J Cancer Res Clin Oncol (2012) 138:195–202 DOI 10.1007/s00432-011-1087-9

ORIGINAL PAPER

Downregulation of EphA2 expression suppresses the growth and metastasis in squamous-cell carcinoma of the head and neck in vitro and in vivo Yong Liu · Changyun Yu · Yuanzheng Qiu · Donghai Huang · Xiaojuan Zhou · Xin Zhang · Yongquan Tian

Received: 12 September 2011 / Accepted: 1 November 2011 / Published online: 16 November 2011 © Springer-Verlag 2011

Abstract Purpose Our previous study has revealed that EphA2 overexpression is signiWcantly associated with aggressive behavior and poor prognosis in patients with squamous-cell carcinoma of the head and neck (SCCHN). However, the function of EphA2 in tumorigenesis and cervical lymph node metastasis of SCCHN has never been elucidated in vivo. Methods EphA2 was knocked down in SCCHN cell lines. CCK-8 assays, Xuorescence-activated cell sorting analysis, invasion and migration assays were performed in vitro. In vivo tumorigenicity assays were performed, and the impact on cervical lymph node metastasis was evaluated. Results The present investigation demonstrated that suppression of EphA2 resulted in a signiWcant inhibition of proliferation, migration, invasion of SCCHN cells in vitro and markedly diminished their tumorigenicity and lymph node metastasis in vivo. Conclusions These results suggest that EphA2 plays a critical role in SCCHN growth and metastasis and may be a

Y. Liu · C. Yu · Y. Qiu · D. Huang · X. Zhang (&) · Y. Tian (&) Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University, Xiangya Road 87, Changsha 410008, Hunan Province, People’s Republic of China e-mail: [email protected] Y. Tian e-mail: [email protected] Y. Liu · C. Yu · Y. Qiu · D. Huang · X. Zhang · Y. Tian Otolaryngology Major Disease Research Key Laboratory of Hunan Province, Changsha 410008, Hunan, China X. Zhou Department of Otolaryngology, No.1 Hospital of Changsha City, Changsha 410013, Hunan, China

promising therapeutic target to prevent the progression of SCCHN. Keywords Head and neck cancer · Squamous-cell carcinoma · Metastasis · Prognosis · EphA2

Introduction Head and neck cancer, mostly of squamous-cell origin, ranks sixth among the most common malignancies worldwide and accounts for approximately 6% of all cases of cancer. Each year, more than 500,000 new cases are diagnosed worldwide (Jemal et al. 2010). Despite the progress in conventional therapies such as surgery, radiation, and chemotherapy, the long-term prognosis for patients with squamous-cell carcinoma of the head and neck (SCCHN) remains unsatisfactory. Lymph node metastasis, which developed in 40–60% of patients with SCCHN, is a major cause of death and a key factor aVecting clinic treatment and prognosis in patients with SCCHN (Mamelle et al. 1994; Mamelle 2000). Thus, the inhibition of invasion and metastasis is of great importance in SCCHN therapies. Eph protein family constitutes the largest group of transmembrane receptor tyrosine kinase (RTKs) identiWed in the genome (Pasquale 1997). Depending on sequence homology and binding aYnity for 2 diVerent types of membraneanchored ephrin ligands, the Eph family can be classiWed as EphA or EphB. Eph kinases have been extensively studied for their roles in embryonic development where they transduce key directional signals for cell positioning, axon guidance, tissue border formation and vascular development (Pasquale 2005, 2008). EphA2, a member of Eph kinases family, has a low expression level on adult epithelial cells (Sulman et al. 1997). Emerging evidence implicates EphA2

