FOXO3a Nuclear Translocation Research Articles

Hydrogen sulfide upregulates KATP channel expression in vascular smooth muscle cells of spontaneously hypertensive rats.

The study was designed to investigate whether H2S could upregulate expression of KATP channels in vascular smooth muscle cells (VSMCs), and by this mechanism enhances vasorelaxation in spontaneously hypertensive rats (SHR). Blood pressure, vascular structure, and vasorelaxation were analyzed. Plasma H2S was detected using polarographic sensor. SUR2B and Kir6.1 expressions were detected in VSMCs of SHR and in A7r5 cells as well as primarily cultured ASMCs using real-time PCR, western blot, immunofluorescence, and confocal imaging. Nuclear translocation of forkhead transcription factors FOXO1 and FOXO3a in ASMCs was detected using laser confocal microscopy, and their binding activity with SUR2B and Kir6.1 promoters was examined by chromatin immunoprecipitation. SHR developed hypertension at 18weeks. They showed downregulated vascular SUR2B and Kir6.1 expressions in association with a decreased plasma H2S level. H2S donor, however, could upregulate vascular SUR2B and Kir6.1 expressions, causing a left shift of the vasorelaxation curve to pinacidil and lowered tail artery pressure in the SHR. Also, H2S antagonized endothelin-1 (ET-1)-inhibited KATP expression in A7r5 cells and cultured ASMCs. Mechanistically, H2S inhibited ET-1-stimulated p-FOXO1 and p-FOXO3a expressions (inactivated forms), but increased their nuclear translocation and the ET-1-inhibited binding of FOXO1 and FOXO3a with Kir6.1 and SUR2B promoters in ASMCs. Hence, H2S promotes vasorelaxation of SHR, at least in part, through upregulating the expression of KATP subunits by inhibiting phosphorylation of FOXO1 and FOXO3a, and stimulating FOXO1 and FOXO3a nuclear translocation and their binding activity with SUR2B and Kir6.1 promoters. H2S increased vascular SUR2B and Kir6.1 expression of SHR, promoting vasorelaxation. H2S antagonized ET-1-inhibited KATP expression in A7r5 cells and cultured ASMCs. H2S inhibited ET-1-induced FOXO1 and FOXO3a phosphorylation in ASMCs. H2S promoted FOXO1 and FOXO3a nuclear translocation and binding with target gene promoters.

Forkhead box transcription factors in cancer initiation, progression and chemotherapeutic drug response.

Forkhead box transcription factors in cancer initiation, progression and chemotherapeutic drug response.

Simvastatin induces mitochondrial dysfunction and increased atrogin-1 expression in H9c2 cardiomyocytes and mice in vivo.

Simvastatin is effective and well tolerated, with adverse reactions mainly affecting skeletal muscle. Important mechanisms for skeletal muscle toxicity include mitochondrial impairment and increased expression of atrogin-1. The aim was to study the mechanisms of toxicity of simvastatin on H9c2 cells (a rodent cardiomyocyte cell line) and on the heart of male C57BL/6 mice. After, exposure to 10μmol/L simvastatin for 24h, H9c2 cells showed impaired oxygen consumption, a reduction in the mitochondrial membrane potential and a decreased activity of several enzyme complexes of the mitochondrial electron transport chain (ETC). The cellular ATP level was also decreased, which was associated with phosphorylation of AMPK, dephosphorylation and nuclear translocation of FoxO3a as well as increased mRNA expression of atrogin-1. Markers of apoptosis were increased in simvastatin-treated H9c2 cells. Treatment of mice with 5mg/kg/day simvastatin for 21days was associated with a 5% drop in heart weight as well as impaired activity of several enzyme complexes of the ETC and increased mRNA expression of atrogin-1 and of markers of apoptosis in cardiac tissue. Cardiomyocytes exposed to simvastatin in vitro or in vivo sustain mitochondrial damage, which causes AMPK activation, dephosphorylation and nuclear transformation of FoxO3a as well as increased expression of atrogin-1. Mitochondrial damage and increased atrogin-1 expression are associated with apoptosis and increased protein breakdown, which may cause myocardial atrophy.

