Upregulation of Eps8 in oral squamous cell carcinoma promotes cell ...

Oncogene (2009) 28, 2524–2534

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ORIGINAL ARTICLE

Upregulation of Eps8 in oral squamous cell carcinoma promotes cell migration and invasion through integrin-dependent Rac1 activation LF Yap1,2,8, V Jenei3,4,8, CM Robinson5, K Moutasim3,4, TM Benn1, SP Threadgold1, V Lopes6, W Wei7, GJ Thomas3,4,9 and IC Paterson1,9 1 Department of Oral and Dental Science, University of Bristol, Bristol, UK; 2Cancer Research Initiatives Foundation, Kuala Lumpur, Malaysia; 3Centre for Tumor Biology, Institute of Cancer, Bart’s and the London School of Medicine and Dentistry, London, UK; 4 Cancer Sciences Division, Southampton University School of Medicine, Southampton, UK; 5School of Dental Sciences, University of Newcastle, Newcastle, UK; 6Edinburgh Dental Institute, University of Edinburgh, Edinburgh, UK and 7Institute for Cancer Studies, University of Birmingham, Birmingham, UK

Oral squamous cell carcinoma (OSCC) is a lethal disease and early death usually occurs as a result of local invasion and regional lymph node metastases. Current treatment regimens are, to a certain degree, inadequate, with a 5-year mortality rate of around 50% and novel therapeutic targets are urgently required. Using expression microarrays, we identified the eps8 gene as being overexpressed in OSCC cell lines relative to normal oral keratinocytes, and confirmed these findings using RT–PCR and western blotting. In human tissues, we found that Eps8 was upregulated in OSCC (32% of primary tumors) compared with normal oral mucosa, and that expression correlated significantly with lymph node metastasis (P ¼ 0.032), suggesting a diseasepromoting effect. Using OSCC cell lines, we assessed the functional role of Eps8 in tumor cells. Although suppression of Eps8 produced no effect on cell proliferation, both cell spreading and migration were markedly inhibited. The latter cell functions may be modulated through the small GTP-ase, Rac1 and we used pull-down assays to investigate the role of Eps8 in Rac1 signaling. We found that avb6- and a5b1integrin-dependent activation of Rac1 was mediated through Eps8. Knockdown of either Eps8 or Rac1, inhibited integrindependent cell migration similarly and transient expression of constitutively active Rac1 restored migration of cells in which Eps8 expression had been suppressed. We also showed that knockdown of Eps8 inhibited tumor cell invasion in an organotypic model of OSCC. These data suggest that Eps8 and Rac1 are part of an integrated signaling pathway modulating integrin-dependent tumour cell motility and identify Eps8 as a possible therapeutic target. Oncogene (2009) 28, 2524–2534; doi:10.1038/onc.2009.105; published online 18 May 2009 Keywords: Eps8; oral cancer; integrin; migration; Rac1; invasion

Correspondence: Professor GJ Thomas, Cancer Sciences Division, Southampton University School of Medicine, Tremona Road, Southampton, UK. E-mail: [email protected] 8 These authors contributed equally to this work. 9 These authors jointly supervised this study. Received 16 June 2008; revised 23 March 2009; accepted 24 March 2009; published online 18 May 2009

Introduction Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy worldwide (Parkin et al., 2005). Cervical lymph node metastasis is the most important prognostic indicator and patients with metastases that show evidence of invasion into the parenchymal tissues of the neck have been shown to have the worst prognosis (Myers et al., 2001; Woolgar et al., 2003). Although the molecular basis for the development of OSCC is starting to be elucidated (Kim and Califano, 2004), the factors that influence invasion and metastasis are poorly understood. The epidermal growth factor receptor pathway substrate 8 gene (Eps8) was originally identified as a substrate for the epidermal growth factor receptor kinase (Fazioli et al., 1993). Eps8 was later shown to form a complex with Sos1, Eps8 SH3 domain-binding protein 1 (E3b1; also known as Abi1) and the p85 regulatory subunit of PI3K, resulting in the activation of Rac1 and subsequent actin remodeling (Scita et al., 1999; Innocenti et al., 2003). More recently, it has been shown that Eps8 is a novel actin capping protein that is essential for cell motility by regulating the growth of actin filaments to generate propulsive force (Disanza et al., 2004). The C-terminal effector region of Eps8 possesses actin filament barbed-end capping activity, recruiting Eps8 to the actin dynamic sites, such as membrane ruffles, where E3b1, Sos1 and p85 also localize (Scita et al., 2001; Innocenti et al., 2003; Croce et al., 2004; Disanza et al., 2004). Eps8 has also been shown to increase the expression and activity of focal adhesion kinase (FAK; Maa et al., 2007), which raises the possibility that Eps8 may be involved in the transduction of integrin-induced signals. Overexpression of Eps8 in fibroblasts has been shown to induce malignant transformation in vitro and promote tumor formation in vivo (Matoskova et al., 1995; Maa et al., 2001), suggesting that Eps8 is an oncogene. Eps8 has also been shown to form a complex with IRSp53, an adaptor protein between Rho-family small GTPases and actin cytoskeleton reorganizing proteins (Miki et al., 2000), in fibroblasts and various cancer cell lines to augment Eps8/E3b1/Sos1 complex