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overexpression in cell transformation, primary tumor initiation, progression, angiogenesis, and metastasis in a variety of cancer models (Ireton and Chen 2005; Duxbury et al. 2004). In nontransformed mammary epithelial cells, ectopic overexpression of EphA2 has been shown to result in a malignant phenotype both in vitro and in vivo contexts (Zelinski et al. 2001). Cancer cells that overexpress EphA2 exhibit increased motility and invasive properties, consistent with a pro-metastatic phenotype (Zantek et al. 1999). EphA2 overexpression is prevalent in numerous solid tumors, including those of breast (Zelinski et al. 2001), ovarian (Lin et al. 2007; Meade-Tollin and Martinez 2007; Thaker et al. 2004; Han et al. 2005), lung (Brannan et al. 2009a, b; Kinch et al. 2003), glioblastoma (Wykosky et al. 2005), prostate (Walker-Daniels et al. 1999) et al. And these excellent clinical investigations reveal that EphA2 overexpression by tumor cells is an indicator of poor prognosis due to its close association with reduced time to disease recurrence and enhanced disease progression and metastatic spread. Therefore, EphA2 represents an attractive target for therapeutic intervention in a broad range of patients with cancer, which deserves closer scrutiny. Recent studies in our laboratory have demonstrated that EphA2 overexpression was signiWcantly associated with SCCHN aggressive behavior and poor prognosis from the analysis of clinical tissue specimens (Liu et al. 2011). Similar results in clinical tissue level have been obtained by other investigators (Wu et al. 2011; Shao et al. 2008; Rivera et al. 2008). However, the function of EphA2 in tumorigenesis and lymph node metastasis of SCCHN has never been elucidated in vitro and in vivo. In the present study, we Wrstly employed lentiviral RNAi system to knockdown EphA2 expression in SCCHN cell lines. The roles of EphA2 in SCCHN cell growth and metastasis both in vitro and in vivo conditions were investigated.

Materials and methods Cell culture and reagents The human SCCHN cell line Tu686 was established from a primary tumor in base of tongue. Derived through repeated in vivo selection in nude mice from a lymph node metastasis from the same patient, M2 was metastatic cell lines capable of generating lymph node metastasis in vivo (Zhang et al. 2002, 2006). These two SCCHN cell lines were kindly provided by Dr. Zhuo (Georgia) Chen (Emory University Winship Cancer Institute, Atlanta, Georgia). All cell lines were maintained as monolayer cultures in Dulbecco’s modiWed Eagle’s medium (DMEM)/F12 medium (1:1) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 IU/ml streptomycin at 37°C

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in a humidiWed atmosphere with 5% CO2. Exponentially growing cells were used for the following experiments. RNA isolation and RT-PCR The details of RT-PCR have been previously described (Liu et al. 2011). BrieXy, total RNA of cells was isolated and transcribed to cDNA, and the PCR cycling conditions was as follows: 95°C for 5 min; then 32 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and an extension at 72°C for 5 min. Primers were designed and synthesized according to Herath et al. (2006) as follows: EphA2 primer, forward 5⬘-GGGACCTG ATGCAGAACATC-3⬘, reverse 5⬘-AGTTGGTGCGGAGCCAGT-3⬘; 18S rRNA forward 5⬘-GAC TCAACACGGGA AACCTC-3⬘, reverse 5⬘-AGC ATGCCAGAGTCTCGTTC-3⬘. The gene expression was presented by the ratio between target gene and 18S rRNA (internal control). Each experiment was done in triplicate and then mean value was calculated. Western blotting All Western blotting analyses were performed as we described previously (Liu et al. 2010). In brief, total protein (50 g/sample) was extracted and separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred onto PVDF membrane (Millipore, Bedford, MA). The blotted membranes were incubated with rabbit poly-antibody against EphA2 (sc-924, dilution 1:400) (Santa Cruz, CA, USA) and then secondary antibody (Beyotime, China) in order. -actin protein was also determined by using speciWc antibody (Beyotime, China) as a loading control. Stable transfection and selection EphA2 shRNA Lentiviral Particles (sc-29304-V, Santa Cruz, CA, USA) is a pool of concentrated, transductionready viral particles containing 3 target-speciWc constructs that encode 19–25 nt (plus hairpin) shRNA designed to knockdown EphA2 gene expression in human cells. Tu686 or M2 cells (2 £ 104) were seeded into 24-well plates and allowed to grow at 80% conXuence. And then medium containing EphA2 or control shRNA Lentiviral Particles was added to these cells supplied with Polybrene (8 g/ml) (sc134220, Santa Cruz, CA, USA). After 12 h, the original medium was replaced with fresh complete medium and the cells were subjected to the selection of stable clones at the presence of Puromycine dihydrochloride (8 g/ml) (sigma) 72 h post-infection. The expression of EphA2 was determined by RT-PCR and Western blotting as described above after 21 days of puromycin selection in DMEM/F12 containing 10% FCS.

