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Cell Cycle, Cell Death, and Senescence

Down-Regulation of Mcl-1 by Small Interfering RNA Sensitizes Resistant Melanoma Cells to Fas-Mediated Apoptosis

¹ Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Pavillon CHUL and Faculté de Médecine, Université Laval, Québec, Canada and ² Institut National de la Sante et de la Recherche Medicale U662, Institut Universitaire d'Hématologie, Hôpital St. Louis; ³ Institut Jacques Monod, Université Paris 7, Paris, France

Requests for reprints: Fawzi Aoudjit, Centre de Recherche en Rhumatologie et Immunologie, CHUQ Pavillon CHUL, 2705 Boulevard Laurier, local T1-49, Ste-Foy, Québec G1V 4G2, Canada. Phone: 1-418-656-4141, ext. 46071; Fax: 1-418-654-2765. E-mail: fawzi.aoudjit{at}crchul.ulaval.ca or Reem Al-Daccak, INSERM U662, Hôpital St. Louis, Batiment Bazin, 1 Avenue Claude Vellefaux, Paris 75010, France. Phone: 331-53722063; Fax: 331-42385345. E-mail: reem.al-daccak{at}stlouis.inserm.fr

Resistance of malignant melanoma cells to Fas-mediated apoptosis is among the mechanisms by which they escape immune surveillance. However, the mechanisms contributing to their resistance are not completely understood, and it is still unclear whether antiapoptotic Bcl-2–related family proteins play a role in this resistance. In this study, we report that treatment of Fas-resistant melanoma cell lines with cycloheximide, a general inhibitor of de novo protein synthesis, sensitizes them to anti-Fas monoclonal antibody (mAb)–induced apoptosis. The cycloheximide-induced sensitization to Fas-induced apoptosis is associated with a rapid down-regulation of Mcl-1 protein levels, but not that of Bcl-2 or Bcl-xL. Targeting Mcl-1 in these melanoma cell lines with specific small interfering RNA was sufficient to sensitize them to both anti-Fas mAb-induced apoptosis and activation of caspase-9. Furthermore, ectopic expression of Mcl-1 in a Fas-sensitive melanoma cell line rescues the cells from Fas-mediated apoptosis. Our results further show that the expression of Mcl-1 in melanoma cells is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and not by phosphatidylinositol 3-kinase/AKT signaling pathway. Inhibition of ERK signaling with the mitogen-activated protein/ERK kinase-1 inhibitor or by expressing a dominant negative form of mitogen-activated protein/ERK kinase-1 also sensitizes resistant melanoma cells to anti-Fas mAb-induced apoptosis. Thus, our study identifies mitogen-activated protein kinase/ERK/Mcl-1 as an important survival signaling pathway in the resistance of melanoma cells to Fas-mediated apoptosis and suggests that its targeting may contribute to the elimination of melanoma tumors by the immune system. (Mol Cancer Res 2008;6(1):42–52)