Autophagy, Epigenetics and T-cell Immunity in Cancer (AETIC)

Research themes

RESEARCH TOPIC

In our research group, we are particularly interested in deciphering the role of autophagy effectors (ATG8 proteins), post-transcriptional regulation (NMD) and epigenetic enzymes (e.g. EZH2 and KDM6B enzymes) during tumor development.

Autophagy and EMT:

EMT (Epithelial to Mesenchymal Transition) is a reversible cellular process which is linked to gene expression reprogramming to for allow epithelial cells to undergo a phenotypic switch to acquire mesenchymal properties. EMT is associated with cancer progression and cancer therapeutic resistance, and it is known that, during EMT, many stress response pathways such as autophagy and NMD are dysregulated. Therefore, the objective of this work was to study the involvement of autophagy, a mechanism of stress response, during tumor progression and Epithelial-Mesenchymal Transition (EMT). We particurlaly focused our study on GABARAPL1, a protein belonging to the ATG8 family, which is a main actor of the autophagy process thanks to its lipidation to the autophagosome membrane and its role in the formation and closure of the autophagosomes. But, since our work and other studies have shown that the ATG8 proteins were involved in the initiation step of the autophagy independently of its conjugation to the autophagosome and in the later stage dependently of its conjugation, we concluded that these proteins present a more complex function than the one initially described. It has also been described that the ATG8 proteins are dysregulated in cancer and that their expression is downregulated in tumors compared to healthy tissues. Previous works have shown that the tumor suppressor role of ATG8s would be linked to their implication in the selective degradation of cellular oncogenic proteins. Indeed, we recently showed, for the first time, that GABARAPL1 can inhibit tumor growth independently of its conjugation to the autophagosome and therefore to its role in selective autophagy. We also found that GABARAPL1 might inhibit EMT, a process described to be involved in the late stage of carcinogenesis, tumor aggressiveness and metastasis, through its implication in the selective degradation of SMAD proteins. These results confirmed a complex role of GABARAPL1 in carcinogenesis, tumor progression or metastasis formation.

Autophagy, NMD and EMT:

To complete the study described above, we aimed at studying the regulation of ATG8 family genes (GABARAP, GABARAPL1, LC3B) by the NMD (Nonsense-Mediated mRNA Decay) during EMT, and to identify molecular links between autophagy and NMD, two pathways described to be involved in tumor development and metastasis formation. We first performed IHC experiments, conducted in a cohort of patients presenting lung adenocarcinomas, and we showed high GABARAPL1 and low UPF1 levels in EMT+ tumors. In vitro, in A549 cells, we observed increased levels of GABARAPL1 correlated with decreased levels of NMD factors. We then confirmed that GABARAPL1 mRNA was indeed targeted by the NMD in a 3’UTR-dependent manner and we identified four overlapping binding sites for UPF1 and eIF4A3, two factors known to be involved in NMD regulation, as potentially involved in the recognition of this transcript by the NMD pathway. Our study suggested, for the first time, that the recognition of autophagy transcripts by the 3’UTR-dependent NMD might be an important mechanism involved in the regulation of autophagy and could represent a promising target in the development of new anti-cancer therapies. We are currently pursuing this project by characterizing the mechanism by which UPF1 and eIF4A3 are able to specifically target long 3’UTR-containing transcripts, and in particular the GABARAPL1 one, for degradation by the NMD.

Autophagy and antigen presentation:

In order to determine the link between autophagy and immune anti-tumor response, we investigated the ability of the ATG8 proteins, GABARAP and GABARAPL1, to address the antigens to MHC-I and -II presentation pathways to efficiently prime T cells. To this end, we designed several plasmids expressing the antigen OVALBUMIN fused to the GABARAP or GABARAPL1 protein. Using these constructions, we then showed that dendritic cells (DCs) overexpressing OVA-GABARAP or OVA-GABARAPL1 fusion proteins were effectively able to address OVA peptides restricted to the MHC-I and II to prime CD8+ OT-I and CD4+ OT-II T cells, respectively. Blocking autophagy in DCs overexpressing GABARAP and GABARAPL1 fusion proteins drastically decreased MHC-II antigen presentation, but not MHC-I-linked antigen presentation. Our findings therefore demonstrated, for the first time, that GABARAP and GABARAPL1 proteins were able to effectively activate MHC-II and I-directed peptide presentation leading to stimulation of both CD4 and CD8 T cells. Since it is admitted that CD4+ T cells play a critical role in cancer immunity and immunotherapy and that it is necessary to improve their activity to increase the efficiency of antitumor immunotherapies, our study could therefore lead, in this context, to the development of an attractive way to stimulate a strong CD4+ T cell response directed against tumor cells.

