Oncogene (2009) 28, 2524–2534 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc 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

1Department 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; 4Cancer 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 Introduction and early death usually occurs as a result of local invasion and regional lymph node metastases. Current treatment Oral squamous cell carcinoma (OSCC) is the sixth most regimens are, to a certain degree, inadequate, with a 5-year common malignancy worldwide (Parkin et al., 2005). mortality rate of around 50% and novel therapeutic targets Cervical lymphnode metastasis is themost important are urgently required. Using expression microarrays, we prognostic indicator and patients withmetastases that identified the eps8 as being overexpressed in OSCC cell show evidence of invasion into the parenchymal tissues lines relative to normal oral keratinocytes, and confirmed of the neck have been shown to have the worst prognosis these findings using RT–PCR and western blotting. In (Myers et al., 2001; Woolgar et al., 2003). Although the human tissues, we found that Eps8 was upregulated in molecular basis for the development of OSCC is starting OSCC (32% of primary tumors) compared with normal oral to be elucidated (Kim and Califano, 2004), the factors mucosa, and that expression correlated significantly with that influence invasion and metastasis are poorly lymph node metastasis (P ¼ 0.032), suggesting a disease- understood. promoting effect. Using OSCC cell lines, we assessed the The epidermal growth factor receptor pathway functional role of Eps8 in tumor cells. Although suppression substrate 8 gene (Eps8) was originally identified as a of Eps8 produced no effect on cell proliferation, both cell substrate for the epidermal growth factor receptor spreading and migration were markedly inhibited. The latter kinase (Fazioli et al., 1993). Eps8 was later shown to cell functions may be modulated through the small GTP-ase, form a complex withSos1, Eps8 SH3 domain-binding Rac1 and we used pull-down assays to investigate the role of protein 1 (E3b1; also known as Abi1) and the p85 Eps8 in Rac1 signaling. We found that avb6-and a5b1- regulatory subunit of PI3K, resulting in the activation of integrin-dependent activation of Rac1 was mediated through Rac1 and subsequent remodeling (Scita et al., Eps8. Knockdown of either Eps8 or Rac1, inhibited integrin- 1999; Innocenti et al., 2003). More recently, it has been dependent cell migration similarly and transient expression shown that Eps8 is a novel actin capping protein that is of constitutively active Rac1 restored migration of cells in essential for cell motility by regulating the growth of which Eps8 expression had been suppressed. We also showed actin filaments to generate propulsive force (Disanza that knockdown of Eps8 inhibited tumor cell invasion in an et al., 2004). The C-terminal effector region of Eps8 organotypic model of OSCC. These data suggest that Eps8 possesses actin filament barbed-end capping activity, and Rac1 are part of an integrated signaling pathway recruiting Eps8 to the actin dynamic sites, such as modulating integrin-dependent tumour cell motility and membrane ruffles, where E3b1, Sos1 and p85 also identify Eps8 as a possible therapeutic target. localize (Scita et al., 2001; Innocenti et al., 2003; Croce Oncogene (2009) 28, 2524–2534; doi:10.1038/onc.2009.105; et al., 2004; Disanza et al., 2004). Eps8 has also been published online 18 May 2009 shown to increase the expression and activity of focal adhesion kinase (FAK; Maa et al., 2007), which raises Keywords: Eps8; oral cancer; integrin; migration; Rac1; the possibility that Eps8 may be involved in the invasion transduction of integrin-induced signals. Overexpression of Eps8 in fibroblasts has been shown to induce malignant transformation in vitro and Correspondence: Professor GJ Thomas, Cancer Sciences Division, promote tumor formation in vivo (Matoskova et al., Southampton University School of Medicine, Tremona Road, 1995; Maa et al., 2001), suggesting that Eps8 is an Southampton, UK. oncogene. Eps8 has also been shown to form a complex E-mail: [email protected] withIRSp53, an adaptor protein between Rho-family 8These authors contributed equally to this work. 9These authors jointly supervised this study. small GTPases and actin cytoskeleton reorganizing Received 16 June 2008; revised 23 March2009; accepted 24 March2009; proteins (Miki et al., 2000), in fibroblasts and various published online 18 May 2009 cancer cell lines to augment Eps8/E3b1/Sos1 complex Eps8 upregulation promotes cell migration and invasion LF Yap et al 2525 formation, resulting in activation of Rac1 (Funato et al., seven OSCCs using quantitative real-time PCR. Com- 2004). Eps8 has been reported to be upregulated in pared to normal mucosa, the expression level of Eps8 primary tumors of the breast, pancreas and colon (Yao was increased in five out of seven tumors analysed et al., 2006; Maa et al., 2007; Welsch et al., 2007), but its (Figure 1d). functional role in epithelial carcinogenesis remains to be