Relationship between the agonist activity of synthetic ligands of TRAIL-R2 and their cell surface binding modes.

Neila Chekkat,Benoit Frisch,Marcella De Giorgi,Cendrine Seguin,Caterina M Lombardo,Sylvie Fournel,Olivier Micheau,Gilles Guichard,Florent Dufour,Danièle Altschuh,Marie-Charlotte Lechner,Yves Nominé

doi:10.18632/oncotarget.24526

Abstract

Tumor Necrosis Factor Receptor Apoptosis Inducing Ligand (TRAIL) appears as an interesting candidate for targeted cancer therapy as it induces apoptosis in cancer cells without toxicity to normal cells. TRAIL elicits apoptosis through agonist death receptor TRAIL-R1 and TRAIL-R2 engagement. Nevertheless, recombinant soluble TRAIL and monoclonal antibodies against these receptors demonstrated insufficient efficacy in clinical trials. This may be explained by the cell-type dependency of the apoptotic response, itself influenced by the effect on ligand binding mode of factors such as the level of receptor oligomerization or glycosylation. To investigate the relation between binding mode and signaling, we used previously described synthetic divalent and monovalent peptides specific for TRAIL-R2. We measured their pro-apoptotic activity on three cancer cell lines sensitive to rhTRAIL induced-apoptosis and monitored their cell-surface binding kinetics. The two divalent peptides bound with strong affinity to TRAIL-R2 expressed on B lymphoma BJAB cells and induced a high degree of apoptosis. By contrast, the same peptides bound weakly to TRAIL-R2 expressed at the surface of the human colon cancer HCT116 or T lymphoma Jurkat cell lines and did not induce their apoptosis. Cross-linking experiments suggest that these differences could be afforded by variations in the TRAIL-R2 oligomerization state at cell surface before ligand addition. Moreover divalent peptides showed a different efficiency in BJAB apoptosis induction, and kinetic distribution analysis of the BJAB binding curves suggested subtle differences in binding mechanisms. Thus our data support a relation between the cell-surface binding mode of the peptides and their pro-apoptotic activity. In this case the precise characterization of ligand binding to the surface of living cells would be predictive of the therapeutic potential of TRAIL-R2 synthetic ligands prior to clinical trials.

Highlights

Tumor Necrosis Factor Receptor Apoptosis Inducing Ligand (TRAIL, TNFSF10) is a type 2 transmembrane protein that binds to two agonist death receptors TRAIL-R1 (DR4, TNFR-SF10A) [1] and TRAIL-R2 (DR5, TNFRSF10B) [2], as well as two decoy receptors TRAIL-R3 (DcR1, TNFR-SF10C) [3], and TRAIL-R4 (DcR2, TNFR-SF10D) [4]
The clustering of multiple TRAIL-R1 or TRAIL-R2 receptors induced by the trimeric ligand is not fully understood in terms of preorganization of the receptors prior to ligand binding [10, 11], of kinetics of the oligomerization process [12] and of the number of receptors required for optimal signal transduction [11, 13], it is commonly accepted that TRAIL-R2 requires a higher degree of oligomerization as compared to TRAIL-R1 [9]
While BJAB cells underwent apoptosis when treated with 1 d and 2 d (Figure 1D, left panel), two divalent TRAILmim/DR5 peptides, Jurkat or HCT116, albeit expressing TRAIL-R2, displayed strong resistance, and limited apoptosis only detected at the highest peptide concentrations (Figure 1D, middle and right panel)

Summary

Introduction

Tumor Necrosis Factor Receptor Apoptosis Inducing Ligand (TRAIL, TNFSF10) is a type 2 transmembrane protein that binds to two agonist death receptors TRAIL-R1 (DR4, TNFR-SF10A) [1] and TRAIL-R2 (DR5, TNFRSF10B) [2], as well as two decoy receptors TRAIL-R3 (DcR1, TNFR-SF10C) [3], and TRAIL-R4 (DcR2, TNFR-SF10D) [4]. TRAIL-R1/TRAIL-R2 receptors are type I transmembrane proteins that trigger apoptosis via their death domain in their intracellular region. When bound to TRAIL-R1 or TRAIL-R2 at the cell surface, TRAIL enables the recruitment of the cytosolic protein FADD (Fas-Associated protein with Death Domain) to the receptors, which, in turn, recruit pro-caspase-8 [5]. Depending on the cell type, caspase-8 can cleave the pro-apoptotic protein Bid to induce the intrinsic pathway of apoptosis [6]. The clustering of multiple TRAIL-R1 or TRAIL-R2 receptors induced by the trimeric ligand is not fully understood in terms of preorganization of the receptors prior to ligand binding [10, 11], of kinetics of the oligomerization process [12] and of the number of receptors required for optimal signal transduction [11, 13], it is commonly accepted that TRAIL-R2 requires a higher degree of oligomerization as compared to TRAIL-R1 [9]

Methods

Results

Conclusion