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CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells

2004; Springer Nature; Volume: 23; Issue: 15 Linguagem: Inglês

10.1038/sj.emboj.7600325

1460-2075

Bryan C. Barnhart, Patrick Legembre, Eric M. Pietras, Concetta Bubici, Guido Franzoso, Marcus E. Peter,

Cancer therapeutics and mechanisms

Article22 July 2004free access CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells Bryan C Barnhart Bryan C Barnhart The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Patrick Legembre Patrick Legembre The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Eric Pietras Eric Pietras The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Concetta Bubici Concetta Bubici The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Guido Franzoso Guido Franzoso The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Marcus E Peter Corresponding Author Marcus E Peter The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Bryan C Barnhart Bryan C Barnhart The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Patrick Legembre Patrick Legembre The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Eric Pietras Eric Pietras The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Concetta Bubici Concetta Bubici The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Guido Franzoso Guido Franzoso The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Marcus E Peter Corresponding Author Marcus E Peter The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA Search for more papers by this author Author Information Bryan C Barnhart1,‡, Patrick Legembre1,‡, Eric Pietras1, Concetta Bubici1, Guido Franzoso1 and Marcus E Peter 1 1The Ben May Institute for Cancer Research, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL, USA ‡These authors contributed equally to this work *Corresponding author. The Ben May Cancer Institute, University of Chicago, 924 East 57th Street, R112, Chicago, IL 60637-5420, USA. Tel.: +1 773 702 4728; Fax: +1 773 702 3701; E-mail: [email protected] The EMBO Journal (2004)23:3175-3185https://doi.org/10.1038/sj.emboj.7600325 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The apoptosis-inducing death receptor CD95 (APO-1/Fas) controls the homeostasis of many tissues. Despite its apoptotic potential, most human tumors are refractory to the cytotoxic effects of CD95 ligand. We now show that CD95 stimulation of multiple apoptosis-resistant tumor cells by CD95 ligand induces increased motility and invasiveness, a response much less efficiently triggered by TNFα or TRAIL. Three signaling pathways resulting in activation of NF-κB, Erk1/2 and caspase-8 were found to be important to this novel activity of CD95. Gene chip analyses of a CD95-stimulated tumor cell line identified a number of potential survival genes and genes that are known to regulate increased motility and invasiveness of tumor cells to be induced. Among these genes, urokinase plasminogen activator was found to be required for the CD95 ligand-induced motility and invasiveness. Our data suggest that CD95L, which is found elevated in many human cancer patients, has tumorigenic activities on human cancer cells. This could become highly relevant during chemotherapy, which can cause upregulation of CD95 ligand by both tumor and nontumor cells. Introduction CD95 ligand (CD95L/FasL), a member of the death ligand family, which also includes TNFα and TRAIL has been viewed as an apoptosis-inducing ligand and members of this family are therefore being considered as potential antitumor reagents (Gardnerova et al, 2000). CD95L is expressed either on cell membranes (mCD95L) or in soluble form (sCD95L) and induces apoptosis when it triggers its cognate receptor CD95 (APO-1/Fas) (Peter et al, 2003). We have previously demonstrated that cells can die in different ways following CD95 activation, either dependent on (Type II) or independent of (Type I) mitochondria (Scaffidi et al, 1998; Barnhart et al, 2003). We recently found that Type I cells correspond to mesenchymal tumors, whereas Type