Aberrant Myosin 1B Expression Promotes Cell Migration and Lymph

Published OnlineFirst November 24, 2014; DOI: 10.1158/1541-7786.MCR-14-0410

Genomics Molecular Cancer Research Aberrant Myosin 1b Expression Promotes Cell Migration and Lymph Node Metastasis of HNSCC Gaku 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 feature tasis-associated molecule, was strongly associated with lymph associated with poor prognosis in patients with head and neck node metastasis. RNA interference (RNAi) of Myo1b in HNSCC squamous cell carcinoma (HNSCC). Here, web-based bioinfor- cells, SAS and HSC4, significantly inhibited migratory and invasive matics meta-analysis was performed to elucidate the molecular abilities through decreased large protrusion formation of cell mechanism of lymph node metastasis of human HNSCC. Prefer- membranes. Finally, Myo1b knockdown in SAS cells significantly ential upregulation of Myosin 1b (MYO1B) transcript in HNSCC inhibited in vivo cervical lymph node metastases in a cervical lymph datasets was identified. Myo1b mRNA was highly expressed in node metastatic mouse model system. human HNSCC cells and patient tissue specimens compared with their normal counterparts as shown by quantitative PCR (qPCR) Implications: Myo1b is functionally involved in lymph node analyses. Immunohistochemistry (IHC)-detected Myo1b expres- metastasis of human HNSCC through enhanced cancer cell sion was significantly correlated with lymph node metastases in motility and is an attractive target for new diagnostic and ther- patients with oral cancer of the tongue. HNSCC with high expres- apeutic strategies for patients with HNSCC. Mol Cancer Res; 13(4); sion of Myo1b and chemokine receptor 4 (CCR4), another metas- 721–31. 2014 AACR.

Introduction initiated by protrusion of the cell membrane. Aberrant regulation of cell migration drives cancer invasion and metastasis. Head and neck squamous cell carcinoma (HNSCC) is the sixth The protrusive structures formed by migrating and invading most common malignancy worldwide (1). Despite improved cancer cells are ﬁlopodia, lamellipodia, and invadopodia/ locoregional control of HNSCC (2), the 5-year survival rate for podosomes, depending on their morphologic, structural, and HNSCC remains relatively unchanged, at under 50% for the past functional characters (10). However, the mechanisms of three decades (3). HNSCC tends to metastasize to lymph nodes human HNSCC cell migration and metastasis remain poorly before distant metastasis (4–6). Because lymph node metastasis understood. is strongly associated with poor prognosis in patients with Here, we investigated the mechanisms of lymph node meta- HNSCC (4, 7–9), understanding the mechanisms that underlie stasis of human HNSCC. Myosin 1b (Myo1b, also named lymph node metastasis of HNSCC is important for improving myosin 1 alpha or Myr1), a member of the myosin family, diagnostic and therapeutic strategies. was found to be upregulated in HNSCC through a web-based Cancer cells use their intrinsic migratory ability to invade bioinformatics meta-analysis. Myo1b,aclassImyosin,isa adjacent tissues and ultimately to metastasize to different widely expressed, single-headed, actin-associated molecular organs. Cell migration is a highly integrated multistep process, motor, associated with cell migration of zebraﬁsh embryo cells (11) and intracellular transport regulated by the formation of post-Golgi carriers (12, 13). However, its role in cancer biology 1Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Shinjuku-ku, Tokyo, has never been reported. Aberrant expression of Myo1b immu- Japan. 2Department of Otolaryngology-Head and Neck Surgery, nohistochemically (IHC) detected was correlated with lymph Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto City, node metastases in patients with HNSCC (n ¼ 31, P ¼ 0.0320). 3 Kyoto, Japan. Division of Diagnostic Pathology, Keio University Myo1b knockdown in human HNSCC cell lines by RNA inter- School of Medicine, Shinjuku-ku, Tokyo, Japan. ference inhibited in vitro migration and invasion abilities of the Note: Supplementary data for this article are available at Molecular Cancer HNSCC cells accompanied by decreased large protrusions Research Online (http://mcr.aacrjournals.org/). formation in cell membrane. Downregulation of Myo1b in G. Ohmura and T. Tsujikawa contributed equally to this article. human HNSCC cell lines resulted in inhibition of metastasis Corresponding Author: Yutaka Kawakami, Division of Cellular Signaling, Insti- to cervical lymph nodes in cervical lymph node metastatic tute for Advanced Medical Research, Keio University School of Medicine, 35 model using nude mice implanted with human HNSCC. These Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: 81-3-5363-3778; results demonstrate that Myo1b is involved in cancer cell Fax: 81-3-5362-9259; E-mail: [email protected] motility and lymph node metastasis of human HNSCC, imply- doi: 10.1158/1541-7786.MCR-14-0410 ing that Myo1b could be a novel diagnostic and therapeutic 2014 American Association for Cancer Research. target for patients with HNSCC.