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Cell proliferation assay Cell Counting Kit-8 (Beyotime, China) was employed to draw cell growth curves according to the manufacturer’s instructions. BrieXy, Cells at 2.5 £ 103/well were cultured in triplicate in 96-well plates with 10% FCS at 37°C in humidiWed 5% CO2 atmosphere for various periods and exposed to fresh media every other day. At 1, 2, 3, 4, 5 and 6 day post-transfection, cell proliferation was measured by absorbance at OD (490 nm). Cell apoptosis and cell cycle analysis by Xow cytometry For Xuorescence-activated cell sorting analysis, after incubation in serum-free medium for 24 h, cells were washed with PBS at 4°C twice and were modulated concentration to 1 £ 106/ml. Annexin V/Cy5 and propidium iodide were added for incubation for 30 min. Fluorescence-activated cell sorting analysis was done on the FACS Calibur instrument (Becton–Dickinson). Migration and matrigel invasion assay The invasiveness of the transfected cells was evaluated in 24-well transfected chambers (Costar, Cambridge, MA) according to the manufacturer’s instructions. BrieXy, Transwell with an 8 m diameter pore membrane was coated with 200 l Matrigel at 200 g/ml and incubated overnight. 3 £ 104 cells in 100 l of serum-free medium were seeded into the upper chamber of the Transwell, and the lower chamber was Wlled with 0.8 mL DMEM/F12 medium containing 12% FBS to induce chemotaxis. After 48 h of incubation at 37°C in humidiWed 5% CO2 atmosphere, the cells were Wxed in methanol and stained with H&E, and the cells that invaded through the pores to the lower surface of the Wlter were counted under a microscope. Three invasion chambers were used per condition. The values obtained were calculated by averaging the total number of cells from three Wlters. For migration assay, wound-healing assay was done. Tu686 or M2 cells (10 £ 104) were seeded on 12-well plates with complete medium and incubated for 24 h to grow to almost conXuence. And then cell monolayer was disrupted with a 10-l pipette tip, and photographs were taken at 0 and 48 h in a phase-contrast microscope. Experiments were carried out in triplicate, and four Welds of each point were recorded. SCCHN metastatic xenograft mouse model The animal experiment was approved by the Institutional Animal Care and Use Committee, Xiangya Hospital, Central South University. A metastatic SCCHN model in mice

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was established as described previously (Zhang et al. 2002, 2006). BrieXy, 4–5-week-old BALB/c athymic nude mice (about 20 g body weight) were housed under speciWc pathogen-free conditions. Each animal was injected with cells (2.5 £ 106) of M2, M2EphA2RNAi¡ or M2EphA2RNAi+ suspended in 0.05 ml of Hanks-buVered saline into the submandibular to mylohyoid muscle. The xenograft tumors were measured three times per week, and all mice were killed 25 days after implantation. After all mice were killed, primary tumor, cervical lymph nodes, and lungs were collected, Wxed immediately in 10% buVered formalin, and embedded in paraYn. Tissue serial sections were stained with hematoxylin–eosin and observed under a microscope. Lymph node metastasis was identiWed by an experienced pathologist. Statistical analysis All statistical analyses were performed with SPSS 17.0 software. Results of quantitative data in this study were expressed as mean § SD. Statistical diVerences between groups were compared using two-tailed ANOVA and t test. A P value < 0.05 was considered to be statistically signiWcant.

