Z-IETD-FMK

mTOR Inhibition Sensitizes ONC201-Induced Anti-Colorectal Cancer Cell Activity

Abstract
We here tested the anti-colorectal cancer (CRC) activity of a first-in-class small molecule TRAIL inducer, ONC201. The potential effect of mTOR on ONC201’s actions was also examined. ONC201 induced moderate cytotoxicity against CRC cell lines (HT-29, HCT-116, and DLD-1) and primary human CRC cells. Significantly, AZD-8055, a mTOR kinase inhibitor, sensitized ONC201-induced cytotoxicity in CRC cells. Meanwhile, ONC201-induced TRAIL/death receptor-5 (DR-5) expression, caspase-8 activation, and CRC cell apoptosis were also potentiated with AZD-8055 co-treatment. Reversely, TRAIL sequestering antibody RIK-2 or the caspase-8 specific inhibitor z-IETD-fmk attenuated AZD-8055 plus ONC201-induced CRC cell death. Further, mTOR kinase-dead mutation (Asp-2338-Ala) or shRNA knockdown significantly sensitized ONC201’s activity in CRC cells, leading to profound cell death and apoptosis. On the other hand, expression of a constitutively-active S6K1 (T389E) attenuated ONC201-induced CRC cell apoptosis. For the mechanism study, we showed that ONC201 blocked Akt but only slightly inhibited mTOR in CRC cells. Co-treatment with AZD-8055 concurrently blocked mTOR activation. These results suggest that mTOR could be a primary resistance factor of ONC201 in CRC cells.

Keywords
Colorectal cancer, ONC201, TRAIL, mTOR, AZD-8055

1. Introduction
The prognosis of metastatic and/or recurrent colorectal cancer (CRC) is often poor. CRC cells frequently exhibit pre-existing and acquired resistance to traditional chemotherapeutic agents. Therefore, research focus has shifted to exploring novel anti-CRC agents.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a valuable therapeutic target for CRC and other human malignancies. TRAIL selectively targets cancer cells. However, the clinical application of TRAIL (or TRAIL-related components) has been hampered by several drawbacks, such as high cost, short half-life, and recurrence. Recently, a first-in-class small molecule inducer of TRAIL, ONC201 (or TIC10), has been developed. ONC201 inhibits Akt and Erk activation, forcing Foxo3a nuclear translocation and TRAIL transcription. Its potential functions in CRC cells have not been extensively studied.

Mammalian target of rapamycin (mTOR) signaling is often hyperactivated in CRC, playing pivotal roles in cancer initiation, survival, progression, and chemoresistance. Whether mTOR also influences ONC201-induced anti-cancer activity has not been investigated. In the present study, we provide pharmacological and genetic evidence to show that mTOR could be a primary resistance factor of ONC201.

2. Materials and Methods
2.1 Chemicals and Reagents
AZD-8055 was purchased from Selleck Chemicals (Shanghai, China). The caspase-8 specific inhibitor z-IETD-fmk was purchased from CalBiochem (La Jolla, CA). The purified mAb specific for TRAIL (RIK-2) was obtained from Santa Cruz Biotech (Santa Cruz, CA). All other antibodies were purchased from Cell Signaling Tech (Shanghai, China). Cell culture reagents were obtained from Gibco (Nanjing, China).

2.2 Culture of Human CRC Cell Lines
Human CRC cell lines (HT-29, DLD-1, and HCT-116) were gifts from Dr. Tian’s group. The cells were cultured in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and necessary antibiotics.

2.3 Primary Colon Cancer Cell Culture
Primary colon cancer cells were gifts from Dr. Pei-hua Lu’s group at Nanjing Medical University. The cells were cultured as described. The study was approved by the institutional review board (IRB) and ethics committee of all authors’ institutions. Written-informed consent was obtained from all patients providing specimens.

2.4 Cell Proliferation MTT Assay
Cells (1×10^4 cells/well) were seeded onto 96-well plates. Following treatment, methylthiazol tetrazolium (MTT, Sigma, 5 mg/mL) was added. DMSO (200 μL/well, Sigma) was used to dissolve the crystals, and the optical density (OD) at 590 nm was recorded.

