aberrant myosin 1b expression promotes cell migration and...

Genomics

Aberrant Myosin 1b Expression Promotes CellMigration and Lymph Node Metastasis of HNSCCGaku Ohmura1,2, Takahiro Tsujikawa1,2, Tomonori Yaguchi1, Naoshi Kawamura1,Shuji Mikami3, Juri Sugiyama1, Kenta Nakamura1, Asuka Kobayashi1, Takashi Iwata1,Hiroshi Nakano2, Taketoshi Shimada2, Yasuo Hisa2, and Yutaka Kawakami1

Abstract

Lymph node metastasis is the major clinicopathologic featureassociated with poor prognosis in patients with head and necksquamous cell carcinoma (HNSCC). Here, web-based bioinfor-matics meta-analysis was performed to elucidate the molecularmechanism of lymph node metastasis of human HNSCC. Prefer-ential upregulation of Myosin 1b (MYO1B) transcript in HNSCCdatasets was identified. Myo1b mRNA was highly expressed inhuman HNSCC cells and patient tissue specimens compared withtheir normal counterparts as shown by quantitative PCR (qPCR)analyses. Immunohistochemistry (IHC)-detected Myo1b expres-sion was significantly correlated with lymph node metastases inpatients with oral cancer of the tongue. HNSCC with high expres-sion of Myo1b and chemokine receptor 4 (CCR4), another metas-

tasis-associated molecule, was strongly associated with lymphnode metastasis. RNA interference (RNAi) of Myo1b in HNSCCcells, SAS andHSC4, significantly inhibitedmigratory and invasiveabilities through decreased large protrusion formation of cellmembranes. Finally, Myo1b knockdown in SAS cells significantlyinhibited in vivo cervical lymphnodemetastases in a cervical lymphnode metastatic mouse model system.

Implications: Myo1b is functionally involved in lymph nodemetastasis of human HNSCC through enhanced cancer cellmotility and is an attractive target for new diagnostic and ther-apeutic strategies for patients with HNSCC. Mol Cancer Res; 13(4);721–31. �2014 AACR.

IntroductionHead and neck squamous cell carcinoma (HNSCC) is the sixth

most common malignancy worldwide (1). Despite improvedlocoregional control of HNSCC (2), the 5-year survival rate forHNSCC remains relatively unchanged, at under 50% for the pastthree decades (3). HNSCC tends to metastasize to lymph nodesbefore distant metastasis (4–6). Because lymph node metastasisis strongly associated with poor prognosis in patients withHNSCC (4, 7–9), understanding the mechanisms that underlielymph node metastasis of HNSCC is important for improvingdiagnostic and therapeutic strategies.

Cancer cells use their intrinsic migratory ability to invadeadjacent tissues and ultimately to metastasize to differentorgans. Cell migration is a highly integrated multistep process,

initiated by protrusion of the cell membrane. Aberrant regu-lation of cell migration drives cancer invasion and metastasis.The protrusive structures formed by migrating and invadingcancer cells are filopodia, lamellipodia, and invadopodia/podosomes, depending on their morphologic, structural, andfunctional characters (10). However, the mechanisms ofhuman HNSCC cell migration and metastasis remain poorlyunderstood.

Here, we investigated the mechanisms of lymph node meta-stasis of human HNSCC. Myosin 1b (Myo1b, also namedmyosin 1 alpha or Myr1), a member of the myosin family,was found to be upregulated in HNSCC through a web-basedbioinformatics meta-analysis. Myo1b, a class I myosin, is awidely expressed, single-headed, actin-associated molecularmotor, associated with cell migration of zebrafish embryo cells(11) and intracellular transport regulated by the formation ofpost-Golgi carriers (12, 13). However, its role in cancer biologyhas never been reported. Aberrant expression of Myo1b immu-nohistochemically (IHC) detected was correlated with lymphnode metastases in patients with HNSCC (n ¼ 31, P ¼ 0.0320).Myo1b knockdown in human HNSCC cell lines by RNA inter-ference inhibited in vitro migration and invasion abilities of theHNSCC cells accompanied by decreased large protrusionsformation in cell membrane. Downregulation of Myo1b inhuman HNSCC cell lines resulted in inhibition of metastasisto cervical lymph nodes in cervical lymph node metastaticmodel using nude mice implanted with human HNSCC. Theseresults demonstrate that Myo1b is involved in cancer cellmotility and lymph node metastasis of human HNSCC, imply-ing that Myo1b could be a novel diagnostic and therapeutictarget for patients with HNSCC.

1Division of Cellular Signaling, Institute for Advanced MedicalResearch, Keio University School of Medicine, Shinjuku-ku, Tokyo,Japan. 2Department of Otolaryngology-Head and Neck Surgery,Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto City,Kyoto, Japan. 3Division of Diagnostic Pathology, Keio UniversitySchool of Medicine, Shinjuku-ku, Tokyo, Japan.

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

G. Ohmura and T. Tsujikawa contributed equally to this article.

Corresponding Author: Yutaka Kawakami, Division of Cellular Signaling, Insti-tute for Advanced Medical Research, Keio University School of Medicine, 35Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: 81-3-5363-3778;Fax: 81-3-5362-9259; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-14-0410

�2014 American Association for Cancer Research.

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Materials and MethodsPatients and clinical samples

A total of 31 patients with tongue cancer for IHC and 7 patientswith HNSCC for quantitative PCR assays, who underwent surgeryat Kyoto Prefectural University of Medicine (Kyoto, Japan)between April 2008 and April 2012, were enrolled in a retrospec-tive study. Data were collected from clinical and pathologicrecords with the written informed consent of individual patientsafter approval by the Ethics Committee of the institutes.

