Autocrine IL-1Β-TRAF6 Signalling Promotes Squamous Cell

ORIGINAL ARTICLE Autocrine IL-1b-TRAF6 signalling promotes squamous cell carcinoma invasion through paracrine TNFa signalling to carcinoma-associated ﬁbroblasts

SI Chaudhry1,2, S Hooper1, E Nye3, P Williamson4, K Harrington5 and E Sahai1

The invasion of squamous cell carcinoma (SCC) is a signiﬁcant cause of morbidity and mortality. Here, we identify an E3 ligase, Traf6 and a de-ubiquitinating enzyme, Cezanne/ZA20D1, as important regulators of this process in organotypic models. Traf6 can promote the formation of Cdc42-dependent F-actin microspikes. Furthermore, Traf6 has a key role in autocrine interleukin- 1b signalling in SCC cells, which in turn is required to drive the expression of tumour necrosis factor a (TNFa). TNFa acts in a paracrine manner to increase the invasion-promoting potential of carcinoma-associated ﬁbroblasts (CAFs). Exogenous TNFa signalling can restore invasion in cells depleted of Traf6. In conclusion, Traf6 has two important roles in SCC invasion: it promotes cell intrinsic Cdc42-dependent regulation of the actin cytoskeleton and enables production of the paracrine signal, TNFa, that enhances the activity of CAFs.

Oncogene (2013) 32, 747--758; doi:10.1038/onc.2012.91; published online 26 March 2012 Keywords: Traf6; Interleukin-1; TNFa; invasion; carcinoma-associated ﬁbroblast

INTRODUCTION cytokine signalling. Both TNFa and IL-1a/b bind to membrane Squamous cell carcinoma (SCC) is an epithelial-derived cancer spanning receptors that then trigger a range of intracellular of variable clinical presentation and histological appearance. signalling pathways. Canonical NFkB signalling is activated by Important tumour-related factors that govern patient prognosis both cytokines. Once the receptors have engaged their ligand, include the tumour size and grade, the depth of invasion, the a multiprotein complex is assembled that ultimately leads to degree of infiltration of neighbouring tissue, and the presence or the activation of IkB kinases (IKKs). Traf2 is a critical adaptor absence of metastases. In particular, the extent of local invasion molecule in the activation of IKK following TNFa stimulation;12 has significant consequences when planning surgery with overall while the K63 ubiquitin linkage E3 ligase, Traf6, performs a management being largely determined by the nature and pattern similar function after IL-1 stimulation.13 The action of Traf6 can of metastatic disease.1--3 A greater understanding of the basic be antagonised by the de-ubiquitinating enzyme (DUB), A20.14 biology of SCC invasion is therefore likely to lead to significant Active IKKs then phosphorylate IkB leading to its ubiquitin- benefits in the treatment of the disease and thus its long-term dependent degradation. The degradation of IkB enables NFkB prognosis. transcription factors to accumulate in the nucleus and drive a Carcinoma invasion is greatly influenced by non-tumour cells broad transcriptional programme. Besides NFkB activation, TNFa within the tumour microenvironment. Fibroblasts within tumours and IL-1a/b also trigger many other changes within the cell, are able to promote cancer cell invasion through the production including alteration of the actin cytoskeleton.15,16 The ubiquitina- of soluble factors and also by remodelling the extra-cellular matrix tion and SUMOylation machinery can also target Rho-family (ECM) as well as the generation of permissive tracks for invasion.4,5 proteins that regulate actin organisation and cell migration.17 Both It is clear that carcinoma-associated fibroblasts (CAFs) have the HECT domain E3 ligase, Smurf1,18 and CRL3, a component of distinct properties from normal fibroblasts.6 However, the mecha- Cullin-RING E3 ligase complexes, target RhoA for degradation.19 nisms by which cancer cells co-opt fibroblasts to promote invasion In contrast, SUMOylation of Rac1 can promote its activity, the are varied and incompletely understood. Paracrine TGFb and formation of actin protusions, and cell migration in response Hedgehog signalling can promote the activation of fibroblasts,4,7 to HGF.20 but genetic blockade of TGFb signalling in fibroblasts can also be We hypothesised that other regulators of ubiquitin and SUMO pro-tumourigenic (Bhowmick et al., 2004).8 More recently cytokine modifications would also regulate F-actin organisation and thus signalling has been implicated in the invasion-promoting proper- cell migration in SCC. Therefore, we undertook a systematic ties of CAFs.9--11 The inflammatory cytokines TNFa (tumour siRNA screen for the effect of depletion of E2 ligases, E3 ligases necrosis factor a), IL-1a/b and IL-6 can all modulate tumourigen- and DUBs on F-actin. Following the analysis of several hundred esis by signalling to fibroblasts. genes, we focused on two that had effects on F-actin organisation. Regulators of ubiquitin and ubiquitin-like modification can We find that the E3 ligase Traf6 and the DUB Cezanne/ZA20D1 affect cancer invasion in many ways, including modulation of have contrasting effects on actin architecture, though they do not