Eps8 upregulation promotes cell migration and invasion LF Yap et al

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formation, resulting in activation of Rac1 (Funato et al., 2004). Eps8 has been reported to be upregulated in primary tumors of the breast, pancreas and colon (Yao et al., 2006; Maa et al., 2007; Welsch et al., 2007), but its functional role in epithelial carcinogenesis remains to be clarified. Using expression microarrays, we have identified Eps8 as being overexpressed in OSCC-derived cell lines compared with normal oral keratinocytes and showed that Eps8 is upregulated in a subset of OSCCs where it correlates significantly with metastatic disease. Knockdown of Eps8 in OSCC cell lines did not effect cell proliferation, but cell spreading and migration were markedly inhibited. We next investigated the effect of Eps8 knockdown on Rac1 signaling and showed that integrin-dependent activation of Rac1 was mediated through Eps8. Knockdown of Eps8 or Rac1, inhibited cell migration similarly and transient expression of constitutively active Rac1 restored the migration of cells which had been transfected with Eps8 RNAi. Finally, we demonstrated that Eps8 promotes OSCC invasion in organotypic assays. Therefore, Eps8 appears to be an important mediator of integrin-dependent tumor cell motility as part of an integrated signaling pathway involving Rac1.

Results Gene expression differences between NKs and OSCC-derived cell lines To identify genes whose transcription is deregulated in OSCC, the gene expression profiles of eight OSCC cell lines (H-series and M9) and three primary cultures of normal oral keratinocytes (NKs) were examined using Affymetrix HG-U133A and HG-U133 Plus 2.0 arrays and the significance of gene expression changes using significance analysis of microarrays analysis. Genes whose expression levels were increased or decreased by two-fold or greater between normal and tumor cells with a Q-value threshold of less than 5% were selected and 309 named genes were identified, with 79 being upregulated (Supplementary Table 1) and 230 being downregulated (Supplementary Table 2) in tumor cells relative to NKs. Two-way hierarchical clustering analysis was used to group the cell lines based on the degree of similarity of their expression patterns using the 309 named genes and the normal and malignant cells formed two distinct groups (Figure 1a). Eps8 is overexpressed in OSCC-derived cell lines and tissues By microarray, the expression of Eps8 was enhanced >5-fold in the OSCC cell lines relative to NKs. Semiquantitative RT–PCR showed that Eps8 mRNA levels were elevated in six out of the eight cell lines (Figure 1b) and Eps8 protein could be detected in the same six cell lines by western blotting but was undetectable in the NKs (Figure 1c). The expression of Eps8 was then examined in five samples of normal oral mucosa and

seven OSCCs using quantitative real-time PCR. Compared to normal mucosa, the expression level of Eps8 was increased in five out of seven tumors analysed (Figure 1d). Immunohistochemical analysis of Eps8 expression in primary OSCC tissues The expression and cellular location of Eps8 was examined in 59 formalin-fixed paraffin-embedded primary OSCCs and nine samples of normal oral mucosa by immunohistochemistry. The clinical characteristics of the tumor samples are listed in Supplementary Table 3. Normal oral epithelium and epithelium adjacent to carcinomas (present in 57 of 59 samples) were consistently negative, whereas endothelium and salivary duct epithelium was always positive, demonstrating the specificity of the Eps8 antibody (Figures 2a–d). Eps8 expression was detected in 19 of 59 (32%) of the tumors examined. The staining was cytoplasmic and ranged from weak and focal to strong and diffuse (Figures 2e–j). Eps8 expression was examined in the context of clinicopathological characteristics of the tumors (Table 1). There was no significant association between the expression of Eps8 protein and the degree of cellular differentiation (P ¼ 0.116) and T category (P ¼ 0.168). By contrast, Eps8 expression correlated significantly with lymph node metastasis (N category; P ¼ 0.032) and was strongly associated with advanced tumor stage, although this association did not reach statistical significance (P ¼ 0.057). Inhibition of Eps8 expression suppresses cell migration and spreading To investigate the functional role of Eps8 in vitro, we transfected OSCC cell lines with Eps8 siRNA (Figure 3a; Supplementary Figure 1). Sequence 3 gave the most efficient knockdown and was used in subsequent experiments. VB6, BICR56 and CA1 express high levels of endogenous Eps8 and we have shown previously that these cells migrate well in vitro (Thomas et al., 2001; Ramsay et al., 2007). Although inhibition of Eps8 expression had no effect on cell growth in any of the cell lines (data not shown), migration of VB6, BICR56 and CA1 cells towards fibronectin was significantly suppressed (VB6, Po0.02; BICR56 Po0.02; CA1 Po0.0003; Figure 3b). No suppression was evident in a further OSCC cell line, H400 (data not shown). Cell spreading on fibronectin, as assessed by changes in cell morphology and lamellipodia formation, was also inhibited by Eps8 knockdown in VB6, BICR56 and CA1 cells (Po0.0004 for all cell lines at 2 h; Figure 3c). Cells transfected with Eps8 siRNA were generally smaller with less extensive protrusions than control transfected cells (Figure 3c). To confirm that the effects of the siRNA were specific, we knocked down the endogenous Eps8 using siRNA specific to the human sequence and restored Eps8 expression using a mouse Eps8 cDNA. Transfection with the mouse Eps8 cDNA rescued the inhibition of cell migration caused by knocking down endogenous Oncogene