clarified. Immunohistochemical analysis of Eps8 expression Using expression microarrays, we have identified in primary OSCC tissues Eps8 as being overexpressed in OSCC-derived cell The expression and cellular location of Eps8 was lines compared withnormal oral keratinocytes and examined in 59 formalin-fixed paraffin-embedded pri- showed that Eps8 is upregulated in a subset of OSCCs mary OSCCs and nine samples of normal oral mucosa where it correlates significantly with metastatic disease. by immunohistochemistry. The clinical characteristics of Knockdown of Eps8 in OSCC cell lines did not effect the tumor samples are listed in Supplementary Table 3. cell proliferation, but cell spreading and migration were Normal oral epithelium and epithelium adjacent to markedly inhibited. We next investigated the effect of carcinomas (present in 57 of 59 samples) were consis- Eps8 knockdown on Rac1 signaling and showed that tently negative, whereas endothelium and salivary duct integrin-dependent activation of Rac1 was mediated epithelium was always positive, demonstrating the through Eps8. Knockdown of Eps8 or Rac1, inhibited specificity of the Eps8 antibody (Figures 2a–d). Eps8 cell migration similarly and transient expression of expression was detected in 19 of 59 (32%) of the tumors constitutively active Rac1 restored the migration of cells examined. The staining was cytoplasmic and ranged which had been transfected with Eps8 RNAi. Finally, from weak and focal to strong and diffuse (Figures 2e–j). we demonstrated that Eps8 promotes OSCC invasion in Eps8 expression was examined in the context of organotypic assays. Therefore, Eps8 appears to be an clinicopathological characteristics of the tumors important mediator of integrin-dependent tumor cell (Table 1). There was no significant association between motility as part of an integrated signaling pathway the expression of Eps8 protein and the degree of cellular involving Rac1. differentiation (P ¼ 0.116) and T category (P ¼ 0.168). By contrast, Eps8 expression correlated significantly withlymphnode metastasis ( N category; P ¼ 0.032) and Results was strongly associated withadvanced tumor stage, although this association did not reach statistical differences between NKs and significance (P ¼ 0.057). OSCC-derived cell lines To identify whose transcription is deregulated in Inhibition of Eps8 expression suppresses cell migration OSCC, the gene expression profiles of eight OSCC cell and spreading lines (H-series and M9) and three primary cultures of To investigate the functional role of Eps8 in vitro, normal oral keratinocytes (NKs) were examined using we transfected OSCC cell lines withEps8 siRNA Affymetrix HG-U133A and HG-U133 Plus 2.0 arrays (Figure 3a; Supplementary Figure 1). Sequence 3 gave and the significance of gene expression changes using the most efficient knockdown and was used in sub- significance analysis of microarrays analysis. Genes sequent experiments. VB6, BICR56 and CA1 express whose expression levels were increased or decreased by high levels of endogenous Eps8 and we have shown two-fold or greater between normal and tumor cells previously that these cells migrate well in vitro (Thomas witha Q-value threshold of less than 5% were selected et al., 2001; Ramsay et al., 2007). Although inhibition of and 309 named genes were identified, with79 being Eps8 expression had no effect on cell growth in any upregulated (Supplementary Table 1) and 230 being of the cell lines (data not shown), migration of VB6, downregulated (Supplementary Table 2) in tumor cells BICR56 and CA1 cells towards fibronectin was sig- relative to NKs. Two-way hierarchical clustering analy- nificantly suppressed (VB6, Po0.02; BICR56 Po0.02; sis was used to group the cell lines based on the degree of CA1 P 0.0003; Figure 3b). No suppression was evident similarity of their expression patterns using the 309 o in a further OSCC cell line, H400 (data not shown). Cell named genes and the normal and malignant cells formed spreading on fibronectin, as assessed by changes in cell two distinct groups (Figure 1a). morphology and lamellipodia formation, was also inhibited by Eps8 knockdown in VB6, BICR56 and Eps8 is overexpressed in OSCC-derived cell lines and CA1 cells (Po0.0004 for all cell lines at 2 h; Figure 3c). tissues Cells transfected withEps8 siRNA were generally By microarray, the expression of Eps8 was enhanced smaller withless extensive protrusions thancontrol >5-fold in the OSCC cell lines relative to NKs. Semi- transfected cells (Figure 3c). quantitative RT–PCR showed that Eps8 mRNA levels To confirm that the effects of the siRNA were specific, were elevated in six out of the eight cell lines (Figure 1b) we knocked down the endogenous Eps8 using siRNA and Eps8 protein could be detected in the same six cell specific to the human sequence and restored Eps8 lines by western blotting but was undetectable in the expression using a mouse Eps8 cDNA. Transfection NKs (Figure 1c). The expression of Eps8 was then with the mouse Eps8 cDNA rescued the inhibition of examined in five samples of normal oral mucosa and cell migration caused by knocking down endogenous

