Notch1 regulates invasion and metastasis of head and neck squamous cell carcinoma by inducing EMT through c-Myc

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Notch1 regulates invasion and metastasis of head and neck squamous cell carcinoma by inducing EMT through c-Myc Naoya Inamura a, Taichi Kimura b,*, Lei Wang b, Hiroko Yanagi a, Masumi Tsuda c, Mishie Tanino c, Hiroshi Nishihara b, Satoshi Fukuda a, Shinya Tanaka b,c a Department of Otolaryngology-Head and Neck Surgery, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638, Japan b Department of Translational Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638, Japan c Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 May 2016 Accepted 15 August 2016 Available online xxx

Objective: As 50% of patients of head and neck squamous carcinoma (HNSCC) exhibit poor prognosis, the identification of new therapeutic targets is required. Recently, there have been several reports about the correlation between Notch1 and HNSCC, but the precise mechanism is still obscure. Therefore, in this study, we examined the involvement of Notch1 in HNSCC by using HNSCC cell lines and surgical specimens. Methods: To investigate the role of Notch1 in HNSCC, we examined the effect of Notch inhibitor DAPT on cell growth, invasion, and tumorigenicity using five HNSCC cell lines in vitro and in vivo. We further examined that the correlation with Notch expression and clinical prognostic factors was evaluated by using 101 HNSCC surgical specimens. Results: DAPT reduced the nuclear expression of Notch and c-Myc and repressed cell growth, EMT-dependent cell invasion in vitro, and tumorigenicity in vivo. An overexpression of Myc enhanced EMT with an increase of Snail and vimentin together with decreased levels of E-cadherin in HSC3 cells. Finally, we discovered that Notch expression was well correlated with MIB-1 index and lymph node metastases. Conclusion: We discovered that Notch1 was strongly correlated with HNSCC growth, invasion, and metastases. Therefore, Notch1 might be a new therapeutic target and a predictive marker of proliferation and metastasis of HNSCC. ß 2016 Published by Elsevier Ireland Ltd.

Keywords: Notch1 Head and neck cancer Squamous cell carcinoma Growth Invasion

1. Introduction Head and neck cancer is the 6th most common cancer worldwide. Head and neck cancer refers to malignancies arising in the mucosal surfaces of the oral cavity, pharynx, and larynx, and 90% is histologically squamous cell carcinomas (SCC) [1]. The most common treatments for HNSCC include

* Corresponding author. Fax: +81 11 706 5902. E-mail address: [email protected] (T. Kimura).

chemotherapy, radiation, and surgery. However, in the advanced stage, prognosis is remarkably poor with a significantly reduced quality of life. Despite new treatment options for HNSCC patients, the prognosis has remained unchanged over the past 40 years [2]. Therefore, defining the molecular mechanisms involved in HNSCC pathogenesis and identification of new drug targets is of critical importance. Recently, it was reported that Notch1 mRNA is up-regulated in both HNSCC cell lines and biopsy specimens and that immunohistochemistry or immunocytochemistry revealed high expression of Notch1 in both HNSCC cell lines and biopsy

http://dx.doi.org/10.1016/j.anl.2016.08.003 0385-8146/ß 2016 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Inamura N, et al. Notch1 regulates invasion and metastasis of head and neck squamous cell carcinoma by inducing EMT through c-Myc. Auris Nasus Larynx (2016), http://dx.doi.org/10.1016/j.anl.2016.08.003

