Small-Molecule ONC201/TIC10 Targets Chemotherapy-Resistant Colorectal Cancer Stem-like Cells in an Akt/Foxo3a/TRAIL-Dependent Manner

Abstract

Self-renewing colorectal cancer stem/progenitor cells (CSC) contribute to tumor maintenance and resistance to therapy. Therapeutic targeting of CSCs could improve treatment response and prolong patient survival. ONC201/TIC10 is a first-in-class antitumor agent that induces TRAIL pathway-mediated cell death in cancer cells without observed toxicity. We have previously described that ONC201/TIC10 exposure leads to transcriptional induction of the TRAIL gene via transcription factor Foxo3a, which is activated by dual inactivation of Akt and ERK. The Akt and ERK pathways serve as important targets in CSCs. Foxo3a is a key mediator of Akt and ERK-mediated CSC regulation. We hypothesized that the potent antitumor effect of ONC201/TIC10 in colorectal cancer involves targeting CSCs and bulk tumor cells. ONC201/TIC10 depletes CD133(+), CD44(+), and Aldefluor(+) cells in vitro and in vivo. TIC10 significantly inhibits colonosphere formation of unsorted and sorted 5-fluorouracil-resistant CSCs. ONC201/TIC10 significantly reduces CSC-initiated xenograft tumor growth in mice and prevents the passage of these tumors. ONC201/TIC10 treatment also decreased xenograft tumor initiation and was superior to 5-fluorouracil treatment. Thus, ONC201/TIC10 inhibits CSC self-renewal in vitro and in vivo. ONC201/TIC10 inhibits Akt and ERK, consequently activating Foxo3a and significantly induces cell surface TRAIL and DR5 expression in both CSCs and non-CSCs. ONC201/TIC10-mediated anti-CSC effect is significantly blocked by the TRAIL sequestering antibody RIK-2. Overexpression of Akt, DR5 knockdown, and Foxo3a knockdown rescues ONC201/TIC10-mediated depletion of CD44(+) cells and colonosphere inhibition. In conclusion, ONC201/TIC10 is a promising agent for colorectal cancer therapy that targets both non-CSCs and CSCs in an Akt-Foxo3a-TRAIL-dependent manner.

Figure 1. ONC201/TIC10 depletes colorectal CSC markers in vitro

(A) ONC201/TIC10 depletes CD44(+) cells. SW480 cells were treated for 72 h with DMSO or 5 μM ONC201/TIC10. CD44(+) cells were detected by flow cytometry. * indicates p<0.005. (B) ONC201/TIC10 depletes 5-Fluorouracil (5-FU) resistant Aldefluor(+) cells. HCT116 cells were treated for 72 h with DMSO or 5 μM ONC201/TIC10 or 40/80 μM 5-FU. Aldefluor(+) cells were detected by flow cytometry. * indicates p<0.001. (C) Flow cytometry histograms for Aldefluor assay of DLD1 cells treated with DMSO or 2.5/5 μM ONC201/TIC10 for 72 h. Aldefluor(+) cells were determined according to the DEAB negative control. Data is represented as side scatter (SS) or count versus Aldefluor (ALDF1). (D) ONC201/TIC10 depletes CD133(+) cells in a dose- and time-dependent manner. DLD1 cells were treated with DMSO (72 h) or 2.5/5/10 μM ONC201/TIC10 (72 h) or 5 μM ONC201/TIC10 (24/48/72 h). CD133(+) cells were detected by flow cytometry. * indicates p < 0.05.

Figure 2. ONC201/TIC10 prevents colonosphere formation in vitro

(A) ONC201/TIC10 prevents colonosphere formation. DLD1 and SW480 cells (10,000 cells/well) were plated for colonosphere formation in the presence of DMSO or 10 μM ONC201/TIC10. Colonospheres (> 60 μm) were counted at 96 h. (B) Representative image (10X magnification) for DLD1 cells in (A). Each division on the scale is 10 μm. (C) Sorted Aldefluor(+) and Aldefluor(−) HCT116 cells (1000 cells/well) were plated for colonosphere formation. Colonospheres (> 60 μm) were counted after 6 days. (D) Sorted Aldefluor(+) HCT116 cells (1000 cells/well) were plated for colonosphere formation in the presence of DMSO or 40 μM 5-FU. Colonospheres (> 60 μm) were counted after 6 days (p value = 0.446). (E) Sorted Aldefluor(+) SW480 cells (10,000 cells/well) were plated for colonosphere formation in the presence of DMSO or 2.5/5/10 μM ONC201/TIC10. Colonospheres (> 60 μm) were counted after 72 h. * indicates p < 0.006. (F) Representative images (20X magnification) for (E). Each division on the scale is 10 μm.