II cells have a more epithelial phenotype (Algeciras-Schimnich et al, 2003). sCD95L was only cytotoxic to Type II/epithelial tumor cells and did not kill Type I/mesenchymal cells, indicating that CD95L has differential effects on tumors depending on the tumor phenotype. Chemotherapeutic drugs can cause upregulation of CD95L and it is believed that this contributes to the elimination of tumor cells by inducing their apoptosis (Friesen et al, 1996). However, many tumor cells are resistant to CD95-mediated apoptosis, especially after therapy (Friesen et al, 1999). Although it has been recognized that CD95 stimulation of certain cells under certain conditions can cause induction of genes that have functions outside of apoptosis (Faouzi et al, 2001; Park et al, 2003) and that CD95 can promote proliferation of T cells (Alderson et al, 1993; Alam et al, 1999; Kennedy et al, 1999), specific tumor-promoting effects of CD95 stimulation for CD95 apoptosis-resistant tumor cells have not been reported. We now have performed an analysis of the responses of a panel of CD95 apoptosis-resistant tumor cell lines to sCD95L, mCD95L or agonistic anti-CD95 antibodies. Our data demonstrate that a significant number of these cells respond to any stimulation of CD95 with increased motility and invasiveness through Matrigel-coated membranes. This activity of CD95 involves activation of at least five different apoptosis-independent pathways of which at least three (activation of caspase-8, NF-κB and Erk1/2) are independently required to different degrees for the increased motility and invasiveness. An analysis of 54 transcription factors demonstrated that AP1, AP2, CREB and NF-κB are activated in CD95-stimulated cells. The effect is selective for CD95 stimulation since treatment of apoptosis-resistant cells with either TNFα or TRAIL only marginally induced motility or invasiveness. Furthermore, we found that one of the genes identified in gene screens, urokinase plasminogen activator, is required for both CD95-induced motility and invasiveness. Our data suggest that elevated levels of CD95L found in cancer patients could contribute to increase tumorigenicity of tumor cells. Results Stimulation of CD95 on CD95 apoptosis-resistant tumor cells induces increased invasiveness It has been previously suggested that sCD95L is very inefficient in inducing apoptosis in tumor cells (Schneider et al, 1998; Tanaka et al, 1998) and although it was shown that sCD95L can activate other nonapoptotic pathways (Ahn et al, 2001) the range of biological responses of tumors cells to this stimulus is unknown. It is well established that as part of carcinogenesis tumors often either inactivate the apoptosis-inducing activity of CD95 by downregulating or inactivating CD95 or its proapoptotic signaling molecules, or by upregulation of antiapoptotic proteins such as Bcl-2 or c-FLIP (Peter et al, 2003). Since increased expression of sCD95L is frequently found in cancer patients and is associated with various forms of cancer (Owen-Schaub et al, 2000), we asked whether CD95 stimulation of CD95 apoptosis-resistant tumor cells provokes protumorigenic responses. To address directly this question, we performed in vitro invasiveness assays (Figure 1). In this assay, tumor cells were placed in the top of two chambers separated by a Matrigel-coated membrane (pore size 8 μm) in serum-free medium. The bottom chamber contained 10% FCS. The anti-CD95 stimulus (either anti-CD95 antibody or CD95 ligand) was added to both the top and bottom chambers. Any response by tumor cells in this assay was therefore the result of stimulation of surface CD95 on the tumor cell ultimately triggering a program of increased motility and invasiveness through the basement membrane mimetic Matrigel. Figure 1.Stimulation of CD95 in CD95 apoptosis-resistant tumor cells induces invasiveness. (A) A 3-day in vitro invasiveness assay of MCF7(FB) and SK-OV-3 cells incubated with anti-APO-1 (αA), LzCD95L (Lz) or sCD95L (S).The inset shows fixed and stained cells that migrated through a Matrigel-coated