Curcumin induces apoptosis in human neuroblastoma cells via inhibition of AKT and Foxo3a nuclear translocation

Neuroblastoma (NB) is one of the most frequent extracranial solid tumors in children. It accounts for 8–10% of all childhood cancer deaths, and there is a need for development of new drugs for its treatment. Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been shown to exert anti-tumor activity on NB, but the specific mechanism by which curcumin inhibits cancer cells proliferation remains unclear. In the present study, we investigated the anti-proliferative effect of curcumin in human LAN5 NB cells. Curcumin treatment causes a rapid increase in reactive oxygen species and a decrease in the mitochondrial membrane potential—events leading to apoptosis activation. Furthermore, curcumin induces decrease in haet shock protein (Hsp)60 and hexokinase II mitochondrial protein levels and increase in the pro-apoptotic protein, bcl-2 associated death promoter (BAD). Moreover, we demonstrate that curcumin modulates anti-tumor activity through modulation of phosphatase and tensin homolog deleted on chromosome 10 and consequential inhibition of the survival Akt cell-signaling pathway. Inhibition of Akt causes its translocation into the cytoplasm and import of Foxo3a into the nucleus where it activates the expression of p27, Bim, and Fas-L pro-apoptotic genes. Together, these results take evidence for considering curcumin as a potential therapeutic agent for patients with NB.

The pterocarpanquinone LQB-118 induces apoptosis in acute myeloid leukemia cells of distinct molecular subtypes and targets FoxO3a and FoxM1 transcription factors.

Acute myeloid leukemia (AML) patients' outcome is usually poor, mainly because of drug resistance phenotype. The identification of new drugs able to overcome mechanisms of chemoresistance is essential. The pterocarpanquinone LQB-118 compound has been shown to have a potent cytotoxic activity in myeloid leukemia cell lines and patient cells. Our aim was to investigate if LQB-118 is able to target FoxO3a and FoxM1 signaling pathways while sensitizing AML cell lines. LQB-118 induced apoptosis in both AML cell lines HL60 (M3 FAB subtype) and U937 (M4/M5 FAB subtype). Cell death occurred independently of alterations in cell cycle distribution. In vivo administration revealed that LQB-118 was not cytotoxic to normal bone marrow-derived cells isolated from mice. LQB-118 induced FoxO3a nuclear translocation and upregulation of its direct transcriptional target Bim, in HL60 cells. However, LQB-118 induced FoxO3a nuclear exclusion, followed by Bim downregulation, in U937 cells. Concomitantly, LQB-118 exposure reduced FoxM1 and Survivin expression in U937 cells, but this effect was more subtle in HL60 cells. Taken together, our data suggest that LQB-118 has a selective and potent antitumor activity against AML cells with distinct molecular subtypes, and it involves differential modulation of the signaling pathways associated with FoxO3a and FoxM1 transcription factors.

MiR-155 regulates lymphocyte homeostasis in LAT mutant mice (LYM4P.749)

