Tissue Factor/FVIIa prevents the extrinsic pathway of apoptosis by regulation of the tumor suppressor Death-Associated Protein Kinase 1 (DAPK1)

Introduction: This study determines the impact of tissue factor (TF)-signaling on the extrinsic pathway of apoptosis in cancer cells and propose death associated protein kinase-1 (DAPK1) as a novel key regulator. Materials and Methods: In MDA-MB-231 breast and PC3 prostate cancer cells, mRNA levels were analyzed by real-time PCR and protein expressions were assessed by flow cytometry or western blot. Caspase-8 and -3 levels, cell size, and changes in nuclear morphology were recorded using the ArrayScan microscope and 84 apoptosis-related genes were screened with the RT2 Profiler (TM) PCR Array. Results: In serum starved MDA-MB-231 cells, a TF/FVIIa-sensitive upregulation of apoptosis markers was recorded. Similarly, TRAIL-induced apoptosis was negatively regulated by TF/FVIIa (10 and 100 nM) and TF/FVIIa/FXa but not by active-site inhibited FVIIa. FVIIa, moreover, decreased the transcription of DAPK1 and thereby diminished the association between DAPK1 and FADD in the caspase-8 activating death-inducing signaling complex (DISC). TF/FVIIa regulation of caspase-8 and DAPK1 was dependent on PI3-kinase/AKT and independent of the protease activated receptors (PAR) 1 and 2. Despite of receptor expression and functional signaling, both PAR-agonist treatment and PAR-blocking antibodies in combination with FVIIa failed to influence the anti-apoptotic signal. Conclusions: We hereby report that TF/FVIIa-induced signaling governs the extrinsic pathway of apoptosis by reducing the levels of DAPK1 in the DISC independently of PAR1 and PAR2. This implies the conceivable involvement of cell surface components other than the PARs and entails the search for TF-dependent regulators of DAPK1 transcription. (C) 2010 Elsevier Ltd. All rights reserved.