In silico gene expression studiesWe used the Oncomine database (Compendia Bioscience;

http://www.oncomine.org) to identify upregulated genes inHNSCC on May 26, 2013 and performed a microarray meta-analysis to compare all genes across 13 different datasets (14–22)that were identified by the following parameters: "mRNA," "can-cer vs. normal analysis," and "head and neck cancer" (excludingnasopharyngeal cancer, salivary gland cancer, and thyroid cancer).To compare Myo1b expression, Myo1b expression fold changeswere limited to P < 0.05.

Cell cultureHuman oral tongue cancer cell lines, SAS and HSC-4, were

purchased from RIKEN Bioresource Center Cell Bank (Tsukuba,Japan). Cells were cultured in RPMI-1640 supplemented with10%FBS, penicillin (50units/mL), and streptomycin (50mg/mL)and were incubated at 37�C in a humidified chamber supple-mented with 5%CO2. Human hypopharyngeal cancer, FaDu andDetroit562, human oral tongue cancer, HSC-3, human Hodgkinlymphoma, L-428, human melanoma, Skmel23, 928mel, andA375mel, human brain tumor, U87MG, human esophageal can-cer, TE4 and TE9, lung cancer, LK2 and EBC1, pancreatic ductaladenocarcinoma, PK59, prostate cancer, PC3, breast cancer,Hs578 and MDA-MB-231, chronic myeloid leukemia, K562,acute myeloid leukemia, HL60, was obtained and cultured aspreviously reported (23–25). Human brain tumor, T98G, humancervical cancer, C33a and HeLa, were purchased from the ATCCand cultured in DMEM as well as in RPMI-1640. Human cervicalcancer, SKG1, was a kind gift from Dr. Daisuke Aoki (KeioUniversity, Tokyo, Japan) and cultured in DMEM as well as inRPMI-1640. SAS, HSC4, HSC3, Detroit526, FaDu, L-428,A375mel, U87MG, T98G, TE4, TE9, C33a, SKG1, HeLa, PK59,Hs578, and MDA-MB-231 were authenticated by short tandemrepeat profiling.

Evaluation of mRNA and protein expressionTotal RNA was isolated from cells using an RNeasy Mini Kit

(Qiagen) with on-column DNase treatment (Qiagen), andTissueRuptor (Qiagen) was used in the total RNA isolationfrom patient tissue samples. cDNA was synthesized usingSuperscript III Reverse Transcriptase and Oligo (dT) 12–18primers (Invitrogen). Quantitative real-time PCR was per-formed using a TaqMan probe (Applied Biosystems), Myo1b(Hs00362654_m1), Snail1 (Hs00195591_m1), Twist1(Hs01675818_s1), CDH1 (Hs01023895_m1), and GAPDH(Hs03929097_g1), on ABI PRISM 7900 HT Sequence DetectionSystem with TaqMan Gene Expression Master Mix (AppliedBiosystems). Target gene expression was standardized toGAPDH levels using SDS Software v2.2 (Applied Biosystems).Relative expression of Myo1b mRNA was shown after normal-

ization to the Myo1b expression level in HSC3 as the baselinevalue (¼1). Western blotting was performed by general proce-dure with rabbit anti-hMyo1b pAb (Sigma-Aldrich) and rabbitanti-GAPDH pAb (Santa Cruz Biotechnology).

Myo1b knockdownsiRNAs specific for human Myo1b (si-Myo1b-1; HSS106714

and si-Myo1b-2; HSS106715, Stealth Select RNAi), as well asscrambled siRNA (Stealth RNAi Negative Control Kit, mediumGC), were purchased from Invitrogen. These siRNAs (final con-centration during transfection, 100 nmol/L) were transfected intocells using Lipofectamine RNAi MAX (Invitrogen) according tothe manufacturer's recommendations. Assays were carried out 72hours after transfection. Lentiviral pGIPZ shRNA vectors targetinghuman Myo1b (sh-Myo1b-1; V3LHS_356101, sh-Myo1b-2;V3LHS_356104) and nonsilencing pGIPZ control vector (sh-NC)were purchased fromOpen Biosystems (Thermo Fisher Scientific,Inc.), and they were integrated with turboGFP and puromycinresistance sequences. Lentivirus particles were produced in 293Tcells according to the manufacturer's instructions. The viral titerwas calculated by evaluating GFP expression in infected 293Tcells. SAS cells were transduced by the lentiviral particles in thesame viral titer. The cells stably expressing shRNA were selected,pooled, and maintained with 2 mg/mL puromycin (Wako).

In vitro migration, invasion, and cell proliferation assaysCellmigration, invasion, or proliferationwas evaluated by real-

time monitoring using the xCELLigence RTCA DP Instrument(Roche Applied Science), as described before (26, 27). Briefly, forTranswell migration and invasion assays, 4� 104 cells suspendedin medium were seeded with plain medium in each upperchamber of a CIM-Plate 16 with 8-mm pores (Roche AppliedScience). The plate was then monitored for 48 hours with 10%FBS-containing medium in the lower chambers. In addition, forinvasion assays, 2.5% BD Matrigel matrix (BD Biosciences) wasplaced on upper chamber. For in vitro cell proliferation assays, 2�104 cells suspended in medium containing 10% FBS were seededper well in an E-plate 16 (Roche Applied Science) and monitoredfor 48 hours. We acquired and analyzed data at some time pointswith RTCA software (version 1.2, Roche Applied Science). Thecorrelation between cell index andmanually counted cell numberwas previously confirmed (24).

Wound-healing assaysCells were grown to confluence in 24-well dishes (BD Falcon).