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Materials and Methods ization to the Myo1b expression level in HSC3 as the baseline value (¼1). Western blotting was performed by general proce- Patients and clinical samples dure with rabbit anti-hMyo1b pAb (Sigma-Aldrich) and rabbit A total of 31 patients with tongue cancer for IHC and 7 patients anti-GAPDH pAb (Santa Cruz Biotechnology). with HNSCC for quantitative PCR assays, who underwent surgery at Kyoto Prefectural University of Medicine (Kyoto, Japan) Myo1b knockdown between April 2008 and April 2012, were enrolled in a retrospec- siRNAs specific for human Myo1b (si-Myo1b-1; HSS106714 tive study. Data were collected from clinical and pathologic and si-Myo1b-2; HSS106715, Stealth Select RNAi), as well as records with the written informed consent of individual patients scrambled siRNA (Stealth RNAi Negative Control Kit, medium after approval by the Ethics Committee of the institutes. GC), were purchased from Invitrogen. These siRNAs (final con- centration during transfection, 100 nmol/L) were transfected into In silico gene expression studies cells using Lipofectamine RNAi MAX (Invitrogen) according to We used the Oncomine database (Compendia Bioscience; the manufacturer's recommendations. Assays were carried out 72 http://www.oncomine.org) to identify upregulated genes in hours after transfection. Lentiviral pGIPZ shRNA vectors targeting HNSCC on May 26, 2013 and performed a microarray meta- human Myo1b (sh-Myo1b-1; V3LHS_356101, sh-Myo1b-2; – analysis to compare all genes across 13 different datasets (14 22) V3LHS_356104) and nonsilencing pGIPZ control vector (sh-NC) fi that were identi ed by the following parameters: "mRNA," "can- were purchased from Open Biosystems (Thermo Fisher Scientific, cer vs. normal analysis," and "head and neck cancer" (excluding Inc.), and they were integrated with turboGFP and puromycin nasopharyngeal cancer, salivary gland cancer, and thyroid cancer). resistance sequences. Lentivirus particles were produced in 293T Myo1b Myo1b To compare expression, expression fold changes cells according to the manufacturer's instructions. The viral titer P < were limited to 0.05. was calculated by evaluating GFP expression in infected 293T cells. SAS cells were transduced by the lentiviral particles in the Cell culture same viral titer. The cells stably expressing shRNA were selected, Human oral tongue cancer cell lines, SAS and HSC-4, were pooled, and maintained with 2 mg/mL puromycin (Wako). purchased from RIKEN Bioresource Center Cell Bank (Tsukuba, Japan). Cells were cultured in RPMI-1640 supplemented with In vitro migration, invasion, and cell proliferation assays 10% FBS, penicillin (50 units/mL), and streptomycin (50 mg/mL) Cell migration, invasion, or proliferation was evaluated by real- fi and were incubated at 37 C in a humidi ed chamber supple- time monitoring using the xCELLigence RTCA DP Instrument mented with 5% CO2. Human hypopharyngeal cancer, FaDu and (Roche Applied Science), as described before (26, 27). Briefly, for Detroit562, human oral tongue cancer, HSC-3, human Hodgkin Transwell migration and invasion assays, 4 104 cells suspended lymphoma, L-428, human melanoma, Skmel23, 928mel, and in medium were seeded with plain medium in each upper A375mel, human brain tumor, U87MG, human esophageal can- chamber of a CIM-Plate 16 with 8-mm pores (Roche Applied cer, TE4 and TE9, lung cancer, LK2 and EBC1, pancreatic ductal Science). The plate was then monitored for 48 hours with 10% adenocarcinoma, PK59, prostate cancer, PC3, breast cancer, FBS-containing medium in the lower chambers. In addition, for Hs578 and MDA-MB-231, chronic myeloid leukemia, K562, invasion assays, 2.5% BD Matrigel matrix (BD Biosciences) was acute myeloid leukemia, HL60, was obtained and cultured as placed on upper chamber. For in vitro cell proliferation assays, 2 – previously reported (23 25). Human brain tumor, T98G, human 104 cells suspended in medium containing 10% FBS were seeded cervical cancer, C33a and HeLa, were purchased from the ATCC per well in an E-plate 16 (Roche Applied Science) and monitored and cultured in DMEM as well as in RPMI-1640. Human cervical for 48 hours. We acquired and analyzed data at some time points cancer, SKG1, was a kind gift from Dr. Daisuke Aoki (Keio with RTCA software (version 1.2, Roche Applied Science). The University, Tokyo, Japan) and cultured in DMEM as well as in correlation between cell index and manually counted cell number RPMI-1640. SAS, HSC4, HSC3, Detroit526, FaDu, L-428, was previously confirmed (24). A375mel, U87MG, T98G, TE4, TE9, C33a, SKG1, HeLa, PK59, Hs578, and MDA-MB-231 were authenticated by short tandem Wound-healing assays repeat profiling. Cells were grown to confluence in 24-well dishes (BD Falcon). Scratch wounds were then made with sterile plastic 200-mL pipette Evaluation of mRNA and protein expression tips. After washing and a change of medium, microscopic images Total RNA was isolated from cells using an RNeasy Mini Kit of healing areas were photographically recorded at 0 and 12 hours (Qiagen) with on-column DNase treatment (Qiagen), and after scratching. Healing area was calculated using Axio Vision LE TissueRuptor (Qiagen) was used in the total RNA isolation software (version 4.7.2.0, Carl Zeiss Imaging Solutions GmbH). from patient tissue samples. cDNA was synthesized using Superscript III Reverse Transcriptase and Oligo (dT) 12–18 Epithelial-to-mesenchymal transition induction by TGF-b1 primers (Invitrogen). Quantitative real-time PCR was per- Cells were prepared with recombinant human TGF-b1 (R&D formed using a TaqMan probe (Applied Biosystems), Myo1b Systems; 240-B, 2 ng/mL) for 24 hours. Their total RNA was then (Hs00362654_m1), Snail1 (Hs00195591_m1), Twist1 isolated. (Hs01675818_s1), CDH1 (Hs01023895_m1), and GAPDH (Hs03929097_g1), on ABI PRISM 7900 HT Sequence Detection Immunohistochemistry System with TaqMan Gene Expression Master Mix (Applied Human HNSCC clinical samples embedded in paraffin blocks Biosystems). Target gene expression was standardized to were cut into 4-mm-thick sections. After deparaffinization, sec- GAPDH levels using SDS Software v2.2 (Applied Biosystems). tions were then pretreated with an antigen retrieval solution, Relative expression of Myo1b mRNA was shown after normal- HistoVTone (Nacalai Tesque), for 20 minutes at 90C.