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Figure 1 Gene expression in OSCC. (a) Two-way hierarchical clustering of gene expression in OSCC cells and normal keratinocytes (NKs). Eight OSCC cell lines and three primary cultures of NKs were clustered based on the expression levels of 309 named genes that were identified to be differentially expressed (> 2-fold; Q-value threshold 5%) between tumor and normal cells by significance analysis of microarrays. Gene expression patterns are represented by a blue to red (lower to higher expression) colour scale. (b) and (c), the expression of Eps8 in OSCC cell lines and NKs, by RT–PCR and western blotting, respectively. (d), the expression of Eps8 was examined in seven primary OSCCs and five normal oral mucosa samples by quantitative real-time PCR. GAPDH was used as internal control for normalization. The results are calculated as log 2 ratio of the expression levels of the OSCCs relative to the average expression level of the normal mucosa. Experiments were performed in triplicate and error bars indicate standard deviations.

Eps8 (Figure 3d). Knockdown of Eps8 using two additional siRNAs (sequences 1 and 2) also inhibited cell migration (Supplementary Figure 1), providing additional confirmation that the effects of the siRNA were specific. Subcellular distribution of Eps8 To examine the subcellular distribution of Eps8, we plated VB6 cells onto matrix-coated glass coverslips and stained for Eps8 using immunoflourescence. In actively spreading cells (o4 h), Eps8 co-localized with actin at the cell plasma membrane (Figure 3e). However, in cells that were fully spread (>4 h) this co-localization was lost, and Eps8 relocated to the cytoplasm (data not shown).

Knockdown of Eps8 and Rac1 inhibit migration to a similar extent We next investigated the relative contributions of Eps8 and Rac1 to OSCC migration using siRNA to knockdown these proteins, either alone or in combination. In VB6, BICR56 and CA1 cells, knockdown of either Eps8 or Rac1 significantly inhibited migration towards fibronectin (Eps8 Po0.005 and Rac1 Po0.005 for all cell lines); knockdown of both proteins did not further inhibit migration (Figure 4a). To confirm whether Eps8-dependent cell migration is modulated through Rac1, we transiently expressed constitutively active Rac1 (V12-Rac1) in VB6, BICR56 and CA1 cells following Eps8 siRNA-treatment. Expression of V12-Rac1 significantly restored migration

Figure 2 Immunohistochemical analysis of Eps8 expression in primary OSCC and normal oral mucosa. (a–d), Photomicrographs demonstrating the specificity and distribution of Eps8 expression. Mucosa adjacent to an OSCC incubated with secondary antibody only (negative control; a) and anti-Eps8 antibody (b). The endothelium of capillaries shows moderate staining intensity whereas the epithelium is negative. OSCC and adjacent lobules of minor salivary gland incubated with secondary antibody only (negative control; c) and anti-Eps8 antibody (d). The salivary gland ducts show strong staining intensity whereas the OSCC is negative. (e–j), Photomicrographs demonstrating the staining intensity and distribution of Eps8 expression in OSCC. OSCC showing strong staining intensity. Strong staining is present at the invasive front (e), within an intra-vascular deposit (f) and within islands showing perineural invasion (g). OSCC showing strong but focal staining (h). OSCC showing moderate staining intensity (i). OSCC showing weak staining intensity (j). Magnification 150. Oncogene


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2528 Table 1 Correlation between Eps8 expression and clinicopathological characteristics of OSCCs Characteristics

Eps8 expression

P-value

Differentiation Poor Moderate Well

7/13 (53%) 7/21 (33%) 5/25 (20%)

0.116

T category T1 T2 T3 T4

4/19 7/22 0/3 8/15

(21%) (31%) (0%) (53%)

0.168

N category N0 N1 N2 N3

6/29 1/7 11/22 1/1

(20%) (14%) (50%) (100%)

0.032

Stage I II III IV

2/13 1/6 1/9 15/31

(15%) (16%) (11%) (48%)