membrane unstimulated (−) or stimulated (+) with anti-APO-1. (B) Cell proliferation in MCF7(FB) cells following stimulation with LzCD95L was determined by counting cells each day following trypsinization. (C) Increase in the total number of migrating (left) and invading (right) MCF7(FB) cells upon stimulation with LzCD95L. Percentages of cells compared to the total number of cells in the well are given. (D) Invasiveness assay of apoptosis-resistant cells (left panel) following stimulation with anti-APO-1 (asterisk), LzCD95L (underlined) or sCD95L. Two apoptosis-sensitive Type I tumor cell lines that are resistant to sCD95L also responded with increased invasiveness (right panel). Cell proliferation was controlled by MTS assay to ensure that the increase in cell number was not due to the increase in proliferation. Selected cell lines were also counted as described in (B). (E) Invasiveness (left) and motility (right) assay of MCF7(FB) cells incubated with anti-APO-1 (αA), CT26L or CT26 cells at the ratios 1:1, 3.3:1 (labeled as 3:1) and 6.7:1 (labeled as 7:1). The ratio of CT26 to MCF7(FB) cells in the invasiveness assays was 6.7:1. (F) PBMCs that had been activated for 16 h with PHA, followed by 6 days with IL-2 (A), or were not stimulated with either PHA or IL-2 (R) were cocultured with MCF7(FB) cells at a 6.7:1 ratio in an invasiveness assay. Where indicated, the PBMCs were preincubated with NOK-1. NOK-1 was maintained in the culture at 10 μg/ml. Download figure Download PowerPoint To test the effects of triggering CD95 on apoptosis-resistant tumor cells under controlled conditions, we first tested MCF7-Fas-Bcl-xL(FB) breast carcinoma cells, which express high levels of CD95 (Algeciras-Schimnich et al, 2003) and are rendered apoptosis resistant by the antiapoptotic protein Bcl-xL (Stegh et al, 2002). These cells are a model for a tumor cell that has acquired apoptosis resistance through upregulation of a Bcl-2 family member (which is frequently found in human tumors). As expected in apoptosis-sensitive MCF7-Fas control cells, CD95 stimulation did not promote invasiveness (Figure 1A) but induced apoptosis (data not shown). Unstimulated MCF7(FB) cells also exhibited low invasiveness. However, when these cells were exposed to either the agonistic anti-CD95 mAb anti-APO-1, leucine zipper-tagged CD95L (LzCD95L) or sCD95L, they migrated through Matrigel. This response was not limited to MCF7(FB) cells since it was also observed in a naturally CD95-resistant ovarian tumor cell line SK-OV-3. Again all three CD95-specific stimuli induced increased invasiveness of SK-OV-3 cells (Figure 1A). Stimulation of CD95 on apoptosis-resistant tumor cells could induce increased proliferation, which might interfere with the quantification of the invasiveness assay. To determine whether tumor cells responded to CD95 with increased proliferation, we counted MCF7(FB) cells plated in Boyden chambers under the exact conditions of an invasiveness assay and counted the total number of cells (invaded and noninvading) at different time points in the absence or presence of LzCD95L (Figure 1B). The cells did not respond to triggering with increased proliferation, indicating that the increased numbers of cells detected in an invasiveness assay on the bottom of the Matrigel-coated membrane was solely due to increased invasiveness and not accelerated growth. This result was also confirmed by performing a 3-day MTS assay and by growing and counting cells under standard conditions (data not shown). We next determined the percentage of cells that responded to stimulation of CD95 with increased motility (as determined in a migration assay, without Matrigel) and/or invasiveness (Figure 1C). We found that the percentage of cells that migrated through the membrane after CD95 stimulation increased from 2 to 12.9% and the percentage of cells invading from 0.5 to 4.8%. The increase in invasiveness induced by CD95L was not limited to MCF7(FB) or SK-OV-3 cells. A total of 14 other cell lines also responded with increased invasiveness when stimulated