Results Knockdown expression of EphA2 by EphA2 shRNA lentiviral particles in SCCHN cells To clarify the correlation of EphA2 expression and SCCHN metastasis, we employed the lentivirus-delivered shRNA to inhibit the expression of EphA2. Stable clones were isolated after selection with 8 g/ml Puromycine dihydrochloride for 4 weeks. Both RT-PCR and Western blotting were carried out to assess the inhibition eYciency. As presented in Fig. 1a, Lenti-shRNAi eVectively infected Tu686 cells. All cells were infected by Lenti-shRNAi in terms of GFP expression as observed under Xuorescent microscope 72 h after infection (Fig. 1a) inhibited the EphA2 mRNA expression more than 80% (Fig. 1b) and resulted in more than 70% inhibition eYciency in protein expression (Fig. 1c). However, the control did not cause detectable changes of mRNA and protein expression of EphA2. Then the Tu686 cells transfected EphA2 shRNA and scramble control shRNA were termed as, for convenience, Tu686EphA2RNAi+ and Tu686EphA2RNAi¡, respectively. Down-regulated EphA2 expression inhibits proliferation of SCCHNcells in vitro In order to clarify the eVect of EphA2 on the proliferation of SCCHN cells in vitro, a CCK-8 assay was performed and cell

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Fig. 1 Knock-down of EphA2 expression by lentiviral vectormediated RNAi in Tu686 cells. a EYcient transduction of Tu686 cells with Lenti-shRNA. Fluorescence photomicrographs of Tu686 cells infected by LentishRNA. Pictures were captured 72 h after infection at a magniWcation of 50. b Decreased mRNA expression of EphA2 as revealed by RT-PCR. For convenience, Tu686 cells infected with control shRNA lentiviral particles was named Tu686EphA2RNAi¡and Tu686 cells infected with EphA2 shRNA lentiviral particles was named Tu686EphA2RNAi+. c Down-regulated protein expression of EphA2 detected by Western blotting. Numbers 1, 2, and 3 refers to Tu686, Tu686EphA2RNAi¡, and Tu686EphA2RNAi+, respectively

growth curve was obtained. As presented in Fig. 2, Tu686EphA2RNAi+ cell grew slowly than both control groups (Tu686 and Tu686EphA2RNAi¡), which indicates that the expression of EphA2 has an inXuence on SCCHN cell growth. Down-regulation of EphA2 induces cell cycle arrest in G0/G1 phase but does not aVect apoptosis of SCCHNcells in vitro To elucidate the mechanism of EphA2-mediated cell growth inhibition in SCCHN cells, Xow cytometry was carried out to monitor cell cycle and apoptosis changes. The results demonstrated that, when compared with the control groups, the percentage of Tu686EphA2RNAi+ cells in G0/G1 phase was signiWcantly increased (75.04 § 1.08 vs. 56.09 § 1.58, 55.04 § 1.38) (P < 0.01), whereas the percentage of cells in S phase (18.33 § 0.77 vs. 32.54 § 1.70, 32.66 § 0.99) and G2/M phase (6.64 § 1.00 vs. 12.30 § 0.76, 11.37 § 0.87) were obviously decreased (both P < 0.01) (Fig. 3a). However, there was no signiWcant diVerence in apoptotic rate between diVerently treated groups (8.61 § 0.90 vs. 8.76 § 1.24, 9.57 § 0.89)

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Fig. 2 The eVect of EphA2 on the proliferation of Tu686 cells. Cell proliferation was measured by the CCK-8 assay every 24 h for 6 days. Results were means of three independent experiments § SD (*P < 0.05; **P < 0.01)

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Fig. 3 The eVect of EphA2 on cell cycle distribution and apoptosis of Tu686 cells. a Tu686, Tu686EphA2 RNAi¡ and Tu686EphA2 RNAi+ cells were stained with propidium iodide and analyzed by Xow cytometry. Proportion of cells in various phases of the cell cycle. The results are means of three independent experiments § SD (P < 0.05). b Cells staining positive for Annexin V-FITC and negative for PI were considered to have undergone apoptosis. Average apoptotic rate of three independent experiments § SD are shown. *P < 0.01, Tu686EphA2 RNAi¡ versus Tu68, Tu686EphA2 RNAi+. Numbers 1, 2, 3 refers to Tu686, Tu686EphA2RNAi¡ and Tu686EphA2RNAi+, respectively