0.057

compared with empty vector controls (Figure 4b; Pp0.005 for all three cell lines). Eps8 is required for both a5b1- and avb6-dependent migration To determine which integrins modulate OSCC cell migration towards fibronectin, we first examined the expression of fibronectin-binding integrins by flow cytometry. VB6, BICR56 and CA1 cells expressed both a5b1 and avb6 (Figure 5a) and migration of the cell lines towards fibronectin was modulated through both integrins (Figure 5b). Although antibodies directed against either a5b1 or avb6 produced some level of suppression when used alone, maximal abrogation of migration required inhibition of both integrins (Figure 5b). To determine whether Eps8 modulated avb6 and/or a5b1 integrin-dependent migration, cells were transfected with siRNAs and treated with specific inhibitory antibodies directed against avb6 or a5b1. Antibodies against both avb6 and a5b1 when used in combination with Eps8 siRNA produced further inhibition of migration; similar results were obtained with Rac1 siRNA (Supplementary Figure 2). These results suggested that Eps8-dependent migration was not modulated through a single integrin. Therefore, we investigated whether Eps8 modulated both avb6- and a5b1-dependent migration. We have shown previously that VB6 cells adhere to, and migrate towards, the latency-associated peptide (LAP) of TGF-b1 solely through avb6 (Thomas et al., 2002). Similarly, H357 cells adhere to, and migrate towards, fibronectin solely through a5b1 (Thomas et al., 2001). Transfection of VB6 cells with siRNAs against Eps8 or Rac1, or treatment of cells with an avb6-blocking antibody resulted in a significant inhibition of migration (Eps8 Po0.005; Rac1 Po0.01; anti-avb6 Po0.004; Figure 5ci). Similarly, the migration of H357 cells Oncogene

towards fibronectin was also significantly inhibited with Eps8 and Rac1 siRNAs or by treatment with cells with an a5b1-blocking antibody (all conditions Po0.001; Figure 5ciii). We also transiently expressed constitutively active Rac1 in VB6 and H357 cells that had been transfected with Eps8 RNAi. Expression of V12-Rac1 significantly restored migration in all cell lines (Figure 5cii and iv; P ¼ 0.02, P ¼ 0.01, respectively). These data confirmed that Eps8 modulates both avb6and a5b1-mediated, Rac1-dependent OSCC migration. avb6 and a5b1 integrin-dependent activation of Rac1 is modulated by Eps8 We have shown previously that Rac1 is activated in VB6 cells upon avb6 ligand-binding (Nystrom et al., 2006) and our migration experiments suggested that Eps8 may regulate both avb6- and a5b1-dependent Rac1 activation. We therefore carried out pull-down assays using VB6 cells plated onto poly-d-lysine (control substrate) or LAP (avb6-specific binding) and H357 cells plated onto polyD-lysine or fibronectin (a5b1-specific binding). Consistent with our previous findings, avb6-specific attachment of VB6 cells to LAP-induced Rac1 activation and this activation was reduced following Eps8 knockdown (by B70%; Figure 5d). Similarly, Rac1 activation was also seen in H357 cells plated on fibronectin (a5b1specific), and this was reduced by B90% following Eps8 knockdown. These data confirmed that Eps8 was required for avb6- and a5b1-dependent Rac1 activation. Eps8 knockdown inhibits OSCC invasion in organotypic culture To study tumor invasion, we have developed an organotypic culture model, which recapitulates the morphology of OSCC and is more physiologically relevant than Transwell assays (Nystrom et al., 2005, 2006). The invasion of VB6 cells into the underlying stroma of organotypic cultures is Rac1-dependent (Nystrom et al., 2006). Consistent with this finding, Eps8 knockdown markedly inhibited invasion of VB6 cells (Figure 6). Invasion of BICR56 cells was also significantly inhibited following Eps8 knockdown (Figure 6). CA1 cells invade poorly in this type of assay, with little invasion observed (data not shown). Discussion Most patients with OSCC are treated by surgery, and over 50% of patients die within 5 years. The identification of genes differentially expressed between normal and malignant cells will lead to a more comprehensive understanding of the molecular events that drive the pathogenesis of the disease and may lead to the identification of novel therapeutic targets. In this study, we used high-density oligonucleotide microarrays to profile gene expression in eight OSCC cell lines and three cultures of normal oral keratinocytes. We found that 309 named genes were differentially expressed in the OSCC cell lines, with a predominance of underexpressed genes relative to overexpressed genes (230 vs 79). The


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Figure 3 Eps8 knockdown inhibits OSCC cell migration and spreading on fibronectin. (a) OSCC cell lines transfected with control (Ctl) or Eps8 siRNA were analysed by western blotting with the indicated antibodies. (b) VB6, BICR56 and CA1 cells were transfected with control or Eps8 siRNA and examined in fibronectin-coated Transwell migration assays. Data are expressed as mean percentage of cells migrating ±s.d. and the results are expressed relative to migration of control siRNA transfected cells ( ¼ 100%). The migration of VB6, BICR56 and CA1 cells was significantly inhibited in cells transfected with Eps8 siRNA compared to controls (VB6, Po0.02; BICR56 Po0.02; CA1 Po0.0003). (c) Following transfection with siRNA cells were plated onto fibronectin-coated glass coverslips and fixed at the indicated time points. Cell spreading, as determined by counting spread and non-spread cells in 7 high power fields was significantly inhibited by Eps8 knockdown (Po0.0004 for each cell line at 2 h). The results represent the percentage spread cells ±s.d. Cells were also stained with phalloidin-TRITC to detect actin and images collected digitally on a confocal microscope. (d) VB6, BICR56 and CA1 cells were transfected with EV or mouse Eps8EGFP following Eps8 knockdown and examined in fibronectin-coated Transwell migration assays. Cells expressing mouse Eps8EGFP following transfection with human Eps8-specific siRNA had their migration restored and did not differ significantly from controls (P ¼ 0.389; P ¼ 0.177; P ¼ 0.131 respectively). (e) VB6 cells were plated onto LAP-coated glass coverslips and fixed at time 1 h (top panel) and 2 h (bottom panel). Cells were immunostained and images collected digitally on a confocal microscope. Micrograph shows nuclei (blue; i and v), Eps8 staining (green; ii and vi), actin (red iii and vii) and merged (iv and viii) images. Eps8 and actin co-localized at the plasma membrane of spreading cells.