through CD95 (Figures 1D and 5C). Again we determined that these responses were not due to an increase in cell proliferation but due to increased invasiveness, by performing 3-day MTS proliferation assays with all tested cells (data not shown). Tumor cells most likely encounter CD95L in two different forms in vivo: sCD95L in the serum and membrane-bound (m)CD95L, for example, on tumor-infiltrating lymphocytes, proinflammatory cells or stromal cells. This could potentially involve cell–cell interaction between tumor cells and CD95L-expressing cells. To test whether cell-bound human mCD95L could trigger an increase in motility and/or invasiveness of CD95 apoptosis-resistant tumors, we incubated MCF7(FB) cells with a cell line expressing authentic unmodified human mCD95L (CT26L; Aoki et al, 2001). When mixed at a ratio of 3.3:1, CT26L cells induced maximal motility of MCF7(FB) cells (Figure 1E) although significant increase of motility was observed when mixed at a ratio of 1:1 or 6.7:1. The activity of the CT26L cells was due to the human mCD95L that they express since it could be significantly inhibited by a neutralizing anti-human CD95L mAb NOK-1. CT26L cells also induced invasiveness, which was not observed with the parental CT26 cells that do not express human CD95L (Figure 1E). In summary, any form of stimulation of CD95 tested, including sCD95L and physiological, unmodified mCD95L, can induce an increase in motility and invasiveness of CD95-resistant tumor cells. To test directly the form of CD95L that tumor cells might encounter in vivo, we incubated MCF7(FB) cells with human peripheral blood mononuclear cells (PBMCs) (Figure 1F). Resting PBMCs did not show an effect on the invasiveness of the tumor cells. However, when the tumor cells were mixed with PBMCs that had been activated with phytohemagglutinin (24 h) followed by stimulation with interleukin 2 (IL-2) for 5 days, which selectively activates T lymphocytes, an increase in invasiveness could be detected (Figure 1F). This activity of the activated PBMCs was primarily due to CD95L since it could substantially be blocked by the CD95L-neutralizing antibody. The majority of CD95 apoptosis-resistant tumor cells respond to anti-CD95 stimulation with activation of NF-κB Two independent Affymetrix gene screens of MCF7(FB) cells stimulated through CD95 identified a number of NF-κB target genes that were upregulated in CD95-stimulated tumor cells (see Figure 6B). Electromobility shift assays (EMSAs) confirmed activation of NF-κB in MCF7(FB) cells when stimulated through CD95 by both the agonistic anti-CD95 antibody anti-APO-1 and two CD95 ligand preparations (Figure 2A). Activation of NF-κB was first detectable 1 h after CD95 stimulation and it progressively increased reaching levels almost as high as those induced by TNFα (Figure 2A). Activation of NF-κB was likely a direct effect as CD95-induced activation of NF-κB was not prevented by cycloheximide pretreatment (Supplementary Figure 1A). Supershift analysis identified the canonical p50/p65 heterodimer as the predominant complex activated by CD95 (Supplementary Figure 1B). Figure 2.CD95-mediated activation of NF-κB contributes to in vitro invasiveness. (A) EMSA analysis of NF-κB activation of MCF7(FB) cells stimulated through CD95 or cells treated with TNFα or LzTRAIL. (B) EMSA analysis of NF-κB activity of cell extracts of MCF7(FB) cells stimulated with LzCD95L for 4 h and after pretreatment with BAY 11-7082 or either GST-TAT-IκBαmut or GST-TAT control (tat). (C) MCF7(FB) cells were treated with 2.5 μM BAY 11-7082 and subjected to invasiveness assay. The NF-κB inhibitor was maintained in the culture for the duration of the assay. (D) Invasiveness assay and (E) migration assay of MCF7(FB) cells incubated with GST-TAT-IκBαmut (mut) or GST-TAT control (t). Download figure Download PowerPoint To assess the significance of the activation of NF-κB in CD95-resistant tumor cells, we monitored NF-κB activation in cell lines of the antitumor drug screening panel of the NCI (NCI60). We previously determined that the