1Tumour Cell Biology Laboratory, Cancer Research UK London Research Institute, London, UK; 2Oral Medicine, UCL Eastman Dental Institute and UCLHT Eastman Dental Hospital, London, UK; 3Experimental Histopathology Laboratory, Cancer Research UK London Research Institute, London, UK; 4Thomas Tatum Head and Neck Unit, St George’s Hospital, London, UK and 5Institute of Cancer Research, London, UK. Correspondence: Dr E Sahai, Tumour Cell Biology Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK. E-mail: [email protected] Received 13 August 2011; revised 25 January 2012; accepted 12 February 2012; published online 26 March 2012 Traf6 promotes SCC invasion SI Chaudhry et al 748 simply antagonise one another. Divergent pathways downstream two other SCC cell lines, Detroit 562 and SCC12 (Supplementary of Traf6 regulate SCC invasion. Cdc42 function is required for Figure 1b). The effects of ZA20D1 depletion were less pronounced Traf6 to promote the formation of F-actin microspikes, while in these cell lines (Supplementary Figure 1b). the regulation of NFkB signalling by Traf6 sustains an autocrine In view of the reported antagonistic relationship of these two IL-1b loop that facilitates cell invasion through the expression of proteins,22 we next tested whether the microspikes resulting from TNFa. Moreover, TNFa participates in paracrine signalling that ZA20D1 depletion were dependent on Traf6 function. When both promotes the activity of CAFs and thereby enhances cancer cell ZA20D1 and Traf6 were simultaneously knocked down the invasion. number of F-actin microspikes were similar to that in ZA20D1- depleted cells (Figure 1e). Although we could not confirm that every cell received equal amounts of both siRNAs, western blot RESULTS and immune-fluorescence analysis indicated that Traf6 was Identification of Traf6 and Cezanne/ZA20D1 as regulators of SCC depleted by at least 90% when co-transfected with ZA20D1 morphology siRNA (data not shown). Indeed, the phenotype of cells depleted To investigate the role of the ubiquitin regulatory machinery in for both Traf6 and ZA20D1 was more of a mixture of the two the control of cancer cell invasion, we hypothesised that key individual phenotypes. These data suggest that de-regulated Traf6 regulators of invasion would affect the F-actin organisation of activity is not responsible for the F-actin microspikes observed in cells. We therefore investigated F-actin architecture in A431 SCC ZA20D1-depleted cells. cells grown on thick collagen-rich deformable matrices following siRNA-mediated depletion of 426 E2 ligases, E3 ligases and Cdc42 is required for Traf6-induced microspikes DUBs. Cells were fixed 72 h after transfection and stained with Having established that Traf6 and ZA20D1 had effects on the actin phalloidin to label F-actin structures. Control cells grew in small cytoskeleton, we then sought to investigate the molecular well-packed groups with F-actin protrusions extending at the mechanism. cDNA encoding Traf6 and ZA20D1 were transiently edges of the clusters. We observed a diverse range of phenotypes transfected into A431 cells and the F-actin organisation examined following depletion of E3 ligases and DUBs using siRNA smart- 48 h later. The majority of Traf6 over-expressing cells showed pools: these ranged from elongated cells to contracted cells with pronounced F-actin microspikes and frequently increased levels of membrane blebs. To identify genes that consistently affected F-actin (Figure 2a). ZA20D1 over-expressing cells did not show F-actin in an unbiased manner, the screen was performed twice striking changes in morphology (Figure 2a). Consequently, we and each time the F-actin organisation was scored by three focused our attention on Traf6. ‘blinded’ observers using standardised nomenclature: this gave a Traf6 was over-expressed in A431 cells and 6 h before fixation total of six scores per gene. A total of 45 genes scored 5/6 or 6/6 cells were treated with inhibitors of pathways previously linked to and these were chosen for further investigation (Supplementary Traf6 and Rho-family regulators of F-actin organisation. Specifi- Figure 1a). cally, IKK, PI-3K, Src-family kinases, RhoA, Rac1 and Cdc42 were To exclude the possibility of ‘off-target’ effects sometimes inhibited.23,24 Co-transfection of Traf6 with a ‘dominant negative’ associated with siRNA reagents, we tested multiple siRNA non-degradable IkB mutant was also used to block canonical oligonucleotides targeting the 45 genes that we identified in NFkB signalling. This analysis indicated that the ability of Traf6 to our initial screen. In all, 29 genes showed consistent phenotypes regulate the formation of actin microspikes was partly dependent (Supplementary Figure 1b). We noted that within this set of genes on IKK-NFkB, PI3 K and Src-family kinase signalling (Figure 2b. there was an E3 ligase, Traf6 and a DUB, ZA20D1/Cezanne Supplementary Figure 2 demonstrates that effective IKK inhibition (hereafter referred to as ZA20D1) that had been reported to 21,22 was achieved). More impressively, we consistently observed that interact with each other. We therefore chose these genes for depletion of Cdc42, but not Rac1 or RhoA, prevented Traf6 from further study. inducing F-actin microspikes (Figure 2c). Figure 1a shows that depletion of Traf6 generates cells with an elongated and spread morphology. The 3D confocal analysis also revealed that these cells were flatter than controls (Figure 1a). In Traf6, IKK and Cdc42 function are required for SCC invasion contrast, depletion of ZA20D1 leads to an increase in the number We next tested the role of Traf6 depletion on SCC invasion in an of F-actin microspikes that frequently protrude into the underlying organotypic invasion assay. SCC cells are cultured on a collagen-rich collagen-rich matrix (Figure 1a). We confirmed that these matrix containing CAFs at an air--liquid interface.25 This model phenotypes could be reproduced with three independent siRNA reproduces several features of the epidermal--dermal architecture of duplexes targeting either Traf6 or ZA20D1 (Figures 1b--d), and a carcinoma and utilises the same collagen-rich deformable matrix that these siRNA effectively depleted Traf6 and ZA20D1 that was used for the cell morphology analysis. While control cells (Figure 1b). Traf6 depletion also led to more elongated cells in invade efficiently in the presence of CAFs, depletion of Traf6 (using

Figure 1. Traf6 and Cezanne/ZA20D1 regulate SCC morphology. (a) (i) Cells depleted for either Traf6 or Cezanne/ZA20D1 using siRNA smart-pools are shown. F-actin is shown in green and reflectance imaging of the thick collagen-rich deformable matrix is shown in white. Upper panels show xy ‘above’ view, lower panels show xz ‘side’ view. Panels are 121 mm wide. (ii) Quantification of the long axis of A431 cells following mock, control, Traf6 or ZA20D1 depletion. Min, max, quartiles and mean are shown, 450 cells analysed for each condition, *indicates Po0.01 one way ANOVA. (iii) Quantification of the percentage of cells with microspikes 47.5 mm, 450 cells analysed for each condition, *indicates Po0.01 t-test. (b) (i) Single confocal sections (146 Â 146 mm) of A431 cells shown depleted for either Traf6 or Cezanne/ZA20D1 using three independent siRNA sequences. F-actin is shown in green, reflectance imaging of matrix is shown in red and nuclei are shown in blue. (ii) Western blot showing Traf6 protein levels in A431 cells following transfection of three independent siRNA sequences. (iii) QRT--PCR showing Cezanne/ZA20D1 mRNA levels in A431 cells following transfection of three independent siRNA sequences. (c) Quantification of the long axis of A431 cells following mock, control, Traf6 or ZA20D1 depletion. Min, max, quartiles and mean are shown, 450 cells analysed for each condition, *indicates Po0.01 one way ANOVA. (d) Quantification of the percentage of cells with microspikes 47.5 mm, 450 cells analysed for each condition, *indicates Po0.01 t-test. (e) (i) Single confocal sections (146 Â 146 mm) of A431 cells shown depleted for either Traf6 or Cezanne/ZA20D1 or both using siRNA smartpools. F-actin is shown in green and nuclei are shown in blue. (ii) Quantification of the long axis of A431 cells following mock, control, Traf6 or ZA20D1 depletion. Min, max, quartiles and mean are shown, 450 cells analysed for each condition, *indicates Po0.01 one way ANOVA. (iii) Quantification of the percentage of cells with microspikes 47.5 mm, 450 cells analysed for each condition, *indicates Po0.01 one way t-test.

