.1) 4 (8.9) .001b 8 (21.6) 11 (29.7) 18 (48.7) .704

Positive 2 (13.3) 13 (86.7) 8 (53.3) 7 (46.7) 4 (28.6) 5 (35.7) 5 (35.7)

VEGFA

Negative 21 (43.8) 27 (56.2) .896 44 (91.7) 4 (8.3) .001b 7 (17.1) 13 (31.7) 21 (51.2) .372

Positive 5 (41.7) 7 (58.3) 7 (53.9) 6 (46.1) 4 (36.4) 3 (27.2) 4 (36.4)

Ki-67

�25% 17 (44.7) 21 (55.3) .418 36 (94.7) 2 (5.3) .007b 6 (18.2) 10 (30.3) 17 (51.5) .726

>25% 9 (34.6) 17 (65.4) 19 (70.4) 8 (29.6) 6 (27.3) 6 (27.3) 10 (45.4)

Abbreviations: Twist1, Twist-related protein 1; VEGFA, vascular endothelial growth factor A.a Percentages are based on the patients who had complete information.b These P values indicate a statistically significant difference.

Figure 2. Signatures of Twist-related protein 1 (TWIST1) and related genes from the University of California-Santa Cruz CancerGenomics Browser are shown. Each column corresponds to a single sample, and each row corresponds to a biomolecular entityrelated to the current study. The genomic heat map was organized according to normal samples versus tumor samples. TheTWIST1 gene is overexpressed in tumor samples but is mostly absent in normal cases. Chr indicates chromosome; CDH1, cadherin1 type 1; CTNNB1, catenin (cadherin-associated protein), b1; VIM, vimentin; VEGFA, vascular endothelial growth factor A; Ki67, Ki-67 antigen.



outcomes (mean follow-up, 157 months). The medianoverall survival for the metastatic group was 11.5 monthsversus 88 months for the nonmetastatic group (long-ranktest; P< .0001). The 5-year overall survival rates inpatients who had aggressive OSCC and patients who hadfavorable outcomes were 23.7% and 84.7%, respectively.The mean disease-free survival in patients who presentedwith distant metastasis was 18.6 months (range, 5-78months; standard deviation, 619 months). A significantlylower survival probability was verified in patients who hadtumors with Twist1 protein overexpression (log-rank test;P 5 .0310) (Fig. 4).

Twist1 Is a Potential Therapeutic Target toInterfere With Oral Squamous Cell CarcinomaInvasiveness

Because Twist1 is strongly expressed in patients withadvanced OSCC, and its expression level is correlated withmetastasis formation and poor survival, we investigated thepotential relevance of this transcription factor as a therapeu-tic target. We addressed the impact of Twist1 manipulationon the EMT phenotype using an NOE cell line, which wasestablished from normal human tongue tissue, and theOSCC1.2 cell line, which was established from a poorlydifferentiated, metastatic, human oral cancer (stageT4N2b) that had vascular, lymphatic, and perineural inva-

sion.23,24 Exposure of NOE cells to TGFb induced a clearEMT phenotype in almost 100% of the population (Fig.5A). In contrast, a majority of OSCC1.2 cells expressed anEMT morphology, and exposure of these cells to TGFb

further induced the morphologic characteristics typical ofEMT; this coincided with Twist1 overexpression anddown-regulation of E-cadherin (Fig. 5B) as well asincreased cell invasion capacity (Fig. 5C). Down-regulationof Twist1 using RNA interference (Fig. 5D) significantlyprevented the oncogenic features of these invasive OSCCcells. Compared with control cells infected with empty viralparticles, down-regulation of Twist1 expression clearlydecreased cell proliferation as well as invasive potentialusing the Boyden chamber assay (P< .005) (Fig. 5E).