Oncogene Eps8 upregulation promotes cell migration and invasion LF Yap et al 2526

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 Knockdown of Eps8 and Rac1 inhibit migration additional siRNAs (sequences 1 and 2) also inhibited to a similar extent cell migration (Supplementary Figure 1), providing We next investigated the relative contributions of Eps8 additional confirmation that the effects of the siRNA and Rac1 to OSCC migration using siRNA to knock- were specific. down these proteins, either alone or in combination. In VB6, BICR56 and CA1 cells, knockdown of either Subcellular distribution of Eps8 Eps8 or Rac1 significantly inhibited migration towards To examine the subcellular distribution of Eps8, we fibronectin (Eps8 Po0.005 and Rac1 Po0.005 for all plated VB6 cells onto matrix-coated glass coverslips and cell lines); knockdown of bothproteins did not further stained for Eps8 using immunoflourescence. In actively inhibit migration (Figure 4a). spreading cells (o4 h), Eps8 co-localized with actin at To confirm whether Eps8-dependent cell migration is the cell plasma membrane (Figure 3e). However, in cells modulated through Rac1, we transiently expressed that were fully spread (>4 h) this co-localization was constitutively active Rac1 (V12-Rac1) in VB6, BICR56 lost, and Eps8 relocated to the cytoplasm (data not and CA1 cells following Eps8 siRNA-treatment. Ex- shown). pression 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 Eps8 upregulation promotes cell migration and invasion LF Yap et al 2527

