Therapeutic potential and molecular mechanism of a novel, potent, nonpeptide, Smac mimetic SM-164 in combination with TRAIL for cancer treatment

Abstract

Smac mimetics are being developed as a new class of anticancer therapies. Because the single-agent activity of Smac mimetics is very limited, rational combinations represent a viable strategy for their clinical development. The combination of Smac mimetics with TNF-related apoptosis inducing ligand (TRAIL) may be particularly attractive because of the low toxicity of TRAIL to normal cells and the synergistic antitumor activity observed for the combination. In this study, we have investigated the combination synergy between TRAIL and a potent Smac mimetic, SM-164, in vitro and in vivo and the underlying molecular mechanism of action for the synergy. Our study shows that SM-164 is highly synergistic with TRAIL in vitro in both TRAIL-sensitive and TRAIL-resistant cancer cell lines of breast, prostate, and colon cancer. Furthermore, the combination of SM-164 with TRAIL induces rapid tumor regression in vivo in a breast cancer xenograft model in which either agent is ineffective. Our data show that X-linked IAP (XIAP) and cellular IAP 1 (cIAP1), but not cIAP2, work in concert to attenuate the activity of TRAIL; SM-164 strongly enhances TRAIL activity by concurrently targeting XIAP and cIAP1. Moreover, although RIP1 plays a minimal role in the activity of TRAIL as a single agent, it is required for the synergistic interaction between TRAIL and SM-164. This study provides a strong rationale to develop the combination of SM-164 and TRAIL as a new therapeutic strategy for the treatment of human cancer.

Figure 1

SM-164 potentiates cell viability inhibition by TRAIL in both TRAIL-sensitive and TRAIL-resistant cancer cell lines of three tumor types. A panel of breast cancer (2LMP, MDA-MB-436, SK-BR-3, MDA-MB-453), prostate cancer (PC-3 and DU-145) and colon cancer (SW620 and SW480) cell lines were treated with TRAIL (T) alone, SM-164 (SM) (100 nM) alone and their combination for 4 days. Cell viability inhibition was determined using a WST-8 assay.

Figure 2

SM-164 enhances TRAIL-induced apoptosis through amplification of caspase-8 mediated apoptotic signals. (A) 2LMP cells were treated with SM-164 at 10 nM at indicated time-points, degradation of cIAP1 and cIAP2 was assessed by Western blotting. (B) 2LMP cells were treated as indicated, and apoptosis was determined using Annexin-V/PI double staining and flow cytometry assay. (C) 2LMP cells were treated as indicated, PARP cleavage and activation of caspases were determined by Western blotting. (D) 2LMP cells were transfected with control siRNA, siRNA against caspase-8, -9 or -3. Cells were treated as indicated and cell viability inhibitory activity was determined by a WST-8 assay.

Figure 3

SM-164 enhances TRAIL-induced anticancer activity by targeting cIAP1 and XIAP, but not by targeting cIAP2. (A) 2LMP cells were transfected with control (CTL) siRNA, or siRNA against XIAP (X), cIAP1 (C1), or cIAP2 (C2) individually or in combination for 48 h, siRNA transfection efficiency was examined by western blotting (upper panel); cells were treated as indicated, cell death induction was determined with trypan blue exclusion assay (middle panel); cell viability inhibition was determined by a WST-8 assay (lower panel). (B) 2LMP cells were transfected with control (CTL) siRNA, and siRNA against XIAP (X), or cIAP1 (C1), individually or in combination for 48 h, siRNA transfection efficiency was examined by western blotting (upper panel); cells were treated for another 60 h, cell viability inhibition was determined by a WST-8 assay (lower panel). (C) HCT116 XIAP wild type (XIAP-WT) and knockout (XIAP-KO) human colon cancer cells were treated for 60 h, cell viability inhibitory activity was determined by a WST-8 assay. (D) HCT116 XIAP knockout (XIAP-KO) cells were transfected with control siRNA or siRNA against cIAP1 (C1) or cIAP2 (C2) individually or in combination for 48 h, siRNA transfection efficiency was examined by western blotting (upper left panel); cells were treated for a further 60 h, cell viability inhibition was determined by a WST-8 assay (upper right panel). HCT116 XIAP knockout (XIAP-KO) cells were transfected with cIAP1 siRNA, cells were treated for a further 60 h, cell viability inhibitory activity was determined by a WST-8 assay (lower panel).