In vivo, mice that were implanted with OSCC1.2cells in the tongue had shorter survival because of fast-growing tumors and respiratory problems caused by lungmetastatic lesions, which were observed in >50% of ani-mals through pathologic examination (Fig. 6A-C).E-cadherin immunostaining was observed in normal epi-thelium but was reduced significantly in the primary tumorand at the metastatic site (Fig. 6D-F). An inverse correla-tion was observed for Twist1 in the same areas (Fig. 6G-I).Equally important, Twist1 down-regulation inhibited tu-mor growth when cells were implanted either orthotopi-cally into the tongues of SCID mice or subcutaneously

Figure 3. Immunohistochemical detection of (A) Twist-related protein 1 (Twist1), (B) E-cadherin, (C) b-catenin, (D) vimentin, (E)vascular endothelial growth factor A (VEGFA), and (F) Ki-67 antigen is illustrated. Nuclear immunoreactivity for (A) the transcrip-tional repressor Twist1 and (F) Ki-67 antigen was easily identified. (B) E-cadherin had sharply demarcated membrane stainingobserved in well differentiated tumor areas. (C) Beta-catenin was affected by the loss of expression in most samples. (D) Intracy-toplasmatic labeling was observed for vimentin, especially in metastatic samples (original magnification 3400 in A and C-F,3200 in B).

Original Article


(P< .05) (Fig. 6J-K). No macroscopic lung metastasis wasobserved after Twist1 down-regulation, whereas the controlmice had to be killed because of lung metastatic lesions.

DISCUSSIONTranscriptional gene expression profiling revealed the pre-dictive power of gene signatures for cancer progres-sion.26,27 In this study, laser-microdissected, invasivetongue carcinomas were evaluated using a cDNA micro-array platform that contained genes critical to the biologyof cancer metastasis. We identified several genes that weredifferentially expressed. Not surprisingly, most of thegenes associated with OSCC invasiveness have beenimplicated in the regulation of cell-matrix interaction,loss of intercellular junctions, cell scattering, cytokinesis,angiogenesis, and migration, all of which are importantprocesses of metastasis development.6,8,28 With regard tothe signature of OSCC progression, it is significant thatwe identified Twist1 as a predominant marker (based onhigh levels of expression). In particular, a significant cor-relation was observed between high Twist1 expression, tu-mor cell proliferation, and lymph node and distantmetastasis. In addition to the prognostic value of Twist1for OSCC clinical cases, our preclinical studies establisheda potential role for Twist1 as a therapeutic target. Wedemonstrated that the inhibition of Twist1 in an OSCC

cell line, which was isolated from metastatic oral cancer,lead to the significant inhibition of cell invasion in vitroand to tumor growth in mice.

Twist1, a highly conserved, basic helix-loop-helixtranscription factor mapped at 7q21.2, has a bifunctionalrole, acting as an activator or a repressor, depending onpost-translational modifications and physiologic con-texts.29,30 Twist1 induces gene transactivation through cis-binding to E-box regulatory regions, which are present inseveral target genes, and this involves complex homodime-rization and heterodimerization mechanisms regulated byprotein phosphorylation.29 In the case of gene repression,Twist1 can repress genes by regulating chromatin remodel-ing through histone acetyltransferase-dependent=histonedeacetylase-dependent mechanisms and through the inhi-bition of DNA binding activity of transcription factors.30

The implication of Twist1 in cell migration is attrib-uted primarily to its ability to contribute to EMT, eg,through the down-regulation of E-cadherin and the up-regulation of mesenchymal markers like vimentin, fibro-nectin, and N-cadherin, as noted above.1–5,7,26,27,31 Pre-vious studies have indicated that Twist1 promotes cellproliferation, migration, and expression of a primitiveECM, thus promoting an undifferentiated state.31 Inaddition, Twist1 contributes to the EMT phenotype,which has been associated with resistance to chemother-apy and relapses.32 We observed that, as noted above (seeResults), several of the EMT-associated genes regulated byTwist1 were altered in our advanced OSCC cases, includ-ing Twist-activated genes like E-cadherin, b-catenin, andVEGFA.