Figure 3. ONC201/TIC10 prevents CSC-mediated xenograft tumor growth and self-renewal in vivo

(A) ONC201/TIC10 prevents CSC-mediated xenograft tumor growth. Sorted DLD1 Aldefluor(+) cells were subcutaneously injected into the right and left flank of athymic nude mice. Upon tumor formation, mice were administered either vehicle or ONC201/TIC10 50 mg/kg (i.p.). (N = 7 per group) Doses (indicated by triangle) were administered post-tumor implantation on day 7, 14 and 22. Tumor growth was monitored until the endpoint. Data is presented as mean ± standard error of mean. * indicates p < 0.05. (B) Representative image for (A). (C) Body weight of mice in (A) was monitored along with tumor growth until the end point. (D) Immunohistochemistry analysis of harvested tumors from (A) for CSC markers and TRAIL. (E) Sorted Aldefluor(+) DLD1 cells were plated for colonosphere culture in the presence of DMSO or 50 μM 5-FU or 5 μM ONC201/TIC10 for 72 h. Viable cells were counted and the indicated number of cells were injected into the right and left flank of hairless SCID mice. Tumor initiation was monitored till Day 80 post tumor implantation.

Figure 4. ONC201/TIC10-mediated anti-CSC effect involves induction of surface TRAIL

(A) ONC201/TIC10 induces surface TRAIL in CSCs. HCT116 cells were treated with DMSO or 5/10 μM ONC201/TIC10 for 72 h and CD133 and TRAIL staining was detected by flow cytometry. * indicates p < 0.04. (B) SW480 cells were treated with DMSO or indicated doses of ONC201/TIC10 for 72 h. CD44 and TRAIL staining was detected by flow cytometry. * indicates p < 0.03. (C) ONC201/TIC10-mediated anti-CSC effect is TRAIL-dependent. DLD1 cells were treated with DMSO or 5 μM ONC201/TIC10 for 72 h in the presence of control IgG or RIK-2 antibody. Aldefluor(+) cells were detected by flow cytometry. Fold depletion is the ratio of % Aldefluor(+) cells in DMSO and ONC201/TIC10 treatment groups. * indicates p < 0.05. (D) DLD1 cells were treated with DMSO or 5 μM ONC201/TIC10 for 72 h in the presence of control IgG or RIK-2 antibody. CD44(+) cells were detected by flow cytometry. * indicates p < 0.04. (E) HCT116 cells were treated with DMSO or 5/10 μM ONC201/TIC10 for 72 h and CD133 and TRAIL staining was detected by flow cytometry. * indicates p < 0.04.

Figure 5. ONC201/TIC10-mediated induction of surface TRAIL in CSCs involves inhibition of Akt and ERK followed by Foxo3a activation

(A) ONC201/TIC10 inhibits Akt, ERK and activates Foxo3a in CSCs. Sorted DLD1 Aldefluor(+) cells were subcutaneously injected into the right and left flank of athymic nude mice. Upon tumor formation, mice were administered either vehicle or ONC201/TIC10 50 mg/kg (i.p.). Tumors were harvested after 72 h of treatment and western blot analysis was performed (B) Western blot analysis of sorted Aldefluor(+) (CSC) and unsorted (non CSC) SW480 cells treated with DMSO or 5 μM ONC201/TIC10 for 72 h (C) Parental (WT) or myristoylated- (myr-) Akt overexpressing HCT116 cells were treated with DMSO or 5 μM ONC201/TIC10 for 72 h. CD44 staining was detected by flow cytometry. Fold depletion is the ratio of % CD44(+) cells in DMSO and ONC201/TIC10 treatment groups. * indicates p < 0.02. (D) WT or myr-Akt overexpressing HCT116 cells were treated with DMSO or 5 μM ONC201/TIC10 for 60 h. Aldefluor(+) cells were detected by flow cytometry. Fold depletion is the ratio of % Aldefluor(+) cells in DMSO and ONC201/TIC10 treatment groups. * indicates p < 0.0005. (E) WT or myr-Akt overexpressing HCT116 cells were treated with DMSO or 5 μM ONC201/TIC10 for 72 h. CD44 and TRAIL staining was detected by flow cytometry. Fold induction is the ratio of % CD44(+)TRAIL(+) cells in ONC201/TIC10 and DMSO treatment groups. * indicates p < 0.05.