Oncogene Eps8 upregulation promotes cell migration and invasion LF Yap et al 2528 Table 1 Correlation between Eps8 expression and clinicopathological towards fibronectin was also significantly inhibited with characteristics of OSCCs Eps8 and Rac1 siRNAs or by treatment withcells with Characteristics Eps8 expression P-value an a5b1-blocking antibody (all conditions Po0.001; Figure 5ciii). We also transiently expressed constitu- Differentiation tively active Rac1 in VB6 and H357 cells that had been Poor 7/13 (53%) 0.116 transfected withEps8 RNAi. Expression of V12-Rac1 Moderate 7/21 (33%) Well 5/25 (20%) significantly restored migration in all cell lines (Figure 5cii and iv; P ¼ 0.02,P¼ 0.01, respectively). T category These data confirmed that Eps8 modulates both avb6- T1 4/19 (21%) 0.168 and a5b1-mediated, Rac1-dependent OSCC migration. T2 7/22 (31%) T3 0/3 (0%) T4 8/15 (53%) avb6 and a5b1 integrin-dependent activation of Rac1 is modulated by Eps8 N category We have shown previously that Rac1 is activated in VB6 N0 6/29 (20%) 0.032 N1 1/7 (14%) cells upon avb6 ligand-binding (Nystrom et al., 2006) and N2 11/22 (50%) our migration experiments suggested that Eps8 may N3 1/1 (100%) regulate both avb6- and a5b1-dependent Rac1 activation. We therefore carried out pull-down assays using VB6 cells Stage plated onto poly-d-lysine (control substrate) or LAP I 2/13 (15%) 0.057 II 1/6 (16%) (avb6-specific binding) and H357 cells plated onto poly- III 1/9 (11%) D-lysine or fibronectin (a5b1-specific binding). Consistent IV 15/31 (48%) withour previous findings, avb6-specific attachment of VB6 cells to LAP-induced Rac1 activation and this compared withempty vector controls (Figure 4b; activation was reduced following Eps8 knockdown (by B70%; Figure 5d). Similarly, Rac1 activation was Pp0.005 for all three cell lines). also seen in H357 cells plated on fibronectin (a5b1- specific), and this was reduced by B90% following Eps8 Eps8 is required for both a5b1- and avb6-dependent knockdown. These data confirmed that Eps8 was required migration for avb6- and a5b1-dependent Rac1 activation. To determine which integrins modulate OSCC cell migration towards fibronectin, we first examined the Eps8 knockdown inhibits OSCC invasion expression of fibronectin-binding integrins by flow cyto- in organotypic culture metry. VB6, BICR56 and CA1 cells expressed both a5b1 To study tumor invasion, we have developed an and avb6 (Figure 5a) and migration of the cell lines organotypic culture model, which recapitulates the towards fibronectin was modulated throughbothinteg- morphology of OSCC and is more physiologically rins (Figure 5b). Although antibodies directed against relevant than Transwell assays (Nystrom et al., 2005, either a5b1oravb6 produced some level of suppression 2006). The invasion of VB6 cells into the underlying when used alone, maximal abrogation of migration stroma of organotypic cultures is Rac1-dependent required inhibition of both integrins (Figure 5b). (Nystrom et al., 2006). Consistent withthisfinding, To determine whether Eps8 modulated avb6 and/or Eps8 knockdown markedly inhibited invasion of VB6 a5b1 integrin-dependent migration, cells were trans- cells (Figure 6). Invasion of BICR56 cells was also fected withsiRNAs and treated withspecific inhibitory significantly inhibited following Eps8 knockdown antibodies directed against avb6ora5b1. Antibodies (Figure 6). CA1 cells invade poorly in this type of against both avb6 and a5b1 when used in combination assay, withlittle invasion observed (data not shown). with Eps8 siRNA produced further inhibition of migration; similar results were obtained withRac1 siRNA (Supplementary Figure 2). These results sug- Discussion gested that Eps8-dependent migration was not modu- lated through a single integrin. Therefore, we Most patients withOSCC are treated by surgery, and investigated whether Eps8 modulated both avb6- and over 50% of patients die within 5 years. The identifica- a5b1-dependent migration. We have shown previously tion of genes differentially expressed between normal that VB6 cells adhere to, and migrate towards, the and malignant cells will lead to a more comprehensive latency-associated peptide (LAP) of TGF-b1 solely understanding of the molecular events that drive the through avb6 (Thomas et al., 2002). Similarly, H357 pathogenesis of the disease and may lead to the cells adhere to, and migrate towards, fibronectin solely identification of novel therapeutic targets. In this study, through a5b1 (Thomas et al., 2001). Transfection of we used high-density oligonucleotide microarrays to VB6 cells withsiRNAs against Eps8 or Rac1, or profile gene expression in eight OSCC cell lines and treatment of cells withan avb6-blocking antibody three cultures of normal oral keratinocytes. We found resulted in a significant inhibition of migration that 309 named genes were differentially expressed in the (Eps8 Po0.005; Rac1 Po0.01; anti-avb6 Po0.004; OSCC cell lines, witha predominance of underexpressed Figure 5ci). Similarly, the migration of H357 cells genes relative to overexpressed genes (230 vs 79). The