OSCC cell lines could be distinguished from normal cells based on the expression pattern of this set of genes. Consistent with previous microarray studies of OSCC, this study showed the deregulated expression of various keratin genes, together with cell adhesion molecules and differentiation markers, supporting the general notion that loss of cell structure and differentiation is a critical step in the development of oral cancer (Al Moustafa et al., 2002; Jeon et al., 2004). Furthermore, the expression of a number of specific genes showed similar changes to those reported in previous studies, namely syndecan-1, stratifin, FAT2, interleukin-1 receptor antagonist, secreted frizzled

related protein 1 and aldehyde dehydrogenase (Alevizos et al., 2001; Al Moustafa et al., 2002; Cromer et al., 2004; Jeon et al., 2004). Our microarray experiments indicated that the expression of Eps8 was >5 fold higher in OSCC cells relative to normal oral keratinocytes. The same series of cell lines were used to validate the results, which demonstrated that Eps8 mRNA and protein levels were elevated in six of eight cell lines (Figure 1). Our data are consistent with the limited number of reports that examined Eps8 expression in tumor-derived cell lines and tissues (Jeon et al., 2004; Yao et al., 2006; Maa et al., 2007; Welsch et al., 2007). We Oncogene


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Figure 4 Eps8-dependent migration is modulated through Rac1. (a) Fibronectin-coated Transwell migration assays were carried out using OSCC cell lines transfected with control (Ctl), Eps8 or Rac1 siRNA. Rac1 and Eps8 knockdown produced similar levels of inhibition of migration (Eps8 Po0.005 and Rac1 Po0.005 for all cell lines) and no further inhibition was observed when a combination of Eps8 and Rac1 siRNA was used. Western blotting confirmed protein knockdown. (b), OSCC cells were treated with Eps8 RNAi, and 24 h later transfected with empty vector (EV) or V12-Rac1. Cells expressing V12-Rac1 following transfection with Eps8 RNAi had their migration significantly restored compared with empty vector controls (Pp0.005 for all three cell lines). Western blotting confirmed protein knockdown.

next examined the expression of Eps8 protein in tissue samples of primary OSCCs and normal oral mucosa and we show for the first time that Eps8 is expressed in a subset (32%) of OSCCs, whereas no Eps8 expression can be detected in either normal oral epithelium or epithelium adjacent to the carcinomas (Figure 2). Eps8 was detected in the cytoplasm of tumor cells, consistent with the known functions of Eps8 as an intracellular regulator of RTK signaling. Further, Eps8 expression was detected in salivary duct epithelium, which is in agreement with the observation that Eps8 is expressed in submandibular glands in developing mouse embryos (Inobe et al., 1999), results which confirm the specificity of the antibody. The percentage of poorly differentiated tumors that expressed Eps8 was higher than that of more well differentiated tumors and Eps8 expression also increased with T category, but these results did not reach statistical significance. However, Eps8 positivity correlated significantly with cervical lymph node metastasis and was strongly associated with advanced tumor stage (Table 1). Our data together with reports that Eps8 is upregulated in cell lines derived from metastases compared with those derived from primary tumors (Yeudall et al., 2005; Welsch et al., 2007), strongly suggests that Eps8 plays a causal role in metastatic dissemination. To examine the functional role of Eps8 in oral carcinogenesis, we used siRNA to examine the effect of Eps8 knockdown on tumor behaviour in vitro. Knockdown of Eps8 in BICR56, CA1 and VB6 OSCC cells had no effect on cell proliferation for up to 72 h post transfection. Oncogene

Our results contrast with data to show that Eps8 overexpression in NIH 3T3 fibroblasts and 32D myeloblasts enhanced the mitogenic responsiveness of these cells to EGF (Fazioli et al., 1993) and the recent observation that Eps8 knockdown reduces the proliferation of colon cancer cells expressing high levels of Eps8 (Maa et al., 2007). We show that Eps8 knockdown inhibits OSCC cell migration and spreading. Our data are consistent with recent reports implicating Eps8 in the regulation of tumor cell migration (Maa et al., 2007; Welsch et al., 2007), and show for the first time that Eps8 is critical for integrindependent, Rac1-mediated tumor cell motility. Eps8 is known to participate in growth factordependent Rac1 activation (Scita et al., 1999, 2001). In this study, we show that Eps8 is also required for integrin-dependent Rac1 activation and for integrindependent cell migration, modulating both a5b1 and avb6-dependent cell motility. We also demonstrate that inhibition of a5b1, avb6, Rac1 or Eps8 suppressed OSCC cell migration to a similar extent and that the expression of constitutively active Rac1 in Eps8depleted OSCC cells restored migration. Together, these data suggest that Eps8 and Rac1 act together within the same pathway to regulate integrin-mediated cell migration. However, overexpression of Eps8 in H103 and H157 OSCC cells (which express relatively low levels of endogenous Eps8), did not lead to an increase in cell migration or Rac1 activation (Supplementary Figure 3), indicating that Eps8 alone is not sufficient to promote tumor cell motility.


