1. ¹INSERM, U753, Laboratoire d'Immunologie des Tumeurs Humaines: Interaction effecteurs cytotoxiques-système tumoral, Institut Gustave Roussy PR1 and IFR 54, Villejuif, France
2. ²INSERM, U520, laboratoire de biologie cellulaire de l'immunité antitumorale, Institut Curie, Paris, France
3. ³CNRS, UMR 8125, laboratoire de Génétique Oncologique, Institut Gustave Roussy PR1, Villejuif, France
4. ⁴Institut Gustave Roussy PR2, Villejuif, France
5. ⁵CNRS, UMR 146, Génétique du développement des mélanocytes, Institut Curie, Orsay, France
6. ⁶Chinese Academy of Sciences, Laboratory of Apoptosis and Cancer Biology, The National Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Beijing, China

Correspondence: Dr M Mehrpour, INSERM, U753, Laboratoire d'Immunologie des tumeurs Humaines : Interaction effecteurs cytotoxiques-système tumoral, Institut Gustave Roussy, PR1, F-94805 Villejuif Cedex, France. E-mail: mehrpour@igr.fr or mehrpour@ioz.ac.cn

Received 22 July 2005; Revised 24 March 2006; Accepted 9 May 2006; Published online 18 September 2006.

Abstract

In order to define genetic determinants of primary and metastatic melanoma cell susceptibility to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we have applied oligonucleotide microarrays to TRAIL-sensitive primary T1 cells and TRAIL-resistant metastatic G1 cells treated or not with TRAIL. T1 and G1 cells are isogenic melanoma cell subclones. We examined 22 000 spots, 4.2% of which displayed differential expression in G1 and T1 cells. Cell susceptibility to TRAIL-mediated apoptosis was found to be correlated with gene expression signatures in this model. Some of the differentially expressed genes were identified as involved in ATP-binding and signaling pathways, based on previously published data. Further analysis provided evidences that c-kit was overexpressed in G1 cells while it was absent in T1 cells. The c-kit inhibitor, imatinib, did not restore TRAIL sensitivity, excluding a role for c-kit in TRAIL resistance in G1 cells. Surprisingly, imatinib inhibited cell proliferation and TRAIL-mediated apoptosis in melanoma cells. We investigated the possible involvement of several molecules, including c-ABL, platelet-derived growth factor receptor (PDGFR), cellular FADD-like interleukin-1 -converting enzyme-like inhibitory protein (c-FLIP)_L/S, Fas-associated DD kinase, p53, p21^WAF1, proteins of B-cell leukemia/lymphoma 2 (Bcl-2) family and cytochrome c. Imatinib did not modulate the expression or activation of its own targets, such as c-ABL, PDGFR and PDGFR, but it did affect the expression of c-FLIP_L, BCL2-associated X protein (Bax) and Bcl-2. Moreover, c-FLIP_L knockdown sensitized T1 cells to TRAIL-mediated apoptosis, with a sensitivity similar to that of cells previously treated with imatinib. More notably, we found that the resistance to TRAIL in G1 cells was correlated with constitutive c-FLIP_L recruitment to the DISC and the inhibition of caspase 8, 3 and 9 processing. Moreover, c-FLIP_L knockdown partly restored TRAIL sensitivity in G1 cells, indicating that the expression level of c-FLIP_L and its interaction with TRAIL receptor2 play a crucial role in determining TRAIL resistance in metastatic melanoma cells. Our results also show that imatinib enhances TRAIL-induced cell death independently of BH3-interacting domain death agonist translocation, in a process involving the Bax:Bcl-X_L ratio, Bax:Bcl-X_L/Bcl-2 translocation, cytochrome c release and caspase activation. Our data indicate that imatinib sensitizes T1 cells by directly downregulating c-FLIP_L, with the use of an alternative pathway for antitumor activity, because PDGFR is not activated in T1 cells and these cells do not express c-kit, c-ABL or PDGFR. Caspase cascade activation and mitochondria also play a key role in the imatinib-mediated sensitization of melanoma cells to the proapoptotic action of TRAIL.