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CD95 and TRAF2 promote invasiveness of pancreatic cancer cells

Anna Trauzold^*, Christian Röder^*, Bence Sipos, Kristin Karsten^*, Alexander Arlt, Ping Jiang^*, Jose Ignacio Martin-Subero, Daniela Siegmund^||, Susanne Müerköster, Laia Pagerols-Raluy^*, Reiner Siebert, Harald Wajant^|| and Holger Kalthoff^*,1

^* Molecular Oncology Unit; Clinic for General Surgery;
Institute of Pathology;
Laboratory of Molecular Gastroenterology, 1st Department of Medicine;
Institute of Human Genetics; University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany; and
^|| Department of Molecular Internal Medicine, Medical Polyclinic, University of Würzburg, Würzburg, Germany

¹Correspondence: Molecular Oncology Unit; Clinic for General Surgery, Arnold-Heller Str. 7, 24105 Kiel, Germany. E-mail: hkalthoff{at}email.uni-kiel.de

SPECIFIC AIMS

TRAF2 is an adaptor molecule that interacts with death receptors, promoting activation of NF-B. To investigate a possible role of TRAF2 in CD95-mediated signaling in pancreatic tumor cells, we 1) investigated expression levels of TRAF2 protein in pancreatic tumor tissues and cell lines, 2) analyzed consequences of TRAF2 overexpression on CD95-mediated apoptosis and invasive properties of pancreatic tumor cells, and 3) evaluated the role of TRAF2 in nonapoptotic CD95 signaling.

PRINCIPAL FINDINGS

1. TRAF2 is overexpressed in pancreatic tumors and pancreatic tumor cell lines
We determined expression levels of TRAF2 in pancreatic tumor cells and found it strongly elevated in pancreatic tumors and tumor cell lines (Fig. 1 A, B, D). By immunohistochemistry, 34 of 36 pancreatic carcinoma tissues showed high levels of TRAF2 whereas ductal cells in normal tissues showed little or no expression (Fig. 1C ). Anti-CD95-resistant cell lines PancTuI, Panc89, and Panc1 expressed highly elevated levels of TRAF2; sensitive Colo357 cells showed very low levels (Fig. 1D ).

View larger version (106K): [in this window] [in a new window]   Figure 1. Expression of TRAF2 in pancreatic tumor cells. Immunohistochemical staining of TRAF2 in poorly (A) and moderately differentiated (B) ductal adenocarcinoma as well as in normal pancreas (C). D) Western blot showing the expression of TRAF2 in pancreatic tumor cell lines. Equal loading was verified by determination of ß-actin expression. For comparison, cell viability (expressed as % of untreated control) after challenge with agonistic anti-CD95 antibodies (CH11, 100 ng/mL) for 24 h is shown as determined by JAM assay. E) Regulation of TRAF2 expression. Cells were seeded (1.5x105/well) into 6-well plates in RPMI medium with 10% FCS. 24 h later cells were treated with UO126 (10 µM), Goe6983 (100 nM), Goe6983 (20 µM), MG132 (2 µM) or left untreated for additional 36 h. The expression of TRAF2 was analyzed by Western blot with ß-actin as loading control.

By interphase FISH analysis we found that differences in TRAF2 expression in pancreatic tumor cell lines are not related to chromosomal imbalances in TRAF2 gene locus. As NF-B, MEK, and PKCs are constitutively activated in pancreatic tumor cells, we tested involvement of corresponding pathways in TRAF2 expression (Fig. 1E ). We saw a strong decrease in TRAF2 protein levels in all cell lines tested using the NF-B inhibitor MG132. Similar results were obtained with two other NF-B inhibitors, sulfasalazine and gliotoxin (data not shown). Inhibition of MEK by UO126 resulted in reduced TRAF2 expression in Panc89 and Colo357 cells but no effect was observed in Panc1 and PancTuI cells. Blocking of PKC activity with Goe6983 at a concentration inhibiting classical and novel PKCs (100 nM) or one inhibiting PKD1/PKCµ (20 µM) showed no influence on TRAF2 expression in cell lines tested.