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specimens [3]. The inhibition of the Notch pathway by using a gamma-secretase inhibitor was reported to prevent the growth of HNSCC in vitro [4]. Furthermore, it was demonstrated that high expression of Notch1 is associated with poor overall survival in HNSCC patients [5]. Notch1 is a member of the Notch family proteins known as Notch1 to Notch4 in mammals. Notch proteins are receptors consisting of an extracellular domain with epidermal growth factor-like (EGF) repeats and an intracellular domain referred as NICD. The notch signaling pathway is regulated by interactions with ligands of physically adjacent cells. In mammals, there are three Delta-like ligands known as Dll1, Dll3, and Dll4, and two Jagged family ligands called Jag1 and Jag2 [6]. When membrane-bound Notch receptors interact with ligands on adjacent cells, Notch is cleaved by TNF-a converting enzyme (TACE) to yield a membrane-associated intermediate fragment called notch extracellular truncation (NEXT). Following endocytosis, Notch fragment is then cleaved by presenilin-dependent g-secretase to release a Notch-derived peptide with NICD which enters the nucleus and activates the transcription of target genes [7]. Notch is a signaling molecule evolutionarily conserved from Drosophilla to vertebrates, involving various developmental processes for cell fate decisions, apoptosis, proliferation, and stem cell self-renewal [7]. Besides a pleiotropic role, the deregulation of Notch also leads to malignancies. Notch is reported to be correlated with the malignancy of T-cell acute lymphoblastic leukemia [8], lung cancer [9], ovarian cancer [10], colon adenocarcinoma [11], pancreatic tumorigenesis [12], and osteosarcoma [13]. Notch1 may act as an oncogene in HNSCC as well as in other malignancies. Although there are several reports about the correlation between Notch1 and HNSCC, the detailed mechanism and the role of Notch1 in HNSCC remain largely unclear. Therefore, we examined the involvement of Notch1 in HNSCC using HNSCC cell lines. Furthermore, we utilized HNSCC surgical specimens by a clinicopathological approach. 2. Materials and methods 2.1. Cell lines and culture The HNSCC cell lines HSC-2, HSC-3, HSC-4, SAS, OSC20, and Ca9-22 were kindly provided by Dr. Masanobu Shindo (Department of Oral Pathology and Biology, Hokkaido University Graduate School of Dental Medicine). Cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U/ml penicillin and streptomycin at 37 8C with 5% CO2. Cells were treated with the indicated concentrations of the g-secretase inhibitor DAPT (Sigma–Aldrich, MO, USA) or the corresponding volume of DMSO as a control. 2.2. Plasmids and transfection pMXs-hc-Myc was purchased by Addgene (MA, USA). The expression plasmid pMXs-GFP was kindly provided by

T. Kitamura (Institute of Medical Science, University of Tokyo, Japan). Cells were transfected with the use of FuGENE HD reagents (Roche, Penzberg, Germany). 2.3. RNA isolation and RT-PCR analysis Total RNA was isolated from cells with the use of RNeasy Mini Kit (Qiagen, CA, USA) and subjected to reverse transcription by SuperScript II reverse transcriptase (Invitrogen, CA, USA). The 100 ng of the resulting first-strand cDNA was used as a template and amplified by PCR using GoTaq Green Master Mix (Promega, WI, USA). Sequences of the oligonucleotide primer sets used for RT-PCR are as follows: Notch1 (50 -TTC AGT GACGGCCACTGTGA-30 and 50 -CAC GTA CAT GAA GTG CAG CT-30 ), Hes-1 (50 -AGG CGG ACA TTCTGGAAATG-30 and 50 -CGG TAC TTC CCC AGC ACA CTT-30 ), c-Myc (50 -GCG TCC TGG GAA GGG AGATCCGGAGC-30 and 50 -TTG AGG GGC ATC GTC GCG GGA GGC TG-30 ), Snail (50 -GCT GCA GGA CTC AAT CCA GA-30 and 50 -ATCTCCGGAGGTGGGATG-30 ), E-cadherin (50 -GTG ACT GAT GCT GAT GCC CCC AAT ACC-30 and 50 -GAC GCA GAA TCA GAA TTA GGA AAG CAA G-30 ), Vimentin (50 -TCC AGC AGC TTC CTG TAG GT-30 and 50 -CCC TCA CCT GTG AAG TGG AT-30 ), and human GAPDH (50 -AGC CAC ATC GCT CAG ACA C-30 and 50 -GCC CAA TAC GAC CAA ATC C-30 ). PCR products were subjected to agarose gel electrophoresis, and the intensity of bands was determined with the use of MultiGauge software (Fujifilm, Tokyo, Japan). Quantitative real-time PCR was done by StepOne Real-time PCR (Applied Biosystems, CA, USA) and the SYBR Green systems (Applied Biosystems). Data were normalized by the expression level of GAPDH in each sample and shown as the relative expression to those of parental cells. 2.4. Antibodies Antibodies were obtained from following sources: antiNotch1 (D1E11) (Cell Signaling Technology, Beverly, MA, USA); anti-c-Myc (D84C12) (Cell Signaling Technology); anti-LaminA/C (636) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Actin antibody (Chemicon International, Temecula, CA, USA); and anti-Ki67 (Clone MIB-1, M7240) (Dako, Tokyo, Japan). 2.5. Immunoblotting Cells were lysed with ice-cold buffer containing 10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM sodium orthovanadate (Na3VO4). Proteins were fractionated by SDSpolyacrylamide gel electrophoresis. The separated proteins were transferred electrophoretically to PVDF membrane (Immobilon-P; Millipore, Billerica, MA, USA). After blocking with 5% dried nonfat milk, the membrane was incubated with antibodies to Notch1 (1:1000 dilution), to c-Myc (1:1000), to LaminA/C (1:200) or to actin (1:5000) at RT for 2 h, followed by peroxidase-labeled secondary antibodies. Immune complexes were detected with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Freiburg, Germany)

