Rôles des caspases initiatrices et des sphingomyéline synthases dans l'apoptose induite par CD95L et TRAIL

La mort cellulaire par apoptose est un mécanisme effecteur de la réponse immunitaire anti-tumorale. La liaison de ligands, dont TRAIL et CD95L, produits par des cellules immunitaires, sur des récepteurs de mort (DR pour Death Receptor), exprimés par les cellules tumorales, permet l'induction de l'apoptose. Ceci met en jeu différents événements dont la formation du DISC (Death Inducing Signalling Complex) et l'activation des caspases. De plus, le taux intracellulaire de céramide, un sphingolipide pro-apoptotique, augmente au cours de cette signalisation. L'objectif de cette thèse a été de clarifier le rôle des caspases initiatrices et du céramide dans l'apoptose induite par CD95L et TRAIL. Le rôle causal de la caspase 10, caspase initiatrice, dans l'induction de mort par CD95L est controversé. Nos travaux indiquent que cette protéase est impliquée dans la mort induite par CD95L en présence ou non de zVAD-fmk, un inhibiteur des caspases. Par ailleurs, nous montrons que la sphingomyéline synthase 1 (SMS1), qui synthétise de la sphingomyéline à partir de céramide, est inhibée de façon caspase-dépendante dans la signalisation de CD95. La SMS1 module l'induction de l'apoptose en réponse à CD95L et TRAIL. Ainsi, la surexpression de la SMS1 confère une protection vis-à-vis de la mort induite par ces ligands, tandis que la diminution de son expression sensibilise des cellules cancéreuses. Ce rôle protecteur dépendrait de la modulation de la formation du DISC et de la voie apoptotique mitochondriale. L'inhibition de cette enzyme pourrait donc permettre de sensibiliser des cellules cancéreuses à la mort induite par des agonistes des DR en modulant plusieurs étapes de l'apoptose.Apoptotic cell death is an effector mechanism involved in immune responses, and, particularly in anti-tumoral immune response. Binding of ligands, such as TRAIL and CD95L, which are produced by immune cells, on DR (Death Receptors) expressed by tumor cells, induces apoptosis. This involves, in particular, DISC (Death Inducing Signalling Complex) formation and caspases activation. Moreover, an increase of intracellular level of ceramide, a pro-apoptotic sphingolipid, has been observed during this signalling and might be involved in cell death induction. The aim of this PhD was to clarify initiator caspases and ceramide roles in CD95L and TRAIL induced apoptosis. Initiator caspase 10 involvement in CD95L-induced cell death is still controversed. Our results indicate that this protease is indeed involved in CD95L-induced cell death as well as in CD95L induced cell death in the presence of zVAD-fmk, a caspase inhibitor. Moreover, we show that sphingomyelin synthase 1 (SMS1), which is responsible for the synthesis of sphingomyelin from ceramide, is inhibited in CD95 signalling, in a caspase-dependent way, and more precisely in a caspase-8-dependent way. SMS1 could modulate CD95L- and TRAIL-induced cell death. Indeed, SMS1 overexpression is protective against CD95L- and TRAIL- induced cell death, whereas down-regulating SMS1 by RNA interference sensitizes cancer cells. Our results point out that SMS1 protective role might depend on DISC formation and apoptotic mitochondrial pathway modulations. Sphingomyelin synthesis inhibition might therefore be an original way to sensitize cancer cells towards DR agonists via putative apoptotic signalling modulations at several steps

Caspase-10-Dependent Cell Death in Fas/CD95 Signalling Is Not Abrogated by Caspase Inhibitor zVAD-fmk

Upon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non-apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase-10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated

LUBAC prevents lethal dermatitis by inhibiting cell death induced by TNF, TRAIL and CD95L

The linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1 and SHARPIN, is required for optimal TNF-mediated gene activation and to prevent cell death induced by TNF. Here, we demonstrate that keratinocyte-specific deletion of HOIP or HOIL-1 (E-KO) results in severe dermatitis causing postnatal lethality. We provide genetic and pharmacological evidence that the postnatal lethal dermatitis in HoipE-KO and Hoil-1E-KO mice is caused by TNFR1-induced, caspase-8-mediated apoptosis that occurs independently of the kinase activity of RIPK1. In the absence of TNFR1, however, dermatitis develops in adulthood, triggered by RIPK1-kinase-activity-dependent apoptosis and necroptosis. Strikingly, TRAIL or CD95L can redundantly induce this disease-causing cell death, as combined loss of their respective receptors is required to prevent TNFR1-independent dermatitis. These findings may have implications for the treatment of patients with mutations that perturb linear ubiquitination and potentially also for patients with inflammation-associated disorders that are refractory to inhibition of TNF alone

Targeting of T/Tn Antigens with a Plant Lectin to Kill Human Leukemia Cells by Photochemotherapy

