Cell death signalling

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TNF-related apoptosis-inducing ligand (TRAIL/APO-2L) is a member of the TNF family that promotes apoptosis, particularly in tumor cell lines. TRAIL treatment also results in significant growth suppression of TRAIL-sensitive human cancer xenografts in mice. While further studies are needed to evaluate the possible cytotoxicity of TRAIL especially for human hepatocytes, indications are increasing that TRAIL may be a novel therapeutic agent for human cancer. Furthermore, studies from various laboratories have recently demonstrated that tumor cells that are resistant to TRAIL can be sensitised by subtoxic concentrations of drugs/cytokines and the sensitised tumor cells are significantly killed by TRAIL. Results from our laboratory indicate that sensitisation of breast tumor cells by interferon-gamma occurs at the initial activation of apical caspase-8 by TRAIL.

We have characterized the human caspase-8 gene promoter and studied the transcriptional regulation of caspase-8 gene expression in MCF-7 breast tumor cells treated with IFN-?. Our findings show that IFN-? induces the up-regulation of caspase-8 mRNA expression through a mechanism involving the action of the IFN-?-inducible transcription factor IRF-1. The human caspase-8 gene promoter lacks recognizable TATA and CAAT boxes, but contains a consensus SP1 binding site. We have identified two major IFN-?-inducible transcriptional start sites in these cells by S1 nuclease mapping and primer extension analysis. Deletion analysis of the promoter defined an 82-bp minimal region responsible for IFN-?-inducible promoter activity. In this region we have identified an IFN-stimulated response element (ISRE) that is important for both the basal and IFN-??enhanced transcriptional activities.

Exogenously expressed IRF-1 in MCF-7 cells up-regulated the activity of a luciferase reporter plasmid containing an 82-bp region of the caspase-8 promoter. These data define a new pathway through which IFN-? might control the sensitivity of tumor cells to death receptor-mediated apoptosis. We have also shown that pre-treatment of different human breast cancer cell lines with the cyclin-dependent kinase inhibitor flavopiridol facilitates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Flavopiridol treatment enhances TRAIL-induced formation of death-inducing signaling complex (DISC) and early processing of procaspase-8. Down-regulation of cellular FLICE-inhibitory proteins (c-FLIPL and c-FLIPS) by the ubiquitin/proteasome system is observed upon flavopiridol treatment. Furthermore, targeting c-FLIP directly with siRNA oligonucleotides also sensitizes various human breast tumor cell lines to TRAIL-induced apoptosis.

Our results indicate that flavopiridol sensitizes breast cancer cells to TRAIL-induced apoptosis by facilitating early events in the apoptotic pathway, and this combination treatment could be regarded as a potential therapeutic tool against breast tumors. Moreover, recent data from our laboratory demonstrate that TRAIL induces cytoprotective autophagy in human epithelial cells. TRAIL-induced autophagy is mediated by the AMP-activated protein kinase (AMPK) that inhibits mammalian target of rapamycin complex 1 (mTORC1), a potent inhibitor of autophagy. Interestingly, the TRAIL-induced AMPK activation depends on transforming growth factor-ß-activating kinase 1 (TAK1). These findings identify TAK1 as an activator of AMPK and cytoprotective autophagy and thereby as a regulator of cellular energy homeostasis and survival. Furthermore, our unpublished observations indicate that in breast epithelial cells transformed by oncogenes TRAIL-induced autophagy is inhibited, suggesting that the regulation by TRAIL of the TAK1-AMPK-mTORC1 autophagy pathway might explain the differential TRAIL sensitivity of normal and malignant cells.