Oncogene Eps8 upregulation promotes cell migration and invasion LF Yap et al 2529

Figure 3 Eps8 knockdown inhibits OSCC cell migration and spreading on fibronectin. (a) OSCC cell lines transfected withcontrol (Ctl) or Eps8 siRNA were analysed by western blotting withtheindicated antibodies. ( b) VB6, BICR56 and CA1 cells were transfected withcontrol 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 withsiRNA 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 withphalloidin-TRITC to detect actin and images collected digitally on a confocal microscope. ( d) VB6, BICR56 and CA1 cells were transfected withEV 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. Micrographshowsnuclei (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 related protein 1 and aldehyde dehydrogenase (Alevizos cells based on the expression pattern of this set of genes. et al., 2001; Al Moustafa et al., 2002; Cromer et al., 2004; Consistent withprevious microarray studies of OSCC, Jeon et al., 2004). this study showed the deregulated expression of various Our microarray experiments indicated that the expres- keratin genes, together with cell adhesion molecules and sion of Eps8 was >5 fold higher in OSCC cells relative to differentiation markers, supporting the general notion normal oral keratinocytes. The same series of cell lines that loss of cell structure and differentiation is a critical were used to validate the results, which demonstrated that step in the development of oral cancer (Al Moustafa et al., Eps8 mRNA and protein levels were elevated in six of 2002; Jeon et al.,2004).Furthermore,theexpressionofa eight cell lines (Figure 1). Our data are consistent with the number of specific genes showed similar changes to those limited number of reports that examined Eps8 expression reported in previous studies, namely syndecan-1, stratifin, in tumor-derived cell lines and tissues (Jeon et al., 2004; FAT2, interleukin-1 receptor antagonist, secreted frizzled Yao et al., 2006; Maa et al., 2007; Welsch et al.,2007).We

Oncogene Eps8 upregulation promotes cell migration and invasion LF Yap et al 2530

Figure 4 Eps8-dependent migration is modulated through Rac1. (a) Fibronectin-coated Transwell migration assays were carried out using OSCC cell lines transfected withcontrol (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 hlater transfected withempty 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 Our results contrast with data to show that Eps8 samples of primary OSCCs and normal oral mucosa and overexpression in NIH 3T3 fibroblasts and 32D myelo- we show for the first time that Eps8 is expressed in a blasts enhanced the mitogenic responsiveness of these cells subset (32%) of OSCCs, whereas no Eps8 expression can to EGF (Fazioli et al., 1993) and the recent observation be detected in either normal oral epithelium or epithelium that Eps8 knockdown reduces the proliferation of colon adjacent to the carcinomas (Figure 2). Eps8 was detected cancer cells expressing high levels of Eps8 (Maa et al., in the cytoplasm of tumor cells, consistent with the known 2007). We show that Eps8 knockdown inhibits OSCC cell functions of Eps8 as an intracellular regulator of RTK migration and spreading. Our data are consistent with signaling. Further, Eps8 expression was detected in recent reports implicating Eps8 in the regulation of tumor salivary duct epithelium, which is in agreement with the cell migration (Maa et al., 2007; Welsch et al.,2007),and observation that Eps8 is expressed in submandibular show for the first time that Eps8 is critical for integrin- glands in developing mouse embryos (Inobe et al., 1999), dependent, Rac1-mediated tumor cell motility. results which confirm the specificity of the antibody. The Eps8 is known to participate in growthfactor- percentage of poorly differentiated tumors that expressed dependent Rac1 activation (Scita et al., 1999, 2001). In Eps8 was higher than that of more well differentiated this study, we show that Eps8 is also required for tumors and Eps8 expression also increased withT integrin-dependent Rac1 activation and for integrin- category, but these results did not reach statistical dependent cell migration, modulating both a5b1 and significance. However, Eps8 positivity correlated signifi- avb6-dependent cell motility. We also demonstrate that cantly withcervical lymphnode metastasis and was inhibition of a5b1, avb6, Rac1 or Eps8 suppressed strongly associated withadvanced tumor stage (Table 1). OSCC cell migration to a similar extent and that the Our data together with reports that Eps8 is upregulated in expression of constitutively active Rac1 in Eps8- cell lines derived from metastases compared withthose depleted OSCC cells restored migration. Together, these derived from primary tumors (Yeudall et al., 2005; Welsch data suggest that Eps8 and Rac1 act together within the et al., 2007), strongly suggests that Eps8 plays a causal same pathway to regulate integrin-mediated cell migra- role in metastatic dissemination. tion. However, overexpression of Eps8 in H103 and To examine the functional role of Eps8 in oral H157 OSCC cells (which express relatively low levels of carcinogenesis, we used siRNA to examine the effect of endogenous Eps8), did not lead to an increase in cell Eps8 knockdown on tumor behaviour in vitro. Knockdown migration or Rac1 activation (Supplementary Figure 3), of Eps8 in BICR56, CA1 and VB6 OSCC cells had no indicating that Eps8 alone is not sufficient to promote effect on cell proliferation for up to 72 hpost transfection. tumor cell motility.