Oncogene (2013) 747 --758 & 2013 Macmillan Publishers Limited Traf6 promotes SCC invasion SI Chaudhry et al 749 three different siRNA duplexes) signiﬁcantly reduces the inva- The data outlined above suggests that Traf6-mediated regula- sion of SCC12 and A431 SCC (Figures 2d--f). Conventional transwell- tion of Cdc42 and F-actin microspikes may contribute to SCC matrigel assays also demonstrated that Traf6 is required for A431 invasion. Additionally, Traf6 is known to be a key regulator of cell invasion (Supplementary Figure 1d). Depletion of ZA20D1 also downstream events in canonical NFkB signalling, which itself has reduced SCC invasion, which further suggests that it has additional been shown to inﬂuence cancer invasion.13,26 We therefore sought functions besides antagonising Traf6 (Supplementary Figure 1d and e). to determine whether Traf6 regulates IKK functions in SCC cells.

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0.2 0.1 Invasion Index (AU) Invasion Index (AU) 0 0 HNCAF - ++++ + HNCAF -++++ + SCC12Ctrl Ctrl Ctrl #2 #3 #B A431Ctrl Ctrl Ctrl #2 #3 #B siRNA siRNA TRAF6 siRNA TRAF6 siRNA Figure 2. Traf6 is required for SCC invasion. (a) Membrane-GFP (control), HA-tagged Traf6 or GFP-Cezanne/ZA20D1 were transfected into A431 cells. 3D reconstructions of the morphology of A431 cells viewed from ‘above’ and ‘the side’ are shown: transfected protein is shown in green and F-actin in red. Panels are 50 mm wide. (b) Membrane-GFP (control), HA-tagged Traf6 and IkB-super-repressor were transfected into A431 cells as indicated. Six hours before fixation the indicated inhibitors were added at 10 mM final concentration (except Dasatinib at 250 nM). The number of transfected cells with F-actin microspikes 47.5 mm is shown. Average of at least three experiments (standard error is shown). (c) Membrane-RFP (control) or HA-tagged Traf6 were transfected into A431 cells together with 80 nM siRNA against RhoAsi#smartpool, Cdc42si#1, Cdc42si#2 or Rac1si#smartpool as indicated. The number of transfected cells with F-actin microspikes 47.5 mm is shown. Average of three experiments (standard error is shown). (d) Representative images (600 Â 280 mm) showing the extent of SCC12 invasion following Traf6 depletion in organotypic cultures containing carcinoma-associated fibroblasts. (e) Quantification of SCC12 invasion in organotypic assays. At least 10 images from at least two independent assays are quantified for each data point. (f) Quantification of A431 invasion in organotypic assays. At least 10 images from at least two independent assays are quantified for each data point.

IKK phosphorylates IkB leading to its degradation. Hence, reduced phosphorylation of IkB and increased the levels of IkB in both IkB phosphorylation along with increased IkB levels indicate lower A431 and SCC12 cells (Figures 3a and b and data not shown). levels of IKK function. Depletion of Traf6 decreased the Depletion of ZA20D1 had no effect.

Oncogene (2013) 747 --758 & 2013 Macmillan Publishers Limited Traf6 promotes SCC invasion SI Chaudhry et al 751 A431 level and in a much greater proportion of cells (Supplementary SCC12 Figure 3b). Quantiﬁcation of Traf6 levels in 50 oral SCC biopsies compared with 9 normal oral mucosa samples is shown in Supplementary Figure 3c. We also analysed a limited number

