PhD in the DesCarTes Team

Introduction : TNF-related apoptosis-inducing ligand (TRAIL or Apo2 or TNFSF10) belongs to the TNF superfamily. When bound to its agonist receptors, TRAIL can induce apoptosis in tumour cells, while sparing healthy cells. Over the last few decades, this tumour selectivity has prompted numerous studies aimed at assessing the anti-tumour potential of TRAIL or its derivatives. Although most of these attempts have failed, new formulations are still being evaluated. However, evidence is accumulating that TRAIL may also trigger a non-canonical signal transduction pathway that is likely to be detrimental to its use in oncology. Thus, our work (Guerrache and Micheau 2024, Dufour et al. 2017, Somasekharan et al. 2013) and those published in the literature (Hartwig et al. 2017, von Karstedt et al. 2015, Davidovich et al. 2023) suggest that TRAIL can induce, via the death domain receptor 5 (DR5) under certain circumstances, tumour cell motility, which could lead to and contribute to tumour metastasis. While the mechanism of TRAIL’s pro-apoptotic signal transduction is well known from a mechanistic point of view, that of the non-canonical pathway is less well understood. 

The main objective of this thesis work will be to identify the interaction partners of DR5 by mass spectrometry, using cell models genetically manipulated to express a DR5 receptor fused to the APEX system, a biochemical labelling system based on a peroxidase (APEX), making it possible to biotinylate DR5 partners in cellulo. This thesis work will be part of a multidisciplinary and multinational European consortium, TRAIL4LIFE, which aims to improve understanding of TNF death domain receptor signalling pathways, including TRAIL (TRAIL/DR), and to translate this knowledge into new diagnostic and therapeutic tools, with a focus on cancer and autoimmune diseases (https://www.trail4life.eu). Funded until 2027 by a Marie Curie MSCA funding as part of the CHIRON project (https://www.trail4life.eu/chiron), the student in charge of the project will have the opportunity to visit our partners to complete his/her thesis project.

Positioning : Our understanding of the non-apoptotic signal transduction pathways of receptors in the TNF family, with the exception of TNFR1 (Micheau and Tschopp 2003), and in particular the non-apoptotic signalling induced by TRAIL or Fas ligand, is more than fragmentary and far from a consensus in the scientific community, unlike our understanding of the mechanisms and players involved in inducing apoptosis.

This is even more obvious with regard to the ability of some of these receptors to transcribe a signal for cell mobility or motility. There is strong experimental evidence to suggest that Fas pro-motile signalling could be induced by a new molecular complex (different from DISC, named MISC for Migration inducing Signalling Complex), associating the Fas receptor, src kinase c-Yes, NADPHox3 and the EGF receptor (Malleter et al., 2013).

However, these experimental data remain limited insofar as they have not yet been convincingly reproduced by independent teams, nor extended to other receptors in the family, including TRAIL receptors, which, unlike TNFR1, display signalling modalities very similar to Fas. The same applies to TRAIL-induced promotile signalling, which has been associated with the activation of kinases such as ROCK1, RAC1 and Src (Azijli et al., 2013; Hartwig et al., 2017; Somasekharan et al., 2013). We have attempted to validate these targets, and to extend our search to other kinases previously associated with non-apoptotic Fas signalling, without much success. The latter emerge neither in mass spectrometry analyses after receptor immunoprecipitation (not shown), nor after immunoblotting. It is important to note here that we assessed their potential involvement using the DR4 -/- and DR5-/- isogenic lines using the TALEN (Dufouret al. 2017) and CRISPR/Cas9 (Radoua et al. 2023) approaches, in order to test their selectivity for DR5. However, for the editions that we were able to control, although the loss of expression of some of these kinases does lead to a reduction in cell motility, as expected, this loss of function is in no way specific to DR5 pro-motile signalling (results not shown).

The APEX approach that will be deployed for this thesis work is already well underway in the team. Our isogenic models reconstituted with our chimeric receptors (DR-APEX) are capable of transducing a pro-apoptotic signal equivalent to the corresponding wild-type receptor, indicating that these chimeric receptors are functional and ready to be used to identify the interaction partners of the DR4 and DR5 receptors. We have also implemented a chemistry-click or optoproteomics approach (Hino and Sakamaoto 2017) that will enable us to validate the screen we wish to set up to achieve our objectives. 

knowledge and skills required :  

• cellular and molecular biology and biochemistry
• cell culture, Immunoblots, flow cytometry
• genetics and bioinformatics
• English

Summary : TRAIL belongs to the TNF superfamily. By binding to its agonist receptors, TRAIL can induce apoptosis in tumour cells, while sparing healthy cells. Over the last few decades, this tumour selectivity has prompted numerous studies aimed at assessing the anti-tumour potential of TRAIL or its derivatives. However, evidence is accumulating that TRAIL may also trigger a non-canonical signal transduction pathway, which could be detrimental to its use in oncology. Our work and that published in the literature suggest that TRAIL can induce tumour cell motility via the death domain 5 (DR5) receptor, which could lead to and contribute to tumour metastasis. While the mechanism of TRAIL pro-apoptotic signal transduction is well known from a mechanistic point of view, that of the non-canonical pathway is less well understood. The main objective of this thesis work will be to identify the interaction partners of DR5 by mass spectrometry, using cell models genetically manipulated to express a DR5 receptor fused to the APEX system, a biochemical labelling system based on a peroxidase (APEX), making it possible to biotinylate DR5 partners in cellulo. This thesis work will be part of a European TRAIL4LIFE consortium, funded until 2027, which aims to improve understanding of TNF death domain receptor signalling pathways, with a focus on cancer and autoimmune diseases (https://www.trail4life.eu/chiron). The student in charge of the project will have the opportunity to visit our partners to carry out part of his/her thesis project.

Area of expertise

Biochimie

Biologie

Key words : TNFRSF ; Signalisation non-apoptotic Signal transduction ; Cell motility ; Cancer