(P > 0.05) (Fig. 3b). These results demonstrate that knockdown of the expression of EphA2 induces G0/G1 phase arrest in SCCHN cells, which leads to the growth-inhibitory properties of Tu686EphA2RNAi+. Down-regulated EphA2 expression inhibits migration and invasion of SCCHN cell lines in vitro In order to evaluate the function of EphA2 on SCCHN cell migration and invasion, scratch-wound assay and Matrigel invasion chambers were performed. As presented in Fig. 4, inhibited EphA2 expression led to signiWcantly decreased migration [Tu686EphA2RNAi+ group, wound closure rate (46.7 § 7.6)% vs. Tu686, Tu686EphA2 RNAi¡ control groups, wound closure rate (88.7 § 4.5)% and (86.0 § 2.6)%), P < 0.01] and invasion (123 § 13 vs. 288 § 20, 303 § 11; P < 0.01) of SCCHN Tu686 cells. These results clearly demonstrate that EphA2 plays a functional role in mediating cell migration and invasion in SCCHN, which are key determinants of SCCHN malignant progression and metastasis. Down-regulated EphA2 expression inhibits tumorigenicity and metastasis of SCCHN in vivo We Wnally used established SCCHN metastatic mouse model to test whether EphA2 could regulate SCCHN

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growth and metastasis in vivo. Firstly, lentivirus-delivered shRNA was applied to inhibit the expression of EphA2 in SCCHN M2 cells and stable clones were isolated after selection with 8 g/ml Puromycine dihydrochloride for 4 weeks. RT-PCR and Western blotting results demonstrated that we successfully inhibited the EphA2 mRNA expression more than 80% (Fig. 5a) and more than 70% inhibition eYciency in protein expression (Fig. 5b). Subsequently, M2, M2EphA2RNAi¡ and M2EphA2RNAi+ cells were injected subcutaneously into the submandibular to mylohyoid muscle, and tumor formation was carefully monitored. After 25 days, all nude mice were killed and the tumor weights were measured. The average tumor volume of mice in M2EphA2RNAi+ group was signiWcantly smaller than that of both groups of M2 and M2EphA2RNAi¡ (P < 0.01; Fig. 5c– e), and the average tumor weight of mice inoculated with M2EphA2RNAi+ cells was 0.26 § 0.10 g, which was much lower than that of mice inoculated with M2 and M2EphA2RNAi¡ cells (0.65 § 0.11 g and 0.54 § 0.12 g, respectively; P < 0.01; Fig. 5d–f). However, no signiWcant diVerence existed in tumor volume and weight between groups of M2 and M2EphA2RNAi¡ (both P > 0.05). These results were consistent with our in vitro results, which indicated that inhibition of EphA2 expression resulted in SCCHN primary tumor growth. By using this SCCHN metastasis animal model, only 1 out of 7 mice injected with M2EphA2RNAi+ cells had identiWable bilateral and 1 mouse had unilateral cervical lymph node metastasis (lymph node metastasis rate 3/14 = 21.4%), which was signiWcantly lower than that in both groups of M2 and M2EphA2RNAi¡ (M2, 3 out of 6 mice had bilateral and 1 mouse had unilateral cervical lymph nodes metastasis, rate 7/12 = 58.3%; M2EphA2RNAi¡, 4 out of 6 mice had bilateral cervical lymph nodes metastasis, rate 8/12 = 66.7%; P < 0.05; Fig. 5g). No lung metastasis was found in all nude mice by serial sections. These data indicated that EphA2 inhibition decreased cervical lymph node metastasis in vivo.