Oncogene Eps8 upregulation promotes cell migration and invasion LF Yap et al 2531

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 withspecific integrin-blocking antibodies prior to Transwell migration assays towards fibronectin. Althoughantibodies directed against avb6ora5b1 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 withcontrol (Ctl), Eps8 or Rac1 siRNA, or treated withintegrin-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 withempty 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 a5b1- dependent, 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 withcontrol or Eps8 siRNA and plated onto avb6ora5b1 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 expression and activation (Maa et al., 2007), which membrane of spreading cells, consistent withprevious suggests an additional mechanism by which Eps8 may observations that Eps8 localizes to the tips of F-actin regulate the activation and localization of Rac1. filaments, filopodia and the leading edge of cancer cells The role of Eps8 in modulating avb6-dependent (Welsch et al., 2007). It is possible that Eps8 may activate tumor cell motility is particularly interesting, because Rac1 directly at the leading edge as a complex with Abi1 upregulation of this integrin has been described in and Sos1. In addition, it has been shown that integrins numerous carcinomas where expression often correlates target Rac1 to the plasma membrane in lipid rafts, witha poor prognosis (Bates et al., 2005; Elayadi et al., localizing GTP-ase activity to areas within the cell where 2007). We have shown previously that around 80–90% actin remodeling is required and, in migrating cells, of OSCC express high levels of avb6, and that it inhibition of FAK suppresses lipid raft internalization, promotes tumor cell migration and invasion (Thomas thereby maintaining Rac1 activation (del Pozo et al., et al., 2001; Nystrom et al., 2006). Furthermore, we have 2000, 2004). Eps8 has been shown to regulate FAK shown recently that avb6-dependent invasion of VB6

Oncogene Eps8 upregulation promotes cell migration and invasion LF Yap et al 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).

RT–PCR and quantitative real-time PCR Semi-quantitative RT–PCR was performed withstandard 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 Figure 6 Eps8 modulates VB6 and BICR56 invasion in organo- 0 typic culture. OSCC cells were transiently transfected withcontrol GACGGCAGGTCAGGTCCAC-3 . Real-time PCR using or Eps8 RNAi and then grown in organotypic culture with the ABI Prism 7000 Sequence Detection System and ABI fibroblasts for 6 days. Eps8 knockdown markedly inhibited SYBR Green PCR kit (Applied Biosystems, Foster City, CA, invasion of VB6 and BICR56 cells. Western blotting confirmed USA) was performed following the manufacturer’s protocol. protein knockdown 7 days post transfection. TheGAPDHprimerswere50-AAGGTGAAGGTCGGAGT-30 and 50-GAAGATGGTGATGGGATTTC-30. cells requires activation of Rac1 (Nystrom et al., 2006). Consistent with this we show that Eps8 knockdown Western blotting suppressed OSCC invasion in organotypic culture, Cells were lysed in 30–50 ml of ice-cold RIPA buffer [0.15 M suggesting that elevated levels of Eps8 in OSCC NaCl, 1% (v/v) Nonidet P40, 0.5% (w/v) sodium deoxycho- promotes tumour invasion. late, 0.1% (w/v) SDS, 50 mM Tris-HCl (pH 8.0)] containing In summary, we report that Eps8 is overexpressed in a protease inhibitors (cocktail set III; Calbiochem, Merck subset of OSCCs, and this expression correlates with the Chemicals, Beeston, Nottingham, UK), 1 mM sodium ortho- vanadate, 2 mM sodium pyrophosphate, 5 mM b-glyceropho- presence of nodal metastasis. We demonstrate that Eps8 sphate and 49.5 mM sodium fluoride. Blots were probed with and Rac1 are part of an integrated signaling pathway mouse monoclonal antibodies against Eps8 (1:5000; Transduc- modulating integrin-dependent tumor cell motility, tion Laboratories, Oxford, UK) or Rac1 (1:3000; Upstate, and that OSCC migration and invasion are inhibited Millipore, Watford, Hertfordshire, UK) and peroxidase- following suppression of Eps8. These data suggest that conjugated sheep anti-mouse IgG (1:1000; Sigma, Gillingham, specific inhibitors of Eps8 could prove useful as Dorset, UK) used as the secondary antibody. Bound therapeutic agents. antibodies were detected using Enhanced Chemiluminescence (Amersham, GE Healthcare, Amersham, Bucks, UK).