Epigenetic and transcriptional regulation of autophagy in breast cancers:

In this study, we studied the link between epigenetics and autophagy in a different biological model, the breast cancer. Indeed, a better evaluation of the autophagy status in breast cancer patients would be very useful to propose new personalized therapies which could combine well described anti-cancer therapies and autophagy inhibitors. Using the conclusions of the analysis of more than 2,000 transcriptomes (TCGA database), and their confirmation in a local cohort of breast cancers using digital PCR, we demonstrated that ATG genes were differentially expressed in different breast cancer subtypes. For example, Luminal A tumors expressed high levels of ATG2B and Beclin-1 while TNBC (triple negative breast cancer) expressed high level of ATG5 and LC3B. Using an in vitro cell model of autophagy induction, we next identified specific genes, such as TXNIP, whose expression could be quantified and used as a new marker of autophagy levels in tissues and therefore adapt the treatment (inducer of inhibitor) according to their autophagy status.

Publications

Recent publications :
1. – Poillet-Perez L, Jacquet M, Hervouet E, Gauthier T, Fraichard A, Borg C, Pallandre JR, Gonzalez BJ, Randani Y, Boyer-Guittaut M, Delage Mourroux R and Despouy G. GABARAPL1 tumor suppressive function is indépendant of its conjuration to autophagosome in MCF-7 breast cancer cells. Oncotarget 2017, Jul 27; 8(34):55998-56020. doi: 10.18632/oncotarget.19639. eCollection 2017 Aug 22. (IF: 5.17)
2. Claude-Taupin A., Fonderflick L., Gauthier T., Mansi L., Pallandre JR, Borg C., Perez V., Monnien F., Algros MP, Vigneron M., Delage-Mourroux R., Peixoto P., Herfs M., Boyer-Guittaut M. and Hervouet E. ATG9A is overexpressed in triple negative breast cancer and its in vitro extinction leads to the inhibition of pro-cancer phenotypes. Cells 2018, Dec 6;7(12). pii: E248. doi:10.3390/cells7120248. (IF:4.8)
3. Grandvallet C., Feugeas J.P., Monnien F., Despouy G., Perez V., Guittaut M., Peixoto P., Hervouet E. Autophagy is associated with a robust specific transcriptional signature in breast cancer subtypes. Genes & Cancer 2020. (IF: 2.5)
4. Baudu T, Parratte C, Perez V, Ancion M, Millevoi S, Hervouet E, Peigney A, Peixoto P, Overs A, Herfs M, Fraichard A, Guittaut M, Baguet A. The NMD Pathway Regulates GABARAPL1 mRNA during the EMT. Biomedicines 2021. Sep 23;9(10):1302. doi: 10.3390/biomedicines9101302. (IF: 6.08)
5. Jacquet M, Hervouet E, Baudu T, Herfs H, Parratte C, Feugeas J-P, Perez V, Reynders C, Ancion M, Vigneron M, Baguet A, Guittaut G, Fraichard A and Despouy G. GABARAPL1 inhibits EMT through SMAD- targeted autophagy degradation. Biology 2021. Sep 24;10(10):956. doi:10.3390/biology10100956. (IF: 5.07)
6. Overs A, Flammang M, Hervouet E, Bermont L, Pretet JL, Borg C, Selmani Z. Detection of specific hypermethylated WIF1 and NPY genes in circulating DNA by Crystal Digital PCR™ is a new powerful tool for colorectal cancer diagnosis and screening. BCM Cancer. 2021 Oct10;21(1):1092

Reviews:
7. Fonderflick L., Adotévi O., Guittaut M., Adami P. and Delage-Mourroux R. “Role of Autophagy in Antigen Presentation and their impact on Cancer Immunotherapy” in the book entitled “Autophagy in Immune Response: Impact on Cancer Immunotherapy”, edited by Salem Chouaib for Elsevier 2020. Invited book chapter
8. Jacquet M., Guittaut M., Fraichard F. and Despouy G. The functions of ATG8 proteins in autophagy and cancer: linked or unrelated? Autophagy 2020. (IF: 16.01)

Composition de l'équipe

Coordinateur : Michaël Guittaut
Régis Delage-Mourroux (PR)
Jean-Paul Feugeas (PU-PH)
Eric Hervouet (MCF)
Gilles Despouy (MCF)
Pascale Adami (MCF)
Annick Fraichard (MCF)
Aurélie Baguet (MCF)
Paul Peixoto (MCF)
Leila Fonderflick (ATER)
Raphaël Comu (ATER)
Elies Re (ATER)
Valérie Perez (Technicienne)
Anne Baudry (ADJT)
Céline Grandvallet-Plantin (PhD)
Timothée Baudu (PhD)
Elodie Renaude (PhD)
Jules Durand (PhD)
Alexis Overs (PhD)
Maud Leroy (M2 SCM)
Sarah Campenet (M2 SCM)