Other mechanisms by which Twist1 can contributeto OSCC aggressiveness may include effects on othergenes known to be associated with cancer metastasis, eg,activation of the cell adhesion protein periostin(POSTN), the inhibition of c-MYC, and p53-dependentapoptosis enhancing cell survival, or the up-regulation ofAKT2. Also, Twist1 can regulate cell motility throughactivation of the promotility Rho GTPase RAC1, becauseit has been demonstrated that Twist1, in cooperation withBMI1, can suppress let-7i expression, which, in turn, canlead to RAC1 activation by the up-regulation of NEDD9and DOCK3.30,33 Although the mechanism underlyingTwist1 overexpression in advanced OSCC remains to beestablished, it has been demonstrated that Twist1 is up-regulated in response to the activation of several factors,including activation of WNT=b-catenin signaling, SRC,STAT3, HIF-1a, and integrin-linked kinase.30 It is note-worthy that WNT=b-catenin was altered in our cohort ofpatients with advanced OSCC. We observed that b-

Figure 4. Overall survival was short for patients with oralsquamous cell carcinoma who had Twist-related protein 1(Twist1) overexpression (log-rank test; P 5.0310). The solidgreen line indicates negative Twist1 expression; dotted redline, Twist1 overexpression (Kaplan-Meier test).



catenin staining was predominately cytoplasmic and=ornuclear in patients with metastatic OSCC. Nuclear trans-location of b-catenin can promote the activation of

E-cadherin transcriptional repressors, including Twist1, amechanism implicated in promoting EMT in various sys-tems of embryonic development and tumor progression

Figure 5. (A) Transforming growth factor b (TGFb)-driven epithelial-mesenchymal transition (EMT) was able to change the mor-phology to fibroblast-like in normal oral epithelial (NOE) cells. OSCC1.2 indicates an oral squamous cell carcinoma cell line. (B)Western-blot analysis confirmed reduced expression of E-cadherin and increased levels of Twist-related protein 1 (Twist1) afterTGFb-driven EMT. C indicates control. (C) An invasion assay was conducted before and after TGFb treatment using the Boydenchamber assay. The bar graph represents the mean number (6standard error [SE]) of invaded cells (P< .005). GAPDH indicatesglyceraldehyde-3-phosphate dehydrogenase. (D) Western blot analysis revealed the efficient down-regulation of Twist1 obtainedby small interfering RNA (siRNA) sequences compared with their matched control cells (C), which expressed the pSR puromycinvector (PSR). (E) The invasion of OSCC1.2-TWIST1-siRNA and the OSCC1.2-PSR control was determined using the Boyden cham-ber assay. The bar graph represents the mean number (6SE) of invaded cells (P< .001).

Original Article


to metastasis.34 In this study, we observed that inactiva-tion of Twist1 conferred the morphologic transition ofOSCC from a fibroblastic to an epithelial appearance,which was accompanied by a gain of epithelial cellmarkers like E-cadherin.

These findings highlight the utility of Twist1 andregulated genes as a molecular signature for invasiveOSCC. Moreover, our results in OSCC preclinical mod-els support the potential of targeting Twist1 as a therapeu-tic target. It has been established that several transcriptionfactors play key roles in the cell signaling that drives me-

tastasis; however, translation of these findings into clinicalapplications has been hampered in part by the difficulty oftargeting the transcription factor surface motifs requiredto achieve high-affinity binding and the difficulty in get-ting small molecules or peptides to reach the nuclear com-partment.35,36 However, recent advances in the design ofsmall molecules and peptides and of nanotechnologies fordrug-delivery approaches are opening new avenues indrug discovery to target transcription factors likeTwist1.37 In summary, this study provides insights intothe relevance of Twist1 and regulated genes for the

Figure 6. An in vivo mouse model reveals normal epithelium from (A) the tongue, (B) the primary tumor, and (C) lung metastasis(hematoxylin and eosin [H&E] stain). (D) E-cadherin immunostaining was observed in normal epithelium but was significantlyreduced (E) in the primary tumor and (F) at the metastatic site. OSCC1.2 indicates an oral squamous cell carcinoma cell line.(G-I) An inverse correlation was observed for Twist-related protein 1 (Twist1) in the same areas. (J,K) Tumor weights are illus-trated at the time mice were killed (n58) from animals implanted with OSCC1.2-puromycin vector control (c) cells or withOSCC1.2-TWIST1-small interfering RNA (siRNA)-expressing cells implanted either (J) orthotopically in the tongue of severecombined-immunodeficiency mice or (K) subcutaneously (P< .05).



progression of poorly differentiated OSCC to metastasisand highlights their potential to be exploited as therapeu-tic targets for advanced OSCC.