Abstract LAT Y136F mutant mice carry a tyrosine-to-phenylalanine mutation in the LAT adapter gene that leads to a lymphoproliferative disease (LPD) initiated by CD4+ helper T cells. In a screen to detect microRNAs (miRs) specifically regulated in LAT Y136F CD4+ T cells, we found that levels of miR-155 were higher in these T cells than in other proliferating CD4+ T cells from wild type C57BL/6 mice. In order to reveal a potential role of miR-155 in LAT Y136F LPD, we crossed LAT Y136F mice to miR-155-deficient mice yielding double mutant (DM) mice. This resulted in a delay and decreased severity of LPD in DM mice. DM CD4+ T cells showed equal or higher proliferation rates than CD4+ T cells from age-matched LAT Y136F mice, however levels of apoptosis in DM CD4+ T cells were higher. One direct target downregulated by miR-155 is the forkhead transcription factor FOXO3a. In DM T cells, increased expression of FOXO3a led to accumulation of the pro-apoptotic factor Bim and enhanced apoptosis. In addition, elevated Pak1 kinase activity in DM T cells led to activation of Jnk leading to phosphorylation of FOXO3a, FOXO3a nuclear translocation and increased FOXO3a activity. Consequently, direct and indirect effects of miR-155 on FOXO3a led to an increase in Bim-mediated apoptosis and decreased LPD in DM mice. Investigation of downstream targets of miR-155 has led to new information on signaling molecules that control LPD and lymphocyte homeostasis.

FoxO3a Is an Antimelanogenic Factor that Mediates Antioxidant-Induced Depigmentation

Forkhead box-O (FoxO) family transcriptional factors control the expression of many genes involved in a variety of cellular processes. Melanogenesis is an oxidizing process; therefore, many antioxidants are used to inhibit melanin production. However, their mechanism of action is poorly understood. In this study, we investigated the role of FoxO3a, which is a key factor in oxidative stress-related cellular responses in melanogenesis. When FoxO3a expression was inhibited, the expression of melanogenic genes and melanin levels increased. In contrast, the overexpression of wild-type FoxO3a and the increased nuclear translocation induced by the phosphoinositide 3-kinase inhibitors or by Akt inhibition reversed these phenomena. This effect was not observed when FoxO3a harbored a deletion in the nuclear localization signal, indicating that its nuclear translocation is important for the regulation of melanogenesis. When antioxidants such as vitamin C, N-acetylcysteine, and Trolox were applied to MNT1 cells, melanin levels decreased in parallel with FoxO3a nuclear translocation, and this effect disappeared with FoxO3a-directed small interfering RNA treatment. Because FoxO3a orchestrates the expression of many genes in order to regulate cellular phenotypes in a variety of environmental states, this gene, a factor involved in melanogenesis regulation, may represent a good target for studying antimelanogenic signaling pathways and for designing pharmacological or antimelanogenic agents that regulate melanin synthesis.

Diverse effects of ANXA7 and p53 on LNCaP prostate cancer cells are associated with regulation of SGK1 transcription and phosphorylation of the SGK1 target FOXO3A.

Tumor suppressor function of the calcium/phospholipid-binding Annexin-A7 (ANXA7) has been shown in Anxa7-deficient mice and validated in human cancers. In the androgen-resistant prostate cancer cells, ANXA7 and p53 showed similar cytotoxicity levels. However, in the androgen-sensitive LNCaP, ANXA7 greatly exceeded the p53-induced cytotoxicity. We hypothesized that the p53 underperformance in LNCaP could be due to the involvement of p53-responsive SGK1 and FOXO3A. In this study, we show that p53 failed to match programmed cell death (PCD) and G1-arrest that were induced by ANXA7 in LNCaP. WT-ANXA7 preserved total FOXO3A expression with no hyperphosphorylation that could enable FOXO3A nuclear translocation and proapoptotic transcription. In contrast, in the p53-transfected LNCaP cells with maintained cell proliferation, the phosphorylated (but not total) FOXO3A fraction was increased implying a predominantly cytoplasmic localization and, subsequently, a lack of FOXO3A proapoptotic transcription. In addition, p53 reduced the expression of aberrant SGK1 protein form in LNCaP. Using Ingenuity Pathway Analysis and p53-signature genes, we elucidated the role of distinct SGK1/FOXO3A-associated regulation in p53 versus ANXA7 responses and proposed that aberrant SGK1 could affect reciprocal SGK1-FOXO3A-Akt regulation. Thus, the failure of the cell growth regulator p53 versus the phospholipid-binding ANXA7 could be potentially attributed to its diverse effects on SGK1-FOXO3A-Akt pathway in the PTEN-deficient LNCaP.

MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen species (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.

Critical Role of FoxO3a in Alcohol-Induced Autophagy and Hepatotoxicity

Autophagy is a lysosomal degradation process that degrades long-lived cellular proteins and damaged organelles as a critical cell survival mechanism in response to stress. We recently reported that acute ethanol induces autophagy, which then reduces ethanol-induced liver injury. However, the mechanisms by which ethanol induces autophagy are not known. In the present study, ethanol treatment significantly increased both mRNA and protein levels of various essential autophagy-related genes in primary cultured mouse hepatocytes and in mouse liver. Both nuclear translocation of FoxO3a and expression of FoxO3a target genes were increased in ethanol-treated primary hepatocytes and mouse liver. Overexpression of a dominant negative form of FoxO3a inhibited ethanol-induced autophagy-related gene expression and enhanced ethanol-induced cell death in primary hepatocytes, which suggests that FoxO3a is a key factor in regulating ethanol-induced autophagy and cell survival. Resveratrol, a well-known SIRT1 agonist, further enhanced ethanol-induced expression of autophagy-related genes, likely via increased deacetylation of FoxO3a. Moreover, acute ethanol-treated Foxo3a(-/-) mice exhibited decreased autophagy-related gene expression, but enhanced steatosis and liver injury, compared with wild-type mice. FoxO3a thus plays a critical role in ethanol-induced autophagy in mouse liver. Modulating the FoxO3a autophagy pathway may offer novel therapeutic approaches for treating alcoholic liver pathogenesis.

Abstract 843: Inhibition of AKT signaling promotes FOXO3a-dependent apoptosis in castration-resistant prostate cancer cells.

Abstract Treatment for aggressive prostate cancers involves hormonal ablation therapy, radical prostatectomy, radiation therapy, chemotherapy or in combination. Despite their initial sensitivity to these treatments, prostate cancer eventually progresses to castration -resistant prostate cancer (CRPC), which is both aggressive and refractory to current therapeutic modalities. Hence, CRPC demands the identification of novel targeted therapies with minimal side effects. We identified Withaferin-A (WA) a major bioactive compound derived from the medicinal plant, Withania somnifera, is extensively used in Asian herbal medicines to treat a variety of ailments, including cancer. Earlier, we reported that WA induced the pro-apoptotic gene, prostate apoptosis response-4 (Par-4) and apoptosis in CRPC cells. These results encouraged us to determine the mechanism of action of WA on CRPC cells. It is known that activated AKT negatively regulates forkhead transcription factor class O family protein (FOXO3a) that regulates genes involved with tumorigenesis, cell cycle arrest or apoptosis. In the present study, we found that FOXO3a is an upstream of Par-4 signaling which transactivates Par-4 function in CRPC cells. Inhibition of AKT activation by WA facilitates nuclear translocation of FOXO3a which in turn activates Par-4 mediated apoptosis in CRPC cells. Our confocal and immuno-precipitation studies confirmed that upon activation. FOXO3a and Par-4 were co-localized in the nucleus. Similar results were found with overexpression of FOXO3a showing transactivation of Par-4 and growth arrest in CRPC cells. Treatment of actinomycin-D or cyclohexmide along with WA, confirmed that FOXO3a regulation on Par-4 function in CRPC cells. On contrary, inhibition of FOXO3a by siRNA resulted in down regulation of Par-4 and apoptosis by WA in CRPC cells suggesting FOXO3a activation is necessary for the induction of Par-4 mediated apoptosis. Promoter bashing studies suggest a direct interaction of FOXO3a with Par-4 promoter; a sequential deletion of FOXO3a DNA binding sites in Par-4 promoter fails to induce Par-4 activation upon WA treatment in CRPC cells. Overall, our studies conclude that Par-4 is a target of FOXO3a, and Par-4 activation is required for induction of apoptosis in CRPC cells. Activation of FOXO3a is an attractive target for the treatment of CRPC and molecules such as WA can be explored further for the treatment of CRPC. Citation Format: Trinath P. Das, Chendil Damodaran. Inhibition of AKT signaling promotes FOXO3a-dependent apoptosis in castration-resistant prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 843. doi:10.1158/1538-7445.AM2013-843