Scratchwoundswere thenmadewith sterile plastic 200-mL pipettetips. After washing and a change of medium, microscopic imagesof healing areaswere photographically recorded at 0 and 12 hoursafter scratching. Healing area was calculated using Axio Vision LEsoftware (version 4.7.2.0, Carl Zeiss Imaging Solutions GmbH).

Epithelial-to-mesenchymal transition induction by TGF-b1Cells were prepared with recombinant human TGF-b1 (R&D

Systems; 240-B, 2 ng/mL) for 24 hours. Their total RNA was thenisolated.

ImmunohistochemistryHuman HNSCC clinical samples embedded in paraffin blocks

were cut into 4-mm-thick sections. After deparaffinization, sec-tions were then pretreated with an antigen retrieval solution,HistoVTone (Nacalai Tesque), for 20 minutes at 90�C.

Ohmura et al.

Mol Cancer Res; 13(4) April 2015 Molecular Cancer Research722




Endogenous peroxidase activity was then blocked by incubatingwith 1% hydrogen peroxide for 30 minutes at room temperature.After blocking in 1% normal goat serum, the specimens wereincubated with primary antibody (rabbit anti-hMyo1b pAb, at1:75; Sigma-Aldrich) at 4�C overnight. This pAb was also used inWestern blotting. Negative controls were incubated with rabbitIgG in place of the primary antibody. The streptavidin–biotinmethod was performed with a Histofine Simple Stain (R) kit(Nichirei) according to the manufacturer's instructions. The sec-tions were developed with 3,30-diaminobenzidine (DAB) in0.003% hydrogen peroxide (Muto Pure Chemicals) and counter-stained lightly with hematoxylin. IHC staining for human CCR4,CCR7, andCXCR4was performed and the results were interpretedas previously reported (24). Anti-hCCR4 mAb (Kyowa HakkoKirin), anti-hCCR7 mAb (BD Biosciences), and anti-hCXCR4mAb (R&D Systems) were used. IHC staining to detect lymphaticinvasion and lymph node metastasis was performed by similarprotocol. Anti-human D2-40 mAb (Dako) and anti-TurboGFPpAb (Evrogen) were used.

Immunostaining was examined by an experienced pathologistand an experienced oncologist who were blinded to patients'clinical outcomes. Immunostaining was classified by stainingintensity and percentage of stained cancer cells, as previouslyreported (28). Briefly, staining intensity was determined as 3(strong), 2 (moderate), 1 (weak), or 0 (absent; Fig. 2A, C andE). Expression levels of Myo1b were semiquantified using an IHCscore (Myo1b IHC score, range: 0–300) calculated bymultiplyingthe staining intensity by the percentage of positive cancer cells.The median value of Myo1b IHC score was used as a cutoff pointto classify Myo1b expression (high and low). The DAB densitieswere evaluated by the ImageJ color deconvolution methodaccording to the reported protocol (29, 30). The Myo1b ImageJscore was calculated by using the average DAB density of 5randomly selected fields in each sample (range, 68.8626–150.0392; median, 113.9958).

Scratch wound assaysFor scratch wound assays, siRNA-treated HSC4, SAS-sh-NC,

SAS-sh-Myo1b-1, or SAS-sh-Myo1b-2were plated on coverslips at1 � 105 cells suspended in 500 mL of RPMI, and 24 hours later,when the cells were confluent, scratchedwith a pipette tip. SAS-sh-NC, SAS-sh-Myo1b-1, and SAS-sh-Myo1b-2 were incubated inRPMI for 60minutes and siRNA-treated HSC4were incubated for120minutes. Theywere then fixedwith 4%paraformaldehyde for10minutes at room temperature, permeabilizedwith 0.3%TritonX-100, blocked with 1% BSA in PBS for 30 minutes at 37�C,processedwith TexasRed-Xphalloidin (Molecular Probes; diluted1:40) with 1% BSA in PBS for 10 minutes at room temperatureand incubated at room temperature for 5 minutes with 40,6-diamidino-2-phenylindole (DAPI; Doujindo Laboratories; dilut-ed 1:100) in PBS. Images were taken with a Zeiss LSM 700 LaserScanning Microscope and analyzed using the LSM Software ZEN2009 (Carl Zeiss). Cells having largeprotrusions of cellmembrane(i.e., larger than their nuclei) into the open area were counted in 4randomly selected lesions.

Animal modelSAS/nude mouse model was used to establish a cervical

lymph node metastatic model, which was modified from ourpreviously reported model (24). Briefly, female nude mice

(BalbC nu/nu) ages 5 to 6 weeks (from CLEA Japan, Tokyo,Japan) were intramuscularly injected with 1 � 106 SAS cellssuspended in 100 mL of RPMI into the right masseter muscle, asdescribed (31). Tumor diameters were measured using a digitalcaliper twice a week. Tumor volume (V) was calculated accord-ing to the formula: V¼ (A� B2)/2, where A and B were the longand short axes, respectively. After sacrifice at day 14, cells wereextracted from tumor tissues, right cervical lymph nodes, andlung. Tissue dissociation, sample preparation, and flow cyto-metric analysis for detecting GFPþ metastatic cells were per-formed as described (32). Briefly, upon sacrifice, tumor andorgans were collected and dissociated individually by mechan-ical disruption and incubation with collagenase for 30 minutesat 37�C. Collagenase activity was stopped, and samples werepassed through a 70-mm cell strainer (BD biosciences). Cellsuspensions were centrifuged and incubated in ice-cold 0.17mol/L NH4Cl for 10 minutes. Finally, cells were washed andresuspended in 1% FBS/PBS and fixed by 2% paraformalde-hyde/PBS. After wash and fixation, cells were analyzed using theGallios and the Kaluza software (Beckman Coulter). Priorapproval from the Institutional Animal Care and Use Com-mittee was obtained in all animal experiments.

