Targeting a novel N-terminal epitope of death receptor 5 triggers tumor cell death

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand receptors death receptor (DR) 4 and DR5 are potential targets for antibody-based cancer therapy. Activation of the proapoptotic DR5 in various cancer cells triggers the extrinsic and/or intrinsic pathway of apoptosis. It has been shown that there are several functional domains in the DR5 extracellular domain. The cysteine-rich domains of DR5 have a conservative role in tumor necrosis factor-related apoptosis-inducing ligand-DR5-mediated apoptosis, and the pre-ligand assembly domain within the N1-cap contributes to the ligand-independent formation of receptor complexes. However, the role of the N-terminal region (NTR) preceding the N1-cap of DR5 remains unclear. In this study, we demonstrate that NTR could mediate DR5 activation that transmits an apoptotic signal when bound to a specific agonistic monoclonal antibody. A novel epitope in the NTR of DR5 was identified by peptide array. Antibodies against the antigenic determinant showed high affinities for DR5 and triggered caspase activation in a time-dependent manner, suggesting the NTR of DR5 might function as a potential death-inducing region. Moreover, permutation analysis showed that Leu(6) was pivotal for the interaction of DR5 and the agonistic antibody. Synthetic wild-type epitopes eliminated the cytotoxicity of all three agonistic monoclonal antibodies, AD5-10, Adie-1, and Adie-2. These results indicate that the NTR of DR5 could be a potential target site for the development of new strategies for cancer immunotherapy. Also, our findings expand the current knowledge about DR5 extracellular functional domains and provide insights into the mechanism of DR5-mediated cell death.

FIGURE 1.

Epitope recognized by AD5-10 is localized in the NTR of DR5. A, AD5-10 recognizes denatured endogenous DR5 (both isoform-A and -B) in Jurkat cells. Whole cell lysate (W), soluble fraction (S), and membrane fraction (P) of Jurkat cells were separated by 12% SDS-PAGE, whereas recombinant soluble DR5-ECD was taken as a positive control. The blot was then probed with AD5-10. B, AD5-10 binds to native membrane DR5. Cells were grown on glass coverslips coated with poly(l-lysine) and collagen. After fixation, cells were incubated with AD5-10 or normal mouse IgG3 isotype control at 4 °C for 60 min and then stained with an FITC-labeled secondary antibody. C, AD5-10 binds to native membrane DR5 on Jurkat cells and HeLa cells. Membrane DR5 on Jurkat cells and HeLa cells was determined by indirect immunofluorescence with AD5-10 and FITC-labeled secondary antibody followed by flow cytometry assay. D, OPAL of DR5-ECD is detected by AD5-10. An array of 60 peptides derived from the DR5-ECD was probed for binding to AD5-10 (panel b). Meanwhile, mouse normal IgG₃ isotype (panel a) was used as a negative control to monitor the nonspecific binding. All the antibodies were added to the cellulose membrane at a final concentration of 0.2 μg/ml. Bound proteins were identified by an HRP-conjugated goat anti-mouse IgG₃ and visualized by ECL. Positive spots indicated definite binding. E, determining the minimal sequence of peptides required for binding to AD5-10 by analyzing the signal strength of the spots. All experiments were repeated at least three times with similar results.

FIGURE 2.

Residues ⁵DLA⁷ are essential for the interaction between DR5 and AD5-10. A, alanine-scanning mutagenesis of the epitope. Two alanine-scanning arrays of peptides ⁻¹LITQQDLAPQQRA¹² (panel a) and QI¹TQQDLAPQQRA¹² (panel b) were probed with 0.2 μg/ml AD5-10. The sequences of the peptides are shown. The first and the last spot corresponded to the parental peptide, whereas the other spots represented an Ala-substituted analogue of wild-type sequence (wt) as indicated above the individual spots. B, permutation array of peptide ⁻¹LITQQDLAPQQRA¹². Each residue in this peptide was replaced, one at a time, by a naturally occurring amino acid. The resulting array of 260 (13 × 20) peptides was probed with 0.2 μg/ml of AD5-10. Spots that displayed the “half-moon” pattern were likely to be caused by imperfections in array synthesis. C, FLAG-tagged full-length DR5. A C-terminal FLAG tag was added to the full-length DR5. D, analysis of point mutation in full-length DR5 molecule. Both wild-type and mutant FLAG-tagged DR5 constructs were introduced into HEK293T/17 cells by transient transfection. Cell lysates were probed by immunoblot analysis using AD5-10 or anti-FLAG mAb. All experiments were repeated at least three times with similar results. TM, transmembrane; w.t., wild type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 3.

Synthetic wild-type epitopes eliminate tumoricidal activity of AD5-10. A, Jurkat cells were treated with the indicated concentrations of AD5-10 or rsTRAIL for 8 h. AD5-10 (B and C) or rsTRAIL (D and E) was preincubated with 10 μm wild-type or mutant epitope or 10 μm control peptides (N1, CRD1A, and random peptide) at 37 °C for 2 h. Jurkat cells were then treated with these mixtures for 8 h. Both rsTRAIL and AD5-10 concentrations shown in the graphs are in logarithmic scale. F, Jurkat cells were treated with 100 ng/ml AD5-10 or 100 ng/ml rsTRAIL in the absence or presence of synthetic wild-type epitopes. G and H, Western blot analysis of caspase-3 activation in Jurkat cells treated with 100 ng/ml AD5-10 or 100 ng/ml rsTRAIL in the absence or presence of synthetic peptides for 2 h. Cell viability was determined using a CCK-8 assay. Values represent the mean ± S.D. of triplicate samples. **, p < 0.01 versus cells incubated with AD5-10 alone. w.t., wild-type; epit., epitope; R.P., random peptide.

