Dual inactivation of Akt and ERK by TIC10 signals Foxo3a nuclear translocation, TRAIL gene induction, and potent antitumor effects

Abstract

Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an antitumor protein that is in clinical trials as a potential anticancer therapy but suffers from drug properties that may limit efficacy such as short serum half-life, stability, cost, and biodistribution, particularly with respect to the brain. To overcome such limitations, we identified TRAIL-inducing compound 10 (TIC10), a potent, orally active, and stable small molecule that transcriptionally induces TRAIL in a p53-independent manner and crosses the blood-brain barrier. TIC10 induces a sustained up-regulation of TRAIL in tumors and normal cells that may contribute to the demonstrable antitumor activity of TIC10. TIC10 inactivates kinases Akt and extracellular signal-regulated kinase (ERK), leading to the translocation of Foxo3a into the nucleus, where it binds to the TRAIL promoter to up-regulate gene transcription. TIC10 is an efficacious antitumor therapeutic agent that acts on tumor cells and their microenvironment to enhance the concentrations of the endogenous tumor suppressor TRAIL.

Conflict of interest statement

Competing interests: W.S.E.-D. is a co-founder and chief scientific advisor of Oncoceutics Inc., a biotech company focused on developing novel small-molecule anticancer therapies targeting p53-deficient tumors.

Figures

Fig. 1

TIC10 is a small molecule that induces TRAIL independent of p53. (A) Chemical structure of TIC10. (B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of TRAIL mRNA concentrations in HCT116 p53−/− cells (48 hours, n = 4). (C) Surface TRAIL induced by TIC10 in a panel of cancer cells (10 μM, 72 hours, n = 3). (D) Surface TRAIL in HCT116 p53−/− cells after TIC10 treatment at the indicated conditions and time points after treatment (n = 3). (E) HCT116 p53−/− TRAIL surface levels measured by flow cytometry at 72 hours after TIC10 treatment initiation (5 μM, n = 3). Cells were treated with TIC10 or dimethyl sulfoxide (DMSO) control for the indicated time of preincubation and then changed to a drug-free medium for the remaining period until analysis at 72 hours. Error bars indicate SD of replicates. *P < 0.05 between the indicated condition and controls.

Fig. 2

TIC10 induces TRAIL-mediated apoptosis in vitro. (A) Cell cycle profiles of HCT116 p53−/−and HFF cells treated with TIC10 (5 μM, 72 hours, n = 3). (B and C) Representative wells (B) and quantification of colony formation assays (C) of cancer cells per well with or without TIC10 (10 μM, 72 hours, n = 3). (D) Parallel experiments as in (C) but with HFF cells that were counted at the 72-hour endpoint (n = 3). (E) Percentage of sub-G1 DNA (fragmented DNA indicative of cells undergoing apoptosis) in HCT116 wild-type (WT), p53−/−, and Bax−/− cells after treatment with DMSO, TIC10 (1, 5, or 10 μM), or rhTRAIL (25 ng/ml) (72 hours, n = 3). (F) Cleaved caspase-3 (CC3) in HCT116 p53−/− cells assayed by immunofluorescence (left panel, 5 μM TIC10, caspase-3 shown in green) or Western blot analysis (right panel) treated with DMSO or TIC10 (1, 2.5, 5, and 10 μM) for 72 hours. (G) Percentage of sub-G1 DNA in TIC10-treated cancer cells with or without preincubation with zVAD-fmk (10 μM, 72 hours, n = 3). (H) Percentage of sub-G1 DNA in MDA- MB-231 cells with stable knockdown of TRAIL by shRNA treated with TIC10 (1, 5, and 10 μM) for 72 hours (n = 3). (I) Percentage of sub-G1 DNA in H460 cells with endogenous DR5 or overexpressing a DR5 construct (DR5ΔDD) with its death domain replaced by enhanced green fluorescent protein (EGFP) (10 μM TIC10, 72 hours, n = 3). (J) Ability of TIC10 (5 μM) or TRAIL (25 ng/ml) to reduce cell viability in HCT116 cells after a 1-hour preincubation at the indicated temperatures (72 hours, n = 3). Error bars indicate SD of replicates. *P < 0.05 compared to control unless otherwise indicated.