Statistical analysisAll analyses were carried out on Microsoft Excel, version 2010

(Microsoft Corp.), Ekuseru-Toukei version 2010 (SSRI). Datawere analyzed using one of the following tests for significance:Student t test, Fisher exact test, or Mann–Whitney U test. P < 0.05was considered statistically significant.

ResultsAberrant overexpression of Myo1b in human HNSCC

To identify molecules involved in lymph node metastasis ofhuman HNSCC, a microarray meta-analysis was performed tocompare all genes across 13 different datasets including 222cancer tissues from HNSCC patient and 135 head and necknormal tissues from healthy donor (Oncomine; see MaterialsandMethods). The overexpressing genes were ranked by median-ranked analyses across each of 13 analyses. Myo1b was identifiedas one of themostly upregulated genes in HNSCC tissues (Fig. 1Aand Supplementary Fig. S1). Interestingly, Myo1b was preferen-tially overexpressed in the HNSCC datasets among various cancertypes by the in silico gene expression analysis (Fig. 1B). AlthoughSERPINH1 (also known as HSP47) was the highest ranked gene,it was previously shown to be involved in cancer progression ofcervical squamous cancer (33). Therefore, in this study, we furtherevaluated the roles ofMyo1b in the HNSCC progression. We haveconfirmed that by quantitative PCR assays on patient samples andcancer cell lines. Myo1b mRNA found to be aberrantly overex-pressed inHNSCC tissues compared with adjacent normal tissuesobtained from the same individuals (Fig. 1C and SupplementaryFig. S2;n¼7,P¼0.0253).Myo1bmRNAwashighly expressed in 3of 5 human HNSCC cell lines, SAS, HSC4, and Detroit562,especially tongue cancer cell lines SAS and HSC4 (Fig. 1D).Analysis on DNA methylation status of Myo1b promoter regionusing The Cancer Genome Atlas (TCGA) did not show significantalterations of DNA methylations in HNSCC (data not shown).Because the expressionofEGFRwhich is expressed inHNSCCcellswas previously reported to be associated with one of the myosinfamily, myosin6, in lung cancer cell line (34), the relationship

Promotion of Lymph Node Metastasis of HNSCC by Myosin 1b

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between EGFR and Myo1b was examined, and it was found thatMyo1b knockdown did not affect cell surface protein expression ofEGFR in SAS andHSC4 cell lines, and stimulation of EGFRdid notincrease the expression of Myo1b mRNA and protein eitherin SAS and HSC4 cell lines (data not shown). Therefore, Myo1bis not involved in the expression and function of EGFR in HNCCcell lines. High Myo1b expression may be involved in the path-ogenesis of HNSCC through aberrant expression, although themechanisms remain to be investigated.

Correlation of Myo1b expression with lymph node metastasesof human HNSCC

Expression of Myo1b protein in human HNSCC was evaluatedby IHC studies, and Myo1b was detected in all 31 human tonguecancer tissues evaluated but was not expressed in adjacent normaltissues (Fig. 2 and Supplementary Fig. S4A and S4B), which isconsistent with results of the gene expression analysis. Threerepresentative samples with different staining patterns asdescribed in the Materials and Methods are shown in Fig. 2 [Fig.

Median rank P Gene

SERPINH16.25E–0798.5

MYO1B0.006143.0

C3orf11.54E–07150.5

WDR665.20E–05179.0

MMP11.00E–02215.0

TPBG3.26E–05230.0

PSMB21.26E–05245.0

MMP123.91E–04263.0

CLPTM1L6.73E–06271.5

STAT19.25E–11276.0

ACTL6A4.80E–04283.0

HOMER31.40E–02296.0

KPNA24.63E–09297.0

CTHRC15.14E–05313.5

SEPT111.61E–06318.0

WDR531.42E–05321.5

ISG154.54E–04324.0

FNDC385.62E–04325.0

RPN 2 4.61E–04326.0

IFI165.67E–04327.0Bladder

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Figure 1.Myo1b is highly expressed preferentially in human HNSCC. Myo1b mRNA is significantly upregulated in HNSCC. A, a microarray meta-analysis was performedusing Oncomine to compare all genes across 13 different datasets, which included 222 patients with HNSCC and 135 healthy individuals (see Materials and Methods).Myo1b was identified as one of the most upregulated genes in HNSCC tissues. The top 20 overexpressed genes identified by meta-analysis are shown. Agene's rank is its median rank across each of the analyses. P value: gene's P value for the median-ranked analysis. Black and white indicates expression level in eachanalysis, and the diagonal line indicates no analysis. B, Myo1b is preferentially overexpressed in HNSCC. Fold changes of Myo1b mRNA expression in variouscancer types are plotted for comparison. C, Myo1bmRNA expression was significantly upregulated in cancer tissues compared with normal tissues from the sameindividuals by quantitative PCR analysis. (n ¼ 7, � , P ¼ 0.0253, Mann–Whitney U test). Relative expression of Myo1b mRNA was standardized by usingGAPDH gene expression as an internal reference, and each value was shown after normalization to theMyo1b expression level in HSC3 as the baseline value (¼1). D,Myo1bmRNA was highly expressed in 3 of 5 human HNSCC cell lines, particularly tongue cancer cell lines, SAS and HSC4 among various cancer cell lines. Relativeexpression of Myo1b mRNA was standardized by using GAPDH gene expression as an internal reference, and each value was shown after normalization tothe Myo1b expression level in HSC3 as the baseline value (¼1): representative of 3 separate experiments.

