Oncogenic epidermal growth factor receptor up-regulates multiple elements of the tissue factor signaling pathway in human glioma cells

Tissue factor (TF) emerges as a common denominator of multiple processes associated with cancer progression and metastasis.^1,–3  Interestingly, TF up-regulation on the surface of cancer cells and the release of microvesicular TF into the blood^4,,–7  are both linked to oncogenic transformation.^3,5  Notably, oncogenic epidermal growth factor receptor (EGFR) and the truncated, ligand-independent EGFRvIII mutant⁸  both up-regulate TF in various cancer settings, including glioblastoma multiforme (GBM),⁷  the most lethal type of primary brain tumor. GBM is associated with florid angiogenesis, thrombotic complications,⁹  and up-regulation of TF,¹⁰  to which both microenvironmental and genetic influences were found to contribute.^7,11,12 

TF is a 47-kDa cell-associated transmembrane protein that acts as the high-affinity receptor for factor VIIa (FVIIa)¹³  and a trigger of both coagulation¹⁴  and intracellular signaling.^2,15  The latter aspect is mediated, at least in part, by the impact of the TF/VIIa complex on the G protein–coupled protease-activated receptors (PARs), either directly (PAR2) or via FXa (PAR1 and PAR2), or thrombin (PAR1).² 

The significance of these events in cancer is underscored by the link between TF/PAR signaling and tumor angiogenesis,^16,17  invasiveness,¹⁸  and progression,¹⁹  all of which are also a function of oncogenic transformation.³  It is therefore of interest to ask whether oncogenic and TF/PAR signaling pathways converge in some fashion in cancer cells and what the consequences are. Here we show that in GBM cells the EGFRvIII oncogene simultaneously up-regulates a cluster of functionally related elements of the TF/PAR pathway, including TF, PAR1, PAR2, and FVII, and renders cancer cells both procoagulant and hypersensitive to TF signaling.

Detailed description of cells, conditions, assays, and reagents is provided as supplemental data (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Cells were maintained in Dulbecco modified Eagle medium with 10% serum.⁷  Treatments included 10nM of FVIIa (Enzyme Research Laboratories) and agonists of PAR1 (PAR1-AP: TFLLR-NH₂) or PAR2 (PAR2-AP: SLIGKV-NH₂), both from Bachem Biosciences.

Cells were treated as indicated, followed by exposure to 5nM FVIIa, 150nM FX, and 5mM CaCl₂ in Tris-buffered saline.²⁰  The chromogenic reaction was triggered by addition of 2mM S-2765 (Chromogenix), stopped with 20 μL of 50% acetic acid, read at 405 nm, and the results expressed as arbitrary units normalized to the protein content.

Total RNA was extracted using TRIzol (Invitrogen), transcribed into cDNA, and amplified using a OneStep RT-PCR Kit (QIAGEN). Aliquots of 0.5 μg were used to carry out reverse transcription at 50°C for 30 minutes, followed by amplification.

Cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis.^5,7  On transfer, the polyvinylidene difluoride membranes were probed with primary antibodies, including: anti–human TF (1:1000; American Diagnostica 4503), β-actin (1:10 000; Invitrogen), PAR1 (1:500; R&D Systems), and PAR2 SAM11 (1:200; Santa Cruz Biotechnology). The signal was visualized using appropriate horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (GE Healthcare).

Single-cell suspensions were washed with phosphate-buffered saline containing 1% bovine serum albumin and 0.1% sodium azide before staining with mouse anti–human TF antibody (1:200; American Diagnostica 4509) and secondary goat anti–mouse antibody (1:200; Alexa Fluor 488 nm).

Cell culture supernatants were processed and assayed as recommended by suppliers of the human vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) immunoassays (R&D Systems and BD-OptEIA).⁷  All experiments were independently reproduced at least 3 times. Data (mean ± SD) were calculated and Student t test used, as indicated (supplemental data).