 Selected Publications:

G.Rodriguez-Tarduchy, M.Collins and A.López-Rivas. Regulation of apoptosis in interleukin-3-dependent hemopoietic cells by interleukin-3 and calcium ionophores. The EMBO Journal (1990) 9, 2997-3002

G. Rodriguez-Tarduchy, M. Collins, I. García and A. López-Rivas. Insulin-like growth factor-I inhibits apoptosis in mouse haemopoietic IL3-dependent cells. J.Immunol. (1992) 149, 535-540

M.K.L. Collins, J. Marvel, P. Malde and A. López-Rivas. Interleukin-3 prevents apoptosis induced by DNA damaging agents. J.Exp.Med. (1992) 176, 1043-1051

M.K.L. Collins and A. López-Rivas. The control of apoptosis in mammalian cells. Trends Biochem. Sci. (1993) 18, 307-309

M.K.L.Collins,G.Perkins,G.Rodriguez-Tarduchy,M.A.Nieto and A. López-Rivas. Growth Factors as Survival Factors: Regulation of Apoptosis. BioEssays (1994) 16, 133-138

M. Izquierdo, M.C. Ruiz-Ruiz and A. López-Rivas. Stimulation of phosphatidylinositol turnover is a key event for Fas-dependent, activation-induced apoptosis in human T lymphocytes. J. Immunol. (1996) 157, 21-28

M. C. Ruiz-Ruiz, G. Robledo, J. Font, M. Izquierdo and A. López- Rivas. Protein Kinase C inhibits CD95(Fas/APO-1)-mediated apoptosis by at least two different mechanisms in Jurkat T cells. J. Immunol. (1999) 163: 4737-4746

M. C. Ruiz-Ruiz and A. López-Rivas. p53-mediated up-regulation of CD95 is not involved in genotoxic drug-induced apoptosis of human breast tumor cells. Cell Death and Differentiation (1999) 6, 271-280

C. Ruiz-Ruiz, C. Muñoz-Pinedo and A. López-Rivas. Interferon-gamma treatment elevates caspase-8 expression and sensitises human breast tumor cells to a death receptor-induced mitochondria-operated apoptotic program. Cancer Res. (2000) 60, 5673-5680

M. Sarker, C. Ruiz-Ruiz and A. López-Rivas. Activation of protein kinase C inhibits TRAIL-induced caspases activation, mitochondrial events and apoptosis in a human leukemic T cell line. Cell Death and Differentiation (2001) 8: 172-181
C. Muñoz-Pinedo, C. Ruiz-Ruiz, C. Ruiz de Almodovar, C. Palacios and A. López-Rivas. Inhibition of glucose metabolism sensitises tumor cells to death receptor-triggered apoptosis through enhancement of DISC formation and apical procaspase-8 processing. J. Biol. Chem. (2003) 278: 12759-12768

C. Ruiz de Almodovar, C. Ruiz-Ruiz, A. Rodríguez, G. Ortiz-Ferrón, J.M. Redondo and A. López-Rivas. TRAIL decoy receptor TRAIL-R3 is upregulated by p53 in breast tumor cells through a mechanism involving an intronic p53 binding site. J. Biol. Chem. (2004) 279: 4093-4101

C. Ruiz-Ruiz, C. Ruiz de Almodóvar, A. Rodríguez, G. Ortiz-Ferrón, J.M. Redondo and A. López-Rivas. The up-regulation of human caspase-8 by interferon-gamma in breast tumor cells requires the induction and action of the transcription factor IRF-1. J. Biol. Chem. (2004) 279:19712-19720

C. Palacios, R. Yerbes and A. López-Rivas. Flavopiridol induces cFLIP degradation by the proteasome and promotes TRAIL-induced early signaling and apoptosis in breast tumor cells. Cancer Research (2006) 66, 8858-8869

Gustavo Ortiz-Ferrón, Stephen W. Tait, Gema Robledo, Evert de Vries, Jannie Borst, and Abelardo López-Rivas. The mitogen-activated protein kinase pathway can inhibit TRAIL-induced apoptosis by prohibiting association of truncated Bid with mitochondria. Cell Death and Differentiation (2006) 13, 1857-1865

Griselda Herrero-Martín, Maria Høyer-Hansen, Celina García-García, Claudia Fumarola, Lone Batholm, Thomas Farkas, Abelardo López-Rivas@ and Marja Jäättelä@. TAK1 activates AMPK and AMPK-dependent cytoprotective autophagy in TRAIL-treated epithelial cells. The EMBO Journal (2009) 28, 677-685