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.
Project description: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.
Project description:Despite the success of antiretroviral therapy (ART), eradication of HIV-1 from brain reservoirs remains elusive. HIV-1 brain reservoirs include perivascular macrophages that are behind the blood-brain barrier and difficult to access by ART. Macrophages express transcription factor FOXO3a and the TNF superfamily cytokine TRAIL, which are known to target HIV-1-infected macrophages for viral inhibition. ONC201 is a novel and potent FOXO3a activator capable of inducing TRAIL. It can cross the blood-brain barrier, and has shown antitumor effects in clinical trials. We hypothesized that activation of FOXO3a/TRAIL by ONC201 will inhibit HIV-1 replication in macrophages. Using primary human monocyte-derived macrophages, we demonstrated that ONC201 dose-dependently decreased replication levels of both HIV-1 laboratory strain and primary strains as determined by HIV-1 reverse transcriptase activity assay. Consistent with data on HIV-1 replication, ONC201 also reduced intracellular and extracellular p24, viral RNA, and integrated HIV-1 DNA in infected macrophages. Blocking TRAIL or knockdown of FOXO3a with siRNA reversed ONC201-mediated HIV-1 suppression, suggesting that ONC201 inhibits HIV-1 through FOXO3a and TRAIL. The anti-HIV-1 effect of ONC201 was further validated in vivo in NOD/scid-IL-2Rgcnull mice. After intracranial injection of HIV-1-infected macrophages into the basal ganglia, we treated the mice daily with ONC201 through intraperitoneal injection for six days. ONC201 significantly decreased p24 levels in both the macrophages and the brain tissues, suggesting that ONC201 suppresses HIV-1 in vivo. Therefore, ONC201 can be a promising drug candidate to combat persistent HIV-1 infection in the brain.
Project description:One of the major problems associated with the chemotherapy of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) that selectively kills tumor cells is decreased drug resistance. This warranted the development of safe novel pharmacological agents that could sensitize the tumor cells to TRAIL. Herein, we examined the role of aldose reductase (AR) in sensitizing cancer cells to TRAIL and potentiating TRAIL-induced apoptosis of human colon cancer cells. We demonstrate that AR inhibition potentiates TRAIL-induced cytotoxicity in cancer cells by upregulation of both death receptor (DR)-5 and DR4. Knockdown of DR5 and DR4 significantly (>85%) reduced the sensitizing effect of the AR inhibitor fidarestat on TRAIL-induced apoptosis. Further, AR inhibition also downregulates cell survival proteins (Bcl-xL, Bcl-2, survivin, XIAP, and FLIP) and upregulates the expression of proapoptotic proteins such as Bax and alters mitochondrial membrane potential, leading to cytochrome c release, caspases-3 activation, and PARP cleavage. We found that AR inhibition regulates AKT/PI3K-dependent activation of forkhead transcription factor FOXO3a. Knockdown of FOXO3a significantly (>80%) abolished AR inhibition-induced upregulation of DR5 and DR4 and apoptosis in colon cancer cells. Overall, our results show that fidarestat potentiates TRAIL-induced apoptosis through downregulation of cell survival proteins and upregulation of death receptors via activation of the AKT/FOXO3a pathway.
Project description:The Abelson Murine Leukemia Virus (A-MuLV) encodes v-Abl, an oncogenic form of the ubiquitous cellular non-receptor tyrosine kinase, c-Abl. A-MuLV specifically transforms murine B cell precursors both in vivo and in vitro. Inhibition of v-Abl by addition of the small molecule inhibitor STI-571 causes these cells to arrest in the G1 phase of the cell cycle prior to undergoing apoptosis. We found that inhibition of v-Abl activity results in upregulation of transcription of the pro-apoptotic TNF-family ligand tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL). Similarly to BCR-Abl-transformed human cells, activation of the transcription factor Foxo3a led to increased TRAIL transcription and induction of a G1 arrest in the absence of v-Abl inhibition, and this effect could be inhibited by the expression of a constitutively active AKT mutant. Multiple pathways act to inhibit FoxO3a activity within Abelson cells. In addition to diminishing transcription factor activity via inhibitory phosphorylation by AKT family members, we found that inhibition of IKKbeta activity results in an increase in the total protein level of FoxO3a. Furthermore overexpression of the p65 subunit of NF-kappaB results in an increase in TRAIL transcription and in apoptosis and deletion of IKKalpha and beta diminishes TRAIL expression and induction. We conclude that in Abelson cells, the inhibition of both NF-kappaB and FoxO3a activity is required for suppression of TRAIL transcription and maintenance of the transformed state.