Fig. 3

TIC10 is a TRAIL-dependent antitumor agent in vivo. (A) Relative tumor size in mice bearing a HCT116 p53−/− xenograft and treated with three doses of TIC10 (intraperitoneal), TRAIL (intravenous), or vehicle (intraperito-neal) administered on days 0, 3, and 6 as indicated by gray vertical bars (n = 10). (B) Bioluminescence imaging of luciferase-expressing HCT116 p53−/−xenografts that received a single intraperitoneal injection of TIC10 or vehicle (n = 6). (C) Relative tumor size in RKO xenografts treated with a single dose of TIC10 (intraperitoneal), TRAIL (intravenous), or vehicle (intraperito-neal; n = 10). Right panel shows near-infrared images of sample mice from each cohort on day 13 after treatment and 3 days after injection with AngioSense 680. (D) Box and whisker plot of tumor volume on day 9 after treatment initiation in MDA-MB-231 xenografts expressing vector or shTRAIL and treated with single doses of TIC10 (intraperitoneal), TRAIL (intravenous), or vehicle (DMSO, intraperitoneal) (n = 8). (E) TIC10 or vehicle administered as a single oral dose in the HCT116 xenograft (n = 6). (F) Overall survival of Eμ-myc mice treated during weeks 9 to 12 with weekly single oral dose of TIC10 (25 mg/kg). Right panel shows hematoxylin and eosin (H&E) staining of axillary lymph nodes from Eμ-myc and WT C57/B6 mice at 14 weeks of age. P value was determined by log-rank test. For relative tumor volume plots, tumor size is expressed relative to the tumor size on day 0, which is defined as the day of treatment initiation. Error bars indicate SD of replicates. *P < 0.05, **P < 0.005, compared to control unless otherwise indicated.

Fig. 4

TIC10 induces TRAIL in tumor and normal cells. (A) H&E and IHC analysis of HCT116 p53−/− xenograft tumors at 2 and 7 days after a single dose of TIC10 on day 0 [100 mg/kg, in-traperitoneally (ip)]. (B) H&E and IHC analysis for TRAIL at the border between tumor and stromal fibroblasts from HCT116 p53−/− xenograft tumors after treatment with TIC10 (100 mg/kg, ip) or vehicle on day 2 after treatment. The yellow dashed lines indicate stromal fibroblasts, and the black dashed lines indicate the tumor. (C) qRT-PCR analysis of TRAIL transcript in HCT116 p53−/− xenograft tumors in athymic nude mice after a single dose of DMSO or TIC10 [25 mg/kg, intravenously (iv)] (n = 4). (D) Histological and TRAIL IHC analysis of normal tissue in athymic nude mice after TIC10 administration on day 0 (100 mg/kg, iv). (E) Coculture of HCT116 p53−/− and HFF cells labeled as red and green, respectively. TIC10 (10 μM)– or DMSO-treated wells are shown immediately before treatment or 3 days after treatment. The bottom two panels were taken at the 3-day endpoint after being counterstained with Hoechst. (F) Surface TRAIL analysis of HFF cells after TIC10 treatment (0, 2.5, 5, or 10 μM from left to right) (72 hours, n = 3). Surface TRAIL is shown relative to cells that were not treated with TIC10. (G) Percentage of sub-G1 DNA in a coculture of HCT116 p53−/− cells and pretreated HFFs (24 hours, n = 3). HFF pretreatment consisted of a 72-hour incubation with TIC10 (10 μM) or DMSO. Coculture was in the presence or absence of the TRAIL-blocking antibody RIK-2. Scale bars, 100 μm. Error bars indicate SD of replicates. *P < 0.05, compared to control unless otherwise indicated.

Fig. 5

TIC10 is effective as an antitumor agent in GBM. (A) Surface TRAIL in GBM cell lines after incubation with TIC10 (5 μM, 72 hours, n = 3). (B) GI50 values extrapolated from cell viability assays of the indicated GBM cell lines at 72 hours after treatment with TIC10 or DMSO (n = 3). (C) Cell viability assay of freshly resected glioblastoma tissue treated with DMSO, TIC10, or temozolomide (TMZ, 10 μM) (72 hours, n = 3). (D) Relative tumor size (compared to day 0) in a subcutaneous xenograft of T98G in mice treated with a single dose of vehicle, TIC10 (30 mg/kg, orally), or bevacizumab (10 mg/kg, iv) on day 0 (n = 8). (E) Overall survival of mice harboring SF767 intracranial tumors treated with a single oral dose of vehicle (n = 8), TIC10 (25 mg/kg, n = 7), bevacizumab (bev) (10 mg/kg, iv, n = 6), or TIC10 and bevacizumab (n = 7) at 2 weeks after implantation. (F) Bioluminescence imaging of a sample control mouse and a TIC10-treated mouse bearing an intracranial xenograft of SF767 glioblastoma. The bioluminescence scale bar on the right applies to all images in the panel. *P < 0.05 between the indicated condition and control.