2.5 Cell Death Assay
Cell death was determined by trypan blue staining. The percentage of trypan blue-positive cells was calculated.

2.6 Clonogenic Assay
CRC cells (3×10^4 cells per dish) were re-suspended in DMEM medium plus 0.5% agar and plated onto a 100-mm Petri dish. The cells were re-fed with drug-containing medium every two days for 10 days. Colonies were stained and manually counted.

2.7 TUNEL Staining Assay of Apoptosis
Apoptosis was quantified using the TUNEL In Situ Cell Apoptosis Detection Kit (Roche). The apoptosis ratio was calculated as the percentage of TUNEL-positive cells.

2.8 Histone-DNA ELISA Assay
Apoptosis was also quantified using the Histone-DNA ELISA PLUS kit (Roche) following the manufacturer’s protocol.

2.9 Caspase-8 Activity Assay
Caspase-8 activity was measured using Ac-IETD-AFC as the substrate. The released AFC was measured via a spectrofluorometer (excitation at 400 nm).

2.10 Western Blots
Cell lysates were prepared, normalized, and subjected to SDS-PAGE. Proteins were transferred onto PVDF membranes and probed with primary and secondary antibodies. Antibody binding was detected using an ECL kit.

2.11 Quantitative Real-Time PCR (qRT-PCR)
Total RNA was extracted using Trizol reagents, and RT-qPCR was performed using SYBR Green and an ABI Prism 7000 system. Primers for TRAIL, DR-5, and GAPDH were used.

2.12 mTOR shRNA Knockdown
Three non-overlapping shRNAs targeting mTOR were designed and transfected into HT-29 cells. Stable clones were selected with puromycin.

2.13 mTOR Kinase-Dead Mutation
A kinase-dead mTOR mutation (Asp-2338-Ala) was introduced into HT-29 cells. Stable clones were selected with neomycin.

2.14 Constitutively Active S6K1 (ca-S6K1) Construct
The ca-S6K1 (T389E) construct was transfected into HT-29 cells. Stable clones were selected with puromycin.

2.15 Statistical Analysis
Data were analyzed using SPSS 20.0. Values are expressed as mean ± SD. A p-value < 0.05 was considered significant. 3. Results 3.1 AZD-8055 Potentiates ONC201-Induced CRC Cell Death ONC201 (5–25 μM) inhibited HT-29 cell proliferation, but its activity was moderate (IC50 > 25 μM). Co-treatment with AZD-8055 dramatically enhanced ONC201’s activity, leading to profound proliferation inhibition and cell death. Similar results were observed in HCT-116, DLD-1, and primary CRC cells.

3.2 AZD-8055 Facilitates ONC201-Induced CRC Cell Apoptosis
ONC201 induced TRAIL and DR-5 expression and caspase-8 activation in HT-29 cells. These effects were potentiated by AZD-8055. Pre-treatment with RIK-2 or z-IETD-fmk attenuated the combo-induced cell death and apoptosis.

3.3 mTOR Knockdown or Mutation Sensitizes CRC Cells to ONC201
mTOR shRNA knockdown or kinase-dead mutation sensitized HT-29 cells to ONC201, enhancing proliferation inhibition and apoptosis. Conversely, ca-S6K1 expression attenuated ONC201-induced apoptosis.

3.4 ONC201 and AZD-8055 Co-Treatment Blocks Akt and mTOR Activation
ONC201 blocked Akt but only partially inhibited mTOR. Co-treatment with AZD-8055 completely blocked mTOR activation.

4. Discussion
ONC201 is a promising anti-cancer agent, but its activity in CRC cells is moderate. We demonstrate that mTOR activation is a primary resistance factor of ONC201. Inhibition of mTOR (pharmacologically or genetically) sensitizes CRC cells to ONC201, suggesting a potential therapeutic strategy for CRC.