To extend our prior studies,⁷  we examined the expression and activity of TF in a panel of GBM cell lines expressing the EGFRvIII oncogene. Although the parental U373 and U87 cells express low to moderate amounts of TF antigen, mRNA, and procoagulant activity, these levels increase dramatically on the enforced expression of EGFRvIII (Figure 1A-C). This correlates with the onset of a highly tumorigenic and angiogenic phenotype, especially in the case of U373 cells, which in the absence of EGFRvIII are nontumorigenic in mice²¹  (and data not shown).

Interestingly, EGFRvIII-expressing GBM cells up-regulate not only TF, but also PAR1, PAR2, and (ectopically) FVII transcripts (Figure 1C). Thus, indolent U373 cells express TF and PAR1 at low levels and no detectable PAR2 transcript, whereas more aggressive U87 cells are positive for TF and PAR2. However, in both cell lines, the EGFRvIII-driven transformation causes a marked increase in the expression of TF, PAR1, PAR2, and FVII (Figure 1C).

Cancer cells were previously found to ectopically produce FVII,²²  and our study provides one possible (oncogenic) mechanism of such a conversion. The coexpression of TF and FVII in GBM cells also raises a possibility that such cells may be able to endogenously generate FXa (from FX), provided that FVII somehow becomes converted to FVIIa.²²  We tested this by examining U373 (TF-low/FVII-negative) and U373vIII (TF-high/FVII-positive) cells in TF procoagulant activity (PCA) assays, in the absence of exogenous FVIIa. Although U373vIII cells generated higher TF PCA values than their U373 counterparts, the ability of both cell lines to generate FXa under these conditions was exceedingly low (Figure 2A vs D, where exogenous FVIIa was added). Hence, the efficient activation of coagulation by these cells depends on the availability of the exogenous FVIIa.

To further examine the consequences of EGFRvIII-dependent TF up-regulation, we verified it at the protein level (Figure 2B) and expressed TF exogenously in U373 cells (Figure 2C). Of several resulting clones, some (TF U373D11 and TF U373G11) produced TF at levels similar to those of U373vIII cells, from which they did not differ in terms of their procoagulant activity in the presence of recombinant FVIIa (Figure 2D).

Because EGFRvIII triggered not only the up-regulation of TF, but also of PAR1 and PAR2 (Figure 2B), we compared the signaling capacity of the TF/PAR pathway²  between parental, EGFRvIII-transformed, and TF-transfected, U373-derived cell lines. The cells were stimulated with FVIIa, PAR1, or PAR2 activating peptides (PAR1/2APs), and interrogated for production of angiogenic factors, such as IL-8¹⁷  and VEGF.^1,7  Indeed, very low/undetectable levels of IL-8 and VEGF were found in culture supernatants of U373 cells, and their control (EV1/2 U373), or TF-expressing transfectants (TF U373D11/G11), and this did not change markedly on stimulation. In contrast, U373vIII cells exhibited both constitutively high levels and a dramatic up-regulation of angiogenic factor production on stimulation with FVIIa, PAR1-AP, and PAR2-AP, although the responses differed between IL-8 and VEGF (Figure 2E-F).

In conclusion, our observations suggest that EGFRvIII and possibly other oncogenes may sensitize cancer cells to coagulation factor signaling, at least in part, because of simultaneous up-regulation of TF and PARs. This may precipitate proangiogenic and other biologic consequences related to the TF/PAR pathway. Targeting this pathway may therefore have therapeutic value in GBM, and the levels of TF, FVII, and PARs could be explored as biomarkers.

This work was supported by the Canadian Cancer Society (J.R.), Canadian Institutes of Health Research (J.R.), and infrastructure funds from Fonds de la recherche en santé du Quebec (J.R.). J.R. is the Jack Cole Chair in Pediatric Oncology.

Contribution: N.M. and D.G. performed experiments; and N.M., D.G., and J.R. wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Janusz Rak, Montreal Children's Hospital Research Institute, 4060 Ste Catherine West, Montreal, QC, H3Z 3Z2, Canada; e-mail: janusz.rak@mcgill.ca.