Please cite this article in press as: Inamura N, et al. Notch1 regulates invasion and metastasis of head and neck squamous cell carcinoma by inducing EMT through c-Myc. Auris Nasus Larynx (2016), http://dx.doi.org/10.1016/j.anl.2016.08.003

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and quantified with an LAS1000 image analyzer (Fuji Film, Tokyo, Japan). 2.6. Subcellular fractionation Cells were lysed in an ice-cold solution containing 20 mM HEPES (Sigma–Aldrich, MO, USA), 1.5 mM MgCl2, 1 mM dithiothreitol (DTT) (Sigma–Aldrich, MO, USA), 0.1% Triton X-100 (Sigma–Aldrich, MO, USA), 1 Complete (Roche, Penzberg, Germany), 20% glycerol, 10 mM NaCl, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM PMSF, and 1 mM Na3VO4. The cytoplasmic fraction was collected after centrifugation at 3000 rpm for 15 min at 4 8C. The resulting nuclear pellet was resuspended for 1 h at 4 8C with an ice-cold buffer containing 20 mM HEPES, 1.5 mM MgCl2, 1 mM DTT, 0.1% Triton X-100, 1 Complete, 20% glycerol, 500 mM NaCl, 0.2 mM EDTA, 1 mM PMSF, and 1 mM Na3VO4. After centrifugation of the extract at 15,000 rpm for 15 min at 4 8C, the supernatant was collected as the nuclear fraction [14]. 2.7. Cell growth assay 1  105 cells were seeded onto 60 mm diameter plates with DMEM with 10% FBS. The numbers of cells were counted 2, 4, and 6 days after seeding using a hemocytometer (Fisher Scientific, Japan) [15].

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Tokyo, Japan). The number of cells invading through the Matrigel membrane was counted in microscopic fields at 40 magnification. To minimize the bias, at least five randomly selected fields were counted [15]. 2.11. Surgical specimens We used 101 cases of primary HNSCC diagnosed from 1992 through 2012 in our faculty for histopathological examination. 73 were men, and 28 were women. Median age at first medical examination was 63 years (range, 21–92 years). Median follow-up time was 58 months (range 0–261 months). The characteristics of the patients are summarized in Table 1. 2.12. Immunohistochemical analysis Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned and stained using anti-Notch1 (D1E11, 1:100 dilution), anti-Ki67 (Clone MIB-1, M7240, 1:100 dilution) antibodies. Immunostaining of Notch1 was evaluated concerning proportion and staining intensity of tumor cells. The positivity of Notch1 staining in HNSCC was evaluated at 0– 100%. Staining intensity was evaluated as none (0), weak (+1), moderate (+2), or strong (+3). The Ki-67 index was calculated in the field of maximal activity as a percentage of positive cells. Mean Ki-67 index of 101 HNSCC was 51.2% (range, 0–95%).

2.8. Colony formation assay 2.13. Statistical analysis 5  104 of cells were plated in 60 mm diameter plates with 3 ml of 0.4% noble agar in DMEM in 10% FBS, overlaying 5 ml of 0.5% bacto agar in DMEM with 10% FBS. We exposed cells to 20 mmol/L DAPT or DMSO, respectively, 48 h after plating. Numbers of colonies were counted under a microscope 19 days after plating.