Antitumoral Activity of Indole-3-carbinol Cyclic tri- and Tetrameric Derivatives Mixture in Human Breast Cancer Cells: In Vitro and In Vivo Studies

Indole-3-carbinol (I3C) and its oligomeric derivatives have been widely studied for their chemopreventive and chemotherapeutic properties. We have previously shown that the I3C cyclic tetrameric derivative CTet inhibits breast cancer cell proliferation in vitro and in xenotrasplanted tumor. Here we report the antitumoral activity of a mixture of tri- and tetrameric cyclic I3C derivatives (CTr/CTet) both in vitro (MCF-7 and MDA-MB-231 breast cancer cell lines) and in vivo in a tumor xenograft model. CTr/CTet mixture avoids the low solubility drawbacks of CTet, thus favouring its solubilization, and reducing purification process, time and costs. CTr/CTet mixture has been shown to inhibit breast cancer cell proliferation (IC50 = 1.3 and 1.6 μg/ml in MCF-7 and MDAMB- 231, respectively) inducing the G0/1 cell cycle phase accumulation. The main molecular events related to CTr/CTet activity are the overexpression of p21, p27 and GADD45A, nuclear translocation of FOXO3a, inhibition of Akt activity and downregulation of estrogen receptor. In vivo, the growth of xenotransplanted tumor has been inhibited and the pro-tumoral low molecular weight cyclin E downregulation has been detected. Our data indicate that CTr/CTet is a potential anticancer combination agent for both hormone-responsive and triple-negative breast tumors.

T63, a new 4-arylidene curcumin analogue, induces cell cycle arrest and apoptosis through activation of the reactive oxygen species–FOXO3a pathway in lung cancer cells

Curcumin (diferuloylmethane) is a natural polyphenol product of the plant Curcuma longa and has a diversity of antitumor activities. T63, a new 4-arylidene curcumin analogue, was reported to inhibit proliferation of lung cancer cells. However, its precise molecular antitumor mechanisms have not been well elucidated. Here, we showed that T63 could significantly inhibit the proliferation of A549 and H460 human lung cell lines via induction of G0/G1 cell cycle arrest and apoptosis. We found that the reactive oxygen species (ROS)-activated FOXO3a cascade plays a central role in T63-induced cell proliferation inhibition. Mechanistically, enhancement of ROS production by T63 induced FOXO3a expression and nuclear translocation through activation of p38MAPK and inhibition of AKT, subsequently elevating the expression of FOXO3a target genes, including p21, p27, and Bim, and then increased the levels of activated caspase-3 and decreased the levels of cyclin D1. Moreover, the antioxidant N-acetylcysteine markedly blocked the above effects, and small interfering RNA-mediated knockdown of FOXO3a also significantly decreased T63-induced cell cycle arrest and apoptosis. In vivo experiments showed that T63 significantly suppressed the growth of A549 lung cancer xenograft tumors, associated with proliferation suppression and apoptosis induction in tumor tissues, without inducing any notable major organ-related toxicity. These data indicated that the novel curcumin analogue T63 is a potent antitumor agent that induces cell cycle arrest and apoptosis and has significant therapeutic potential for lung cancer.