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Figure 5 Eps8 modulates integrin-dependent Rac1 activation and migration. (a) FACS analysis showed VB6, BICR56 and CA1 cells express both avb6 and a5b1 fibronectin receptors. Results represent mean fluorescence (arbitrary units, log scale). (b) OSCC cells were incubated with specific integrin-blocking antibodies prior to Transwell migration assays towards fibronectin. Although antibodies directed against avb6 or a5b1 produced some level of suppression when used alone, maximal abrogation of migration required inhibition of both integrins in combination. (c) VB6 and H357 cells were transfected with control (Ctl), Eps8 or Rac1 siRNA, or treated with integrin-blocking antibodies (VB6, avb6; H357, a5b1) prior to performing Transwell cell migration assays towards either LAP (VB6) or fibronectin (H357). In addition, following Eps8 knockdown, cells were transfected with empty vector (EV) or V12-Rac1. Western blotting confirmed protein knockdown. (i) Migration of VB6 towards LAP was avb6-dependent, and significantly inhibited by Eps8 or Rac1 knockdown. (ii) VB6 cells expressing V12-Rac1 following transfection with Eps8 RNAi had their migration significantly restored, but not when transfected with empty vector control (P ¼ 0.02). (iii) Migration of H357 towards fibronectin was a5b1dependent, and significantly inhibited by Eps8 or Rac1 knockdown. (iv) H357 cells expressing V12-Rac1 following transfection with Eps8 RNAi had their migration significantly restored, but not when transfected with empty vector control (P ¼ 0.01). (d) OSCC cells were transfected with control or Eps8 siRNA and plated onto avb6 or a5b1 integrin-specific substrates (VB6, LAP; H357, fibronectin, FN) or poly-D-lysine (PDL). Pull-down assays showed that avb6- or a5b1-induced Rac1 activation was suppressed following Eps8 knockdown (by 70 and 90%, respectively). Western blotting confirmed protein knockdown.

In this study, Eps8 co-localized with actin at the plasma membrane of spreading cells, consistent with previous observations that Eps8 localizes to the tips of F-actin filaments, filopodia and the leading edge of cancer cells (Welsch et al., 2007). It is possible that Eps8 may activate Rac1 directly at the leading edge as a complex with Abi1 and Sos1. In addition, it has been shown that integrins target Rac1 to the plasma membrane in lipid rafts, localizing GTP-ase activity to areas within the cell where actin remodeling is required and, in migrating cells, inhibition of FAK suppresses lipid raft internalization, thereby maintaining Rac1 activation (del Pozo et al., 2000, 2004). Eps8 has been shown to regulate FAK

expression and activation (Maa et al., 2007), which suggests an additional mechanism by which Eps8 may regulate the activation and localization of Rac1. The role of Eps8 in modulating avb6-dependent tumor cell motility is particularly interesting, because upregulation of this integrin has been described in numerous carcinomas where expression often correlates with a poor prognosis (Bates et al., 2005; Elayadi et al., 2007). We have shown previously that around 80–90% of OSCC express high levels of avb6, and that it promotes tumor cell migration and invasion (Thomas et al., 2001; Nystrom et al., 2006). Furthermore, we have shown recently that avb6-dependent invasion of VB6 Oncogene


2532 cell lines and three primary cultures of NKs. Differentially expressed genes were identified using significance analysis of microarrays with a fold change threshold of two and a Q-value threshold of 5% (Tusher et al., 2001). Two-way hierarchical clustering analysis was performed using dChip (http:// www.dchip.org).

Figure 6 Eps8 modulates VB6 and BICR56 invasion in organotypic culture. OSCC cells were transiently transfected with control or Eps8 RNAi and then grown in organotypic culture with fibroblasts for 6 days. Eps8 knockdown markedly inhibited invasion of VB6 and BICR56 cells. Western blotting confirmed protein knockdown 7 days post transfection.

cells requires activation of Rac1 (Nystrom et al., 2006). Consistent with this we show that Eps8 knockdown suppressed OSCC invasion in organotypic culture, suggesting that elevated levels of Eps8 in OSCC promotes tumour invasion. In summary, we report that Eps8 is overexpressed in a subset of OSCCs, and this expression correlates with the presence of nodal metastasis. We demonstrate that Eps8 and Rac1 are part of an integrated signaling pathway modulating integrin-dependent tumor cell motility, and that OSCC migration and invasion are inhibited following suppression of Eps8. These data suggest that specific inhibitors of Eps8 could prove useful as therapeutic agents.