Ohmura et al.





2A, Sample 1 (T2N2bM0): staining intensity 3, 95% Myo1bþ

cancer cells, Myo1b IHC score 285; Fig. 2C, Sample 2 (T1N0M0):staining intensity 2, 75% Myo1bþ cancer cells, Myo1b IHC score150; Fig. 2E, sample 3 (T1N0M0): staining intensity 1, 75%Myo1bþ cancer cells, Myo1b IHC score 75; Fig 2B, D, and F showcontrol staining with rabbit IgG].Myo1b protein was expressed incytoplasmof cancer cells and tended to increase in tumor borders.Myo1b protein expression in cervical lymph node metastasisappeared similar to that in primary lesions (data not shown).

To evaluate the clinicopathologic roles of Myo1b in humanHNSCC, correlations between Myo1b expression (Myo1b IHCscore) and various clinicopathologic features were examined.Myo1b expression levels (high and low) were classified by themedian value of the Myo1b IHC score. In addition, we alsoevaluated the DAB intensity of Myo1b IHC using ImageJ (seeMaterials andMethods) and confirmed the significant correlationbetween the results by the Myo1b IHC score and the ImageJanalyses (Supplementary Fig. S3). High Myo1b expression wassignificantly correlated with lymphatic invasion and lymph node

metastasis (Table 1, Supplementary Fig. S4C and S4D, n¼ 31, P¼0.032), although other clinicopathologic factors, including T-stage and histologic type, showed no significant correlation. HighMyo1b expression was not correlated with expression of CCR4,CXCR4, or CCR7, which are known to correlate with lymph nodemetastasis in HNSCC (24, 35–37), suggesting that Myo1b andthese chemokine receptors were independent factors for lymphnode metastasis (Table 2). Interestingly, cancer cells with bothhigh Myo1b and CCR4þ status was strongly correlated withlymph node metastasis (Table 3, P ¼ 0.0006). Further multivar-iate analysis on their relationships in lymph node metastasis,confirmation of diagnostic power of the Myo1b and CCR4 com-bination, using increased number of HNSCC samples is required.These results, togetherwithprevious reports indicating theMyo1binvolvement in motility of non-cancer cells (11), demonstratethat Myo1b is involved in migration, lymphatic invasion, andlymph node metastasis.

Knockdown of Myo1b inhibited migration and invasion ofhuman HNSCC cells through reduced formation of largeprotrusions in cell membrane

We evaluated the functional role of Myo1b in cell motility ofhuman HNSCC cell lines. Myo1b expression was downregulatedby 2 different Myo1b-specific siRNAs (si1 and si2) and shRNAs(sh1 and sh2; Fig. 3A–C, Western blotting). These siRNAs orshRNAs significantly inhibited cell migration and invasion ofSAS andHSC4when evaluated by the in vitro Transwell migrationand invasion assays using the xCELLigence RTCA DP Instrument(Fig. 3D, E, G, and H; Supplementary Fig. S5A and S5B) withoutaffecting cell proliferation (Fig. 3F and I; Supplementary Fig. S5C).These observations were also confirmed by wound-healing assayswith theMyo1b-knockdown SAS and HSC4 (Fig. 3J and K). Theseresults indicate that Myo1b is functionally involved in cell migra-tion and invasion of human HNSCC.

Because epithelial-to-mesenchymal transition (EMT) is one ofthe mechanisms of enhanced cancer cell migration and invasion,relationships between Myo1b expression and some EMT-relatedmolecules were evaluated. However, Myo1b expression did notaffect expression of EMT-governing transcription factors such asSnail1and Twist1, or EMT-related molecules such as CDH1 (E-cadherin), as shown using quantitative PCR analysis (Supplemen-tary Fig. S6A–S6F). In addition, TGF-b1–induced EMT in SAS andHSC4 did not result in Myo1b induction (Supplementary Fig.S6G–S6J). Therefore, Myo1b regulates cell motility of humanHNSCC not depending on the major EMT transcription factors.

To investigate themechanismof enhancedHNSCCmotility viaMyo1b expression, possible Myo1b regulation of membraneprotrusion formation, including filopodia, lamellipodia, andinvadopodia/podosomes, was evaluated because they are knownto be involved in cancer cell migration, and Myo1b was reportedto localize at membranes of these protrusions (38, 39). Large

Table 1. Correlation between Myo1b and clinicopathologic features

Myo1b IHC scoreClinicopathologic factors Low (<140) High (�140) P

SexFemale 4 5 1.0000Male 11 11

Age, y<70 10 7 0.2852�70 5 9

T stage�2 13 12 0.6539�3 2 4

Histologic type/differentiationWell 12 13 1.0000Moderate/poorly 3 3

Lymphatic invasionAbsent 10 4 0.0320�

Present 5 12Venous invasionAbsent 15 12 0.1012Present 0 4

Preoperative chemotherapyNot performed 8 9 1.0000Performed 7 7

Lymph node metastasisNegative 11 5 0.0320

�

Positive 4 11

NOTE: Myo1b expression was significantly correlated with lymphatic invasionand cervical lymph node metastases.� , P < 0.05 by the Fisher exact test.

Table 3. Correlation between lymph node metastasis and cancer cellexpression of Myo1b and CCR4

Lymph nodemetastasis

Myo1b high andCCR4 positive

Theothers P

Positive 10 5 0.0006��

Negative 1 15

NOTE: HumanHNSCCwith high expression of bothMyo1b andCCR4are stronglyassociated with lymph node metastasis.�� , P < 0.01 by the Fisher exact test.