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Promotion of Lymph Node Metastasis of HNSCC by Myosin 1b

Endogenous peroxidase activity was then blocked by incubating (BalbC nu/nu) ages 5 to 6 weeks (from CLEA Japan, Tokyo, with 1% hydrogen peroxide for 30 minutes at room temperature. Japan) were intramuscularly injected with 1 106 SAS cells After blocking in 1% normal goat serum, the specimens were suspended in 100 mLofRPMIintotherightmassetermuscle,as incubated with primary antibody (rabbit anti-hMyo1b pAb, at described (31). Tumor diameters were measured using a digital 1:75; Sigma-Aldrich) at 4C overnight. This pAb was also used in caliper twice a week. Tumor volume (V) was calculated accord- Western blotting. Negative controls were incubated with rabbit ing to the formula: V ¼ (A B2)/2, where A and B were the long IgG in place of the primary antibody. The streptavidin–biotin and short axes, respectively. After sacrifice at day 14, cells were method was performed with a Histofine Simple Stain (R) kit extracted from tumor tissues, right cervical lymph nodes, and (Nichirei) according to the manufacturer's instructions. The sec- lung. Tissue dissociation, sample preparation, and flow cyto- þ tions were developed with 3,30-diaminobenzidine (DAB) in metric analysis for detecting GFP metastatic cells were per- 0.003% hydrogen peroxide (Muto Pure Chemicals) and counter- formed as described (32). Briefly, upon sacrifice, tumor and stained lightly with hematoxylin. IHC staining for human CCR4, organs were collected and dissociated individually by mechan- CCR7, and CXCR4 was performed and the results were interpreted ical disruption and incubation with collagenase for 30 minutes as previously reported (24). Anti-hCCR4 mAb (Kyowa Hakko at 37C. Collagenase activity was stopped, and samples were Kirin), anti-hCCR7 mAb (BD Biosciences), and anti-hCXCR4 passed through a 70-mm cell strainer (BD biosciences). Cell mAb (R&D Systems) were used. IHC staining to detect lymphatic suspensions were centrifuged and incubated in ice-cold 0.17 invasion and lymph node metastasis was performed by similar mol/L NH4Cl for 10 minutes. Finally, cells were washed and protocol. Anti-human D2-40 mAb (Dako) and anti-TurboGFP resuspended in 1% FBS/PBS and fixed by 2% paraformalde- pAb (Evrogen) were used. hyde/PBS. After wash and fixation, cells were analyzed using the Immunostaining was examined by an experienced pathologist Gallios and the Kaluza software (Beckman Coulter). Prior and an experienced oncologist who were blinded to patients' approval from the Institutional Animal Care and Use Com- clinical outcomes. Immunostaining was classified by staining mittee was obtained in all animal experiments. intensity and percentage of stained cancer cells, as previously reported (28). Briefly, staining intensity was determined as 3 Statistical analysis (strong), 2 (moderate), 1 (weak), or 0 (absent; Fig. 2A, C and All analyses were carried out on Microsoft Excel, version 2010 E). Expression levels of Myo1b were semiquantified using an IHC (Microsoft Corp.), Ekuseru-Toukei version 2010 (SSRI). Data score (Myo1b IHC score, range: 0–300) calculated by multiplying were analyzed using one of the following tests for significance: the staining intensity by the percentage of positive cancer cells. Student t test, Fisher exact test, or Mann–Whitney U test. P < 0.05 The median value of Myo1b IHC score was used as a cutoff point was considered statistically significant. to classify Myo1b expression (high and low). The DAB densities were evaluated by the ImageJ color deconvolution method according to the reported protocol (29, 30). The Myo1b ImageJ Results score was calculated by using the average DAB density of 5 Aberrant overexpression of Myo1b in human HNSCC randomly selected fields in each sample (range, 68.8626– To identify molecules involved in lymph node metastasis of 150.0392; median, 113.9958). human HNSCC, a microarray meta-analysis was performed to compare all genes across 13 different datasets including 222 cancer tissues from HNSCC patient and 135 head and neck Scratch wound assays normal tissues from healthy donor (Oncomine; see Materials For scratch wound assays, siRNA-treated HSC4, SAS-sh-NC, and Methods). The overexpressing genes were ranked by median- SAS-sh-Myo1b-1, or SAS-sh-Myo1b-2 were plated on coverslips at ranked analyses across each of 13 analyses. Myo1b was identified 1 105 cells suspended in 500 mL of RPMI, and 24 hours later, as one of the mostly upregulated genes in HNSCC tissues (Fig. 1A when the cells were confluent, scratched with a pipette tip. SAS-sh- and Supplementary Fig. S1). Interestingly, Myo1b was preferen- NC, SAS-sh-Myo1b-1, and SAS-sh-Myo1b-2 were incubated in tially overexpressed in the HNSCC datasets among various cancer RPMI for 60 minutes and siRNA-treated HSC4 were incubated for types by the in silico gene expression analysis (Fig. 1B). Although 120 minutes. They were then fixed with 4% paraformaldehyde for SERPINH1 (also known as HSP47) was the highest ranked gene, 10 minutes at room temperature, permeabilized with 0.3% Triton it was previously shown to be involved in cancer progression of X-100, blocked with 1% BSA in PBS for 30 minutes at 37C, cervical squamous cancer (33). Therefore, in this study, we further processed with Texas Red-X phalloidin (Molecular Probes; diluted evaluated the roles of Myo1b in the HNSCC progression. We have 1:40) with 1% BSA in PBS for 10 minutes at room temperature confirmed that by quantitative PCR assays on patient samples and and incubated at room temperature for 5 minutes with 40,6- cancer cell lines. Myo1b mRNA found to be aberrantly overex- diamidino-2-phenylindole (DAPI; Doujindo Laboratories; dilut- pressed in HNSCC tissues compared with adjacent normal tissues ed 1:100) in PBS. Images were taken with a Zeiss LSM 700 Laser obtained from the same individuals (Fig. 1C and Supplementary Scanning Microscope and analyzed using the LSM Software ZEN Fig. S2; n ¼ 7, P ¼ 0.0253). Myo1b mRNA was highly expressed in 3 2009 (Carl Zeiss). Cells having large protrusions of cell membrane of 5 human HNSCC cell lines, SAS, HSC4, and Detroit562, (i.e., larger than their nuclei) into the open area were counted in 4 especially tongue cancer cell lines SAS and HSC4 (Fig. 1D). randomly selected lesions. Analysis on DNA methylation status of Myo1b promoter region using The Cancer Genome Atlas (TCGA) did not show significant Animal model alterations of DNA methylations in HNSCC (data not shown). SAS/nude mouse model was used to establish a cervical Because the expression of EGFR which is expressed in HNSCC cells lymph node metastatic model, which was modified from our was previously reported to be associated with one of the myosin previously reported model (24). Briefly, female nude mice family, myosin6, in lung cancer cell line (34), the relationship