Discussion Recent studies about EphA2 in other types of carcinoma have raised our great interest to investigate its potential roles in SCCHN. In our previous publication (Liu et al. 2011), close associations between the expression of EphA2 protein and several clinicopathological parameters, such as tumor site, T classiWcation, clinical stage, recurrence and lymph node metastasis, have been clearly conWrmed in a relative large SCCHN tissue specimens. Moreover, overexpression of EphA2 predicts a poor prognosis in patients with SCCHN. Therefore, the role of EphA2 in tumorigenesis and lymph node metastasis of

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Fig. 4 Knockdown of the expression of EphA2 inhibited the migration and invasion of Tu686 in vitro. Numbers 1, 2, and 3 refers to Tu686, Tu686EphA2RNAi¡, and Tu686EphA2RNAi+, respectively. a Would healing assay demonstrated that knockdown of the expression of EphA2 inhibited cell migration of Tu686. Photographs were captured at 0 h and 24 h after wound induction at 100£ magniWcation. Representative of three independent experiments. b Graphical representation of the percentage of migration of Tu686 cells. Data were shown as

mean § SD from three independent experiments. c Transwell invasion assay showed that knockdown of the expression of EphA2 inhibited cell invasion of Tu686. Photographs were taken at 200£ magniWcation. Representative of three independent experiments. d Graphical representation of the numbers of invaded Tu686 cells per microscopic Welds. Data were shown as mean § SD from three independent experiments. *P < 0.05, Tu686 versus Tu686EphA2RNAi+; **P < 0.05, Tu686EphA2 RNAi¡ versus Tu686EphA2 RNAi+

SCCHN was further been elucidated both in vitro and in vivo. In the current investigation, EphA2 shRNA Lentiviral Particles were successfully employed to knockdown the expression of EphA2 in SCCHN cells. The CCK-8 assay revealed an inhibition of cell growth in vitro by inducing cell cycle arrest in G0/G1 phase, but not apoptotic rate, when SCCHN cells were transfected with Lenti-EphA2 shRNA. Furthermore, inhibition of EphA2 markedly diminished the tumorigenecity of SCCHN in vivo. This result indicates a critical role of EphA2 in proliferation of SCCHN cancer cells, which is similar to its suggested roles in other reported cancers (Bogan et al. 2009; Brantley-Sieders et al. 2008; Lu et al. 2008; Margaryan et al. 2009; Zhou et al. 2008). Since EphA2 has long been recognized as an oncogene and a potential tumor marker in tumorigenesis for other carcinomas, it is not surprising to present an association between the expression of EphA2 and proliferation of SCCHN cells. Cell migration and invasion play critical roles in cancer metastasis. Our in vitro data have clearly indicated that EphA2 inXuenced the migration and invasion of SCCHN cells. Therefore, a SCCHN metastatic xenograft mouse model, established by our team previously (Zhang et al. 2002, 2006) was applied to explore its in vivo role in metastasis. The present study also demonstrated that sub-

mandibular injection of SCCHN M2 cell lines could successfully induce lymph node metastasis. No signiWcant reduction of cervical lymph node metastasis after the injection of M2EphA2RNAi¡, yet the injection with M2EphA2RNAi+ caused apparent inhibition of cervical lymph node metastasis in vivo. This suggests the value of targeting EphA2 as a promising therapeutic strategy to inhibiting lymph node metastasis in patients with SCCHN. Such Wnding is of particular importance, since cervical lymph node metastasis occurs frequently in patients with SCCHN and severely inXuences the treatment and prognosis of SCCHN (Mamelle 2000; Mamelle et al. 1994). Until now, the underlying mechanisms about how EphA2 modulates tumor metastasis are not fully elucidated. In summary, this is the Wrst study that identiWes the role of EphA2 in the growth and metastasis of SCCHN in vivo. Our results demonstrate that decreased expression of EphA2 could signiWcantly inhibit cell growth and lymph node metastasis of SCCHN, which implicates that EphA2 may be a valuable therapeutic target for the patients with SCCHN. Furthermore, as increasing evidence indicates that EphA2 regulates tumorigenesis and cancer progression in a wide range of other human malignancies, diverse therapy strategies have been developed to target this pathway, including, RNA interference (Landen et al. 2005), memetic peptide (Koolpe et al. 2002), monoclonal