Fig. 6

TIC10-induced TRAIL and cytotoxicity are Foxo3a-dependent. (A) Transcriptional changes associated with FOXO signaling from gene expression profiling of HCT116 p53−/− cells at 48 hours after TIC10 treatment (10 μM) normalized to DMSO-treated cells (n = 3). P < 0.05 between DMSO and TIC10 treatment groups for all of these changes. (B) Immunofluorescence of Foxo3a in HCT116 cells with and without TIC10 treatment (48 hours, 10 μM). (C) Western blot analysis of whole-cell lysates (W) and cytoplasmic (C) and nuclear (N) extracts from HCT116 cells treated with DMSO or TIC10 (48 hours, 10 μM). β-Actin and lamin B1 are shown as cytoplasmic and nuclear loading controls, respectively. (D) ChIP assay for TIC10-induced translocation of Foxo3a to the TRAIL promoter at 48 hours after TIC10 treatment in HCT116 p53−/− cells (0, 2.5, 5, or 10 μM from left to right). (E) Flow cy-tometric analysis of cell surface TRAIL induced by TIC10 (10 μM) with or without transient knockdown of Foxo1 and/or Foxo3a in HCT116 p53−/−cells with siRNA (72 hours, n = 3). The cell surface TRAIL ratio refers to the amount of surface TRAIL in treated cells relative to that in cells that were not treated with TIC10. Confirmation of knockdown is shown by Western blot analysis (right). (F) Percentage of sub-G1 DNA in HCT116 cells with or without stable knockdown of Foxo3a and with or without TIC10 treatment (10 μM, 72 hours, n = 3). (G) Tumor volume of HCT116 xenograft with or without stable knockdown of Foxo3a by shRNA after a single oral dose of vehicle or TIC10 (25 mg/kg) on day 0 (n = 10). (H) IHC analysis and TUNEL staining of HCT116 tumors with or without stable knockdown of Foxo3a 3 days after a single dose of TIC10 (25 mg/kg, orally). Scale bars, 100 μm. Error bars indicate SD of replicates. *P < 0.05, compared to control unless otherwise indicated.

Fig. 7

TIC10 inactivates Akt and ERK to induce TRAIL through Foxo3a. (A) Western blot analysis of HCT116 p53−/− cells treated with TIC10 (2.5, 5, and 10 μM) for 72 hours. (B) Time course of TIC10-induced effects determined by den-sitometry of Western blot analysis of HCT116 p53−/− cells treated with TIC10 (5 μM) or DMSO (n = 3). Data are expressed relative to the control sample for each time point and normalized to Ran as a loading control. TRAIL was quantified by flow cytometry as a parallel experiment (n = 3). (C) Western blot analysis of HCT116 p53−/− xenograft tumors in athymic nude mice after a single dose of DMSO or TIC10 (25 mg/kg, iv) (D) Western blot analysis of TIC10-induced effects on Foxo3a in DLD-1 human colon cancer, MDA-MB-468 human breast cancer, and T98G human GBM cell lines (10 μM, 72 hours). (E) Flow cytometric analysis of TRAIL in HCT116 p53−/− cells after incubation with 10 μM A6730 (Akt inhibitor), U0126 monoethanolate (MEK inhibitor), or both (48 hours), with or without stable knockdown of Foxo3a (n = 3). (F) qRT-PCR analysis of TRAIL mRNA at 48 hours after transient knockdown of Akt and/or ERK in HCT116 p53−/−cells (n = 3). For siERK and siAkt combination, P < 0.05 compared to all other conditions. (G) Confirmation of Akt and ERK knockdown by Western blot analysis. (H) Western blot analysis of B-Raf/MEK/ERK signaling in HCT116 cells treated with TIC10 at the indicated concentrations (48 hours). (I) Western blot analysis of Raf expression and phosphorylation in HCT116 cells treated with TIC10 at the indicated concentrations (48 hours). Error bars indicate SD of replicates. *P < 0.05, compared to control unless otherwise indicated.

Fig. 8

TIC10 up-regulates TRAIL through inhibition of Akt and MEK/ERK and activation of Foxo3a. TIC10 causes inactivation of the prosurvival kinases MEK, ERK, and Akt. ERK and Akt normally phosphorylate Foxo3a at S253 and S294, respectively. These phosphorylation events create docking sites for 14-3-3 proteins that bind Foxo3a and sequester it in the cytoplasm, thereby inhibiting its activity as a transcription factor. Through its actions on Akt and ERK, TIC10 inhibits Foxo3a phosphorylation, which allows Foxo3a to translocate to the nucleus and bind to the TRAIL promoter that harbors a FOXO binding site. This binding stimulates TRAIL gene transcription and translation, increasing the amount of TRAIL on the cell surface.