Control siRNA Cez/ZA#2 Cez/ZA#3 Cez/ZA#A TRAF6 #2 TRAF6 #3 TRAF6 #B of patient matched oral mucosa and SCC samples, examples of which are shown in Supplementary Figure 3d. Elevated levels Control siRNA TRAF6 #2 TRAF6 #3 TRAF6 #B of Traf6 in carcinoma tissue were observed compared with that of normal tissue (controls for antibody specificity on frozen sections are shown in Supplementary Figure 3e). Analysis of 10 vulval SCC biopsies also revealed elevated levels of Traf6 expression in some TRAF6 samples (Supplementary Figure 3f and g). TRAF6 Traf6 participates in autocrine IL-1 signalling to promote cancer cell invasion 0.7 0.8 0.6 0.7 Traf6 mediates signals from a range of extra-cellular cues * * * 0.5 0.6 including IL-1, LPS and dsRNA. The effect of Traf6 depletion in 0.5 0.4 the absence of exogenous ligands suggests a basal level of Traf6 0.4 0.3 signalling in standard culture conditions. Figure 4a shows that 0.3 0.2 both IL-1a and IL-1b were able to promote SCC invasion in the 0.2 presence of CAFs. IL-1a and IL-1b also efficiently induced IkB Invasion Index (AU) 0.1 Invasion Index (AU) 0.1 degradation in SCC cells but had no effect on CAFs (Supplemen- 0 0 HNCAF (-) (+) (+) (+)HNCAF (-) (+)(+) (+) (+) tary Figure 4 and data not shown). In contrast, neither LPS or dI:C SCC12 CtrlCtrl Ctrl Cdc42 SCC12 Ctrl Ctrl DMSO 1μM 10μM (a dsRNA mimetic) had any effect on invasion or NFkB signalling siRNA siRNA IKKi IKKi (Supplementary Figure 5 and data not shown). This indicates that Figure 3. Role of IKK and Cdc42 in SCC invasion. (a) A431 cells only the carcinoma cells in our model system are able to respond were depleted for either Cezanne/ZA20D1 or Traf6 using multiple to IL-1 ligands and led us to exclude a role for TLR2 and TLR4. independent siRNA. At 48 h after transfection the levels and Given the ability of exogenous IL-1a and IL-1b to promote phosphorylation of IkB was determined by western blot analysis. carcinoma invasion, we investigated if they were endogenously b-Tubulin and Traf6 levels are shown as controls for protein loading expressed in SCC cells and CAFs. Figure 4b shows that both IL-1a and transfection efficiency, respectively. (b) SCC12 cells were and b are predominantly expressed by SCC cells, suggesting that depleted for Traf6 using multiple independent siRNA. At 48 h after autocrine IL-1 to Traf6 signalling may be occurring. The transfection the levels of IkB were determined by western blot significantly greater efficiency of IL-1b PCR (compare y-axis scales analysis. b-Tubulin and Traf6 levels are shown as controls for protein in Figure 4b) suggests that it is the predominant isoform loading and transfection efficiency, respectively. (c) The ability of SCC12 cells to invade into carcinoma-associated fibroblast condi- expressed in A431 and SCC12 cells. tioned matrix following the depletion of Cdc42 is shown. At least 10 Positive feedback is a common feature of autocrine loops. We images from at least two independent assays are quantified for each tested whether IL-1b treatment increased the expression levels of data point. (d) The ability of SCC12 cells to invade into carcinoma- either IL1a or b. IL-1b was robustly induced following IL-1b associated fibroblast conditioned matrix in the presence of IKK stimulation in A431 cells, whereas IL-1a levels remained inhibitor. At least 10 images from at least two independent assays unchanged (Figure 4c and data not shown). Similar results were are quantified for each data point. . Mean and s.e.m. are shown. obtained in SCC12 cells (Supplementary Figure 6a and data not *Indicates Po0.01 relative to control þ HNCAF. shown). This indicates that IL-1b is involved in a positive feedback loop. We next examined whether Traf6 or ZA20D1 were involved in the maintenance of this positive feedback loop. Depletion of Traf6 reduced the levels of IL-1b mRNA in both basal conditions We next tested whether the function of IKK or Cdc42 is required and following IL-1b stimulation (Figure 4c and Supplementary for the invasion of SCC. To exclude any indirect effects of IKK Figure 6b). These argue that Traf6 contributes to autocrine IL-1 inhibitors on fibroblasts, we used organotypic invasion assays with signalling, even in unstimulated cells. We directly tested the CAF-conditioned matrices from which the CAF had been removed. importance of IL-1 autocrine signalling by depleting the IL-1 The ability of SCC cells treated with either siRNA or inhibitors to receptor in SCC12 cells in organotypic invasion assays. Figures 4d invade into these matrices was tested. Similar to previous 5 and e show that depletion of IL-1R leads to a significant reduction findings, siRNA-mediated depletion of Cdc42 in SCC12 cells in cancer cell invasion thereby confirming the involvement of an caused a pronounced reduction in invasion (Figure 3c). Figure 3d IL-1 autocrine loop in this setting. shows that IKK inhibition reduced SCC invasion in a dose- dependent manner (Supplementary Figure 2 confirms the efficacy of the IKK inhibitors). Together, this suggests that IKK function is TNFa promotes CAF function required for SCC invasion, even though its inhibition only slightly NFkB signalling regulates the expression of a broad range of reduces the formation of Traf6-driven F-actin microspikes. cytokines. We therefore hypothesised that autocrine IL-1b4Traf6 signalling might contribute to the invasion of SCC by regulating the expression of other cytokines. We found that IL-1b could increase Traf6 is over-expressed in SCC TNFa mRNA, but not IL-6 mRNA (Figure 5a and data not shown). Our analyses indicate that the alteration of Traf6 expression leads Thus, we investigated the role of TNFa in our organotypic to changes in SCC cell morphology and that reduced Traf6 levels co-culture invasion system. Both cancer cells and HNCAF express inhibit SCC invasion. Consequently, we evaluated TRAF6 expres- TNFa receptor mRNA, however, only the cancer cells expressed sion in human SCC samples. Supplementary Figure 3a confirms significant levels of TNFa mRNA (Figure 5a). These data were that Traf6 can be specifically detected in formalin-fixed paraffin- confirmed by ELISA analysis of TNFa protein levels (Figure 5b). embedded material. In normal oral mucosa Traf6 was largely Furthermore, IL-1b efficiently induces TNFa expression in SCC cells, restricted to the basal layer of cells within the epidermis. However, but not CAFs (Figure 4d). Both the induced and basal levels of TNFa in numerous oral SCC samples Traf6 was expressed to a higher were reduced in Traf6-depleted cells (Figures 5c and d and

& 2013 Macmillan Publishers Limited Oncogene (2013) 747 --758 Traf6 promotes SCC invasion SI Chaudhry et al 752 2

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IL-1R#2 Figure 4. Traf6 is required to maintain autocrine IL-1 signalling and TNFa expression. (a) Quantification of SCC12 invasion in organotypic assays in the presence of either 1 ng/ml IL-1a or IL-1b. At least 10 images from at least two independent assays are quantified for each data point. (b) Analysis of IL-1a and IL-1b mRNA levels in A431, SCC12 and head and neck carcinoma-associated fibroblasts. IL-1 mRNA is normalised to GAPDH levels: one representative experiment out of three is shown. (c) Analysis of IL-1b levels in A431 cells following combinations of IL-1b treatment for 60 min and depletion of either Traf6 or Cezanne/ZA20D1 using siRNA smart-pools. Average of two experiments performed in triplicate---error bars represent half the range of values. (d) Quantification of SCC12 invasion following depletion of IL-1R using two independent sets of siRNA in organotypic assays. At least 10 images from at least two independent assays are quantified for each data point. (e) Representative images (3000 Â 750 mm) showing the extent of SCC12 invasion following IL-1R depletion in organotypic cultures containing carcinoma-associated fibroblasts.

Supplementary Figure 6c); these data demonstrate that autocrine We therefore tested whether TNFa could specifically affect the IL-14Traf6 signalling is required for the expression of TNFa. invasion-promoting ability of CAFs by adding TNFa to cultures Further, the data suggest that Traf6 may promote invasion by only during the period of fibroblast-dependent matrix remodel- regulating the production of TNFa by SCC cells. Figure 5e ling. Significantly, the deficit in invasion observed in the absence demonstrates that TNFa can signal to CAFs and that a TNFa of paracrine signals from SCC12 cells could be rescued by blocking antibody, infliximab (Remicade), is able to inhibit TNFa supplying additional TNFa to the CAFs. function. Crucially, treatment of organotypic cultures with infliximab A key invasion-promoting function of CAFs is remodelling of reduced SCC invasion in a dose-dependent manner (Figures 5f and the ECM. In view of this, we examined whether TNFa modulates g), confirming a role for TNFa and raising the possibility that it may the ability of CAFs to remodel the ECM. This can be measured act as a paracrine signal from cancer cells to CAFs. by the ability of CAFs to contract collagen-rich gels (Figure 6b), Previous work has shown that CAFs can remodel the ECM and and gel contraction was significantly increased in the presence of facilitate cancer cell invasion even when they are not co-cultured 31 ng/ml TNFa (compare columns 3 and 4). Microscopic analysis with cancer cells.5 In this context, soluble paracrine signals also confirmed that CAFs treated with TNFa had a greater ability produced by cancer cells, such as TNFa, that may promote the to remodel the ECM, with more prominent areas of matrix invasion-stimulating activity of CAFs are absent. Figure 6a shows degradation being observed around the CAFs (indicated by the that the ability of CAFs to facilitate SCC invasion when they are not red arrows in Figure 6c). A similar pattern of matrix remodelling ‘co-cultured’ with cancer cells is reduced (compare columns 2 and degradation was induced by the addition of SCC-conditioned and 3). This supports the argument that soluble factors produced media. The effect of conditioned media was reduced by the by SCC12 cells influence the ability of CAFs to drive invasion. addition of infliximab (Figure 6c panels 3 and 4 and Figure 6d).

