Blanc SVSE 2 - Sciences de la vie, de la santé et des écosystèmes : Biologie cellulaire, développement

Overcoming cancer cell resistance to death receptor ligands by modulating plasma membrane sphingolipids. – SphingoDR

Overcoming cancer cell resistance to death receptor ligands by modulating plasma membrane sphingolipids

Our aim is to overcome cancer cell resistance to death signals (triggered by the so-called death receptors) by modifying some lipids (i.e., the sphingolipids) of the plasma membrane.

Modification of the tumor cell lipid composition to trigger cell death.

Background: Sphingolipids (SLs) are ubiquitous ceramide-derived lipids, which modulate membrane functions, cancer cell growth and death. Death receptor (DR) ligands such as CD95L and TRAIL play a crucial role in anti-cancer immune response. DR engagement triggers apoptotic cell death, which is associated with alterations in SL metabolism and ceramide production. Resistance to DR ligands is a hallmark of cancer cells. <br />Our project aims at (i) understanding mechanisms of cancer cell resistance to DR ligands and (ii) evaluating the possibility to (re)sensitize cancer cells by inhibiting the synthesis of sphingomyelin and glucosylceramide from ceramide. Collaborative studies between the 4 partners indicate that SL-metabolizing enzymes (SMS1 and 2, GCS) and their metabolites can regulate DR signalling, including CD95L-induced apoptosis and CD95 distribution in resistant cancer cells. <br />Working hypothesis: The relative content of plasma membrane SLs is able to modulate : (i) the biophysical and biochemical properties of the membrane, (ii) DR distribution within the lipid rafts, (iii) DISC formation, and (iv) the apoptotic response to DR ligands. Our study will be carried out in cancer cell lines of various histological origins to evaluate the role of various SL-metabolizing enzymes on DR membrane distribution and apoptotic signalling. Clinical perspectives: To decipher SL functions in the regulation of cancer cell death and tumor progression is essential to identify novel pharmacological targets for future anticancer therapies.

Project: Task 1: modulation by sphingolipids (SLs) of epithelial to mesenchymal transition (EMT): EMT, an essential process for tumor progression, regulates CD95 signalling. We will evaluate the effects of modulating SL levels on EMT.
Task 2: development of novel approaches to manipulate SL metabolism: Sphingomyelin and glucosylceramide syntheses will be inhibited by RNA interference or by novel pharmacological approaches that will be developed. Alternatively, those SL-metabolizing enzymes will be overexpressed using retroviral vectors.
Task 3: how does SL metabolism influence membrane fluidity, death receptor (DR) distribution and signalling?: We will evaluate the effects of SL level modulation on membrane fluidity, DR distribution and cancer cell sensitivity to DR ligands (ie., CD95L and TRAIL).
Task 4: can modulation of SL metabolism and chemotherapy synergize to increase the sensitivity of tumor cells to DR ligands?: We will evaluate whether the SL metabolism alterations elicited by chemotherapy are involved (and/or act in synergy) in the sensitization to TRAIL.
Task 5: can modulation of SL metabolism affect the sensitivity of cancer cells to DR ligands in mouse models of tumorigenesis? The role of SL metabolites in tumor growth and response to DR ligands will be evaluated in mice grafted with cancer cells.

We observed that during the cell dedifferentiation process called epithelial to mesenchymal transition (EMT), cancer cells lose the expression of an enzyme, ceramide synthase 6 (CerS6), which regulates C16:0-ceramide production. We show that, during EMT cancer cells display an increased plasma membrane fluidity, leading to cell motility. Our collaborative studies demonstrate that the loss of Cers6 expression during EMT participates to lipid bilayer structuration, thus modulating cancer cell motility and metastatic spread. Our current objective is to determine how the non-apoptotic CD95 signalling can modulate SL metabolism to increase membrane fluidity and promote motility.
Moreover, we have clarified the mechanisms of production and action of ceramide in CD95L-induced leukemic cell death.
We have also developed novel tools to chemically manipulate SL metabolism. Thanks to multidisciplinary approaches, we have identified Jaspine B, a natural compound that inhibits sphingomyelin biosynthesis, as a new ligand and putative antagonist of the CERT protein, which transports ceramide. We are currently trying to identify novel CERT antagonists.

A variety of methods and tools have been set up, including those to identify novel inhibitors and putative antagonists of proteins involved in SL metabolism. Moreover, various vectors have been generated to perform future experiments. Among the studies to be done in the future, we will investigate the molecular mechanisms by which SLs overcome cancer cell resistance to anticancer drugs. Finally, we will carry out experiments in mice with established tumors to decipher whether the lipid alterations detected in cell culture and applied in mice can modify the tumor progression.