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A Median rank P Gene B Upregulated Downregulated 98.5 6.25E–07 SERPINH1 Prostate 143.0 0.006 MYO1B 150.5 1.54E–07 C3orf1 Pancreatic 179.0 5.20E–05 WDR66 Ovarian 215.0 1.00E–02 MMP1 Melanoma 230.0 3.26E–05 TPBG Lung 245.0 1.26E–05 PSMB2 263.0 3.91E–04 MMP12 Liver 271.5 6.73E–06 CLPTM1L Kidney

276.0 9.25E–11 STAT1 Head and Neck 283.0 4.80E–04 ACTL6A Gastric 296.0 1.40E–02 HOMER3 297.0 4.63E–09 KPNA2 Esophageal 313.5 5.14E–05 CTHRC1 Colorectal 318.0 1.61E–06 SEPT11 Cervical 321.5 1.42E–05 WDR53 Breast 324.0 4.54E–04 ISG15 325.0 5.62E–04 FNDC38 Brain 326.0 4.61E–04 RPN 2 Bladder 327.0 5.67E–04 IFI16 −20246 −4 −6 −8 −10

C D 10 HNSCC * 60 8 50 6 40 4 Other SCC 30 Other cancers /mRNA (HSC3 = 1)

2 20 /mRNA (HSC3 = 1)

Relative expression of

Myo1b 0 10 Relative expression of LK2 TE4 TE9 PC3 SAS Myo1b L428 K562 C33a SKGI HeLa HL60 FaDu PK59 T98G EBC1 HSC3 0 HSC4 Hs578 928mel Normal Cancer U87MG Skmel23 A375mel Detroit562 MDA-MB-231

Figure 1. Myo1b is highly expressed preferentially in human HNSCC. Myo1b mRNA is significantly upregulated in HNSCC. A, a microarray meta-analysis was performed using 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. A gene'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 each analysis, and the diagonal line indicates no analysis. B, Myo1b is preferentially overexpressed in HNSCC. Fold changes of Myo1b mRNA expression in various cancer types are plotted for comparison. C, Myo1b mRNA expression was significantly upregulated in cancer tissues compared with normal tissues from the same individuals by quantitative PCR analysis. (n ¼ 7, , P ¼ 0.0253, Mann–Whitney U test). Relative expression of Myo1b mRNA was standardized by using GAPDH gene expression as an internal reference, and each value was shown after normalization to the Myo1b expression level in HSC3 as the baseline value (¼1). D, Myo1b mRNA was highly expressed in 3 of 5 human HNSCC cell lines, particularly tongue cancer cell lines, SAS and HSC4 among various cancer cell lines. Relative expression of Myo1b mRNA was standardized by using GAPDH gene expression as an internal reference, and each value was shown after normalization to the Myo1b expression level in HSC3 as the baseline value (¼1): representative of 3 separate experiments.

between EGFR and Myo1b was examined, and it was found that Correlation of Myo1b expression with lymph node metastases Myo1b knockdown did not affect cell surface protein expression of of human HNSCC EGFR in SAS and HSC4 cell lines, and stimulation of EGFR did not Expression of Myo1b protein in human HNSCC was evaluated increase the expression of Myo1b mRNA and protein either by IHC studies, and Myo1b was detected in all 31 human tongue in SAS and HSC4 cell lines (data not shown). Therefore, Myo1b cancer tissues evaluated but was not expressed in adjacent normal is not involved in the expression and function of EGFR in HNCC tissues (Fig. 2 and Supplementary Fig. S4A and S4B), which is cell lines. High Myo1b expression may be involved in the path- consistent with results of the gene expression analysis. Three ogenesis of HNSCC through aberrant expression, although the representative samples with different staining patterns as mechanisms remain to be investigated. described in the Materials and Methods are shown in Fig. 2 [Fig.