Materials and methods Cell lines and tissues Details and culture conditions for the OSCC cell lines, normal oral keratinocytes (NK) and fibroblasts have been described earlier (Prime et al., 1990; Nystrom et al., 2006; Thirthagiri et al., 2007; Marsh et al., 2008). Paraffin-embedded archival tissue samples were obtained from patients OSCC (n ¼ 59). Paraffin-embedded samples of normal oral mucosa (n ¼ 9) were used as controls. Approval for this study was obtained from the Ethics Committee of the United Bristol Healthcare Trust. Expression microarray analysis Total RNA was extracted from cells using the RNeasy Total RNA Mini Kit (Qiagen, Crawley, West Sussex, UK). The cDNA was synthesized, in vitro transcribed, labeled and hybridized to Affymetrix HG-U133A or HG-U133 Plus 2.0 chips, using standard Affymetrix protocols. The scanned images of microarray chips were analysed using the Affymetrix GeneChip Operating Software (Affimetrix, High Wycombe, Buckinghameshire, UK). Probes on HG-U133A and HGU133 Plus 2.0 chips were matched using the matchprobes package of the Bioconductor (http://www.bioconductor.org) project. Probe level quantile normalization and robust multiarray analysis (Irizarry et al., 2003) on the raw CEL files were performed using the Affymetrix package of Bioconductor project. Gene expression was compared between eight OSCC Oncogene

RT–PCR and quantitative real-time PCR Semi-quantitative RT–PCR was performed with standard protocols. Single-stranded cDNA was synthesized using Superscript II (200 units/ml; Invitrogen, Paisley, UK) prior to PCR. The primers were: Eps8 50 -CAGAATCCTAGTGCTG CAGA-30 and 50 -CTGCCGTTCATCACCATTGA-30 ; GAPDH 50 -CCACCCATGGCAAATTCCATGGCA-30 and 50 -CTA GACGGCAGGTCAGGTCCAC-30 . Real-time PCR using the ABI Prism 7000 Sequence Detection System and ABI SYBR Green PCR kit (Applied Biosystems, Foster City, CA, USA) was performed following the manufacturer’s protocol. The GAPDH primers were 50 -AAGGTGAAGGTCGGAGT-30 and 50 -GAAGATGGTGATGGGATTTC-30 . Western blotting Cells were lysed in 30–50 ml of ice-cold RIPA buffer [0.15 M NaCl, 1% (v/v) Nonidet P40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 50 mM Tris-HCl (pH 8.0)] containing protease inhibitors (cocktail set III; Calbiochem, Merck Chemicals, Beeston, Nottingham, UK), 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 5 mM b-glycerophosphate and 49.5 mM sodium fluoride. Blots were probed with mouse monoclonal antibodies against Eps8 (1:5000; Transduction Laboratories, Oxford, UK) or Rac1 (1:3000; Upstate, Millipore, Watford, Hertfordshire, UK) and peroxidaseconjugated sheep anti-mouse IgG (1:1000; Sigma, Gillingham, Dorset, UK) used as the secondary antibody. Bound antibodies were detected using Enhanced Chemiluminescence (Amersham, GE Healthcare, Amersham, Bucks, UK). Immunohistochemistry Eps8 immunostaining was examined using a biotin–streptavidin peroxidase method (StrAviGen, Biogenex, San Ramon, CA, USA). Antigen retrieval was by microwaving the sections in 0.01 M citrate buffer (pH 6.0) for 30 min and endogenous peroxidase activity quenched using 3% (v/v) hydrogen peroxide for 15 min. Sections were washed in PBS (3 2 min) and then incubated with primary anti-Eps8 mouse monoclonal antibody (1:20) in PBS containing 1% (v/v) normal goat serum. Sections were washed in PBS (3 2 min) and the biotinylated secondary antibody ‘Multilink’ (1:50 dilution in PBS) was applied for 30 min. After further washes in PBS (3 2 min), peroxidase-conjugated streptavidin ‘Label’ (1:50 dilution in PBS) was added to the sections for a further 30 min. After final rinses in PBS (3 2 min), the sections were covered with two drops of freshly prepared diaminobenzide reagent and incubated for 6 min. Following a wash in tap water, the sections were counterstained with Mayer’s haematoxylin [1% (w/v) haematoxylin, 0.11 M ammonium aluminium sulphate, 0.001 M sodium iodate, 0.0048 M citric acid, 0.3 M chloral hydrate] for 40 s, washed in tap water for 5 min, dehydrated in graded alcohols, cleared in xylene and mounted in XAM. Omission of the primary antibody acted as a negative control. Modulation of Rac1 activity and Eps8 overexpression Cells were transfected with EGFP-tagged, constitutively active Rac1 (V12Rac1-GFP (construct made by J Monypenny, GKT, London, UK), EGFP-tagged mouse Eps8 (generously