Oncogene (2013) 747 --758 & 2013 Macmillan Publishers Limited Traf6 promotes SCC invasion SI Chaudhry et al 753

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0.9 0.8 * 0.7 0.6 control 0.5 0.4 0.3 Infliximab 10μg/ml Invasion Index (AU) 0.2 0.1 0 Infliximab 31 μg/ml HNCAF- + + + Infliximab --10 31 (μg/ml) Figure 5. TNFa is required for SCC invasion. (a) QRT--PCR analysis of TNFa and TNFa receptor mRNA levels in SCC12 and HNCAF. (b) ELISA analysis showing TNFa secreted by SCC12 or HNCAF cultured in the presence or absence of 1 ng/ml IL-1b. Mean and s.e.m. are shown. *Indicates Po0.01 relative to control þ IL-1b.(c) Analysis of TNFa mRNA in A431 cells following combinations of IL-1b treatment for 60 min and depletion of Traf6 or using siRNA. Average of two experiments performed in triplicate---error bars represent half the range of values. (d) ELISA analysis showing TNFa secreted by SCC12 or A431 following combinations of IL-1b treatment for 60 min and depletion of Traf6 or using siRNA. Mean and s.e.m. are shown. *Indicates Po0.01 relative to control þ IL-1b.(e) Western blots showing the modulation of IkB levels following TNFa treatment of HNCAF cells in the absence or presence of infliximab. (f) Representative images (600 Â 240 mm) of organotypic invasion assays using SCC12 cells and HNCAF with increasing doses of infliximab (Remicade). (g) Quantification of organotypic invasion assays using SCC12 cells and HNCAF with increasing doses of infliximab. At least 10 images from at least two independent assays are quantified for each data point. Mean and s.e.m. are shown. *Indicates Po0.01 relative to control þ HNCAF.

Thus, TNFa acts as a paracrine signal from SCC cells to CAFs to co-ordinated pro-invasive programme that involves the expres- promote matrix remodelling. sion of TNFa and enhanced fibroblast-dependent remodelling This data demonstrates a role for TNFa in promoting cancer cell of the ECM. invasion and confirms that Traf6 is required for TNFa expression by SCC cells (Figure 4d). Considered together, this predicts that the exogenous provision of TNFa should restore carcinoma invasion in DISCUSSION organotypic co-cultures containing SCC cells depleted of Traf6. The invasion of SCC and its propensity for metastasis determines Figure 6e shows the results of invasion assays containing SCC12 patient prognosis, morbidity and ultimately mortality. To learn cells depleted of Traf6 with and without daily stimulation with more about this process, we screened for ubiquitin-linked TNFa. In the control assays, TNFa leads to a marginal increase in regulators of SCC invasion. This work shows that Traf6 is critical invasion. In the setting of Traf6-depleted SCC cells, however, for two reasons: first, it enables canonical NFkB signalling to drive exogenous TNFa caused a striking ‘rescue’ of invasion. Collectively, paracrine signalling to CAFs and second, it can regulate the these results indicate that Traf6 is a critical factor in autocrine formation of Cdc42-dependent F-actin microspikes (Figure 7). IL-1 signalling within SCC cells. This in turn helps to maintain a Blockade of either activity reduces SCC invasion, suggesting that

& 2013 Macmillan Publishers Limited Oncogene (2013) 747 --758 Traf6 promotes SCC invasion SI Chaudhry et al 754 1 0.9 Gel contraction images 0.8 0.7 * 0.6 0.5 0.8 0.4 * 0.3 0.6 Invasion Index (AU) 0.2 0.4 0.1 0 0.2 HNCAF - + (+) (+) Gel contraction/A.U. 0 SCC12 + + + + HNCAF - - + + paracrine - + - - interactions TNFα - + - + TNFα - - - +

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+ cond. media Area of remodelled ECM

control

+ TNFalpha+ infliximab + cond. media + cond. media + inflimixab + cond. media + infliximab

0.9 0.8 0.7 0.6 *** 0.5 0.4 0.3

Invasion Index (AU) 0.2 0.1 0 HNCAF + + + + ++++ SCC12 Ctrl #2#3 #B Ctrl #2 #3 #B siRNA siRNA TRAF6 siRNA TRAF6 siRNA - --- 3131 31 31 Figure 6. TNFa mediates paracrine signalling from cancer cells to CAFs. (a) Quantification of organotypic assays using SCC12 and HNCAF: ‘ þ ’ indicates the presence of HNCAF at the same time as SCC12 cells ‘( þ )’ indicates the presence of HNCAF before the addition of SCC12 cells. HNCAF and SCC12 were co-cultured to allow paracrine signalling by soluble factors---second column, or HNCAF and SCC12 were cultured sequentially to prevent paracrine signalling by soluble factors---third column. Finally, HNCAF and SCC12 were cultured sequentially and exogenous TNFa was added during the period of HNCAF culture. (b) Upper images show ECM contraction by HNCAF in the absence or presence of 31 ng/ml TNFa. Lower histogram shows quantification of multiple contraction assays. Error bars are standard deviation. (c) Images (146 Â 146 mm) show HNCAF (green) in collagen-rich deformable matrices (white) in the presence or absence of TNFa (31 ng/ml), SCC12- conditioned media and/or infliximab (Remicade) (31 mg/ml). Red arrows indicate area of degraded matrix. (d) Quantification of the average area of matrix degradation surrounding CAFs in the conditions indicated. Average of 450 cells from three independent experiments. Error bars indicate s.e.m. and *indicates Po0.01. One-way non-parametric ANOVA. (e). Quantification of SCC12 invasion following depletion of Traf6 using three independent siRNA in the presence or absence of 31 ng/ml TNFa organotypic assays. At least 10 images from at least two independent assays are quantified for each data point. Mean and s.e.m. are shown. *Indicates Po0.01 relative to control þ HNCAF.

