ONC201 activates ER stress to inhibit the growth of triple-negative breast cancer cells

Abstract

ONC201 was previously identified as a first-in-class antitumor agent and small-molecule inducer of the TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) gene that induces apoptosis in cancer cells. ONC201 has a safety profile and is currently in phase II clinical trials for the treatment of various malignancies. In the current study, we examine the effect of ONC201 on triple-negative breast cancer cells (TNBC), a subtype of breast cancer that is sensitive to TRAIL. We find that ONC201 inhibits the growth of TNBC cells including TNBC cells that have developed acquired TRAIL resistance. However, TNBC cells that have developed acquired ONC201 resistance are cross-resistant to TRAIL. Mechanistically, ONC201 triggers an integrated stress response (ISR) involving the activation of the transcription factor ATF4. Knockdown of ATF4 impairs ONC201-induced apoptosis of TNBC cells. Importantly, the activation of ATF4 is compromised in ONC201-resistant TNBC cells. Thus, our results indicate that ONC201 induces an ISR to cause TNBC cell death and suggest that TNBC patients may benefit from ONC201-based therapies.

Keywords: ATF4; ONC201; TRAIL; apoptosis; triple-negative breast cancer.

Figure 1. ONC201 induces apoptosis in TRAIL-sensitive and -resistant MDA231 cells

MTT and colony formation analyses of MDA231P and MDA231R cells treated with TRAIL A. or ONC201 B. For MTT assays, MDA231P and MDA231R cells were left untreated or treated with 100, 250, 500, 750 ng/ml TRAIL or 5, 10, 25, 50 μM ONC201 for 72 h. For colony formation assays, 500 cells/well were seeded in 6-well plates. The next day, cells were left untreated or treated with 50, 250 ng/ml TRAIL or 2, 10 μM ONC201 for 72 h and then grown in medium without TRAIL or ONC201 for 12 days, followed by crystal violet staining. Data represent mean ± S.D. (error bars) of three independent experiments. ***, p<0.001, statistically significant. Western blot analyses of caspase-8, caspase-9, caspase-3 and PARP, treated with TRAIL C. or ONC201 D. MDA231P and MDA231R cells were left untreated or treated with 250 and 500 ng/ml TRAIL for 24 h or 10, and 25 μM ONC201 for 72 h. β-Actin was used as a loading control.

Figure 2. ONC201-resistant TNBC cells are insensitive to TRAIL

MTT analyses of MDA468P and MDA468R-ONC201 cells treated with ONC201 A. or TRAIL B. Colony formation analyses of MDA468P and MDA468R-ONC201 cells treated with ONC201 C. or TRAIL D. For MTT assays, MDA468P and MDA468R-ONC201 cells were left untreated or treated with 20, 50, and 100 ng/ml TRAIL or 1, 2, and 5 μM ONC201 for 72 h. Data represent mean ± S.D. (error bars) of three independent experiments. *, P<0.05; ***, p<0.001, statistically significant. For colony formation assays, 500 cells/well were seeded in 6-well plates. After 24 h, cells were left untreated or treated with 20, 100 ng/ml TRAIL or 1, 5 μM ONC201 for 72 h, and then grown in medium without TRAIL or ONC201 for 12 days, followed by crystal violet staining.

Figure 3. ONC201 activates ER stress pathways

A. Western blot analyses of caspase-8, caspase-3, DR5, TRAIL, Akt, p-Akt, ERK and p-ERK. MDA231 and MDA468 cells were left untreated or treated with indicated concentrations of ONC201 for 72 h. β-Actin was used as a loading control. B. Western blot analyses of IRE1, p-IRE1, eIF2α, p-eIF2α, ATF4 and CHOP. MDA231 or MDA468 cells were left untreated or treated with ONC201 at indicated concentrations for 72 h. C, qRT-PCR analyses of the expression of sXBP1, usXBP1, total XBP1 and ATF4 in MDA231 and MDA468 cells treated with ONC201 (10 μM and 5 μM, 48 h) as compared with untreated cells. Fold changes were normalized relative to GAPDH.

Figure 4. ATF4 knockdown decreases ONC201-induced growth inhibition

A. Western blot analysis of ATF4 and CHOP in MDA231 and MDA468 cells transfected with siRNA against ATF4 or non-target siRNA, followed by treatment with ONC201 (10 μM, 24 h). B. MTT assays of MDA231 and MDA468 cells transfected with non-target siRNA or siRNA against ATF4 and then treated with ONC201 at the indicated concentrations for 72 h.

Figure 5. ONC201-resistant TNBC cells have a defect in the activation of ATF4

Western blot analyses of IRE1, p-IRE1, eIF2α, p-eIF2α, ATF4, and CHOP. MDA231P and MDA231R-ONC201 cells A. or MDA468P and MDA468R-ONC201 cells B. were left untreated or treated with the indicated concentrations of ONC201 for 72 h. β-Actin was used as a loading control.

Figure 6. ONC201-resistant TNBC cells are sensitive to conventional chemotherapeutics

MTT of parental and ONC201-resistant MDA231 A. and MDA468 B. cells treated with TAXOL or cisplatin. MDA231P, MDA231R-ONC201, MDA468P and MDA468R-ONC201 cells were left untreated or treated with the indicated concentrations of TAXOL or cisplatin for 72 h. Data represent mean ± S.D. (error bars) of three independent experiments.