Neuroprotective effects of phenolic antioxidant tBHQ associate with inhibition of FoxO3a nuclear translocation and activity

The Forkhead transcription factor, FoxO3a induces genomic death responses in neurones following translocation from the cytosol to the nucleus. Nuclear translocation of FoxO3a is triggered by trophic factor withdrawal, oxidative stress and the stimulation of extrasynaptic NMDA receptors. Receptor activation of phosphatidylinositol 3-kinase (PI3K)–Akt signalling pathways retains FoxO3a in the cytoplasm, thereby inhibiting the transcriptional activation of death-promoting genes. We hypothesized that phenolic antioxidants such as tert-Butylhydroquinone (tBHQ), which is known to stimulate PI3K–Akt signalling, would inhibit FoxO3a translocation and activity. Treatment of cultured cortical neurones with NMDA increased the nuclear localization of FoxO3a, reduced the phosphorylation of FoxO3a, increased caspase activity and up-regulated Fas ligand expression. In contrast the phenolic antioxidant, tBHQ, caused retention of FoxO3a in the cytosol coincident with enhanced PI3K- dependent phosphorylation of FoxO3a. tBHQ-induced nuclear exclusion of FoxO3a was associated with reduced FoxO-mediated transcriptional activity. Exposure of neurones to tBHQ inhibited NMDA-induced nuclear translocation of FoxO3a, prevented NMDA-induced up-regulation of FoxO-mediated transcriptional activity, blocked caspase activation and protected neurones from NMDA-induced excitotoxic death. Collectively, these data suggest that phenolic antioxidants such as tBHQ oppose stress-induced activation of FoxO3a and therefore have potential neuroprotective utility in neurodegeneration.

Activity of the selective IκB kinase inhibitor BMS-345541 against T-cell acute lymphoblastic leukemia

Several lines of evidence suggest that the IκB kinase (IKK)/nuclear factor-κB (NFκB) axis is required for viability of leukemic cells and is a predictor of relapse in T-cell acute lymphoblastic leukemia (T-ALL). Moreover, many anticancer agents induce NFκB nuclear translocation and activation of its target genes, which counteract cellular resistance to chemotherapeutic drugs. Therefore, the design and the study of IKK-specific drugs is crucial to inhibit tumor cell proliferation and to prevent cancer drug-resistance. Here, we report the anti-proliferative effects induced by BMS-345541 (a highly selective IKK inhibitor) in three Notch1-mutated T-ALL cell lines and in T-ALL primary cells from pediatric patients. BMS-345541 induced apoptosis and an accumulation of cells in the G2/M phase of the cell cycle via inhibition of IKK/NFκB signaling. We also report that T-ALL cells treated with BMS-345541 displayed nuclear translocation of FOXO3a and restoration of its functions, including control of p21Cip1 expression levels. We demonstrated that FOXO3a subcellular re-distribution is independent of AKT and ERK 1/2 signaling, speculating that in T-ALL the loss of FOXO3a tumor suppressor function could be due to deregulation of IKK, as has been previously demonstrated in other cancer types. It is well known that, differently from p53, FOXO3a mutations have not yet been found in human tumors, which makes therapeutics activating FOXO3a more appealing than others. For these features, BMS-345541 could be used alone or in combination with traditional therapies in the treatment of T-ALL.

JNK- and Akt-mediated Puma expression in the apoptosis of cisplatin-resistant ovarian cancer cells

BH3 (Bcl-2 homology domain 3)-only proteins have an important role in the cisplatin resistance of cells. However, the effect of BH3-only proteins on cisplatin-resistant ovarian cancer cells has not been thoroughly elucidated. Our results from the present study indicate that Puma plays a critical role in the apoptosis of chemo-resistant ovarian cancer cells treated with BetA (betulinic acid). The reduction of Puma expression inhibits Bax activation and apoptosis. However, p53 gene silencing has little effect on Puma activation. Further experiments demonstrated that Akt-mediated FoxO3a (forkhead box O3a) nuclear translocation and the JNK (c-Jun N-terminal kinase)/c-Jun pathway only partially trigger Puma induction and apoptosis, whereas dominant-negative c-Jun expression with FoxO3a reduction completely inhibits Puma expression and cell death. Furthermore, our results suggest that JNK regulates the Akt/FoxO3a signalling pathway. Therefore the dual effect of JNK can efficiently trigger Puma activation and apoptosis in chemoresistant cells. Taken together, our results demonstrate the role of Puma in BetA-induced apoptosis and the molecular mechanisms of Puma expression regulated by BetA during ovarian cancer cell apoptosis. Our findings suggest that the JNK-potentiated Akt/FoxO3a and JNK-mediated c-Jun pathways co-operatively trigger Puma expression, which determines the threshold for overcoming chemoresistance in ovarian cancer cells.