Oncogene (2013) 747 --758 & 2013 Macmillan Publishers Limited Traf6 promotes SCC invasion SI Chaudhry et al 755 HNCAF SCC system. In the absence of Traf6, the expression of both IL-1b and TNFa by SCC cells is reduced. The effect of IL-1b is limited to the

TNFα IL-1β SCC cells in our system and serves to maintain and possibly amplify the expression of itself and TNFa. It is also probable that Traf6 supports the expression of other cytokines that have been 36 TRAF6 reported to be IL-1 regulated, including IL-8. The critical role of ? Traf6 in driving TNFa expression is illustrated by the ability of Cdc42 exogenous TNFa to rescue carcinoma invasion in Traf6-depleted IKK IKK Actin microspikes cells. Significantly, TNFa produced by cancer cells acts in a paracrine manner to enhance the function of CAFs. The matrix NFkB NFkB remodelling abilities of CAFs are promoted by the addition of exogenous TNFa. This is possibly the result of both an increase in Matrix Invasive transcriptional the production of MMPs and an increase in the contractile remodelling programme function of the CAF cytoskeleton.11,37 --39 Increased matrix remodelling by CAFs in turn leads to an increase in cancer cell invasion. The mechanisms involved in the transformation of normal Figure 7. Model summarising the roles of TRAF6, IL-1b and TNFa in fibroblasts to invasion-promoting CAFs are incompletely under- SCC invasion. TRAF6 enables canonical NFB signalling to drive an stood. TGFb has been widely implicated in this process but its invasive transcriptional programme including TNFa and IL-1b and inhibition does not affect the HNCAF used in this study (Hooper can also regulate the formation of F-actin microspikes in SCC data not shown). Erez et al.9 proposed that IL-1b is a critical factor cells (outlined in dark blue). Blockade of either activity reduces SCC in the NFkB-dependent activation of CAFs, but the CAFs used in invasion. TNFa produced by SCC cells acts in a paracrine manner to increase the invasion-promoting potential of carcinoma-associated our study are not responsive to either IL-1a or IL-1b. Interestingly, fibroblasts (outlined in red). our work demonstrates that the expression of TNFa by cancer cells is an important strategy to co-opt stromal fibroblasts to undertake tumour-promoting functions. Inflammation associated regulation of F-actin and NFkB co-operate to promote invasion. with wound healing is usually self-limiting; however, in chronic Both TNFa and IL-1b are transcriptionally controlled by Traf6 as inflammation the response is sustained. Similarly, although a result of NFkB signalling. This probably occurs through the well paracrine signalling by TNFa promotes CAF function, the removal documented K63 ubiquitin ligase function of Traf6.13,27 The ability of paracrine cues does not completely abolish the invasion- of exogenous TNFa to rescue invasion in Traf6-depleted cultures promoting ability of CAFs (compare column 2 and 3 Figure 6a). suggests that regulation of TNFa transcription is the predominant Interestingly, we find that continued invasion-promoting abilities invasion-promoting function of Traf6. However, we noted that of CAF are dependent on IKK (data not shown), but TNFa TNFa could induce changes in F-actin organisation in both control independent. We do not know the upstream activator in this and Traf6-depleted cells (data not shown). These changes might context: our data exclude IL-1 and LPS. provide a mechanism to compensate for the lack of F-actin Anti-inflammatory agents such as COX-2 inhibitors have microspikes in Traf6-depleted cells. The mechanism by which shown promise as chemo-prevention agents in certain clinical Traf6 might modulate Cdc42 function is unclear. Traf6 can form contexts and are well documented to reduce cancer cell oligomers and has been reported to be a pleiotropic scaffold invasion.40,41 Our data also suggests that blocking either IL-1 or molecule, influencing a variety of signalling pathways including TNFa signalling may reduce carcinoma invasion, in part through Akt, Src-family kinases and p38.23,24,28 Our analysis confirms some targeting stromal fibroblasts. This is of particular interest as role for PI3 kinase, Src-family kinases and IKK downstream of Traf6; both IL-1R antagonists such as anakinra (Kineret) and TNFa although no single pathway was absolutely required for Traf6 to blockers such as infliximab (Remicade) are in routine clinical use regulate the cytoskeleton. for the treatment of several chronic inflammatory conditions, We also found that the DUB ZA20D1 is required for SCC including rheumatoid arthritis, psoriasis and Crohn’s disease. invasion (Supplementary Figure 1). We believe that the reduced Indeed, we find that infliximab reduces SCC invasion in our invasion of cells lacking ZA20D1 is not caused by their increased system, indicating that it could potentially be exploited as an anti- number of F-actin microspikes, which are usually pro-invasive invasive cancer drug. This would be of considerable benefit to structures, but by other unidentified defects. ZA20D1 can patients with SCC of the head and neck in which metastatic associate with Traf6 and is possibly an antagonist to Traf6.22 spread along the cervical chain of lymph nodes is associated with However, Traf6 depletion has a minimal effect on the formation of a markedly worse clinical prognosis. In the future, it would be F-actin microspikes induced by ZA20D1 depletion (Figure 1) and interesting to validate these findings in in vivo models of ZA20D1 depletion has no effect on the levels of phosphorylation metastasis. The future role of IL-1 and TNFa antagonists in cancer of IKKa/b and IkB (Figure 4 and data not shown). We did observe a chemotherapy will depend on a number of factors. First, a greater slight increase in IL-1b mRNA levels in ZA20D1-depleted cells, understanding of the nature of IL-1 and TNFa-associated which is consistent with the findings of Enesa et al.29 This may epithelial--stromal interactions in early and late-stage malig- reflect the greater sensitivity of the mRNA analysis compared to nancy is required. Second, criteria for identifying the patients the IKKa/b and IkB analysis or alternatively that IL-1b mRNA can and the tumours most likely to respond to such therapy are be induced in an IKKa/b/IkB-independent manner. Considered needed. Third, anti-TNFa agents have complex side-effects42 collectively, ZA20D1 most likely has additional functions besides and their efficacy and safety in the setting of the medically antagonising Traf6.30,31 compromised cancer patient must be evaluated further. Previous studies have identified an important role for both In conclusion, siRNA-based screening of ubiquitin and ubiquitin- TNFa and IL-1a/b in the development and progression of SCC. like regulators has identified a key role for Traf6 in SCC invasion. However, these studies have primarily focused on signalling Furthermore, we have revealed the interconnected but distinct by these factors to either leukocytes or cancer cells.32 --35 Here, roles of autocrine IL-1 signalling in carcinoma cells and paracrine we describe interlinked but distinct roles for IL-1b and TNFa in TNFa signalling from carcinoma cells to CAFs in promoting an organotypic model of SCC. Initially, we demonstrated a invasion. This work suggests new strategies for reducing cancer requirement for Traf6 in the SCC (epithelial) compartment of our cell invasion using anti-inflammatory agents.