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Fig. 5 EVect of EphA2 expression on the growth and metastasis of inoculated M2 cells. Groups of Balb/c nude mice were inoculated with 2.0 £ 106 M2, M2EphA2RNAi¡ (n = 6) or M2EphA2RNAi+ (n = 7) and the development of solid SCCHN tumors were monitored every 3 days. The mice were killed 25 days post-inoculation and their tumor weights were measured. Numbers 1, 2, and 3 refers to M2, M2EphA2RNAi¡, and M2EphA2RNAi+, respectively. a Decreased mRNA expression of EphA2 as revealed by RTPCR. b Down-regulated protein expression of EphA2 detected by Western blotting. c Photographs of killed nude mice at 25 days after inoculation with M2 cells. d The dynamics of SCCHN tumor growth. e The image of individual tumors. f Quantitative measurement of all tumor weights. Data are expressed as mean § SD of each group. g Comparison of numbers of cervical lymph nodes metastasis. * and ** both P < 0.05 M2EphA2RNAi+ versus control M2 or M2EphA2RNAi¡

antibody (Carles-Kinch et al. 2002; Landen et al. 2006). However, a deeper understanding of the precise mechanisms about how EphA2 regulates tumor growth and metastasis is urgently required, which will inspire us to Wnd new ways to prevent the progression of patients with SCCHN. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No:81172558, 81071757, 30872852, 30901664), the Key Program of Natural Science Foundation of Hunan Province (2010TP4012-1), the Research Fund for the Doctoral Program of Higher Education of China (20100162110036, 20090162110065) and the Fundamental Research Funds for the Central Universities. ConXict of interest

We declare that we have no conXict of interest.

References Bogan C, Chen J, O’Sullivan MG et al (2009) Loss of EphA2 receptor tyrosine kinase reduces ApcMin/+ tumorigenesis. Int J Cancer 124:1366–1371 Brannan JM, Dong W, Prudkin L et al (2009a) Expression of the receptor tyrosine kinase EphA2 is increased in smokers and predicts poor survival in non-small cell lung cancer. Clin Cancer Res 15:4423–4430 Brannan JM, Sen B, Saigal B et al (2009b) EphA2 in the early pathogenesis and progression of non-small cell lung cancer. Cancer Prev Res (Phila) 2:1039–1049 Brantley-Sieders DM, Zhuang G, Hicks D et al (2008) The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signaling. J Clin Invest 118:64–78

123

202 Carles-Kinch K, Kilpatrick KE, Stewart JC et al (2002) Antibody targeting of the EphA2 tyrosine kinase inhibits malignant cell behavior. Cancer Res 62:2840–2847 Duxbury MS, Ito H, Zinner MJ et al (2004) EphA2: a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma. Oncogene 23:1448–1456 Han L, Dong Z, Qiao Y et al (2005) The clinical signiWcance of EphA2 and Ephrin A-1 in epithelial ovarian carcinomas. Gynecol Oncol 99:278–286 Herath NI, Spanevello MD, Sabesan S et al (2006) Over-expression of Eph and ephrin genes in advanced ovarian cancer: ephrin gene expression correlates with shortened survival. BMC Cancer 6:144 Ireton RC, Chen J (2005) EphA2 receptor tyrosine kinase as a promising target for cancer therapeutics. Curr Cancer Drug Targets 5:149–157 Jemal A, Siegel R, Xu J et al (2010) Cancer statistics, 2010. CA Cancer J Clin 60:277–300 Kinch MS, Moore MB, Harpole DH Jr (2003) Predictive value of the EphA2 receptor tyrosine kinase in lung cancer recurrence and survival. Clin Cancer Res 9:613–618 Koolpe M, Dail M, Pasquale EB (2002) An ephrin mimetic peptide that selectively targets the EphA2 receptor. J Biol Chem 277:46974– 46979 Landen CN Jr, Chavez-Reyes A, Bucana C et al (2005) Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res 65:6910–6918 Landen CN Jr, Lu C, Han LY et al (2006) EYcacy and antivascular eVects of EphA2 reduction with an agonistic antibody in ovarian cancer. J Natl Cancer Inst 98:1558–1570 Lin YG, Han LY, Kamat AA et al (2007) EphA2 overexpression is associated with angiogenesis in ovarian cancer. Cancer 109:332– 340 Liu Y, Xie C, Zhang X et al (2010) Elevated expression of HMGB1 in squamous-cell carcinoma of the head and neck and its clinical signiWcance. Eur J Cancer 46:3007–3015 Liu Y, Zhang X, Qiu Y et al (2011) Clinical signiWcance of EphA2 expression in squamous-cell carcinoma of the head and neck. J Cancer Res Clin Oncol 137:761–769 Lu C, Shahzad MM, Wang H et al (2008) EphA2 overexpression promotes ovarian cancer growth. Cancer Biol Ther 7:1098–1103 Mamelle G (2000) Selective neck dissection and sentinel node biopsy in head and neck squamous cell carcinomas. Recent Results Cancer Res 157:193–200 Mamelle G, Pampurik J, Luboinski B et al (1994) Lymph node prognostic factors in head and neck squamous cell carcinomas. Am J Surg 168:494–498 Margaryan NV, Strizzi L, Abbott DE et al (2009) EphA2 as a promoter of melanoma tumorigenicity. Cancer Biol Ther 8:279–288 Meade-Tollin L, Martinez JD (2007) Loss of p53 and overexpression of EphA2 predict poor prognosis for ovarian cancer patients. Cancer Biol Ther 6:288–289