2. Overexpression of TRAF2 not only protects cells from CD95-mediated apoptosis but enhances invasion of pancreatic tumor cells
We investigated whether TRAF2 could inhibit CD95-mediated apoptosis and found that stable introduction of a TRAF2 expression construct into CD95-sensitive Colo357 cells efficiently inhibited CD95-mediated apoptosis. A block of the apoptotic signal transduction had occurred at the level of the death-inducing signaling complex (DISC), as no caspase 8-activation and no Bid cleavage was detected after exposure to agonistic anti-CD95 antibodies. Conversely, down-regulation of TRAF2 by siRNA rendered resistant Panc89 cells sensitive to CD95-mediated apoptosis.

Overexpression of TRAF2 in Colo357 cells resulted in a strongly enhanced secretion of matrix metalloproteinases (MMP-2 and MMP-9), urokinase-type plasminogen activator (uPA), and IL-8, molecules linked to the invasive potential of pancreatic tumor cells. Using an in vitro invasion assay, we demonstrated that Colo357/TRAF2 cells were much more invasive than mock-transfected control cells.

3. Stimulation of CD95 enhances the invasive potential of pancreatic tumor cells
We recently reported the activation of NF-B upon CD95 ligation in apoptosis-resistant PancTuI cells. As TRAF2 is involved in death receptor-mediated NF-B activation, we investigated the CD95-induced NF-B activation in TRAF2 overexpression and control Colo357 cells. NF-B was indeed activated upon CD95 ligation in Colo357 cells and the level of NF-B activity was increased in Colo357/TRAF2 cells compared with vector control cells. Similar results were obtained for the transcription factor AP-1. Moreover, the activity of both transcription factors was constitutively higher in Colo357/TRAF2 than in control cells.

We next studied the effects of CD95 ligation on the expression of these proteins in Colo357/TRAF2 cells. Real-time RT-PCR analysis revealed strong up-regulation of IL-8 and uPA transcripts 5 h after CD95 stimulation in TRAF2-transfectants compared with control cells (Fig. 2 A). Using ELISA tests, we determined IL-8 and uPA levels in cell culture supernatants. Figure 2B shows that CD95 stimulation of TRAF2 and vector control cells induced secretion of IL-8 and uPA. The effects of CD95 stimulation were clearly enhanced in TRAF2-overexpressing cells. Blocking of caspases by z-VAD-fmk partially inhibited secretion of both proteins, suggesting that CD95-induced uPA and IL-8 secretion partially depends on active caspases. To prove the hypothesis that CD95 ligation could increase the invasive potential, we performed an invasion assay using Colo357 cells cultured in conditioned supernatants of cells untreated or treated with CD95L. To inhibit residual CD95L, supernatants were incubated with neutralizing anti-CD95L-antibodies (NOK-1) before its addition to the cells. Figure 2C shows that the invasive potential of Colo357/TRAF2 cells was strongly enhanced when these cells were cultured in conditioned supernatants of Colo357/TRAF2 cells treated with CD95L vs. supernatants derived from untreated Colo357/TRAF2 cells.

View larger version (50K): [in this window] [in a new window]   Figure 2. CD95-induced expression and secretion of IL-8 and uPA in Colo357/TRAF2 cells. Colo357/vector cells (vc) and Colo357/TRAF2 cells (TRAF2) were seeded in six-well plates at a density of 2.7 x 105/well in 3 mL of culture medium. After 24 h (time point 0) cells were treated with 1 mL of medium (cell culture supernatant from each cell line) supplemented with CD95L (100 ng/mL), CD95L (100 ng/mL) + zVAD (50 µM) or left untreated. After 18 h supernatants were collected and clarified by centrifugation. To study IL-8 and uPA expression at the transcriptional level, cells were stimulated for 5 h and total RNA was prepared. A) Quantitative real-time RT-PCR with specific primers for IL-8 and uPA. Linearity of the calibration curve is shown in the inset. Expression is calculated as % of the respective untreated control cells (vc). B) IL-8 and uPA secretion measured by IL-8 and uPA-ELISA tests. Concentrations of IL-8 and uPA were normalized to the cell numbers determined in parallel at time point 0 h. Data represent means ± SD of 3 independent experiments. C) CD95-induced invasion of Colo357/TRAF2 cells. KiF5 fibroblasts were seeded into a 24-well plate at a density of 1.5 x 105 cells/well, grown for 3 days and treated with DMSO. 2 x 104 cells/well of Colo357/TRAF2 cells were seeded on the fibroblasts monolayer and allowed to adhere for 24 h. Medium was replaced with conditioned medium from Colo357/TRAF2 cells treated for 18 h with CD95L (100 ng/mL) and neutralized with NOK-1 antibody (4 µg/mL, right panel). As control (left panel), cells were grown in conditioned medium from untreated Colo357/TRAF2 cells. After an additional 48 h, cells were washed with PBS, stained with Trypan blue, washed again with PBS and photographed. Results shown are representative for 3 experiments each performed in duplicate.