All calculations were performed by use of the statistical software Excel-Toukei 2012 for Windows (Social Survey Table 1 Patient characteristics. No. (n = 101)

2.9. Xenoglaft propagation 5  104 HSC-3 cells were subcutaneously injected into 6week-old female nude mice, BALB/cA Jcl-nu/nu (Clea Japan, Inc., Tokyo, Japan). Mice treated with HSC-3 cells were injected intraperitoneally with 100 ml vehicle control (10% ethanol, 90% corn oil) or 20 mg/kg DAPT (dissolved in 10% ethanol and 90% corn oil) on days 1, 4, 7, 10, 13, 16, and 19. Mice were euthanized 22 days after cell injection. All animal procedures were carried out according to the protocol approved by the institutional Animal Care and Use Committee at ******** University Graduate School of Medicine. 2.10. Invasion assay 7  104 HSC-3 cells were seeded in the upper chambers (24well chambers) of the filter Bio Coat Matrigel Invasion Chamber (BD Biosciences, Franklin Lakes, NJ, USA) in 500 ml of DMEM with 10% FBS, and the bottom chambers contained 750 ml of DMEM with 10% FBS. The non-invading cells on the upper surface of the filters were removed by wiping with a cotton swab. Cells at the bottom side of the membranes were fixed with ethanol, and stained with 0.04% Crystal Violet (TCI,

Median age (range) Sex Male Female Histology Well differentiated Moderately differentiated Poor differentiated Tumor staging T1 T2 T3 T4 Lymph node metastasis Clinical N0 (Subclinical N+: cN0 and pN+ or late cervical LN metastasis) Clinical N+ Tumor sites Tongue Gingiva Buccal mucosa Floor of mouth Survival Alive Death

%

63 (21–92) 73 28

72.3% 27.7%

62 31 8

61.4% 30.7% 7.9%

30 50 9 12

29.7% 49.5% 8.9% 11.9%

84 (19)

83.2% (18.8%)

17

16.8%

86 11 2 2

85.1% 10.9% 2.0% 2.0%

82 19

81.2% 18.8%

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Research Information, Tokyo, Japan). Comparisons between experimental groups were made by Student’s t-test or Welch’s t-test. The correlations between Notch1 scores and T staging and Ki-67 index and lymph node metastasis were analyzed by use of Spearman’s rank test and Mann– Whitney’s U test. 2.14. Ethical requirements The study using human samples was performed with the approval of the Internal Review Board on Ethical Issues of ******** University Hospital and Graduate School of Medicine, Sapporo, Japan.

3. Results 3.1. Notch1 expression in human HNSCC cell lines To evaluate Notch1 mRNA expression in human HNSCC cell lines, we performed semiquantitative RT-PCR of HNSCC cell lines. Notch1 mRNA was detectable in HNSCC cell lines at various levels (Fig. 1A). In SAS, OSC20, HSC3, and HSC4, Notch1 was clearly detectable, while small amounts of Notch1 bands were found in Ca9-22 and HSC2. Notch1 protein was also detectable in HNSCC cell lines at various levels by Western blotting as higher levels in OSC20, HSC3, and HSC4, and lower levels in HSC2 (Fig. 1B). To assess Notch1 protein

Fig. 1. Notch1 expression in human HNSCC cell lines. (A) Semiquantitative RT-PCR analysis of Notch1 expression in human HNSCC cell lines. GAPDH was loading control. (B) Immunoblot analysis using anti-Notch1 antibodies on lysates from HNSCC cell lines. Actin was loading control. 20 mg of proteins were loaded in each lane. (C) Immunohistochemical analysis of Notch1 expression in HNSCC cell lines.