Materials and methods Immunohistochemistry Eps8 immunostaining was examined using a biotin–streptavi- Cell lines and tissues din peroxidase method (StrAviGen, Biogenex, San Ramon, Details and culture conditions for the OSCC cell lines, normal CA, USA). Antigen retrieval was by microwaving the sections oral keratinocytes (NK) and fibroblasts have been described in 0.01 M citrate buffer (pH 6.0) for 30 min and endogenous earlier (Prime et al., 1990; Nystrom et al., 2006; Thirthagiri peroxidase activity quenched using 3% (v/v) hydrogen et al., 2007; Marsh et al., 2008). peroxide for 15 min. Sections were washed in PBS (3 Â 2 min) Paraffin-embedded archival tissue samples were obtained and then incubated with primary anti-Eps8 mouse monoclonal from patients OSCC (n ¼ 59). Paraffin-embedded samples of antibody (1:20) in PBS containing 1% (v/v) normal goat normal oral mucosa (n ¼ 9) were used as controls. Approval serum. Sections were washed in PBS (3 Â 2 min) and the for this study was obtained from the Ethics Committee of the biotinylated secondary antibody ‘Multilink’ (1:50 dilution in United Bristol Healthcare Trust. PBS) was applied for 30 min. After further washes in PBS (3 Â 2 min), peroxidase-conjugated streptavidin ‘Label’ (1:50 dilution in PBS) was added to the sections for a further 30 min. Expression microarray analysis After final rinses in PBS (3 Â 2 min), the sections were covered Total RNA was extracted from cells using the RNeasy Total withtwo drops of freshlyprepared diaminobenzide reagent RNA Mini Kit (Qiagen, Crawley, West Sussex, UK). The and incubated for 6 min. Following a washin tap water, the cDNA was synthesized, in vitro transcribed, labeled and sections were counterstained withMayer’s haematoxylin[1% hybridized to Affymetrix HG-U133A or HG-U133 Plus 2.0 (w/v) haematoxylin, 0.11 M ammonium aluminium sulphate, chips, using standard Affymetrix protocols. The scanned 0.001 M sodium iodate, 0.0048 M citric acid, 0.3 M chloral images of microarray chips were analysed using the Affymetrix hydrate] for 40 s, washed in tap water for 5 min, dehydrated in GeneChip Operating Software (Affimetrix, High Wycombe, graded alcohols, cleared in xylene and mounted in XAM. Buckinghameshire, UK). Probes on HG-U133A and HG- Omission of the primary antibody acted as a negative control. U133 Plus 2.0 chips were matched using the matchprobes package of the Bioconductor (http://www.bioconductor.org) project. Probe level quantile normalization and robust multi- Modulation of Rac1 activity and Eps8 overexpression array analysis (Irizarry et al., 2003) on the raw CEL files were Cells were transfected withEGFP-tagged, constitutively active performed using the Affymetrix package of Bioconductor Rac1 (V12Rac1-GFP (construct made by J Monypenny, project. Gene expression was compared between eight OSCC GKT, London, UK), EGFP-tagged mouse Eps8 (generously

Oncogene Eps8 upregulation promotes cell migration and invasion LF Yap et al 2533 provided by G Scita, University of Milan) or vector control For Eps8 staining, cells were permeabilized with0.1% (pEGFP-C2; Invitrogen). Cells at 50% confluency in 6-well Triton X-100 for 10 min, followed by incubation for 60 min in dishes were transfected with 2 mg of DNA using Fugene PBS containing 0.1% BSA. Eps8 primary antibody was used (Roche Diagnostics, West Sussex, UK). 24 h post-transfection, at a dilution of 1:100. Actin was visualized withTRITC- cells were used in migration assays and analysed by confocal conjugated phalloidin (5 ng/ml; Sigma). Nuclei were visualized microscopy to determine transfection efficiency, which typi- using DAPI (Invitrogen). Coverslips were washed three times cally was between 70–95%. for 5 min in washbuffer and mounted withMOWIOL 4–88 (0.1 g/ml of Citifluor mounting medium; Novabiochem, Merck RNA interference Chemicals, Beeston, Nottingham, UK) and viewed with a To knockdown Eps8, pre-designed siRNA oligonucleotides confocal laser-scanning microscope (Zeiss LSM510, Zeiss, (Applied Biosystems) were used, together with control (ran- Welwyn Garden City, Hertfordshire, UK). dom) siRNA. 5 104 cells per well were seeded into 6-well  Determination of Rac1 activity plates, cultured for 16 h and then transfected with the relevant Rac1 activation assays were carried out as described earlier siRNA (30 nM) using Oligofectamine transfection reagent (Nystrom et al., 2006). Briefly, cells were plated onto 6-cm (Invitrogen). The sequences of the three EPS8 siRNAs are plates coated withpoly- D-lysine (100 mg/ml), LAP (0.5 mg/ml) (1) 50GGGCAAAGUGUGGACUCAAtt30 (2) 50GGAGA or fibronectin (10 mg/ml) and lysed on ice after 4 h. Cleared cell GGGUGUUUUAACGCtt30 and (3) 50GGCCCUUUAUG lysates were incubated withGST-PAK-CRIB beads for 1 hat AACAAAGGtt30. RNAi SMART pool reagents targeting 4 1C. Samples of lysates pre- and post-incubation withthe Rac1 or control (random) sequences were obtained from GST-PAK-CRIB beads were analysed by western blotting. Dharmacon (Chicago, IL, USA) and used at 100 nM. Rescue experiments were performed by specifically knocking down human Eps8 with the siRNA 50AGAGCCAACCCAG Flow cytometry AACAAGtt30 and rescuing withEGFP-tagged mouse Eps8 Flow cytometry was performed using anti-a5b1/-avb6 anti- cDNA or the empty vector (pEGFP-C2). bodies and FITC-conjugated secondary antibody (DAKO, Ely, Cambridgeshire, UK), as described (Thomas et al., 2001). Negative controls used secondary antibody only and was Cell migration assays subtracted from the results. Labelled cells were scanned on a Cells were transfected withsiRNAs and after 48 hhaptotactic FACSCaliber cytometer (BD Biosciences, Oxford, UK) and migration assays were carried out using fibronectin-coated (10 mg/ analysed using CellQuest software, acquiring 1  104 events. ml) or TGF-b1 latency-associated peptide (LAP)-coated (0.5 mg/ ml) polycarbonate filters (8 mm pore size, Transwell, Costar, Organotypic culture Corning, Schiphol-Rijk, The Netherlands), which were per- Organotypic cultures were prepared, as described (Nystrom formed as described (Thomas et al., 2001). Cells were plated et al., 2006). Gels comprised a 50:50 mixture of Matrigel into the upper chamber and allowed to migrate for 24 h. Cells (Becton-Dickinson, Oxford, UK) and type I collagen (Upstate) migrating to the lower chamber were trypsinized and counted on containing 5  105/ml HFFF2 or HFF fibroblasts, to which a Casy 1 counter (Scha¨ rfe System GmbH, Reutlingen, Germany). were added 5  105 OSCC cells that had been transfected with In some experiments, the cells further transfected with V12Rac1- Eps8 or control RNAi 24 hpreviously. After six days thegels GFP or Eps8-GFP 24 hafter transfection withsiRNA. For were bisected, fixed in formal-saline and processed to paraffin. blocking experiments, anti-a5b1or-avb6 (6.3G9) antibodies Sections (4 mm) were stained withH&E. (10 mg/ml) were added to the cells for 30 min before plating. Statistical analysis Cell spreading assay and confocal microscopy For the immunohistochemical studies, comparisons between Glass coverslips were coated withfibronectin (10 mg/ml) or groups were by Fisher’s exact test. For migration assays, LAP (0.5 mg/ml) in a-MEM for 1 hat 37 1C, washed with PBS statistical differences between experimental groups were then blocked with 0.1% BSA at 37 1C for 30 min. Cells were evaluated by Student’s t-test. 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 Conflict of interest were fixed in 4% paraformaldehyde. Spread cells were counted in seven representative high power fields at different time The authors declare no conflict of interest. points. Non-spread cells were defined as small round cells with little or no membrane protrusions, whereas spread cells were Acknowledgements defined as large cells withextensive visible lamellipodia (Dormond et al., 2001). Results represent the percentage of The Cancer Research Initiatives Foundation, Malaysia and spread cells in seven high power fields ±s.d. The Health Foundation supported the research.