FUNDING SUPPORTThis work was supported by the Fundacao de Amparo a Pesquisado Estado de Sao Paulo (FAPESP), the Canadian Institutes forHealth Research (CIHR), and in part by the Canadian Cancer Soci-ety and the Quebec Breast Cancer Foundation.

CONFLICT OF INTEREST DISCLOSURESThe authors made no disclosures.

REFERENCES1. Su HH, Chu ST, Hou YY, et al. Spindle cell carcinoma of the oral

cavity and oropharynx: factors affecting outcome. J Chin Med Assoc.2006;69:478-483.

2. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics,2002. CA Cancer J Clin. 2005;55:74-108.

3. Dutton JM, Graham SM, Hoffman HT. Metastatic cancer to thefloor of mouth: the lingual lymph nodes. Head Neck. 2002;24:401-405.

4. Howell GM, Grandis JR. Molecular mediators of metastasis in headand neck squamous cell carcinoma. Head Neck. 2005;27:710-717.

5. Cavallaro U, Christofori G. Multitasking in tumor progression: sig-naling functions of cell adhesion molecules. Ann N Y Acad Sci.2004;1014:58-66.

6. Thompson EW, Newgreen DF, Tarin D. Carcinoma invasion andmetastasis: a role for epithelial-mesenchymal transition? Cancer Res.2005;65:5991-5995.

7. Larue L, Bellacosa A. Epithelial-mesenchymal transition in develop-ment and cancer: role of phosphatidylinositol 30 kinase/AKT path-ways. Oncogene. 2005;24:7443-7454.

8. Boyer B, Valles AM, Edme N. Induction and regulation ofepithelial-mesenchymal transitions. Biochem Pharmacol. 2000;60:1091-1099.

9. Chin D, Boyle GM, Kane AJ, et al. Invasion and metastasis markersin cancers. Br J Plast Surg. 2005;58:466-474.

10. Califano J, van der Riet P, Westra W, et al. Genetic progressionmodel for head and neck cancer: implications for field cancerization.Cancer Res. 1996;56:2488-2492.

11. Pimenta Amaral TM, da Silva Freire AR, et al. Predictive factors ofoccult metastasis and prognosis of clinical stages I and II squamouscell carcinoma of the tongue and floor of the mouth. Oral Oncol.2004;40:780-786.

12. Ziober AF, Falls EM, Ziober BL. The extracellular matrix in oralsquamous cell carcinoma: friend or foe? Head Neck. 2006;28:740-749.

13. Wahi PN, Cohen B, Luthra UK, et al. Histological Typing of Oraland Oropharyngeal Tumours. Geneva, Switzerland: World HealthOrganization; 1971.

14. Brentani RR, Carraro DM, Verjovski-Almeida S, et al. Gene expres-sion arrays in cancer research: methods and applications. Crit RevOncol Hematol. 2005;54:95-105.

15. Zhang B, Schmoyer D, Kirov S, et al. GOTree Machine (GOTM):a web-based platform for interpreting sets of interesting genes usingGene Ontology hierarchies [serial online]. BMC Bioinformatics.2004;5:16.

16. Sanborn JZ, Benz SC, Craft B, et al. The UCSC Cancer GenomicsBrowser: update 2011. Nucleic Acids Res. 2011;39(database issue):D951-D959, 2011.

17. Bustin SA, Beaulieu JF, Huggett J, et al. MIQE precis: practicalimplementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments [serial online]. BMCMol Biol. 2010;21:74.