& 2013 Macmillan Publishers Limited Oncogene (2013) 747 --758 Traf6 promotes SCC invasion SI Chaudhry et al 756 MATERIALS AND METHODS added to feed each organotypic culture from beneath the bridge thus Cell lines allowing the epithelial layer to grow at an air--liquid interface. Media was A431 cells (human vulval SCC) were grown in DMEM with 10% FCS in changed daily throughout the duration of the assay. One week following gel preparation, the cultures were fixed in either 4% paraformaldehyde 10% CO2 at 37 1C. SCC12 cells (cutaneous SCC cell line) were cultured in FAD 85 medium supplemented with 10% FCS, 5 mg/ml insulin (Invitrogen, with 0.25% gluteraldehyde in PBS or 10% normal buffered formalin. After Paisley, UK), 0.5 mg/ml hydrocortisone and 10 ng/ml EGF. Human HNCAF 4 h, specimens were bisected, transferred to 70% EtOH and embedded in (head and neck CAFs) were cultured in DMEM with 10% FCS and insulin- paraffin wax on their cut edge. The 4-mm sections taken at 50 mm intervals were stained with haematoxylin and eosin for quantitative analysis. The transferrin-selenium in 10% CO2 at 37 1C. processing, embedding and staining of samples was performed by the Cancer Research UK Experimental Histopathology Laboratory. The Invasion Transient DNA transfections Index was subsequently calculated by using the formula 1À(Non-invading A431 cells were transfected with FuGENE 6 transfection reagent (Roche, Area/Invading Area), where 0 represented as no invasion. Welwyn Garden City, UK). Briefly, sub-confluent cells were washed and the media replaced with Opti-MEM. The previously prepared DNA-liposome Transwell-Matrigel invasion assays mix (6 ml FuGENE 6 þ 1.5 mg DNA for 6 well plates or 2.25 ml FuGENE 6 þ 0.75 mg DNA for 24 well plates) was then pipetted onto the cells and A431 cells were trypsinized 1 day after transfection (described above) and B20 000 cells were plated into the upper chamber of a 24 well BD Bioscience swirled briefly. After 5 h of incubation in 10% CO2 at 37 1C the transfection mix was removed, the cells washed with PBS and fresh DMEM with 10% Matrigel invasion chamber (#354480, Oxford, UK). When cells had adhered the FCS added. pUNO-hTRAF6-HA was purchased from InvivoGen (Autogen media in the upper chamber was changed to serum-free DMEM and the Bioclear UK Ltd, Calne, UK). Cezanne/ZA20D1 plasmids were a gift from lower chamber was filled with DMEM containing 10% FCS and 10 ng/ml Paul Evans (Hammersmith Hospital, UK) and IkB super-repressor plasmid EGF. Three days later the cells were fixed and stained with TRITC-phalloidin was a gift from Krishna Rajalingam (Frankfurt, Germany). (described above). Confocal microscopy was used to observe the number of cells above and below the filter. These were then manually counted when siRNA transfections the images were viewed using ImageJ software (http://rsbweb.nih.gov/ij/). Cells were cultured in standard conditions.5,43 A431 and SCC12 cells were transfected using DharmaFECT 2 (Dharmacon, Epsom, UK). HNCAF were ELISA procedure transfected using DharmaFECT 1 (Dharmacon). The final concentration of A431 or SCC12 cells were stimulated with 1 ng/ml IL-1b for 4 h. The siRNA in all transfections was 100 nM for screens and organotypic assays, supernatant was cleared by centrifugation. Levels of TNF-a were measured and 75 nM for all other assays. In all, 0.5, 2 and 5 ml DharmaFECT reagent by ELISA as per the protocol using commercial ELISA-based kits (R&D was used for transfecting 48, 24 and 6 well plates, respectively. TRAF6#2 systems #DTA00C, Oxford, UK). 50-GGACAAAGUUGCUGAAAUCUU-30; Dharmacon D-004712-02; TRAF6#3 50-GUUCAUAGUUUGAGCGUUAUU-30; Dharmacon D-004712-03 TRAF6#B Matrix remodelling assays 0 0 5 -UACGCGAACGUGGAAGUCAAU-3 ; Qiagen S103050145 (Crawley, UK); The contraction of Collagen-Matrigel matrices was measured as previously 0 0 ZA20d1#2 5 -GAAUCUAUCUGCCUUUGGAUU-3 ; Dharmacon D-008670-02 described. Briefly, 5 Â 105 HNCAF were seeded in the same Collagen- 0 0 ZA20d1#3 5 -GAAGGAGAAUACCAAGGAAUU-3 ; Dharmacon D-008670-03 Matrigel mixture used for organotypic invasion assays. After 72 h the 0 0 ZA20d1#A 5 -UUCCUCCGAUUUGUUUCAAUU-3 ; Qiagen S100764869. matrices were photographed and the degree of gel contraction was then assessed by using Image J software to measure the surface area of the gel Antibody methods relative to its well. The Contraction Index was subsequently calculated by Immunofluorescence. Cells were fixed with 4% paraformaldehyde for using the formula 1 --(Gel Area/Well Area). Microscopic analysis was carried 10 min. For visualisation, cells were permeabilised with 0.25% Triton X-100 by seeding 2.5 Â 104 HNCAF in 50 ml of the same Collagen-Matrigel mixture for 10 min and stained with TRITC phalloidin, DAPI and the following antibodies: used for organotypic invasion assays. These matrices were overlaid with naive Traf6 (Abcam#33915 - 1:100, Cambridge, UK), anti-HA epitope (12CA5 - 1:100), FAD 85 medium supplemented with 10% FCS, 5 mg/ml insulin (Invitrogen), Cy5 Donkey anti-Rabbit (Jackson Stratech, Newmarket, UK) and Alexa 555 0.5 mg/ml hydrocortisone and 10 ng/ml EGF or the same media conditioned Donkey anti-Mouse (Molecular Probes, Paisley, UK). Western blotting: by SCC12 cells over a 36 h period. In some cases either 31 ng/ml TNFa or SDS-PAGE and transfer to Protran nitrocellulose membrane was carried out 31 mg/ml infliximab (Remicade, Janssen Biotech, Horsham, PA, USA) was also using standard techniques. Membranes were blocked with 5% BSA before added. Infliximab was a gift of Martin Gore, The Royal Marsden Hospital, UK. incubation with the following antibodies TRAF6 1:2000 #33915 Abcam, IBa 1:1000 #4814 Cell Signaling Technology (Hitchin, UK), Phospho-IBa (Ser32) Traf6 immunohistochemistry 1:1000 #2859 Cell Signaling Technology in TBS 0.1%Tween20. þ Formalin-fixed paraffin-embedded material: paraffin-embedded tissue oral SCC tissue arrays (#OR601) were purchased from US Biomax Inc. (Rockville, Organotypic invasion assays MD, USA). Following dehydration, endogenous peroxidase activity was 5,44 The organotypic culture system was set up as previously described. To blocked using 1.6% hydrogen peroxide (H2O2) in PBS for 10 min followed make 1 ml of organotypic matrix the following reagents were mixed: by washing in distilled water. Antigen retrieval was performed by Matrigel (11.2 mg/ml) 200 ml, Rat tail collagen type I (9.03 mg/ml) 400 ml, immersing samples in sodium citrate buffer and microwaving three times 5 Â aMEM with sodium bicarbonate 80 ml, FCS 100 ml FAD medium with for 5 min at full power. Sections were then cooled to room temperature in 10% FCS 120 ml and 5 Â 105 HNCAF suspended in FAD medium with 10% running water and washed in PBS before blocking in goat serum diluted to FCS 100 ml. A total volume of 900 ml of this solution was aliquoted into wells 10% in 1% BSA for 30 min. Slides were incubated with primary antibody in of a 24 well plate and allowed to polymerise for 30 min at 37 1C. Once the 1% BSA for 1 h at room temperature. After washing in PBS-Tween20 gels were set, 1 ml of complete media was added per well and the gels left solution for 1 min, biotinylated goat anti-rabbit secondary antibody (1:250 to equilibrate at 37 1C for 24 h before changing the media daily thereafter. in 1% BSA) was then applied to the sections for 45 min, also at room A fibroblast-free collagen gel mix was also used to coat sterile nylon discs temperature. Samples were washed as above and developed microscopi- (Millipore, Watford, UK). After incubating at 37 1C for 20 min, the gel coated cally with VECTASTAIN Elite ABC Reagent and Vector DAB substrate (Vector discs were fixed in 1% glutaraldehyde for 1 h, washed four times in PBS, Laboratories Ltd, Peterborough, UK). A final wash in water was performed twice in complete media and then stored at 4 1C. At 72 h, 5 Â 105 SCC12 before the sections were counterstained lightly with haematoxylin, cells were seeded on to each gel in complete media. The next day, the gels dehydrated, cleared and mounted. Omission of the primary antibody were lifted on to individual collagen coated nylon discs resting on stainless acted as a negative control. TRAF6 immunostained sections were viewed at steel bridges placed in six well plates. Sufficient complete media was then Â 20 magnification and three images per sample captured. Digital images