Photochemotherapy is used both for solid tumors and in extracorporeal treatment of various hematologic disorders. Nevertheless, its development in oncology remains limited, because of the low selectivity of photosensitizers (PS) towards human tumor cells. To enhance PS efficiency, we recently covalently linked a porphyrin (TrMPyP) to a plant lectin (Morniga G), known to recognize with high affinity tumor-associated T and Tn antigens. The conjugation allowed a quick uptake of PS by Tn-positive Jurkat leukemia cells and efficient PS-induced phototoxicity. The present study was performed: (i) to evaluate the targeting potential of the conjugate towards tumor and normal cells and its phototoxicity on various leukemia cells, (ii) to investigate the mechanism of conjugate-mediated cell death. The conjugate: (i) strongly increased (×1000) the PS phototoxicity towards leukemic Jurkat T cells through an O-glycan-dependent process; (ii) specifically purged tumor cells from a 1∶1 mixture of Jurkat leukemia (Tn-positive) and healthy (Tn-negative) lymphocytes, preserving the activation potential of healthy lymphocytes; (iii) was effective against various leukemic cell lines with distinct phenotypes, as well as fresh human primary acute and chronic lymphoid leukemia cells; (iv) induced mostly a caspase-independent cell death, which might be an advantage as tumor cells often resist caspase-dependent cell death. Altogether, the present observations suggest that conjugation with plant lectins can allow targeting of photosensitizers towards aberrant glycosylation of tumor cells, e.g. to purge leukemia cells from blood and to preserve the normal leukocytes in extracorporeal photochemotherapy

Stress Management: Death Receptor Signalling and Cross-talks with the Unfolded Protein Response in Cancer

International audienceThroughout tumour progression, tumour cells are exposed to various intense cellular stress conditions owing to intrinsic and extrinsic cues, to which some cells are remarkably able to adapt. Death Receptor (DR) signalling and the Unfolded Protein Response (UPR) are two stress responses which both regulate a plethora of outcomes ranging from proliferation, differentiation, migration, cytokine production to induction of cell death. Both signalling are major modulators of physiological tissue homeostasis and their dysregulation is involved in tumorigenesis and metastastic process. The molecular determinants of the control between the different cellular outcomes induced by DR signalling and the UPR in tumour cells and their stroma and their consequences on tumorigenesis are starting to be unravelled. Herein, I summarize the main steps of DR signalling in relation to its cellular and pathophysiological roles in cancer. I then highlight how the UPR and DR signalling control common cellular outcomes and also cross-talk, providing potential opportunities to further understand the development of malignancies

A novel optinEuRin stress connection in glaucoma

International audienceThe endoplasmic reticulum (ER) is the organelle where the production and shaping of most secreted and transmembrane proteins happens. ER function is finely regulated to prevent accumulation of misfolded proteins generating ER stress. ER stress is common in both healthy and pathological situations due to multiple intrinsic and extrinsic factors including acute demand in protein synthesis, hypoxia or impaired protein folding caused by gene mutations. Sayyad et al. found that the M98K mutation of optineurin sensitizes glaucoma retinal ganglion cells to ER stress-induced cell death. This is associated with an autophagy-dependent elevation of ER stress sensor expression

Therapeutic approaches targeting CD95L/CD95 signaling in cancer and autoimmune diseases

International audienceCell death plays a pivotal role in the maintenance of tissue homeostasis. Key players in the controlled induction of cell death are the Death Receptors (DR). CD95 is a prototypic DR activated by its cognate ligand CD95L triggering programmed cell death. As a consequence, alterations in the CD95/CD95L pathway have been involved in several disease conditions ranging from autoimmune diseases to inflammation and cancer. CD95L-induced cell death has multiple roles in the immune response since it constitutes one of the mechanisms by which cytotoxic lymphocytes kill their targets, but it is also involved in the process of turning off the immune response. Furthermore, beyond the canonical pro-death signals, CD95L, which can be membrane-bound or soluble, also induces non-apoptotic signaling that contributes to its tumor-promoting and pro-inflammatory roles. The intent of this review is to describe the role of CD95/CD95L in the pathophysiology of cancers, autoimmune diseases and chronic inflammation and to discuss recently patented and emerging therapeutic strategies that exploit/block the CD95/CD95L system in these diseases

Death sentence: The tale of a fallen endoplasmic reticulum

International audienceEndoplasmic Reticulum (ER) stress signaling is an adaptive mechanism triggered when protein folding demand overcomes the folding capacity of this compartment, thereby leading to the accumulation of improperly folded proteins. This stress signaling pathway is named the Unfolded Protein Response (UPR) and aims at restoring ER homeostasis. However, if this fails, mechanisms orienting cells towards death processes are initiated. Herein, we summarize the most recent findings connecting ER stress and the UPR with identified death mechanisms including apoptosis, necrosis, pyroptosis, ferroptosis, and autophagy. We highlight new avenues that could be investigated and controlled through actionable mechanisms in physiology and pathology