2533 provided by G Scita, University of Milan) or vector control (pEGFP-C2; Invitrogen). Cells at 50% confluency in 6-well dishes were transfected with 2 mg of DNA using Fugene (Roche Diagnostics, West Sussex, UK). 24 h post-transfection, cells were used in migration assays and analysed by confocal microscopy to determine transfection efficiency, which typically was between 70–95%. RNA interference To knockdown Eps8, pre-designed siRNA oligonucleotides (Applied Biosystems) were used, together with control (random) siRNA. 5 104 cells per well were seeded into 6-well plates, cultured for 16 h and then transfected with the relevant siRNA (30 nM) using Oligofectamine transfection reagent (Invitrogen). The sequences of the three EPS8 siRNAs are (1) 50 GGGCAAAGUGUGGACUCAAtt30 (2) 50 GGAGA GGGUGUUUUAACGCtt30 and (3) 50 GGCCCUUUAUG AACAAAGGtt30 . RNAi SMART pool reagents targeting Rac1 or control (random) sequences were obtained from Dharmacon (Chicago, IL, USA) and used at 100 nM. Rescue experiments were performed by specifically knocking down human Eps8 with the siRNA 50 AGAGCCAACCCAG AACAAGtt30 and rescuing with EGFP-tagged mouse Eps8 cDNA or the empty vector (pEGFP-C2). Cell migration assays Cells were transfected with siRNAs and after 48 h haptotactic migration assays were carried out using fibronectin-coated (10 mg/ ml) or TGF-b1 latency-associated peptide (LAP)-coated (0.5 mg/ ml) polycarbonate filters (8 mm pore size, Transwell, Costar, Corning, Schiphol-Rijk, The Netherlands), which were performed as described (Thomas et al., 2001). Cells were plated into the upper chamber and allowed to migrate for 24 h. Cells migrating to the lower chamber were trypsinized and counted on a Casy 1 counter (Scha¨rfe System GmbH, Reutlingen, Germany). In some experiments, the cells further transfected with V12Rac1GFP or Eps8-GFP 24 h after transfection with siRNA. For blocking experiments, anti-a5b1 or -avb6 (6.3G9) antibodies (10 mg/ml) were added to the cells for 30 min before plating. Cell spreading assay and confocal microscopy Glass coverslips were coated with fibronectin (10 mg/ml) or LAP (0.5 mg/ml) in a-MEM for 1 h at 37 1C, washed with PBS then blocked with 0.1% BSA at 37 1C for 30 min. Cells were resuspended in serum-free a-MEM containing 0.1% BSA and incubated at 37 1C. At given times, unattached cells were removed by rinsing the wells with warm PBS. Attached cells were fixed in 4% paraformaldehyde. Spread cells were counted in seven representative high power fields at different time points. Non-spread cells were defined as small round cells with little or no membrane protrusions, whereas spread cells were defined as large cells with extensive visible lamellipodia (Dormond et al., 2001). Results represent the percentage of spread cells in seven high power fields ±s.d.

For Eps8 staining, cells were permeabilized with 0.1% Triton X-100 for 10 min, followed by incubation for 60 min in PBS containing 0.1% BSA. Eps8 primary antibody was used at a dilution of 1:100. Actin was visualized with TRITCconjugated phalloidin (5 ng/ml; Sigma). Nuclei were visualized using DAPI (Invitrogen). Coverslips were washed three times for 5 min in wash buffer and mounted with MOWIOL 4–88 (0.1 g/ml of Citifluor mounting medium; Novabiochem, Merck Chemicals, Beeston, Nottingham, UK) and viewed with a confocal laser-scanning microscope (Zeiss LSM510, Zeiss, Welwyn Garden City, Hertfordshire, UK). Determination of Rac1 activity Rac1 activation assays were carried out as described earlier (Nystrom et al., 2006). Briefly, cells were plated onto 6-cm plates coated with poly-D-lysine (100 mg/ml), LAP (0.5 mg/ml) or fibronectin (10 mg/ml) and lysed on ice after 4 h. Cleared cell lysates were incubated with GST-PAK-CRIB beads for 1 h at 4 1C. Samples of lysates pre- and post-incubation with the GST-PAK-CRIB beads were analysed by western blotting. Flow cytometry Flow cytometry was performed using anti-a5b1/-avb6 antibodies and FITC-conjugated secondary antibody (DAKO, Ely, Cambridgeshire, UK), as described (Thomas et al., 2001). Negative controls used secondary antibody only and was subtracted from the results. Labelled cells were scanned on a FACSCaliber cytometer (BD Biosciences, Oxford, UK) and analysed using CellQuest software, acquiring 1 104 events. Organotypic culture Organotypic cultures were prepared, as described (Nystrom et al., 2006). Gels comprised a 50:50 mixture of Matrigel (Becton-Dickinson, Oxford, UK) and type I collagen (Upstate) containing 5 105/ml HFFF2 or HFF fibroblasts, to which were added 5 105 OSCC cells that had been transfected with Eps8 or control RNAi 24 h previously. After six days the gels were bisected, fixed in formal-saline and processed to paraffin. Sections (4 mm) were stained with H&E. Statistical analysis For the immunohistochemical studies, comparisons between groups were by Fisher’s exact test. For migration assays, statistical differences between experimental groups were evaluated by Student’s t-test. Conflict of interest The authors declare no conflict of interest. Acknowledgements The Cancer Research Initiatives Foundation, Malaysia and The Health Foundation supported the research.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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