Project description:Cytokine-provided survival signals are known to suppress apoptosis through inhibition of mitochondrial pathways that involve Bcl-2 family members. Here we show that in hematopoietic cells, cytokines also regulate death receptor-mediated pathways. We demonstrate that hematopoietic cytokines such as IL-3 and erythropoietin in normal cells, as well as BCR-ABL oncoprotein in transformed cells, inhibit transcription of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Using small interfering RNAs, we show that the inhibition of TRAIL function is sufficient to partially rescue cytokine-deprived cells from apoptosis. Finally, we demonstrate that cytokine and BCR-ABL suppression of TRAIL transcription is mediated through phosphorylation and inhibition of the forkhead FOXO3a transcription factor. BCR-ABL-induced inhibition of TRAIL transcription in hematopoietic cells may provide a novel mechanism for tumorigenicity in chronic myeloid leukemia.
Project description:The primary cause of non-melanoma skin cancer (NMSC) is ultraviolet B (UVB) radiation. We have shown previously that mTORC2 inhibition sensitizes keratinocytes to UVB-induced apoptosis mediated by the transcription factor FOXO3a. FOXO3a is a key regulator of apoptosis and a tumor suppressor in several cancer types. Activation of FOXO3a promotes apoptosis through the coordinated expression of a variety of target genes, including TRAIL and NOXA. We hypothesized that in the setting of mTORC2 inhibition, the UVB-induced expression of these factors would lead to apoptosis in a FOXO3a-dependent manner. Using spontaneously immortalized human keratinocytes (HaCaT cells), we observed that both TRAIL and NOXA expression increased in cells exposed to UVB and the TOR kinase inhibitor Torin 2. Similar to knockdown of FOXO3a, NOXA knockdown reversed the sensitization to UVB-induced apoptosis caused by mTORC2 inhibition. In contrast, loss of TRAIL by either knockdown or knockout actually enhanced expression of nuclear FOXO3a, which maintained apoptosis. These surprising results are not due to faulty death receptor signaling in HaCaT cells, as we found that the cells undergo extrinsic apoptosis in response to treatment with recombinant TRAIL. Even more striking, TRAIL knockout cells were sensitized to recombinant TRAIL-induced apoptosis compared to wild-type HaCaT cells, with the largest increase occurring in the presence of mTORC2 inhibition. Taken together, these studies provide strong evidence that mTORC2 controls UVB-induced apoptosis by regulating NOXA expression downstream of FOXO3a. Moreover, FOXO3a transcriptional activation by mTORC2 inhibitors may be a valuable target for prevention or therapy of NMSC, especially in cases with low endogenous TRAIL.
Project description:BACKGROUND:Dysregulation of the metabolome is a hallmark of primary brain malignancies. In this work we examined whether metabolic reprogramming through a multi-targeting approach causes enhanced anti-cancer activity in glioblastoma. METHODS:Preclinical testing of a combined treatment with ONC201/TIC10 and 2-Deoxyglucose was performed in established and primary-cultured glioblastoma cells. Extracellular flux analysis was used to determine real-time effects on OXPHOS and glycolysis. Respiratory chain complexes were analysed by western blotting. Biological effects on tumour formation were tested on the chorioallantoic membrane (CAM). RESULTS:ONC201/TIC10 impairs mitochondrial respiration accompanied by an increase of glycolysis. When combined with 2-Deoxyglucose, ONC201/TIC10 induces a state of energy depletion as outlined by a significant decrease in ATP levels and a hypo-phosphorylative state. As a result, synergistic anti-proliferative and anti-migratory effects were observed among a broad panel of different glioblastoma cells. In addition, this combinatorial approach significantly impaired tumour formation on the CAM. CONCLUSION:Treatment with ONC201/TIC10 and 2-Deoxyglucose results in a dual metabolic reprogramming of glioblastoma cells resulting in a synergistic anti-neoplastic activity. Given, that both agents penetrate the blood-brain barrier and have been used in clinical trials with a good safety profile warrants further clinical evaluation of this therapeutic strategy.