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expression and subcellular localization in human HNSCC cell lines, we performed immunohistochemical analysis of HNSCC cell lines (Fig. 1C). Staining intensity of Notch1 protein was also detectable in HNSCC cell lines at various levels. It was confirmed that higher staining intensity was found in SAS, OSC20, and HSC3 cells, and lower intensity in HSC2. In several cell lines, mRNA expression level (Fig. 1A) was not correlated with protein level (Fig. 1B and C). 3.2. Effect of DAPT on Notch1 signaling in HNSCC cell lines To evaluate the function of Notch1 in HNSCC, we used gsecretase inhibitor, DAPT. We confirmed whether DAPT inhibits g-secretase dependent cleavage of Notch1 and proper nuclear translocation of NICD. To examine the effect of the DAPT on expression of Notch1, we carried out Western blot analysis (Fig. 2A). Since Notch1 controls transcription of target genes through the nuclear translocation of NICD, it was decided to evaluate the activity of Notch1 signaling by measuring the NICD protein levels obtained from nuclear extracts. In the presence of DAPT, nuclear NICD was reduced in HNSCC cell lines. To assess the effect of the DAPT on inhibition of Notch1, we carried out immunohistochemical analysis (Fig. 2B). In the presence of DAPT, reduction in the levels of Notch1 staining intensity was observed in HNSCC cell lines. To analyze the effect of the DAPT on Hes-1, a transcriptional target of Notch signaling [16], we conducted a semi-quantitative RT-PCR analysis (Fig. 2C). In the presence of DAPT, reduced levels of Hes-1 mRNA in a DAPT-dose dependent manner was observed in HNSCC cell lines. Therefore, in the HNSCC cell lines, DAPT was confirmed to effectively block the signal of Notch1. 3.3. In vitro and in vivo growth reduction by DAPT in HNSCC cell lines To evaluate the effect on the transforming activity by Notch1, we evaluated proliferative capacity, anchorageindependent growth ability, and tumorigenicity by using Notch inhibitor. We analyzed the growth curve of each of the HNSCC cell lines with DAPT (Fig. 3A). The treatment of HNSCC cell lines with DAPT led to cell growth inhibition with DAPT dosedependency. To assess the effect on the anchorage-independent growth ability, we performed a colony formation assay with DAPT (Fig. 3B). In HSC3, a significant decrease in both number and size of the colony was observed with DAPT. This effect was also seen in OSC20 (data not shown). For assessment of tumorigenicity, we performed Xenograft propagation assay with DAPT (Fig. 3C), and the tumorigenicity of DAPT administration group was lower than that of the nonadministration group in case of HSC3 cell injection. Therefore, it has been shown that Notch1 is involved closely with transforming activity in HNSCC cell lines. 3.4. Inhibition of invasion via c-Myc dependent EMT by DAPT in HSC3 To evaluate the effect of Notch1 on tumor invasion, we analyzed the Matrigel invasion assay with DAPT (Fig. 4A). Numbers of invaded HSC3 cells were obviously decreased by

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DAPT administration. This result was also seen in OSC20 (data not shown). Therefore, DAPT is strictly related to the inhibition of invasion in HNSCC. To examine whether the inhibition of EMT, which is closely involved in tumor invasion [17], is involved in the suppression of invasion by DAPT in HNSCC, we analyzed the effect of DAPT on the expression level of EMT-related molecules such as EMT-related transcription factor Snail, Vimentin as a mesenchymal marker, and Ecadherin as an epithelial marker by real time RT-PCR in HSC3 cells (Fig. 4B). Expression levels of Snail and Vimentin were significantly decreased, and E-cadherin was significantly increased by DAPT in HSC3 cells, suggesting inhibition of EMT. As c-Myc is related to invasion of mammary epithelial cells [18], and is an important direct target of Notch1 [19], we examined whether Notch1 signaling affects invasion via cMyc. We confirmed that the expression levels of c-Myc of nuclear extract were reduced by DAPT in HSC3 cells (Fig. 4C). To ensure the implication of c-Myc in invasion, we established c-Myc-overexpressing HSC3 cells (HSC3-c-Myc). Control was GFP-overexpressing HSC3 cells (HSC3-GFP). Numbers of invaded HSC3-c-Myc cells were increased compared with control in Matrigel invasion assay in the presence of either DMSO or DAPT, because a sufficient amount of c-Myc was maintained even in the presence of DAPT (Fig. 4D and E). We confirmed that Notch1 signal controls invasion via c-Myc in HSC3. To investigate whether the control of invasion via c-Myc is due to the regulation of EMT, we examined the expression levels of mRNA of EMT-associated molecules in HSC3-c-Myc by real time RT-PCR (Fig. 4F). In HSC3-GFP cells as a control, mRNA of Snail and Vimentin was significantly reduced by DAPT, and E-cadherin was increased. On the other hand, in HSC3-c-Myc cells, mRNA of Snail and Vimentin was significantly increased, and E-cadherin was significantly reduced compared to control (HSC3-GFP, DAPT+) even with DAPT administration. Therefore, it was observed that Notch1 signaling affects EMT related molecules via c-Myc in HSC3. 3.5. Correlation of Notch1 expression with MIB-1 labeling index in human HNSCC specimens To investigate whether Notch1 is involved in proliferation and invasion in clinical HNSCC specimens, immunocytochemical staining of Notch1and Ki-67 was performed in 101 human HNSCC samples. A summary of the characteristics of the 101 human HNSCC samples is shown in Table 1. The study included 101 patients with a median age of 60.5 years. The median follow-up period was 58 months (range, 0–261 months). Subclinical lymph node metastasis was defined as that in patients who were clinically (at the first medical examination time point) lymph node-negative HNSCC, and lymph node metastasis was detected either at the operation, at the pathological evaluation, or after radical treatment of primary lesion. Lymph node metastasis was detected in 19 (21.3%) among 89 patients with clinically lymph node-negative HNSCC. We presented the typical histology of scoring of Notch1 and Ki-67 (Fig. 5).