References

Alevizos I, Mahadevappa M, Zhang X, Ohyama H, Kohno Y, Posner ison between matched primary normal epithelial and squamous M et al. (2001). Oral cancer in vivo gene expression profiling assisted carcinoma cells. Oncogene 21: 2634–2640. by laser capture microdissection and microarray analysis. Oncogene Bates RC, Bellovin DI, Brown C, Maynard E, Wu B, Kawakatsu H 20: 6196–6204. et al. (2005). Transcriptional activation of integrin b6 during Al Moustafa AE, Alaoui-Jamali MA, Batist G, Hernandez-Perez M, the epithelial-mesenchymal transition defines a novel prognostic Serruya C, Alpert L et al. (2002). Identification of genes associated indicator of aggressive colon carcinoma. J Clin Invest 115: withheadand neck carcinogenesis by cDNA microarray compar- 339–347.

Oncogene Eps8 upregulation promotes cell migration and invasion LF Yap et al 2534 Croce A, Cassata G, Disanza A, Gagliani MC, Tacchetti C, lines: relevance to malignant transformation. Mol Cell Biol 15: Malabarba MG et al. (2004). A novel actin barbed-end-capping 3805–3812. activity in EPS-8 regulates apical morphogenesis in intestinal cells of Miki H, Yamaguchi H, Suetsugu S, Takenawa T. (2000). IRSp53 is an Caenorhabditis elegans. Nat Cell Biol 6: 1173–1179. essential intermediate between Rac and WAVE in the regulation of Cromer A, Carles A, Millon R, Ganguli G, Chalmel F, Lemaire F membrane ruffling. Nature 408: 732–735. et al. (2004). Identification of genes associated withtumorigenesis Myers JN, Greenberg JS, Mo V, Roberts D. (2001). Extracapsular and metastatic potential of hypopharyngeal cancer by microarray spread. A significant predictor of treatment failure in patients with analysis. Oncogene 23: 2484–2498. squamous cell carcinoma of the tongue. Cancer 92: 3030–3036. del Pozo MA, Price LS, Alderson NB, Ren XD, Schwartz MA. (2000). Nystrom ML, Thomas GJ, Hart IR, Stone M, McKenzie IC, Marshall Adhesion to the extracellular matrix regulates the coupling JF. (2005). Development of a quantitative method to analyse tumor of the small GTPase Rac to its effector PAK. EMBO J 19: cell invasion in organotypic culture. J Pathol 205: 468–475. 2008–2014. Nystrom ML, McCullochD, Weinreb PH, Violette SM, SpeightPM, del Pozo MA, Alderson NB, Kiosses WB, Chiang HH, Anderson RG, Marshall JF et al. (2006). Cyclooxygenase-2 inhibition suppresses Schwartz MA. (2004). Integrins regulate Rac targeting by inter- alphavbeta6 integrin-dependent oral squamous carcinoma invasion. nalization of membrane domains. Science 303: 839–842. Cancer Res 66: 10833–41082. Disanza A, Carlier MF, Stradal TE, Didry D, Frittoli E, Confalonieri Parkin DM, Bray F, Ferlay J, Pisani P. (2005). Global cancer statistics S et al. (2004). Eps8 controls actin-based motility by capping the 2002. CA Cancer J Clin 55: 74–108. barbed ends of actin filaments. Nat Cell Biol 6: 1180–1188. Prime SS, Nixon SV, Crane IJ, Stone A, Matthews JB, Maitland NJ Dormond O, Foletti A, Paroz C, Ruegg C. (2001). NSAIDS inhibit et al. (1990). The behaviour of human oral squamous cell carcinoma avb3 integrin-mediated and Cdc/Rac-dependent endothelial-cell in cell culture. J Pathol 160: 259–269. spreading, migration and angiogenesis. Nat Med 7: 1041–1047. Scita G, Nordstrom J, Carbone R, Tenca P, Giardina G, Gutkind S Elayadi AN, Samli KN, Prudkin L, Liu YH, Bian A, Xie XJ et al. et al. (1999). EPS8 and E3B1 transduce signals from Ras to Rac. (2007). A peptide selected by biopanning identifies the integrin avb6 Nature 401: 290–293. as a prognostic biomarker for non-small cell lung cancer. Cancer Scita G, Tenca P, Areces LB, Tocchetti A, Frittoli E, Giardina G et al. Res 67: 5889–5895. (2001). An effector region in Eps8 is responsible for the activation of Fazioli F, Minichiello L, Matoska V, Castagnino P, Miki T, Wong the Rac-specific GEF activity of Sos-1 and for the proper WT et al. (1993). Eps8, a substrate for the epidermal growth factor localization of the Rac-based actin-polymerizing machine. J Cell receptor kinase, enhances EGF-dependent mitogenic signals. Biol 154: 1031–1044. EMBO J 12: 3799–3808. Ramsay AG, Keppler MD, Jazayeri M, Thomas GJ, Parsons M, Funato Y, Terabayashi T, Suenaga N, Seiki M, Takenawa T, Miki H. Violette S et al. (2007). HS1-associated protein X-1 regulates (2004). IRSp53/Eps8 complex is important for positive regulation of carcinoma cell migration and invasion via clathrin-mediated Rac and cancer cell motility/invasiveness. Cancer Res 64: endocytosis of integrin alphavbeta6. Cancer Res 67: 5275–5284. 5237–5244. Thirthagiri E, Robinson CM, Huntley S, Davies M, Yap LF, Prime SS Innocenti M, Frittoli E, Ponzanelli I, Falck JR, Brachmann SM, et al. (2007). Spindle assembly checkpoint and centrosome Di Fiore PP et al. (2003). Phosphoinositide 3-kinase activates Rac abnormalities in oral cancer. Cancer Lett 258: 276–285. by entering in a complex withEps8, Abi1, and Sos-1. J Cell Biol 160: Thomas GJ, Hart IR, Speight PM, Marshall JF. (2002). Binding of 17–23. TGF-beta1 latency-associated peptide (LAP) to alpha(v)beta6 Inobe M, Katsube K, Miyagoe Y, Nabeshima Y, Takeda S. (1999). integrin modulates behaviour of squamous carcinoma cells. Br J Identification of EPS8 as a Dvl1-associated molecule. Biochem Cancer 87: 859–867. Biophys Res Commun 266: 216–221. Thomas GJ, Lewis MP, Hart IR, Marshall JF, Speight PM. (2001). Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. AlphaVbeta6 integrin promotes invasion of squamous carcinoma (2003). Summaries of Affymetrix GeneChip probe level data. cells through up-regulation of matrix metalloproteinase-9. Int J Nucleic Acids Res 31: e15. Cancer 92: 641–650. Jeon GA, Lee JS, Patel V, Gutkind JS, Thorgeirsson SS, Kim EC et al. Tusher VG, Tibshirani R, Chu G. (2001). Significance analysis of (2004). Global gene expression profiles of human head and neck microarrays applied to the ionizing radiation response. Proc Natl squamous carcinoma cell lines. Int J Cancer 112: 249–258. Acad Sci USA 98: 5116–5121. Kim M, Califano J. (2004). Molecular Pathology of Head and Neck Welsch T, Endlich K, Giese T, Buchler MW, Schmidt J. (2007). Eps8 is Cancer. Int J Cancer 112: 545–553. increased in pancreatic cancer and required for dynamic actin-based Maa MC, HsiehCY, Leu TH. (2001). Overexpression of p97Eps8 cell protrusions and intercellular cytoskeletal organization. Cancer leads to cellular transformation: implication of pleckstrin homology Lett 255: 205–218. domain in p97Eps8-mediated ERK activation. Oncogene 20: Woolgar JA, Rogers SN, Lowe D, Brown JS, Vaughan ED. 106–112. (2003). Cervical lymphnode metastasis in oral cancer: the Maa MC, Lee JC, Chen YJ, Chen YJ, Lee YC, Wang ST et al. (2007). importance of even microscopic extracapsular spread. Oral Oncol EPS8 facilitates cellular growthand motility of colon cancer cells by 39: 130–137. increasing the expression and activity of focal adhesion kinase. Yao J, Weremowicz S, Feng B, Gentleman RC, Marks JR, Gelman R J Biol Chem 282: 19399–19409. et al. (2006). Combined cDNA array comparative genomic Marsh D, Dickinson S, Neill GW, Marshall JF, Hart IR, Thomas GJ. hybridization and serial analysis of gene expression analysis of (2008). alphavbeta6 integrin promotes invasion of morphoeic breast tumor progression. Cancer Res 66: 4065–4078. basal cell carcinoma through stromal modulation. Cancer Res 68: Yeudall WA, Miyazaki H, Ensley JF, Cardinali M, Gutkind JS, Patel 3295–3303. V. (2005). Uncoupling of epidermal growthfactor-dependent Matoskova B, Wong WT, Salcini AE, Pelicci PG, Di Fiore PP. proliferation and invasion in a model of squamous carcinoma (1995). Constitutive phosphorylation of Eps8 in tumor cell progression. Oral Oncol 41: 698–708.

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

Oncogene