Table 2. Correlation between Myo1b and other metastasis-related molecules

Myo1b IHC scoreOther metastasis-related molecules Low (<140) High (�140) P

CCR4Positive 9 11 0.7160Negative 6 5

CCR7Positive 8 7 0.7244Negative 7 9

CXCR4Positive 3 4 1.0000Negative 12 12

NOTE: No correlation was observed between Myo1b expression and othermolecules associated with lymph node metastasis of human HNSCC, chemo-kine receptors CCR4, CXCR4, and CCR7. P value by Fisher exact test.






protrusions formation in cell membrane of SAS with or withoutMyo1b shRNA transfectionwas evaluated in scratchwound assays.SAS treated with control shRNA showed large protrusions forma-tion of cell membrane that were larger than their nuclei 1 hourafter scratching (Fig. 4A and C), whereas large protrusions for-mation was reduced in the Myo1b knockdown SAS (Fig. 4B andD). Similar results were shown in HSC4 with or without Myo1bsiRNA transfection (Fig. 4F and G). Cells having large protrusionof cell membrane were counted by using confocal microscopy,and significant reduction in the number of cells with largeprotrusion was observed in both SAS and HSC4 transduced withMyo1b-specific siRNAs and shRNAs (Fig. 4E andH). Lamellipodiawas observed in some large protrusions of cell membrane. Filo-podia formation inMyo1b knockdown SASwas not changed (datanot shown). Therefore, Myo1b augments cancer cell motilitypossibly through formation of large protrusion in cell membrane.

Myo1b knockdown inhibits cervical lymph node metastases ofhuman HNSCC in cervical lymph node metastatic model usingnude mice implanted with human HNSCC cells

To investigate the roles ofMyo1b in lymph nodemetastasis, wedeveloped a cervical lymph nodemetastatic model by using nudemice implantedwithMyo1b-expressing human tongue cancer cell

line, SAS. SAS-shRNA-Myo1b GFPþ and SAS-shRNA-NCGFPþ celllines were constructed by lentiviral transduction into SAS withGFP gene and either Myo1b-specific shRNA or control shRNA.More than 97% of each cell line expressed GFP (data not shown).These cell lines were injected into right masseter muscle of nudemice as cervical lymph node metastatic model. There was nodifference in in vivo tumor growth between the Myo1b-specificshRNA-transduced and control shRNA-transduced SAS (Fig. 5A),consistent with no difference of their in vitro cell proliferation(Supplementary Fig. S5D). Byflow cytometric analysis, significantdecrease of GFPþ cancer cells was detected in the draining lymphnodes ofmice implantedwith SAS transducedwithMyo1b-specificshRNA cells compared with SAS transduced with control shRNAcells (Fig. 5B and C). Moreover, presence of focal metastaticlesions in cervical lymph nodes was also confirmed by IHCmethod (Supplementary Fig. S7). However, the GFPþ cells werenot detected in lungs. These results indicate that Myo1b knock-down in human HNSCC cells reduces lymph node metastasis.

DiscussionThe roles of myosin family motor proteins in cancer cell

characteristics have not yet been well-investigated (34, 40, 41),

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Figure 2.Myo1b is expressed in tongue cancer tissues.Expression of Myo1b protein in humanHNSCC was evaluated by IHC studies, andMyo1b was detected in all 31 human tonguecancer tissues evaluated but was notexpressed in adjacent normal tissues. A, C, E,three representative immunostaining resultsof tongue cancer tissues expressing variousMyo1b levels with rabbit anti-Myo1b pAb areshown among 31 patients evaluated. TheMyo1b IHC score was calculated as describedin the Materials and Methods. Dotted linesindicate tumor border. A, sample 1 (T2N2bM0)has intensity of 3 and is 95% Myo1bþ cancercells, so its Myo1b IHC score is 285. C, sample 2(T1N0M0) has intensity of 2 and is 75%Myo1bþ

cancer cells, so its Myo1b IHC score is 150.E, sample 3 (T1N0M0) has intensity of 1 and is75% Myo1bþ cancer cells, so its Myo1b IHCscore is 75. B, D, F, the same sections werestained by control rabbit IgG. Bar, 100 mm.

Ohmura et al.





although the role of myosin family motor proteins—particularlymyosin 2—in cancer motility through cross-linking actin fila-ments has previously been reported (42, 43). In this article, wehave revealed that aberrant overexpression of Myo1b in humanHNSCC enhanced lymph node metastasis, possibly throughincreased cell motility and that Myo1b may be an attractive

molecule for the development of new diagnostic and therapeuticstrategies for patients with HNSCC.

Although we attempted to identify the mechanisms of Myo1boverexpression in human HNSCC, it remains to be investigated.No altered DNA methylation in the Myo1b promoter region wasfound, and stimulation of EGFR, which is known to be amplified