- Mechanisms of production and action of ceramide in CD95L-induced leukemic cell death (Lafont et al., 2012) (see illustration below).
- Identification of Jaspine B, a natural anhydrophytosphingosine, which inhibits sphingomyelin synthesis, as a new ligand and putative antagonist of CERT protein (Combemale et al., 2013 RSC Advances, 3, 18970-18984).
- Identification of the major role of ceramide synthase-6 in C16:0-sphingolipid synthesis in human cancer cells and EMT process (Edmond et al., submitted for publication; see illustration below).
- Identification of cl-CD95L as a pro-metastatic factor in breast cancers (Malleter et al., Cancer Res., 2013).

Sphingolipids (SLs) are ubiquitous ceramide-derived lipids, which play a dual function as modulators of membrane function and mediators of cancer cell growth and cell death. Death receptor (DR) ligands (e.g., CD95L and TRAIL) play crucial roles in anticancer immune surveillance. Many cancer cells resist DR-mediated apoptosis. Activation of these DRs triggers apoptosis that is accompanied by changes in SL metabolism, leading to the production of ceramide. Our project aims at understanding the molecular mechanisms which control the resistance of tumor cells towards DR ligands, and at evaluating the possibility to (re)sensitize cancer cells to CD95L and TRAIL by inhibiting the synthesis of complex SLs, i.e., sphingomyelin (SM) and glucosylceramide (GlcCer), from ceramide. Ongoing collaborative work between the 4 partners indicate that SM synthases 1 and 2 (SMS), GlcCer synthase (GCS) and their metabolic products may modulate DR-triggered cell signaling. Indeed, (i) SMS over-expression in HeLa cells inhibits apoptosis in response to CD95L (Lafont et al., in press) and TRAIL (unpublished); (ii) most of CD95L-resistant tumor cells exhibit higher SMS and GCS activities than CD95L-sensitive cells do (unpublished); (iii) whereas in resistant cells, CD95 is found enriched into lipid rafts, which contain high levels of SM and glycolipids, CD95 is partitioned outside lipid raft membranes in sensitive cells (unpublished).
Working hypothesis: The relative composition of SM, GlcCer and ceramide in the plasma membrane may modulate (i) its biophysical and biochemical properties, (ii) the partition of DRs at the plasma membrane level, (iii) Death inducing signaling complex (DISC) formation, and (iv) the apoptotic response to DR ligands. Our study will focus on GCS and on recently identified proteins (CERT, SMS1 and SMS2) required for GlcCer and SM synthesis, respectively, to evaluate their roles in DR partitioning and DR-induced apoptosis. Our study will be carried out on cancer cell lines of various histological origins.
Project: Task 1: SL modulation of Epithelial to Mesenchymal Transition (EMT) by SLs: EMT acts as an essential process in cancer progression and modulates CD95 signal. The role of SM/GlcCer/ceramide on the EMT will be evaluated.
Task 2: development of novel approaches to manipulate SL metabolism: the biosynthesis of SM and GlcCer will be inhibited using RNA interference strategy as well as novel pharmacological tools to be developed. Conversely, CERT, SMS1, SMS2 or GCS will be over-expressed using retroviral vectors.
Task 3: how does SL metabolism influence membrane fluidity, DR distribution and signaling?: we will address the impact of SM/GlcCer/ceramide levels on membrane fluidity, the membrane distribution of DRs, and the sensitivity of tumor cells to CD95L or TRAIL.
Task 4: can modulation of SL metabolism and chemotherapy synergize to increase the sensitivity of tumor cells to DR ligands?: we will explore whether the alterations of SL metabolism are involved in (and/or synergize with) chemotherapy-induced priming and activation of the TRAIL apoptotic signaling pathway.
Task 5: can modulation of SL metabolism affect the sensitivity of cancer cells to DR ligands in mouse models?: The role of SL metabolites in tumor formation/growth and response to DR ligands in vivo will be evaluated after transplantation to mice.
This proposal seeks to bring scientists from different background and expertise in various fields together in a unique, original and timely program. Deciphering the role of SLs in the regulation of cancer cell death and tumor progression should be of particular interest for the identification of novel and original targets and future therapeutic intervention

Project coordinator

Monsieur Thierry Levade (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - Délégation régionale Midi-Pyrénées Limousin) – thierry.levade@inserm.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

INSERM INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - Délégation régionale Midi-Pyrénées Limousin
UPS-LSPCMIB UNIVERSITE TOULOUSE III [PAUL SABATIER]
Université Rennes-1 UNIVERSITE DE RENNES I
INSERM INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - ADR DE STRASBOURG - ADR 16
INSERM GRAND EST

Help of the ANR 520,000 euros
Beginning and duration of the scientific project: - 36 Months

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