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Table 1. Correlation between Myo1b and clinicopathologic features Table 3. Correlation between lymph node metastasis and cancer cell Myo1b IHC score expression of Myo1b and CCR4 Clinicopathologic factors Low (<140) High (140) P Lymph node Myo1b high and The Sex metastasis CCR4 positive others P Female 4 5 1.0000 Positive 10 5 0.0006 Male 11 11 Negative 1 15 Age, y NOTE: Human HNSCC with high expression of both Myo1b and CCR4 are strongly <70 10 7 0.2852 associated with lymph node metastasis. 70 5 9 , P < 0.01 by the Fisher exact test. T stage 2 13 12 0.6539 324 metastasis (Table 1, Supplementary Fig. S4C and S4D, n ¼ 31, P ¼ Histologic type/differentiation 0.032), although other clinicopathologic factors, including T- Well 12 13 1.0000 stage and histologic type, showed no significant correlation. High Moderate/poorly 3 3 Myo1b expression was not correlated with expression of CCR4, Lymphatic invasion CXCR4, or CCR7, which are known to correlate with lymph node Absent 10 4 0.0320 – Present 5 12 metastasis in HNSCC (24, 35 37), suggesting that Myo1b and Venous invasion these chemokine receptors were independent factors for lymph Absent 15 12 0.1012 node metastasis (Table 2). Interestingly, cancer cells with both þ Present 0 4 high Myo1b and CCR4 status was strongly correlated with Preoperative chemotherapy lymph node metastasis (Table 3, P ¼ 0.0006). Further multivar- Not performed 8 9 1.0000 iate analysis on their relationships in lymph node metastasis, Performed 7 7 fi Lymph node metastasis con rmation of diagnostic power of the Myo1b and CCR4 com- Negative 11 5 0.0320 bination, using increased number of HNSCC samples is required. Positive 4 11 These results, together with previous reports indicating the Myo1b NOTE: Myo1b expression was significantly correlated with lymphatic invasion involvement in motility of non-cancer cells (11), demonstrate and cervical lymph node metastases. that Myo1b is involved in migration, lymphatic invasion, and , P < 0.05 by the Fisher exact test. lymph node metastasis.

þ 2A, Sample 1 (T2N2bM0): staining intensity 3, 95% Myo1b cancer cells, Myo1b IHC score 285; Fig. 2C, Sample 2 (T1N0M0): Knockdown of Myo1b inhibited migration and invasion of þ staining intensity 2, 75% Myo1b cancer cells, Myo1b IHC score human HNSCC cells through reduced formation of large 150; Fig. 2E, sample 3 (T1N0M0): staining intensity 1, 75% protrusions in cell membrane þ Myo1b cancer cells, Myo1b IHC score 75; Fig 2B, D, and F show We evaluated the functional role of Myo1b in cell motility of control staining with rabbit IgG]. Myo1b protein was expressed in human HNSCC cell lines. Myo1b expression was downregulated cytoplasm of cancer cells and tended to increase in tumor borders. by 2 different Myo1b-specific siRNAs (si1 and si2) and shRNAs Myo1b protein expression in cervical lymph node metastasis (sh1 and sh2; Fig. 3A–C, Western blotting). These siRNAs or appeared similar to that in primary lesions (data not shown). shRNAs significantly inhibited cell migration and invasion of To evaluate the clinicopathologic roles of Myo1b in human SAS and HSC4 when evaluated by the in vitro Transwell migration HNSCC, correlations between Myo1b expression (Myo1b IHC and invasion assays using the xCELLigence RTCA DP Instrument score) and various clinicopathologic features were examined. (Fig. 3D, E, G, and H; Supplementary Fig. S5A and S5B) without Myo1b expression levels (high and low) were classified by the affecting cell proliferation (Fig. 3F and I; Supplementary Fig. S5C). median value of the Myo1b IHC score. In addition, we also These observations were also confirmed by wound-healing assays evaluated the DAB intensity of Myo1b IHC using ImageJ (see with the Myo1b-knockdown SAS and HSC4 (Fig. 3J and K). These Materials and Methods) and confirmed the significant correlation results indicate that Myo1b is functionally involved in cell migra- between the results by the Myo1b IHC score and the ImageJ tion and invasion of human HNSCC. analyses (Supplementary Fig. S3). High Myo1b expression was Because epithelial-to-mesenchymal transition (EMT) is one of significantly correlated with lymphatic invasion and lymph node the mechanisms of enhanced cancer cell migration and invasion, relationships between Myo1b expression and some EMT-related molecules were evaluated. However, Myo1b expression did not Table 2. Correlation between Myo1b and other metastasis-related molecules affect expression of EMT-governing transcription factors such as Myo1b IHC score Snail1 Twist1 CDH1 E- Other metastasis-related molecules Low (<140) High (140) P and , or EMT-related molecules such as ( cadherin CCR4 ), as shown using quantitative PCR analysis (Supplemen- Positive 9 11 0.7160 tary Fig. S6A–S6F). In addition, TGF-b1–induced EMT in SAS and Negative 6 5 HSC4 did not result in Myo1b induction (Supplementary Fig. CCR7 S6G–S6J). Therefore, Myo1b regulates cell motility of human Positive 8 7 0.7244 HNSCC not depending on the major EMT transcription factors. Negative 7 9 CXCR4 To investigate the mechanism of enhanced HNSCC motility via Positive 3 4 1.0000 Myo1b expression, possible Myo1b regulation of membrane Negative 12 12 protrusion formation, including filopodia, lamellipodia, and NOTE: No correlation was observed between Myo1b expression and other invadopodia/podosomes, was evaluated because they are known molecules associated with lymph node metastasis of human HNSCC, chemo- to be involved in cancer cell migration, and Myo1b was reported kine receptors CCR4, CXCR4, and CCR7. P value by Fisher exact test. to localize at membranes of these protrusions (38, 39). Large