Project description:ONC201/TIC10 is a small-molecule inducer of the TRAIL gene under current investigation as a novel anticancer agent. In this study, we identify critical molecular determinants of ONC201 sensitivity offering potential utility as pharmacodynamic or predictive response markers. By screening a library of kinase siRNAs in combination with a subcytotoxic dose of ONC201, we identified several kinases that ablated tumor cell sensitivity, including the MAPK pathway-inducer KSR1. Unexpectedly, KSR1 silencing did not affect MAPK signaling in the presence or absence of ONC201, but instead reduced expression of the antiapoptotic proteins FLIP, Mcl-1, Bcl-2, cIAP1, cIAP2, and survivin. In parallel to this work, we also conducted a synergy screen in which ONC201 was combined with approved small-molecule anticancer drugs. In multiple cancer cell populations, ONC201 synergized with diverse drug classes, including the multikinase inhibitor sorafenib. Notably, combining ONC201 and sorafenib led to synergistic induction of TRAIL and its receptor DR5 along with a potent induction of cell death. In a mouse xenograft model of hepatocellular carcinoma, we demonstrated that ONC201 and sorafenib cooperatively and safely triggered tumor regressions. Overall, our results established a set of determinants for ONC201 sensitivity that may predict therapeutic response, particularly in settings of sorafenib cotreatment to enhance anticancer responses.
Project description:Glioblastoma is the most frequent primary brain tumor in adults. Current therapeutic options are sparse and the prognosis of patients suffering from this disease is grim. Abundance in intratumoral heterogeneity among different deregulated signaling pathways is a hallmark of glioblastoma and likely accounts for its recurrence and resistance to treatment. Glioblastomas harbor a plethora of deregulated pathways driving tumor formation and growth. In this study, we show that TIC10/ONC201, a promising compound that is currently in planned clinical development, along with Bcl-2/Bcl-xL inhibition by ABT263 yields a strong synergistic antiproliferative effect on pediatric, adult, proneural glioblastoma and glioma stem-like cells. On the molecular level, treatment with TIC10/ONC201 results in a posttranslational decrease of the anti-apoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), through modulation of the chaperone Bag3 and the deubiquitinase Usp9X. Consistently, the combination treatment of TIC10/ONC201 and ABT263 required the presence of functional BAX and BAK to drive intrinsic apoptosis, but is surprisingly independent of the extrinsic apoptotic pathway. Moreover, the expression of Noxa protein was required for efficient apoptosis induction by TIC10/ONC201 and ABT263. Importantly, the drug combination of TIC10/ONC201 and the BH3-mimetic, ABT263, led to a regression of tumors in vivo, without any notable toxicity and side effects. Overall, TIC10/ONC201 along with Bcl-2/Bcl-xL inhibition holds significant promise as a novel potential approach for the treatment of recalcitrant tumors such as glioblastoma.
Project description:To gain a greater understanding of oral squamous cell carcinoma (OSCC) we investigated the actions of all-trans-retinoic acid (RA; a retinoid), bexarotene (a pan-RXR agonist), and forkhead box (FOX) transcription factors in human OSCC-derived cell lines. RA and bexarotene have been shown to limit several oncogenic pathways in many cell types. FOXO proteins typically are associated with tumor suppressive activities, whereas FOXM1 acts as an oncogene when overexpressed in several cancers. RA and/or bexarotene increased the transcript levels of FOXO1, FOXO3A, and TRAIL receptors; reduced the transcript levels of FOXM1, Aurora kinase B (AURKB), and vascular endothelial growth factor A (VEGFA); and decreased the proliferation of OSCC-derived cell lines. Also, RA and/or bexarotene influenced the recruitment of FOXO3A and FOXM1 to target genes. Additionally, FOXM1 depletion reduced cell proliferation, decreased transcript levels of downstream targets of FOXM1, and increased transcript levels of TRAIL receptors. Overexpression of FOXO3A decreased proliferation and increased binding of histone deacetylases (HDACs) 1 and 2 at the FOXM1, AURKB, and VEGFA promoters. This research suggests novel influences of the drugs RA and bexarotene on the expression of FOXM1 and FOXO3A in transcriptional regulatory pathways of human OSCC.