FIGURE 4.

MAPK pathway is investigated in chimera signaling. Before treatment, all cells were starved in serum-free medium for 24 h and then cultured in fresh assay medium. Then NIH-chimera cells were treated with 500 ng/ml AD5-10 or 500 ng/ml rsTRAIL for the indicated times. Lysates were probed for protein phosphorylation by Western blot using the respective specific antibodies. A, schematic diagram depicting DR5e/ErbB2i and derived chimeric receptors. B, expression of surface chimeras in NIH3T3 cells. NIH3T3 cells were transfected with chimeric constructs and stained with PE-labeled anti-DR5 antibody (FAB 71908) or AD5-10 and TRITC-labeled secondary antibody followed by flow cytometry. C and D, immunoblot analysis of the activation of ERK in NIH3T3 chimeras treated with 500 ng/ml AD5-10 (C) or rsTRAIL (D) for the indicated times. E and F, analyses of phosphorylated ERK1/2 and phosphorylated MEK1/2 levels in NIH3T3 wild-type or NIH3T3 chimera-transfected cells with 500 ng/ml AD5-10 (E) and/or rsTRAIL (F) treatment for 15 min. p-, phosphorylated; ICD, intracellular domain. All experiments were repeated at least three times with similar results.

FIGURE 5.

Antibodies against the epitope ⁻¹LITQQDLAPQQRA¹² possess tumoricidal activities. A, binding affinity of Adie-1 and Adie-2 to the DR5 epitope recognized by AD5-10 (left), soluble DR5-ECD (middle), and soluble DR4-ECD (right). Synthetic immunizing peptide or recombinant soluble DR4/5 was immobilized on a 96-well plate. The plate was then incubated with the indicated concentration of AD5-10, Adie-1, and Adie-2. The binding affinities were then measured by enzyme-linked immunosorbent assay. Values represent the mean ± S.D. of triplicate samples. B, alanine-scanning mutagenesis analysis of the recognition pattern of anti-DR5 mAbs. An array of peptide ⁻¹LITQQDLAPQQRA¹² was probed with 0.2 μg/ml AD5-10, Adie-1, or Adie-2, respectively. The first and the last spot corresponded to the parental peptide, whereas the other spots represented an Ala-substituted analogue of wild-type (wt) sequence as indicated above the individual spots. C, tumoricidal function of Adie-1 and Adie-2 compared with AD5-10. Jurkat or HCT116 cells were incubated with 500 ng/ml anti-DR5 mAbs or rsTRAIL for 8 h. Cell viability was determined by a CCK-8 assay. D, Hoechst 33258 staining. HCT116 cells were cultured with 500 ng/ml anti-DR5 mAbs or rsTRAIL for 4 h. After fixation, cells were stained with Hoechst 33258 (1 μg/ml), and nuclear condensation and fragmentation of chromatin were observed by fluorescence microscopy (20×). The excitation wavelength was 380 nm. E, Jurkat cells and HCT116 cells were incubated with 500 ng/ml anti-DR5 mAbs or rsTRAIL for the indicated times. The activation of caspase-8, -9, and -3 was tested using the respective antibodies. F, Western blot analysis of caspase-3 activation in Jurkat cells treated with 500 ng/ml anti-DR5 mAbs or rsTRAIL in the absence or presence of synthetic wild-type epitope for 2 h. All experiments were repeated at least three times with similar results. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 6.

Agonistic anti-DR5 mAbs and TRAIL induce DISC formation in Jurkat cells and H460 cells. A and B, analysis of TRAIL- and agonist anti-DR5 mAbs-induced DISC in Jurkat cells (A) and H460 cells (B). Cells were treated with anti-DR5 mAbs or “TRAIL-FLAG + anti-FLAG” for indicated time before cell lysis, although AD5-10 was added to the cells after cell lysis in unstimulated cells. The samples were pre-cleared with-Sepharose 4B (GE Healthcare) and immunoprecipitated (IP) overnight at 4 °C with protein G-Sepharose beads and analyzed by Western blotting. All experiments were repeated at least three times with similar results. IB, immunoblot; WCE, whole cell extract.

FIGURE 7.

Computational simulation of AD5-10 epitope recognition. A, overall structure of AD5-10-epitope complex, side view (left), and top view (right). B, comparison of the wild-type core epitope (QDLAP, left) and the mutant epitope (QDAAP, right) in the binding pocket of AD5-10. C, ligand interaction plot for core epitope bound to AD5-10. All the diagrams were generated using the molecular operating environment 2006.08 (Chemical Computing Group, Montreal) Ligand Interactions Module.

FIGURE 8.

Differences between TRAIL- and AD5-10-induced signaling pathways. In the initiation stage of the caspase cascade, FADD and caspase-8 are recruited to DR5 (and possibly DR4) DISC. c-FLIP is involved in DISC formation in non-small cell lung cancer. Moreover, unlike TRAIL and other reported agonistic antibodies for DR5, AD5-10 particularly induces ROS accumulation and time-dependent reduction of GSH and GSSG content in the intrinsic mitochondrial pathway of Jurkat leukemia cells. ROS scavengers, antioxidant (N-acetyl-l-cysteine (NAC)) and GSH, suppress AD5-10-triggered cell death by inhibiting the release of apoptosis-inducing factor (AIF) and endonuclease G (EndoG), which contribute to the fragmentation of the nuclear DNA. Under ROS stress, JNK was triggered simultaneously and a subsequent secondary activation of JNK was observed; NF-κB was also activated during this time course.