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A Anti hMyo1b B Negative control

Tumor Figure 2. Myo1b is expressed in tongue cancer tissues. Intensity 3, Myo1b+ 95%, IHC score 285 Expression of Myo1b protein in human HNSCC was evaluated by IHC studies, and C D Myo1b was detected in all 31 human tongue cancer tissues evaluated but was not expressed in adjacent normal tissues. A, C, E, three representative immunostaining results of tongue cancer tissues expressing various Myo1b levels with rabbit anti-Myo1b pAb are Tumor shown among 31 patients evaluated. The Myo1b IHC score was calculated as described in the Materials and Methods. Dotted lines indicate tumor border. A, sample 1 (T2N2bM0) has intensity of 3 and is 95% Myo1bþ cancer cells, so its Myo1b IHC score is 285. C, sample 2 þ + (T1N0M0) has intensity of 2 and is 75% Myo1b Intensity 2, Myo1b 75%, IHC score 150 cancer cells, so its Myo1b IHC score is 150. E, sample 3 (T1N0M0) has intensity of 1 and is E F þ 75% Myo1b cancer cells, so its Myo1b IHC score is 75. B, D, F, the same sections were stained by control rabbit IgG. Bar, 100 mm.

Tumor

+ Intensity 1, Myo1b 75%, IHC score 75

þ þ protrusions formation in cell membrane of SAS with or without line, SAS. SAS-shRNA-Myo1b GFP and SAS-shRNA-NC GFP cell Myo1b shRNA transfection was evaluated in scratch wound assays. lines were constructed by lentiviral transduction into SAS with SAS treated with control shRNA showed large protrusions forma- GFP gene and either Myo1b-specific shRNA or control shRNA. tion of cell membrane that were larger than their nuclei 1 hour More than 97% of each cell line expressed GFP (data not shown). after scratching (Fig. 4A and C), whereas large protrusions for- These cell lines were injected into right masseter muscle of nude mation was reduced in the Myo1b knockdown SAS (Fig. 4B and mice as cervical lymph node metastatic model. There was no D). Similar results were shown in HSC4 with or without Myo1b difference in in vivo tumor growth between the Myo1b-specific siRNA transfection (Fig. 4F and G). Cells having large protrusion shRNA-transduced and control shRNA-transduced SAS (Fig. 5A), of cell membrane were counted by using confocal microscopy, consistent with no difference of their in vitro cell proliferation and significant reduction in the number of cells with large (Supplementary Fig. S5D). By flow cytometric analysis, significant þ protrusion was observed in both SAS and HSC4 transduced with decrease of GFP cancer cells was detected in the draining lymph Myo1b-specific siRNAs and shRNAs (Fig. 4E and H). Lamellipodia nodes of mice implanted with SAS transduced with Myo1b-specific was observed in some large protrusions of cell membrane. Filo- shRNA cells compared with SAS transduced with control shRNA podia formation in Myo1b knockdown SAS was not changed (data cells (Fig. 5B and C). Moreover, presence of focal metastatic not shown). Therefore, Myo1b augments cancer cell motility lesions in cervical lymph nodes was also confirmed by IHC þ possibly through formation of large protrusion in cell membrane. method (Supplementary Fig. S7). However, the GFP cells were not detected in lungs. These results indicate that Myo1b knock- Myo1b knockdown inhibits cervical lymph node metastases of down in human HNSCC cells reduces lymph node metastasis. human HNSCC in cervical lymph node metastatic model using nude mice implanted with human HNSCC cells To investigate the roles of Myo1b in lymph node metastasis, we Discussion developed a cervical lymph node metastatic model by using nude The roles of myosin family motor proteins in cancer cell mice implanted with Myo1b-expressing human tongue cancer cell characteristics have not yet been well-investigated (34, 40, 41),

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A B C

Myo1b

GAPDH

SAS siNC si1 si2 HSC4 si2si1siNC SAS shNC sh1 sh2

D SAS E SAS F SAS n.s.

n.s. 1 1 1 * ** 0.5 ** 0.5 ** 0.5 cell index/36 h cell index/28 h cell index/28 h Relative invasion Relative migration

0 0 Relative proliferation 0 siNC si2si1 siNC si2si1 siNC si2si1

G H I HSC4 HSC4 HSC4 n.s. n.s. 1 1 1 ** ** ** 0.5 0.5 0.5 ** cell index/14 h cell index/14 h cell index/14 h Relative invasion 0 Relative proliferation 0 Relative migration 0 siNC si2si1 siNC si2si1 siNC si2si1 J K 600 SAS 600 HSC4

500 ** 500

400 ** 400 * 300 300 ** 200 200

100 100 Wound-healing area ( µ m) Wound-healing area ( µ m) 0 0 shNC sh1 sh2 siNC si1 si2