Figure 4

Ablation of cIAP1 markedly enhances TRAIL-DISC formation, and facilitates the intracellular interaction of RIP1 and caspase-8. (A) 2LMP cells were treated with a mixture of Flag-tagged TRAIL and anti-Flag M2 IgG with or without pretreatment of 100 nM of SM-164 for 5, 10 and 30 min. Cells were lysed and the associated proteins in the lysates were pulled down with sepharose 4B beads, and subjected to Western blotting as indicated (left panel); Expression of DR4, DR5, procaspase-8, RIP1 and cIAP1 in the whole cell lysates was examined by Western blotting (right panel). (B). Secondary signaling complexes were immunoprecipitated with an RIP1 antibody from DISC-depleted lysates, and analyzed by western blotting assay. (C) 2LMP cells transfected with siRNA against IAPs were treated with a mixture of Flag-tagged TRAIL and anti-Flag M2 IgG for 15 min. Cells were lysed and the associated proteins in the lysates were pulled down with sepharose 4B beads, and subjected to western blotting as indicated (upper panel); Secondary signaling complexes were immunoprecipitated with an RIP1 antibody from DISC-depleted lysates, and analyzed by Western blotting (middle panel); expression of DR4, DR5, procaspase-8, RIP1 and cIAP1 in the cell lysates were examined by Western blotting (lower panel).

Figure 5

RIP1 plays an essential role in TRAIL-sensitization by SM-164 but not in the single-agent activity of TRAIL. (A)-(C). 2LMP cells were transfected with control siRNA, and siRNA against RIP1 for 48 h. (A). Transfected cells were treated for a further 24 h and cell death induction was determined with trypan blue exclusion assay; (B) Transfected cells were treated for a further 24 h and cell lysates were subjected to western blotting analysis as indicated; (C). Transfected cells were treated for a further 60 h and cell viability was determined by a WST-8 assay. (D) Jurkat RIP1 wild type (RIP1-WT) and RIP1 knockout (RIP1-KO) cells were treated with SM-164 alone, TRAIL alone or their combination for 48 h and cell viability was determined by a WST-8 assay.

Figure 6

SM-164 induces cIAP1 degradation in tumor tissues and dramatically enhances the in vivo antitumor activity of TRAIL and the combination of SM-164 and TRAIL achieves tumor regression without toxicity to animals (A) Nude mice, bearing established 2LMP xenograft tumors (200-300 mm³), were treated with a single dose of SM-164 alone, TRAIL alone, or their combination, or vehicle (VEH). Tumor tissues were harvested at the time points indicated. Activation of caspases and PARP cleavage, and cIAP1 degradation in tumor tissues were analyzed by Western blot. (B) Apoptosis in tumor tissues was examined by TUNEL staining (left panels) and scored under microscope (right panel). At least 1,000 cells were counted. (C) Tumor tissues were harvested at 24 h time point and examined by H&E staining. Xenograft tumor tissues treated with SM-164 were characterized with cell shrinkage, nuclear pyknosis and chromatin condensation. (D) Antitumor activity of SM-164 alone, TRAIL alone and their combination in the 2LMP xenograft model. Nude mice (8-12 per group), bearing established 2LMP xenograft tumors, were treated with SM-164 at 5 mg/kg, i.v. alone, TRAIL at 10 mg/kg, i.p. alone, their combination, or VEH, daily, 5 days a week for 2 weeks. The mean of tumor volume ± SEM (left panel) and mean animal body ± SEM (right panel) were shown for each group, respectively.