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J Cancer Res Clin Oncol (2012) 138:195–202 Pasquale EB (1997) The Eph family of receptors. Curr Opin Cell Biol 9:608–615 Pasquale EB (2005) Eph receptor signalling casts a wide net on cell behaviour. Nat Rev Mol Cell Biol 6:462–475 Pasquale EB (2008) Eph-ephrin bidirectional signaling in physiology and disease. Cell 133:38–52 Rivera RS, Gunduz M, Nagatsuka H et al (2008) Involvement of EphA2 in head and neck squamous cell carcinoma: mRNA expression, loss of heterozygosity and immunohistochemical studies. Oncol Rep 19:1079–1084 Shao Z, Zhang WF, Chen XM et al (2008) Expression of EphA2 and VEGF in squamous cell carcinoma of the tongue: correlation with the angiogenesis and clinical outcome. Oral Oncol 44:1110–1117 Sulman EP, Tang XX, Allen C et al (1997) ECK, a human EPH-related gene, maps to 1p36.1, a common region of alteration in human cancers. Genomics 40:371–374 Thaker PH, Deavers M, Celestino J et al (2004) EphA2 expression is associated with aggressive features in ovarian carcinoma. Clin Cancer Res 10:5145–5150 Walker-Daniels J, CoVman K, Azimi M et al (1999) Overexpression of the EphA2 tyrosine kinase in prostate cancer. Prostate 41:275– 280 Wu Z, Doondeea JB, Moghaddas Gholami A et al. (2011) Quantitative chemical proteomics reveals new potential drug targets in head and neck cancer. Mol Cell Proteomics [Epub ahead of print] Wykosky J, Gibo DM, Stanton C et al (2005) EphA2 as a novel molecular marker and target in glioblastoma multiforme. Mol Cancer Res 3:541–551 Zantek ND, Azimi M, Fedor-Chaiken M et al (1999) E-cadherin regulates the function of the EphA2 receptor tyrosine kinase. Cell Growth DiVer 10:629–638 Zelinski DP, Zantek ND, Stewart JC et al (2001) EphA2 overexpression causes tumorigenesis of mammary epithelial cells. Cancer Res 61:2301–2306 Zhang X, Liu Y, Gilcrease MZ et al (2002) A lymph node metastatic mouse model reveals alterations of metastasis-related gene expression in metastatic human oral carcinoma sublines selected from a poorly metastatic parental cell line. Cancer 95:1663–1672 Zhang X, Su L, Pirani AA et al (2006) Understanding metastatic SCCHN cells from unique genotypes to phenotypes with the aid of an animal model and DNA microarray analysis. Clin Exp Metastasis 23:209–222 Zhou Z, Yuan X, Li Z et al (2008) RNA interference targeting EphA2 inhibits proliferation, induces apoptosis, and cooperates with cytotoxic drugs in human glioma cells. Surg Neurol 70:562–568 (discussion 568–569)