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Figure 3.Myo1b knockdown inhibits in vitro migration and invasion of human HNSCC cell lines. Myo1b knockdown using siRNA and shRNA inhibits in vitro cell migrationand invasion of human HNSCC. A–C, decrease of Myo1b protein by Myo1b-specific siRNAs and shRNAs in human HNSCC cell lines, SAS and HSC4, wasconfirmed by Western blotting. GAPDH is an internal control. D–I, these siRNAs significantly inhibited cell migration and invasion of SAS and HSC4. SAS andHSC4 transfected with siRNAs were used for Transwell migration, invasion, and proliferation assays. Migration, invasion, and proliferation were evaluated byreal-time monitoring for 48 hours using the xCELLigence RTCA DP instrument as described in Materials and Methods. Transfections were as follows: si negativecontrol (siNC): scrambled siRNA; si1 or si2: siRNAs specific for humanMyo1b (si-Myo1b-1 and si-Myo1b-2, respectively). Results show cell index at representative timepoints (28 hours for SAS migration and invasion, 38 hours for SAS proliferation, 14 hours for HSC4 migration, invasion, and proliferation) compared witheach cell index of siNC (1), shown asmean� SD (n¼ 4; n.s., not significant; � , P < 0.05; �� , P < 0.01; Student t test), representative of 3 separate experiments. J and K,these observations were also confirmed by wound-healing assays with Myo1b knockdown SAS and HSC4. SAS and HSC4, were transfected with shRNAs andsiRNAs, respectively, and used for wound-healing assays. Transfections were as follows: shNC, control shRNA; sh1 or sh2, shRNAs specific for human Myo1b(sh-Myo1b-1 or sh-Myo1b-2, respectively); siNC, scrambled siRNA; si1 or si2, siRNAs specific for human Myo1b (si-Myo1b-1 and si-Myo1b-2, respectively). Resultsshow wound-healing area after 12 hours incubation as mean � SD (n ¼ 9 or 12; � , P < 0.05; �� , P < 0.01; Student t test), about SAS: representative of3 separate experiments and HSC4: 1 experiment.






in HNSCC, did not change Myo1b expression (data not shown).Interestingly, our in silico gene expression analysis and quantita-tive PCR analysis demonstrated preferential overexpression ofMyo1b in HNSCC, and some of the other squamous cell carcino-mas, including esophageal cancer and cervical cancer, whichsuggest that squamous cell type–related mechanisms may con-tribute toMyo1b overexpression. In contrast toMyo1b overexpres-sion in HNSCC, downregulation of Myo1b expression wasobserved in certain types of cancers (Fig. 1B), suggesting that therole ofMyo1b in metastasis may be different among cancer types.

Further investigation is needed to evaluate the function ofMyo1bin other cancers.

The functional roles of Myo1b in cancer biology have notpreviously been known, although its association with cell migra-tion of zebrafish embryo cells (11) and intracellular transportregulated by the formation of post-Golgi carriers were reported(12, 13). Myo1b was reported to localize at plasma membranestructures, including filopodia, ruffles, and lamellipodia (38,39, 44). Our in vitro study indicated that knockdown of Myo1binhibited HNSCC cell migration and invasion through decreased

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Figure 4.Myo1b knockdown inhibits formation of large protrusion in cell membrane in both SAS and HSC4 cell lines. Myo1b knockdown reduces large protrusionof cell membrane in HNSCC cell lines in scratch wound assays. A–D, large protrusions formation in cell membrane of SAS cell line with or without Myo1bshRNA transfectionwas evaluated in scratchwound assays (seeMaterials andMethods). SAS treatedwith control shRNA showed large protrusions of cellmembranethat were larger than their nuclei 1 hour after scratching (A and C), whereas large protrusions formation in cell membrane was reduced in the Myo1b knockdownSAS (B and D). Transfections were as follows: shNC, control shRNA; sh1 or sh2, shRNAs specific for human Myo1b (sh-Myo1b-1 or sh-Myo1b-2, respectively).Representative large protrusion is larger than cell nucleus in SAS-shNC (C); small protrusion is smaller than cell nucleus in SAS-sh2 (D). E, cells having largeprotrusionof cell membrane were counted by using confocal microscopy, and a significant reduction in the number of cells with large protrusion was observed in SAStransduced with Myo1b-specific shRNA. F and G, the similar results were shown in HSC4. Transfections were as follows: siNC, scrambled siRNA; si1 or si2, siRNAsspecific for human Myo1b (si-Myo1b-1 and si-Myo1b-2, respectively). HSC4-siNC formed large protrusion of cell membrane (F), whereas HSC4-si2 formed fewlarge protrusions (G). H, significant reduction of number of cellswith large protrusionwas observed inHSC4 transducedwithMyo1b-specific siRNA.White, phalloidin;red, DAPI.White arrows indicate large protrusion of cell membrane. Bar, 200mm(A, B, F, G) and 20mm(CandD). Data are shown asmeans� SD from4optical areas(n ¼ 4; � , P < 0.05, ��, P < 0.01; Student t test), about SAS: representative of 3 separate experiments and HSC4: representative of 2 separate experiments.

Ohmura et al.





large protrusion formation of cell membrane. Recently, somemyosin family proteins such as myosin 2 were reported to behighly expressed in cancer cells and to be correlated with cancercell motility and metastasis partly through cross-linking actinfilaments (34, 41, 42). Although myosin 6 was reported to beinvolved in polarized delivery of vesicles containing EGFR intoleading edges of cancer cells (34), Myo1b knockdown did notaffect cell surface protein expression of EGFR (data not shown).We also evaluated the relationship between Myo1b and intracel-lular transport of various molecules other than EGFR (e.g., HLA,IL6, IL8, and CCL2) but no difference was observed (data notshown). Further study on possible Myo1b-associated traffickingchanges of molecules involved in cell migration may be required(12, 13).