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 migration and invasion of human HNSCC. A–C, decrease of Myo1b protein by Myo1b-specific siRNAs and shRNAs in human HNSCC cell lines, SAS and HSC4, was confirmed by Western blotting. GAPDH is an internal control. D–I, these siRNAs significantly inhibited cell migration and invasion of SAS and HSC4. SAS and HSC4 transfected with siRNAs were used for Transwell migration, invasion, and proliferation assays. Migration, invasion, and proliferation were evaluated by real-time monitoring for 48 hours using the xCELLigence RTCA DP instrument as described in Materials and Methods. Transfections were as follows: si negative control (siNC): scrambled siRNA; si1 or si2: siRNAs specific for human Myo1b (si-Myo1b-1 and si-Myo1b-2, respectively). Results show cell index at representative time points (28 hours for SAS migration and invasion, 38 hours for SAS proliferation, 14 hours for HSC4 migration, invasion, and proliferation) compared with each cell index of siNC (1), shown as mean SD (n ¼ 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 and siRNAs, 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). Results show wound-healing area after 12 hours incubation as mean SD (n ¼ 9or12;, P < 0.05; , P < 0.01; Student t test), about SAS: representative of 3 separate experiments and HSC4: 1 experiment. although the role of myosin family motor proteins—particularly molecule for the development of new diagnostic and therapeutic myosin 2—in cancer motility through cross-linking actin fila- strategies for patients with HNSCC. ments has previously been reported (42, 43). In this article, we Although we attempted to identify the mechanisms of Myo1b have revealed that aberrant overexpression of Myo1b in human overexpression in human HNSCC, it remains to be investigated. HNSCC enhanced lymph node metastasis, possibly through No altered DNA methylation in the Myo1b promoter region was increased cell motility and that Myo1b may be an attractive found, and stimulation of EGFR, which is known to be amplified

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C A

SAS-shNC B D

SAS-sh2

E 80 SAS

40 F * 20 ** protrusion (%) Cells with large

0 shNC sh1 sh2

HSC4-siNC H HSC4 G 80 60 *

40 **

HSC4-si2 protrusion (%) Cells with large

0 siNC si1 si2

Figure 4. Myo1b knockdown inhibits formation of large protrusion in cell membrane in both SAS and HSC4 cell lines. Myo1b knockdown reduces large protrusion of 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 Myo1b shRNA transfection was evaluated in scratch wound assays (see Materials and Methods). SAS treated with control shRNA showed large protrusions of cell membrane that were larger than their nuclei 1 hour after scratching (A and C), whereas large protrusions formation in cell membrane was reduced in the Myo1b knockdown SAS (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 large protrusion of cell membrane were counted by using confocal microscopy, and a significant reduction in the number of cells with large protrusion was observed in SAS transduced with Myo1b-specific shRNA. F and G, the similar results were shown in HSC4. Transfections were as follows: siNC, scrambled siRNA; si1 or si2, siRNAs specific for human Myo1b (si-Myo1b-1 and si-Myo1b-2, respectively). HSC4-siNC formed large protrusion of cell membrane (F), whereas HSC4-si2 formed few large protrusions (G). H, significant reduction of number of cells with large protrusion was observed in HSC4 transduced with Myo1b-specific siRNA. White, phalloidin; red, DAPI. White arrows indicate large protrusion of cell membrane. Bar, 200 mm (A, B, F, G) and 20 mm (C and D). Data are shown as means SD from 4 optical 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.

in HNSCC, did not change Myo1b expression (data not shown). Further investigation is needed to evaluate the function of Myo1b Interestingly, our in silico gene expression analysis and quantita- in other cancers. tive PCR analysis demonstrated preferential overexpression of The functional roles of Myo1b in cancer biology have not Myo1b in HNSCC, and some of the other squamous cell carcino- previously been known, although its association with cell migra- mas, including esophageal cancer and cervical cancer, which tion of zebrafish embryo cells (11) and intracellular transport suggest that squamous cell type–related mechanisms may con- regulated by the formation of post-Golgi carriers were reported tribute to Myo1b overexpression. In contrast to Myo1b overexpres- (12, 13). Myo1b was reported to localize at plasma membrane sion in HNSCC, downregulation of Myo1b expression was structures, including filopodia, ruffles, and lamellipodia (38, observed in certain types of cancers (Fig. 1B), suggesting that the 39, 44). Our in vitro study indicated that knockdown of Myo1b role of Myo1b in metastasis may be different among cancer types. inhibited HNSCC cell migration and invasion through decreased

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Promotion of Lymph Node Metastasis of HNSCC by Myosin 1b

A B 900 50 * sh-Myo1b

) n.s. 40 3 sh-NC 600 30

20 cells/100,000 cells

300 + Tumor volume (mm

GFP 10

0 0 121086420 sh-NC sh-Myo1b Days after implantation C Non–tumor bearing sh-NC sh-Myo1b 0.00% 0.05% 0.01%