Oncogene (2013) 747 --758 & 2013 Macmillan Publishers Limited Traf6 promotes SCC invasion SI Chaudhry et al 757 were loaded into NIS Elements software (Nikon UK Ltd, Kingston upon 15 Wojciak-Stothard B, Entwistle A, Garg R, Ridley AJ. Regulation of TNF-alpha- Thames, UK) and 10 representative immunostained areas were selected for induced reorganization of the actin cytoskeleton and cell-cell junctions by analysis per sample. TRAF6 immunostaining was cytosolic in nature and Rho, Rac, and Cdc42 in human endothelial cells. J Cell Physiol 1998; 176: therefore cell nuclei were not included in the regions analysed. The mean 150 --165. absolute pixel intensity within each region of interest was measured and 16 Pritchard S, Guilak F. Effects of interleukin-1 on calcium signaling and the increase normalised to a ‘white balanced’ area of the image by subtracting the of filamentous actin in isolated and in situ articular chondrocytes. Arthritis Rheum background pixel intensity. This normalised value was then expressed on a 2006; 54: 2164 --2174. 17 Ridley AJ. Rho GTPases and actin dynamics in membrane protrusions and vesicle scale of 0 to 1, with 0 representing absolute whiteness and 1 absolute trafficking. Trends Cell Biol 2006; 16: 522 --529. blackness. The mean and standard error of the 10 measurements was 18 Wang HR, Zhang Y, Ozdamar B, Ogunjimi AA, Alexandrova E, Thomsen GH et al. calculated to give an overall measure of mean pixel intensity for TRAF6 Regulation of cell polarity and protrusion formation by targeting RhoA for per sample. degradation. Science 2003; 302: 1775 --1779. Frozen sections: frozen sections of SCC of the head and neck and 19 Chen Y, Yang Z, Meng M, Zhao Y, Dong N, Yan H et al. Cullin mediates matched normal tissue samples were collected at the St George’s degradation of RhoA through evolutionarily conserved BTB adaptors to Hospital, London, in accordance with NHS Research Ethics Committee control actin cytoskeleton structure and cell movement. Mol Cell 2009; 35: (ref 06/Q0403/125) and the St George’s Healthcare NHS Trust Research 841 --855. Office (ref 07.0003). Samples were prepared for confocal microscopy as 20 Castillo-Lluva S, Tatham MH, Jones RC, Jaffray EG, Edmondson RD, Hay RT et al. SUMOylation of the GTPase Rac1 is required for optimal cell migration. Nat Cell described in ‘Antibody methods’ section. Images were acquired using Biol 2010; 12: 1078 --1085. an LSM510 confocal microscope. 21 Evans PC, Smith TS, Lai MJ, Williams MG, Burke DF, Heyninck K et al. A novel type of deubiquitinating enzyme. J Biol Chem 2003; 278: 23180 --23186. 22 Evans PC, Taylor ER, Coadwell J, Heyninck K, Beyaert R, Kilshaw PJ. Isolation CONFLICT OF INTEREST and characterization of two novel A20-like proteins. Biochem J 2001; 357(Pt 3): The authors declare no conflict of interest. 617 --623. 23 Wang KZ, Wara-Aswapati N, Boch JA, Yoshida Y, Hu CD, Galson DL et al. TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras ACKNOWLEDGEMENTS and is mediated by an interaction with cytoplasmic Src. J Cell Sci 2006; 119(Pt 8): 1579 --1591. We thank the Cancer Research UK for funding. We thank St George’s Thomas Tatum 24 Yang WL, Wang J, Chan CH, Lee SW, Campos AD, Lamothe B et al. Head and the Neck Unit staff involved in tissue collection, Martin Gore for providing The E3 ligase TRAF6 regulates Akt ubiquitination and activation. Science 2009; reagents, and lab members for their advice and discussions. 325: 1134 --1138. 25 Nystrom ML, Thomas GJ, Stone M, Mackenzie IC, Hart IR, Marshall JF. 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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2013) 747 --758 & 2013 Macmillan Publishers Limited

Autocrine IL-1&Beta;-TRAF6 Signalling Promotes Squamous Cell

Autocrine IL-1Β-TRAF6 Signalling Promotes Squamous Cell