18. Vandesompele J, De Preter K, Pattyn F, et al. Accurate normaliza-tion of real-time quantitative RT-PCR data by geometric averagingof multiple internal control genes [serial online]. Genome Biol. 2002;3:RESEARCH0034.

19. Pfaffl MW. A new mathematical model for relative quantification inreal-time RT-PCR [serial online]. Nucleic Acids Res. 2001;29:e45.

20. Kononen J, Bubendorf L, Kallioniemi A, et al. Tissue microarraysfor high-throughput molecular profiling of tumor specimens. NatMed. 1998;4:844-847.

21. Tomizawa Y, Wu TT, Wang KK. Epithelial mesenchymal transitionand cancer stem cells in esophageal adenocarcinoma originating fromBarrett’s esophagus. Oncol Lett. 2012;3:1059-1063.

22. Al Moustafa AE, Alaoui-Jamali MA, Batist G, et al. Identification ofgenes associated with head and neck carcinogenesis by cDNA micro-array comparison between matched primary normal epithelial andsquamous carcinoma cells. Oncogene. 2002;21:2634-2640.

23. Balys R, Alaoui-Jamali M, Hier M, et al. Clinically relevant oralcancer model for serum proteomic eavesdropping on the tumourmicroenvironment. J Otolaryngol. 2006;35:157-166.

24. Mlynarek AM, Balys RL, Su J, et al. A cell proteomic approach forthe detection of secretable biomarkers of invasiveness in oral squa-mous cell carcinoma. Arch Otolaryngol Head Neck Surg. 2007;133:910-918.

25. Xu Y, Bismar TA, Su J, et al. Filamin A regulates focal adhesiondisassembly and suppresses breast cancer cell migration and invasion.J Exp Med. 2010;207:2421-2437.

26. Campo-Trapero J, Cano-Sanchez J, Palacios-Sanchez B, et al.Update on molecular pathology in oral cancer and precancer.Anticancer Res. 2008;28:1197-1205.

27. Ziober AF, Patel KR, Alawi F, et al. Identification of a gene signa-ture for rapid screening of oral squamous cell carcinoma. ClinCancer Res. 2006;12:5960-5971.

28. Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol. 2006;7:131-142.

29. Qin Q, Xu Y, He T, et al. Normal and disease-related biologicalfunctions of Twist1 and underlying molecular mechanisms. Cell Res.2012;22:90-106.

30. Franco HL, Casasnovas J, Rodriguez-Medina JR, et al. Redundantor separate entities?—roles of Twist1 and Twist2 as molecularswitches during gene transcription. Nucleic Acids Res. 2011;39:1177–1186.

31. Alami J, Williams BR, Yeger H. Differential expression ofE-cadherin and beta catenin in primary and metastatic Wilms’stumours. Mol Pathol. 2003;56:218-225.

32. Ansieau S, Morel AP, Hinkal G, et al. TWISTing an embryonictranscription factor into an oncoprotein. Oncogene. 2010;29:3173-3184.

33. Yang WH, Lan HY, Huang CH, et al. RAC1 activation mediatesTwist1-induced cancer cell migration. Nat Cell Biol. 2012;14:366-374.

34. Medici D, Hay ED, Olsen BR. Snail and Slug promote epithelial-mesenchymal transition through beta-catenin-T-cell factor-4-dependentexpression of transforming growth factor-beta3. Mol Biol Cell. 2008;19:4875-4887.

35. Secchiero P, Bosco R, Celeghini C, et al. Recent advances in thetherapeutic perspectives of Nutlin-3. Curr Pharm Des. 2011;17:569-577.

36. Whitesell L, Lindquist S. Inhibiting the transcription factor HSF1 asan anticancer strategy. Expert Opin Ther Targets. 2009;13:469-478.

37. Fontan L, Yang C, Kabaleeswaran V, et al. MALT1 small moleculeinhibitors specifically suppress ABC-DLBCL in vitro and in vivo.Cancer Cell. 2012;22:812-824.

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