majority of these cell lines are resistant to CD95-mediated apoptosis, and many of them express high levels of surface CD95, suggesting that they developed mechanisms to withstand apoptotic CD95 stimulation (Algeciras-Schimnich et al, 2003; data not shown). A total of 13 of these cell lines were selected randomly and examined for NF-κB activation upon CD95 triggering. Most cell lines (11/13, 85%) activated NF-κB in response to one or more of the CD95-specific stimuli with only two lines exhibiting no CD95-induced NF-κB complexes (Supplementary Figure 2A). Our recent analysis of the NCI60 cells demonstrated that Type I but not Type II cells (both of which are apoptosis sensitive to crosslinked agonistic anti-CD95 antibodies or highly aggregated CD95L) are resistant to the toxic effects of soluble CD95L (sCD95L) (Algeciras-Schimnich et al, 2003). We now demonstrate that sCD95L induces NF-κB activation in the prototype Type I tumor cell lines SKW6.4 and H9 (Supplementary Figure 2B), and in seven of eight cell lines randomly picked from the 11 NCI60 we identified as Type I cells (Supplementary Figure 2C). In summary, the majority of all CD95 apoptosis-resistant cell lines tested, and even the CD95-sensitive Type I cell lines stimulated with sCD95L, from a variety of histological origins responded to CD95 stimulation with activation of the NF-κB pathway. It has been demonstrated previously that the NF-κB pathway can block apoptosis induced by death receptors as well as promote tumor progression (Orlowski and Baldwin, 2002) and NF-κB has been shown to be activated in cells after triggering of CD95 (Ponton et al, 1996; Packham et al, 1997). However, these studies focused on only a few cell lines and the functional relevance of this activation remained unknown. Our data now suggest that activation of NF-κB may be involved in the increased invasiveness of CD95-stimulated tumor cells. CD95-induced invasiveness requires activation of NF-κB We next tested whether the CD95-induced invasiveness was dependent on activation of NF-κB. Incubation of MCF7(FB) cells with the NF-κB inhibitor Bay 11-7082 (Mori et al, 2002) or a cell-permeable glutathione S-transferase (GST) fusion protein of a nondegradable IκBα mutant protein (GST-TAT-IκBαmut) blocked activation of NF-κB (Figure 2B) and CD95-induced invasiveness (Figure 2C and D). A migration assay demonstrated that activation of NF-κB was also at least in part responsible for increased motility (Figure 2E). CD95L is more potent in inducing invasiveness than TNFα or TRAIL The data so far suggested that activation of NF-κB was involved in CD95-induced motility and invasiveness. CD95 triggering resulted in activation of the canonical NF-κB p50/p65 heterodimer that is also activated by TNFα stimulation (Figure 2A; Tang et al, 2001). However, in both the EMSA analysis and gene reporter assays using a κB-driven CAT reporter construct, TNFα was more active in inducing NF-κB than CD95L (Figure 3A) without toxicity (as monitored by MTS assays, data not shown). We therefore tested the activity of TNFα and the CD95L-related death ligand TRAIL in inducing invasiveness of MCF7(FB) cells (Figure 3B). TNFα and TRAIL only marginally induced invasiveness in these cells. When compared side by side, both TNFα and LzCD95L induced activation of the IκB kinase complex as evidenced by activation of its subunit IKKβ (Figure 3C). The activation of NF-κB through CD95 was somewhat delayed compared to TNFα-induced NF-κB activation, suggesting a different pathway upstream of the IKK complex. We identified NEMO to be an essential component in the pathway of both death receptors since a peptide that blocks interaction of NEMO with the IKK complex (May et al, 2000) efficiently prevented activation of NF-κB as evidenced by both EMSA (Supplementary Figure 1C) and an ELISA-based assay that detects activated p65 (Supplementary Figure 1D). We therefore determined the role of the receptor proximal component of the TNF pathway, RIP, in CD95-mediated activation of NF-κB by testing RIP-deficient Jurkat cells. RIP