Lymph node Side scatter

0.00% 0.00% 0.00%

Lung

GFP

Figure 5. Myo1b knockdown inhibits cervical lymph node metastasis of human HNSCC cell line implanted in nude mice. Myo1b knockdown reduced cervical lymph node metastasis in cervical lymph node metastatic 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) or control shRNA. Each cell line expressed GFP in more than 97% cells (data not shown). These cell lines were implanted into masseter muscle of nude mice. A, there was no difference in in vivo tumor growth between the Myo1b-specific shRNA-transduced and control shRNA–transduced SAS. Tumor volume (mean SD) of transplanted SAS-sh-Myo1b and SAS-sh-NC in vivo (n ¼ 7, 6; n.s., not significant; Student t test), representative of 3 separate experiments. The average tumor volume þ of SAS-sh-Myo1b was 608 mm3 and SAS-sh-NC was 603 mm3. B and C, by flow cytometric analysis, a significant decrease of GFP cancer cells was detected in the draining lymph nodes of mice implanted with SAS transduced with Myo1b-specific shRNA cells compared with SAS transduced with control shRNA cells; 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 were plotted (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 cervical þ lymph node (top) and lung (bottom) samples. Each depicted number shows the percentage of GFP cells among total cells. Left, non–tumor-bearing mouse; middle, SAS-sh-NC–bearing mouse; right, SAS-sh-Myo1b–bearing mouse. Representative dot plots are shown. large protrusion formation of cell membrane. Recently, some Our clinicopathologic analyses indicate that Myo1b expression myosin family proteins such as myosin 2 were reported to be in primary HNSCC tissues is a potential diagnostic marker to highly expressed in cancer cells and to be correlated with cancer predict lymph node metastases and possibly subsequent prog- cell motility and metastasis partly through cross-linking actin nosis of patients with HNSCC, as lymph node metastases is the filaments (34, 41, 42). Although myosin 6 was reported to be major factor in overall survival of patients with HNSCC. This may involved in polarized delivery of vesicles containing EGFR into enable better clinical management, including decisions for pro- leading edges of cancer cells (34), Myo1b knockdown did not phylactic neck dissection or postsurgical chemoradiotherapy. affect cell surface protein expression of EGFR (data not shown). Moreover, combinatorial uses of other predictive markers such We also evaluated the relationship between Myo1b and intracel- as chemokine receptors may further improve the accuracy of lular transport of various molecules other than EGFR (e.g., HLA, predicting power. In fact, cancer cells with both high-Myo1b þ IL6, IL8, and CCL2) but no difference was observed (data not expression and CCR4 status are strongly associated with high shown). Further study on possible Myo1b-associated trafficking occurrence of lymph node metastasis in this study (Table 3, P ¼ changes of molecules involved in cell migration may be required 0.0006). Further analyses including multivariate analysis with (12, 13). increased numbers of patients are warranted for confirmation of

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the diagnostic powers of Myo1b expression along with other Development of methodology: G. Ohmura, T. Tsujikawa, T. Yaguchi, markers such as chemokine receptor for lymph node metastasis N. Kawamura, S. Mikami, J. Sugiyama, K. Nakamura, A. Kobayashi and subsequent survival of patients with HNSCC. Acquisition of data (provided animals, acquired and managed patients, Myo1b provided facilities, etc.): G. Ohmura, T. Tsujikawa, T. Yaguchi, N. Kawamura, Because knockdown inhibited lymph node metastases, S. Mikami, J. Sugiyama, K. Nakamura, H. Nakano, T. Shimada, Y. Kawakami possibly due to decreased cancer cell motility, Myo1b could Analysis and interpretation of data (e.g., statistical analysis, biostatistics, also be a therapeutic target for prevention of lymph node metas- computational analysis): G. Ohmura, T. Tsujikawa, T. Yaguchi, S. Mikami, tasis and subsequent improvement of overall survival. Recently, J. Sugiyama, Y. Hisa, Y. Kawakami pentachloropseudilin, a low-molecular-weight chemical, was Writing, review, and/or revision of the manuscript: G. Ohmura, T. Tsujikawa, reported to be a reversible and allosteric inhibitor of class 1 T. Yaguchi, S. Mikami, Y. Kawakami Administrative, technical, or material support (i.e., reporting or organizing myosin motor activity (45). As Myo1b is expressed in various data, constructing databases): G. Ohmura, T. Tsujikawa, K. Nakamura, normal tissues, systemic administration of Myo1b inhibitors such T. Iwata, Y. Hisa, Y. Kawakami as low-molecular-weight chemicals and siRNAs may cause severe Study supervision: G. Ohmura, T. Tsujikawa, T. Yaguchi, J. Sugiyama, adverse effects. Therefore, a preferential delivery method to cancer Y. Kawakami cells may be required. Otherwise, upstream or downstream molecules in the Myo1b-related cell mobilization axis may also Acknowledgments be targets for HNSCC. Further studies are required to develop The authors thank Dr. Shigeki Ohta for technical advice, Dr. Masato Oka- moto for development of the in vivo tumor model, Dr. Hiroyuki Aburatani for therapeutic strategies that target Myo1b-related malignant DNA methylation analysis, Dr. Ryuta Nakao for the examination of lymphatic features. invasion, Kenji Morii for flow cytometric analysis, Miyuki Saito for IHC staining, In summary, we have shown that aberrant overexpression of and Misako Horikawa and Ryoko Suzuki for preparation of the article. Myo1b in human HNSCC augments cancer cell motility via enhanced large protrusion formation of cell membrane and Grant Support promotes lymph node metastasis. Therefore, Myo1b is an attrac- This work was supported by Grants-in-Aid for Scientific Research (23240128 tive target for the development of new diagnostic and therapeutic and 26221005) from the Japan Society for Promotion of Science, a research strategies for patients with HNSCC. program of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct) from the Ministry of Education, Culture, Sports, Science fl and Technology of Japan. Disclosure of Potential Con icts of Interest The costs of publication of this article were defrayed in part by the payment of No potential conflicts of interest were disclosed. page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: G. Ohmura, T. Tsujikawa, T. Yaguchi, H. Nakano, Received July 23, 2014; revised October 23, 2014; accepted November 12, T. Shimada, Y. Hisa, Y. Kawakami 2014; published OnlineFirst November 24, 2014.

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Aberrant Myosin 1b Expression Promotes Cell Migration and Lymph Node Metastasis of HNSCC

Gaku Ohmura, Takahiro Tsujikawa, Tomonori Yaguchi, et al.

Mol Cancer Res 2015;13:721-731. Published OnlineFirst November 24, 2014.

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