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Autocrine IL-1Β-TRAF6 Signalling Promotes Squamous Cell

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Autocrine IL-1Β-TRAF6 Signalling Promotes Squamous Cell Oncogene (2013) 32, 747 --758 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc 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).
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