was required for TNFα-induced activation of NF-κB but dispensable for CD95-induced NF-κB activation (Figure 3D). In summary, our data indicate that NF-κB activation is required but not sufficient for tumor cells to demonstrate increased motility and invasiveness, suggesting involvement of other pathways that either emanate in the pathway somewhere upstream of the IKK complex or that act in parallel to the NF-κB pathway. Figure 3.CD95L is more efficient than TNFα or TRAIL in inducing increased motility and invasiveness of MCF7(FB) cells. (A) CAT reporter gene analysis of MCF7(FB) cells transfected with an NF-κB promoter (κB) or mutated NF-κB promoter (mut) and treated with LzCD95L (Lz), anti-APO-1 (A), sCD95L or TNFα (for 4 h) and assayed over time. (B) A 3-day invasiveness assay of MCF7(FB) cells stimulated with 1 μg/ml LzCD95L, 1000 U/ml TNFα or 1 μg/ml LzTRAIL. (C) MCF7(FB) cells were incubated for the indicated times with LzCD95L or TNFα. Cell lysates were subjected to IKKβ immunoprecipitation followed by in vitro kinase assay (IKA) using GST-IκBα as the substrate. Phosphorylated IκBα was resolved and exposed to film. W, Western blot. (D) Jurkat cells deficient for RIP expression stimulated with either anti-APO-1/zVAD-fmk (A), LzCD95L/zVAD-fmk (Lz) or TNFα for 4 h were subjected to EMSA. P, parental Jurkat cells; R, RIP-deficient cells. Download figure Download PowerPoint Requirement for the Erk signaling pathway for CD95-induced invasiveness CD95 activates the three major MAP kinase pathways under certain conditions (Toyoshima et al, 1997; Desbarats et al, 2003). Western blot analyses using pairs of antiphospho site-directed antibodies and their corresponding panspecific antibodies against the three major components of the MAPK pathways, Erk1/2, JNK1/2 and p38, determined that all three MAP kinases are activated upon stimulation of CD95 in MCF7(FB) cells (Figure 4A). Treatment of MCF7(FB) cells with LzCD95L induced activation of the Erk1/2 MAP kinases, which was completely prevented by the MEK1/2 inhibitor PD98059 (Figure 4B). Interestingly, addition of caspase inhibitors did not inhibit Erk activation, suggesting that these events occur independently. This conclusion is consistent with the finding that activation of Erk through CD95 is independent of its death domain (DD) (Desbarats et al, 2003). When we treated MCF7(FB) cells with the NEMO-inhibiting peptide, which resulted in a profound inhibition of NF-κB activation in CD95-stimulated cells (Figure 4C, see also Supplementary Figure 1C), no effect was seen on activation of Erk, indicating that CD95-induced activation of Erk is also independent of activation of NF-κB. To determine the functional consequences of Erk activation, we tested the effects of PD98059 in the in vitro invasiveness assay (Figure 4D). PD98059 treatment significantly inhibited invasiveness, indicating that like activation of NF-κB Erk activation contributes to CD95-induced invasiveness. The p38 inhibitor SB203580 had no effect on invasiveness (while it completely abolished activation of p38, data not shown), excluding a role of p38 in the CD95-induced invasiveness (Figure 4D). A contribution of JNK kinases to the in vitro invasiveness could not be determined since inhibition of JNK reduced viability of these cells (as assessed by an MTS assay, data not shown). Additionally, we did not detect activation of PI3 kinase in CD95-stimulated MCF7(FB) cells, and the PI3 kinase inhibitors wortmannin and LY294002 did not inhibit CD95-induced invasiveness (data not shown). To test whether the MAP kinase pathway acts independently or in conjunction with NF-κB, we determined the effects of PD98059 on an NF-κB-driven CAT reporter assay. PD98059 caused only a minor reduction of reporter gene activity (Figure 4E). Figure 4.All three MAP kinase pathways are activated by LzCD95L in MCF7(FB) cells and activation of Erk is involved in CD95-induced invasiveness. (A) MCF7(FB) cells were stimulated with LzCD95L for indicated times and lysates of the cells were subjected to Western blot analysis for