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Fig. 2. Effect of DAPT on Notch signaling in HNSCC cell lines. (A) Immunoblot analysis using anti-Notch1 antibodies on nuclear extracts from Ca9-22, OSC20, HSC3, and HSC4 treated by 10 mM DAPT or DMSO respectively. Actin was loading control. 30 mg of proteins were loaded in each lane. C: Cytoplasmic fraction. N: Nuclear extract. (B) Immunohistochemical analysis of Notch1 expression in SAS, OSC20, HSC3, and HSC4 treated by DAPT or DMSO respectively. (C) Upper panels were semiquantitative RT-PCR analysis of Notch1 expression in SAS, Ca9-22, HSC3, and HSC4 treated by DAPT or DMSO respectively. DMSO was used for dissolving DAPT served as the control. Lower graphs were quantification of the reduced bands of upper panels. Normalization of intensity was performed by the intensity of each GAPDH amounts.

For objective evaluation of Notch1and Ki-67 by immunohistochemistry, we introduced the scoring system described in Section 2. Table 2 summarizes the association of Notch1 and Ki-67 expression with the patient’s T and N stage (classified by

UICC and AJCC Cancer Staging) [20]. Tumors expressing a high level of Notch1 were not related to the T stage, while those that were closely related to the ratio of Ki-67-positive cells were involved in cell proliferation. Patients with tumors

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Fig. 3. In vitro and in vivo growth reduction by DAPT treatment in HNSCC cell lines. (A) Growth curves of HSC3, Ca9-22 and OSC20 treated by DAPT or DMSO. Data represent the number of viable cells and are means  s.d. of values from three independent experiments. *p < 0.05, **p < 0.005 (Student’s t-test) versus the samples treated by DMSO. (B) Effect of treatment by DAPT on anchorage-independent growth of HSC3 cells. HSC3 cells (2  105) treated by DAPT or DMSO were plated in 0.4% soft agar and incubated for 19 d, after which the number of colonies in the indicated size ranges was determined by microscopy. Left, representative micrographs; right, quantitative data of colony number and size. (C) For xenograft propagation, 5  104 cells were subcutaneously injected into nude mice (control, n = 5; DAPT, n = 5). Statistical analysis of mouse xenografts is shown. Data represent the weight of xenografts and are means  s.d. of values from five independent experiments. p < 0.01, (Student’s t-test) versus the samples treated by DMSO.

expressing a high level of Notch1 were significantly correlated with lymph node metastasis even if they were in the category of subclinical. Therefore, expression of Notch1 may be involved in the proliferation and invasion of tumors in clinical HNSCC patients.