CONCLUSIONS

We identified the protein TRAF2 as a key player in pancreatic cancer pathophysiology (Fig. 3 ). We observed overexpression of TRAF2 in the majority of pancreatic tumors and tumor cell lines, a phenomenon that could not be explained by genetic aberrations as shown by FISH analyses. Instead, pharmacological inhibitor studies suggested a role of the MEK and NF-B pathway in TRAF2 up-regulation.

View larger version (28K): [in this window] [in a new window]   Figure 3. Model of constitutive and stimulation-dependent effects of TRAF2 overexpression in a pancreatic tumor cell. TRAF2 increases the constitutive activity of transcription factors NF-B and AP1 and by up-regulating the secretion of proteolytic enzymes (MMPs and uPA) promotes cell invasiveness. After CD95 triggering TRAF2 efficiently inhibits apoptosis by switching the apoptotic pathway toward NF-B/AP1 activation leading to further enhanced invasiveness.

Constitutively active NF-B has been shown to be involved in protecting against apoptosis in pancreatic tumor cells. Since activation of the NF-B pathway in neoplastic cells can be caused by changes on the genomic level, we performed interphase FISH analyses but found no chromosomal aberrations affecting the RELA or NFKB1 loci in any cell lines studied. Besides genomic changes, autocrine stimulation or deregulated signaling may result in permanent transcription factor activity. We propose that overexpression of TRAF2 is responsible for constitutive NF-B activation in apoptosis-resistant pancreatic tumor cells.

Our results demonstrated that pancreatic tumor cells expressing high levels of TRAF2 and active NF-B were resistant to CD95-mediated apoptosis. Stable overexpression of TRAF2 in apoptosis-sensitive Colo357 cells led to enhanced constitutive NF-B activity and apoptosis resistance. Inhibition of CD95-mediated cell death was shown to occur at the level of caspase-8 activation. It is likely that increased constitutive NF-B activity further enhanced activation of NF-B after CD95 ligation, as well as elevated c-FLIP levels in TRAF2 overexpressing Colo357 cells responsible for the phenotype of apoptosis resistance.

Another major biological effect of TRAF2 overexpression in pancreatic tumor cells is the promotion of invasiveness. Colo357/TRAF2 cells secreted higher amounts of MMP-2, -9, uPA, and IL-8 and displayed strongly enhanced invasive properties than control Colo357/vector cells.

The effects attributed to TRAF2 overexpression, uPA secretion, and apoptosis resistance could be inhibited by siRNA treatment of TRAF2 overexpressing Colo357/TRAF2 cells or Panc89 cells showing high constitutive expression of TRAF2.

Little is known about nonapoptotic CD95 signaling. Triggering of CD95 may result in activation of NF-B, JNK, p38, and ERK in a highly regulated and cell type-dependent manner. We recently demonstrated CD95-mediated activation of NF-B in apoptosis-resistant PancTuI cells expressing high levels of TRAF2. Accordingly, an enforced expression of TRAF2 in sensitive Colo357 cells only rendered them resistant to CD95-mediated apoptosis, but also resulted in CD95-mediated activation of AP-1 and NF-B, followed by an increased secretion of IL-8 and uPA. These effects were only marginal in control cells, indicating a key role of TRAF2 in rescuing these cells from CD95-induced cell death and in promoting the invasive phenotype.

Analyses of clinical specimens of pancreatic cancer support the presented model of anti-apoptosis and invasiveness via CD95L. Pancreatic cancer cells have been shown to express CD95 and CD95L, and progression of pancreatic carcinomas has been associated with increased serum levels of the soluble form of CD95L. Activation of CD95 on the surface of cancer cells most likely is a common event. Our findings of an extremely high incidence of TRAF2 overexpression in pancreatic cancer tissues and the impact of conversion of death signaling toward enhanced malignancy may explain the devastating clinical prognosis of this cancer.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2984fje; doi: 10.1096/fj.04-2984fje

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