Our clinicopathologic analyses indicate thatMyo1b expressionin primary HNSCC tissues is a potential diagnostic marker topredict lymph node metastases and possibly subsequent prog-nosis of patients with HNSCC, as lymph node metastases is themajor factor in overall survival of patients with HNSCC. This mayenable better clinical management, including decisions for pro-phylactic neck dissection or postsurgical chemoradiotherapy.Moreover, combinatorial uses of other predictive markers suchas chemokine receptors may further improve the accuracy ofpredicting power. In fact, cancer cells with both high-Myo1bexpression and CCR4þ status are strongly associated with highoccurrence of lymph node metastasis in this study (Table 3, P ¼0.0006). Further analyses including multivariate analysis withincreased numbers of patients are warranted for confirmation of

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Figure 5.Myo1b knockdown inhibits cervical lymph node metastasis of human HNSCC cell line implanted in nude mice. Myo1b knockdown reduced cervical lymphnodemetastasis in cervical lymph nodemetastatic model using nude mice implanted with SAS cell line. SAS-shRNA-Myo1b GFPþ (SAS-sh-Myo1b) and SAS-shRNA-NC GFPþ (SAS-sh-NC) cell lines were constructed by lentiviral transfection into SAS with GFP gene and either human Myo1b-specific shRNA (sh-Myo1b-2) orcontrol shRNA. Each cell line expressedGFP inmore than 97% cells (data not shown). These cell lineswere implanted intomassetermuscle of nudemice. A, therewasno difference in in vivo tumor growth between the Myo1b-specific shRNA-transduced and control shRNA–transduced SAS. Tumor volume (mean � SD) oftransplanted SAS-sh-Myo1b and SAS-sh-NC in vivo (n¼ 7, 6; n.s., not significant; Student t test), representative of 3 separate experiments. The average tumor volumeof SAS-sh-Myo1b was 608 mm3 and SAS-sh-NC was 603 mm3. B and C, by flow cytometric analysis, a significant decrease of GFPþ cancer cells was detectedin the draining lymph nodes of mice implanted with SAS transduced with Myo1b-specific shRNA cells compared with SAS transduced with control shRNAcells; however, the GFPþ cells were not detected in lung tissues. B, numbers of GFPþ cells per 1 � 105 cells derived from tumor-draining cervical lymph node wereplotted (n ¼ 7, 6; �, P ¼ 0.0455; Mann–Whitney U test), representative of 3 separate experiments. C, GFPþ cells were detected by flow cytometry in cervicallymph node (top) and lung (bottom) samples. Each depicted number shows the percentage of GFPþ cells among total cells. Left, non–tumor-bearingmouse;middle,SAS-sh-NC–bearing mouse; right, SAS-sh-Myo1b–bearing mouse. Representative dot plots are shown.






the diagnostic powers of Myo1b expression along with othermarkers such as chemokine receptor for lymph node metastasisand subsequent survival of patients with HNSCC.

Because Myo1b knockdown inhibited lymph node metastases,possibly due to decreased cancer cell motility, Myo1b couldalso be a therapeutic target for prevention of lymph node metas-tasis and subsequent improvement of overall survival. Recently,pentachloropseudilin, a low-molecular-weight chemical, wasreported to be a reversible and allosteric inhibitor of class 1myosin motor activity (45). As Myo1b is expressed in variousnormal tissues, systemic administration ofMyo1b inhibitors suchas low-molecular-weight chemicals and siRNAs may cause severeadverse effects. Therefore, a preferential deliverymethod to cancercells may be required. Otherwise, upstream or downstreammolecules in the Myo1b-related cell mobilization axis may alsobe targets for HNSCC. Further studies are required to developtherapeutic strategies that target Myo1b-related malignantfeatures.

In summary, we have shown that aberrant overexpression ofMyo1b in human HNSCC augments cancer cell motility viaenhanced large protrusion formation of cell membrane andpromotes lymph node metastasis. Therefore, Myo1b is an attrac-tive target for the development of new diagnostic and therapeuticstrategies for patients with HNSCC.

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

Authors' ContributionsConception and design: G. Ohmura, T. Tsujikawa, T. Yaguchi, H. Nakano,T. Shimada, Y. Hisa, Y. Kawakami

Development of methodology: G. Ohmura, T. Tsujikawa, T. Yaguchi,N. Kawamura, S. Mikami, J. Sugiyama, K. Nakamura, A. KobayashiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Ohmura, T. Tsujikawa, T. Yaguchi, N. Kawamura,S. Mikami, J. Sugiyama, K. Nakamura, H. Nakano, T. Shimada, Y. KawakamiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Ohmura, T. Tsujikawa, T. Yaguchi, S. Mikami,J. Sugiyama, Y. Hisa, Y. KawakamiWriting, review, and/or revision of the manuscript: G. Ohmura, T. Tsujikawa,T. Yaguchi, S. Mikami, Y. KawakamiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. Ohmura, T. Tsujikawa, K. Nakamura,T. Iwata, Y. Hisa, Y. KawakamiStudy supervision: G. Ohmura, T. Tsujikawa, T. Yaguchi, J. Sugiyama,Y. Kawakami

AcknowledgmentsThe authors thank Dr. Shigeki Ohta for technical advice, Dr. Masato Oka-

moto for development of the in vivo tumor model, Dr. Hiroyuki Aburatani forDNA methylation analysis, Dr. Ryuta Nakao for the examination of lymphaticinvasion, KenjiMorii forflow cytometric analysis,Miyuki Saito for IHC staining,and Misako Horikawa and Ryoko Suzuki for preparation of the article.

Grant SupportThis work was supported byGrants-in-Aid for Scientific Research (23240128

and 26221005) from the Japan Society for Promotion of Science, a researchprogram of the Project for Development of Innovative Research on CancerTherapeutics (P-Direct) from theMinistry of Education, Culture, Sports, Scienceand Technology of Japan.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 23, 2014; revised October 23, 2014; accepted November 12,2014; published OnlineFirst November 24, 2014.

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2015;13:721-731. Published OnlineFirst November 24, 2014.Mol Cancer Res Gaku Ohmura, Takahiro Tsujikawa, Tomonori Yaguchi, et al. Lymph Node Metastasis of HNSCCAberrant Myosin 1b Expression Promotes Cell Migration and

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aberrant myosin 1b expression promotes cell migration and...

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