phosphorylation of indicated MAP kinases. Ratios of intensity of phosphorylated versus unphosphorylated kinase bands were determined by densitometry. (B) Kinetics of phosphorylation of p42/p44 Erk in LzCD95L-treated MCF7(FB) cells. As indicated (30 min stimulated), cells were preincubated with the following inhibitors: 20 μM PD98059 (PD), 40 μM zVAD (Z) or 40 μM zIETD (I). (C) MCF7(FB) cells were preincubated for 3 h with 140 μM of control peptide (C) or NEMO binding domain (NBD) peptide (N) and then stimulated for 2 h with LzCD95L. Nuclear extracts were purified and subjected to an EMSA (top panel). In parallel, cells were lysed and phosphorylation of Erk was analyzed by Western blotting (bottom panels). (D) Invasiveness assay of MCF7(FB) cells preincubated with PD98059 or p38 inhibitor SB203580. Effectiveness of the SB inhibitor was tested using phosphospecific antibodies (not shown). (E) CAT reporter assay of LzCD95L-stimulated MCF7(FB) cells following preincubation with PD98059. Download figure Download PowerPoint Figure 5.CD95-induced invasiveness requires caspase-8 activity. (A) Invasiveness assay of MCF7(FB) cells in the presence of zVAD-fmk (z) or zIETD-fmk (I). (B) κB-driven CAT reporter assay of MCF7(FB) cells preincubated with zVAD-fmk (z) or zIETD-fmk (I). (C) Invasiveness assay of NCI60 cells stimulated through CD95 in the presence of 20 μM PD98059 (PD) or 40 μM zIETD-fmk (I). Download figure Download PowerPoint Figure 6.CD95 stimulation activates a transcriptional program. (A) MCF7(FB) cells were stimulated with LzCD95L as indicated and subjected to a Transignal DNA/Protein array to determine activation of transcription factors. The assay was performed three times and representative data are shown. Left, transcription factors that were activated in all three assays; right, transcription factors that were not significantly activated in any of the three assays. (B) MCF7(FB) cells were stimulated with anti-APO-1 and protein A for 8 h followed by isolation of RNA and gene chip analysis. The screen was performed twice (scr. 1 and scr. 2) using different MCF7(FB) clones. Only genes induced more than two-fold in both screens are shown. Prepro uPA could not be detected in the first screen due to a bad chip area. If a gene appeared more than once only the more highly induced signal is shown. Cells were treated with PD98059 and BAY 11-7082 in the second screen and percent induction of genes in the presence of these inhibitors is indicated. Genes that are inhibited more than 25% are shown in pink and more than 50% in red. (C) Semiquantitative RT–PCR of 8 h stimulated MCF-7(FB) cells treated with 40 μM zIETD-fmk (I) or zVAD-fmk (z) for the genes indicated. PCR products were subjected to agarose gel electrophoresis. Download figure Download PowerPoint Involvement of active caspase-8 in CD95-induced invasiveness We recently demonstrated that MCF7(FB) cells process caspase-8 despite their resistance to CD95-mediated apoptosis (Stegh et al, 2002). Active caspase-8 could therefore play a role in the increased invasiveness observed upon CD95 stimulation. Treatment of these cells with the oligo-specific caspase inhibitor zVAD-fmk or the caspase-8 selective inhibitor zIETD-fmk completely blocked CD95-induced invasiveness in MCF7(FB) cells (Figure 5A). Inhibition of caspases had no effect on CD95-induced increase in NF-κB DNA binding (data not shown). Furthermore, it also did not prevent NF-κB-dependent transcriptional activation (Figure 5B), suggesting that caspase-8 acts downstream or in parallel to the transcriptional activation induced by activated NF-κB. To determine the contribution of both the MAP kinase pathway and caspase-8 activation to the increase in invasiveness in untransfected tumor cells, we pretreated six of the NCI60 cells with either PD98059 or zIETD-fmk followed by stimulation with sCD95L (Figure 5C). In all cases, we detected increased invasiveness, which was blocked by inhibition of MAP kinases. In contrast, inhibition of caspase-8 significantly inhibited invasiveness of most but not all cells.