4. Discussion When membrane-bound receptors interact with ligands of Delta-like and Jagged family on an adjacent cell, Notch is cleaved by protease of gamma-secretase to release NICD from

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Fig. 4. Inhibition of invasion via c-Myc dependent EMT by DAPT treatment in HSC3. (A) Transwell invasion assay. HSC3, treated by DAPT or DMSO, was incubated for 24 h in a 24-well transwell plate. The migrated cells to lower insert membrane were stained with 0.04% Crystal Violet. Data represent means  SD of four separate samples (significant versus DMSO, p < 0.01). (B) Quantitative real-time RT-PCR analysis of Snail, E-cadherin and Vimentin in HSC3 cells treated by DAPT or DMSO. *p < 0.05, **p < 0.01 (Student’s t-test). (C) Immunoblot analysis using anti-Notch1 and c-Myc antibodies on nuclear extracts from HSC3 treated by 10 mM DAPT or DMSO respectively. Actin was loading control. 30 mg of proteins were loaded in each lane. C: Cytoplasmic fraction. N: Nuclear extract. (D) Immunoblot analysis of c-Myc protein on lysates from HSC3-c-Myc and HSC3-GFP treated by 10 mM DAPT or DMSO respectively. c-Myc overexpressing HSC3 cells (c-Myc) were transfected with expression vector of c-Myc. Control was GFP overexpressing HSC3 cells (GFP). Actin was loading control. 30 mg of proteins were loaded in each lane. (E) Transwell invasion assay. HSC3-c-Myc and HSC3-GFP treated by DAPT or DMSO were incubated for 24 h in a 24-well transwell plate. The migrated cells to lower insert membrane were stained with 0.04% Crystal Violet. Data represent means  SD of four separate samples (significant versus DMSO, p < 0.01). (F) Quantitative real-time RT-PCR analysis of Snail, E-cadherin and Vimentin in HSC3-c-Myc and HSC3-GFP cells treated by DAPT or DMSO. *p < 0.05, **p < 0.01 (Student’s t-test).

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Fig. 5. Correlation of Notch1 expression with Ki-67 labeling index in human HNSCC samples. Representative staining of Notch1 (upper panels) and Ki-67 (lower panels). Each picture (upper panels) depicts Notch1 Intensity Score (IS) as follows: IS0, IS1, IS2, IS3. Percentages of Notch1 expression-positive tumor cells are as follows: 0–24%, 25–49%, 50–74%, 75–100%. Each picture (lower panels) depicts Ki-67 index as follows: 0–24%, 25–49%, 50–74%, 75–100%.

the membrane, freeing NICD to enter the nucleus. Notch signaling regulates the transcription of target genes which are involved in developmental processes and proliferation [4]. In this study, Notch1 expression was detectable in HNSCC at various levels, and Notch1 expression level was correlated with malignant phenotypes. In previous studies about Notch signaling that used DAPT, inhibitory effects of Notch signaling were evaluated with quantification of target genes transcription such as Hes1, without quantification of NICD directly [21,22,4]. However, the results described in these studies may not precisely demonstrate the role of Notch because DAPT inhibits all signals which are regulated by g-secretase. Therefore, we confirmed that DAPT inhibits g-secretasedependent cleavage of Notch1 and proper nuclear translocation of NICD by directly measuring the NICD protein levels obtained from the nuclear extracts. In our study, it was observed that the expression level of Notch1 tends to depend on cell density and that increasing cell confluency is associated with higher Notch1 expression (data not shown). These findings suggest that not only the quantification of transcriptional levels of target genes as mRNA, but also directly measuring the NICD protein levels, is also important for the accurate analysis of Notch signaling. Confluency-dependent Notch1 expression and consequent activation of its target genes in HNSCC cell lines may be concerned with the number of physically adjacent cells

expressing the ligand, and if so, the heterogeneity of Notch1, Delta and Jagged expression may exist in HNSCC. In general, Notch plays important roles on maintenance and differentiation of tissue stem cells [23,24], and it was also reported that Notch signaling is closely related to the maintenance of cancer stem cells of brain [25–27] and breast [28]. Therefore, it is necessary to analyze the association between the Notch signaling and the mechanism of cancer stem cell maintenance. It has been reported that the Notch signaling pathway involved in proliferation is correlated with the RAS/MAPK pathway and the PI3K/Akt pathway [29,30]. However, after having also examined the molecules associated with the RAS/MAPK pathway and PI3K/Akt pathway in this study, we have not obtained any significant correlation (data not shown). c-Myc is generally known as a molecule involved in proliferation [31]. Recently, an important factor of c-Myc for invasion was reported as existing also in human breast tumor [32]. Furthermore, it has been reported that multiple roles of c-Myc are regulated by the Notch in various malignant tumors [33–35]. Therefore, we examined the inhibitory effect of c-Myc expression by DAPT administration and confirmed that inhibition of Notch signaling causes the suppression of c-Myc expression in HNSCC. EMT plays a role in invasion and metastasis in various cancers including HNSCC [17,36], and is related with c-Myc in mammary epithelial cells [18]. These reports and our results

Please cite this article in press as: Inamura N, et al. Notch1 regulates invasion and metastasis of head and neck squamous cell carcinoma by inducing EMT through c-Myc. Auris Nasus Larynx (2016), http://dx.doi.org/10.1016/j.anl.2016.08.003

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ANL-2178; No. of Pages 11 N. Inamura et al. / Auris Nasus Larynx xxx (2016) xxx–xxx

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Table 2 Immunohistochemical findings of Notch1 and Ki-67 expression in human HNSCC samples. No. of cases (%) Percentage of expression positive tumor cells

Notch1 staining intensity Score 0 Score 1 Score 2 Score 3 Ki-67

0%

1–24%

25–49%

50–74%

>75%

11 (10.8) 0 (0) 0 (0) 0 (0)

0 (0) 12(11.9) 7 (6.9) 2 (2.0)

0 (0) 14 (13.9) 12 (11.9) 2 (2.0)

0 (0) 7 (6.9) 11 (10.9) 5 (5.0)

0 3 6 9

3 (3.8)

21 (27.2)

9 (11.6) No. (n = 101)

11 (14.3) Notch1 staining intensity P value

Tumor staging Percentage of Ki-67-positive tumor cells Lymph node metastasis Clinical N+ Subclinical N+ (cN0 and pN+ or late cervical LN metastasis)

a

101 101

0.447 <0.001a

17 19

0.017b <0.001b

(0) (3.0) (5.9) (8.9)

33 (42.9) Percentage of Notch1positive tumor cells

Rs 0.076 0.817

P value a

0.333 <0.001a

Rs 0.097 0.502

0.005b <0.001b

Abbreviations: Rs, Spearman’s rank correlation coefficient. a Spearman’s rank test. b Mann–Whitney’s U test.

made us investigate whether Notch affects the EMT and invasion via c-Myc in HNSCC. c-Myc down-regulation caused by inhibition of Notch1 reduced EMT and invasion. Furthermore, it was observed that c-Myc overexpression under the DAPT treatment partially recovers invasion and EMT. Therefore, it was revealed that regulation of invasion by Notch1 is induced through the EMT followed by transcriptional regulation of c-Myc. In clinicopathological analysis, there was no significant correlation between Notch1 and T stage in this study. We have evaluated the Ki-67 labeling index directly involved in cell proliferation [37], and examined the correlation with Notch1 expression. Thus, in HNSCC, the T stage may not be a suitable indicator for proliferative capacity. Notch1 staining is correlated with clinical lymph node metastasis, which is detectable at first medical examination, as shown in a previous report [38]. Furthermore, we newly discovered the correlation with the subclinical, which is undetectable at first medical examination and secondary lymph node metastases after primary locoregional treatment. These results suggested that the staining of Notch1 may be an important diagnostic marker for decisions of treatment procedures, such as determining prophylactic therapy, and the intensity and range of neck dissection and/or radiation. Therefore, it will be necessary to perform further investigation, such as the prospective clinical trial in the cases with high Notch1 expression. Conflict of interest No potential conflicts of interest were disclosed. Acknowledgments We thank Miho Kimura for technical assistance with immunohistochemistry, and Dr. Masanobu Shindo for providing with the HNSCC cell lines.

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Please cite this article in press as: Inamura N, et al. Notch1 regulates invasion and metastasis of head and neck squamous cell carcinoma by inducing EMT through c-Myc. Auris Nasus Larynx (2016), http://dx.doi.org/10.1016/j.anl.2016.08.003