Calpain 2 Regulates Akt-FoxO-p27Kip1 Protein Signaling Pathway in Mammary Carcinoma

Calpain 2 Regulates Akt-FoxO-p27Kip1 Protein Signaling Pathway in Mammary Carcinoma

Abstract 1935: Potent anti-tumor effects of TIC10 require Foxo3a and TRAIL gene upregulation

Abstract TNF-related apoptosis-inducing ligand (TRAIL; Apo2L) is an endogenous protein that induces tumor-selective apoptosis and is currently being tested in clinical trials as an anti-tumor agent. Despite promising preclinical activity, recombinant human TRAIL suffers from efficacy-limiting properties such as short serum half-life, stability, cost, and limitations in delivery, e.g. to the central nervous system. We hypothesized that induction of the human TRAIL gene by small molecules may overcome these limitations of TRAIL-based protein therapy. A cell-based luciferase reporter screen using the human TRAIL promoter identified TIC10 as a p53-independent small molecule inducer of the TRAIL gene. TIC10 induces TRAIL on the surface of normal and tumor cells, in sera of non-tumor-bearing mice, and throughout the tumor microenvironment. Co-culture experiments suggest that TRAIL upregulation by normal cells can contribute to the anti-tumor response of TIC10 through a TRAIL-mediated bystander effect. TIC10-induced TRAIL causes tumor-specific and TRAIL-mediated cell death as indicated by inhibition of cytotoxic effects following TRAIL gene knockdown in vitro and in vivo. Expression profiling of TIC10-induced transcriptional changes revealed altered expression levels of Foxo3a target genes, including DR5. We found that TIC10 causes the nuclear translocation of Foxo3a, but not Foxo1, to upregulate TRAIL gene transcription as determined by chromatin-immunoprecipitation assays. Western blot analysis indicated that TIC10 potently inhibits the phosphorylation of Foxo3a at Ser253 and Ser294 that normally docks the transcription factor to 14-3-3 proteins in the cytosol. Dual inhibition of Akt and ERK by TIC10, by other individual small molecule kinase inhibitors in combination or targeted siRNA inhibited the phosphorylation of these critical serine residues and synergistically induced Foxo3a-dependent TRAIL and TRAIL-mediated cell death. TIC10 is a promising first-in-class anti-tumor agent with a broad range of anti-tumor effects in preclinical animal models including othrotopic GBM, immunocompetent lymphoma, xenografted colon, lung, or breast cancers, and exhibits synergy with taxanes as well as bevacizumab among other agents. TIC10 appears to have no measurable toxic effects in mice including evaluation of serum chemistries, liver function studies, LDH, tissue histology, weight of the animals or behavioral changes. We are pursuing the clinical translation of these important findings that indicate a favorable and wide therapeutic index for a novel anti-tumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1935. doi:1538-7445.AM2012-1935

Foxo/Atrogin induction in human and experimental myositis

Skeletal muscle atrophy can occur rapidly in various fasting, cancerous, systemic inflammatory, deranged metabolic or neurogenic states. The ubiquitin ligase Atrogin-1 (MAFbx) is induced in animal models of these conditions, causing excessive myoprotein degradation. It is unknown if Atrogin upregulation also occurs in acquired human myositis. Intracellular β-amyloid (Aβi), phosphorylated neurofilaments, scattered infiltrates and atrophy involving selective muscle groups characterize human sporadic Inclusion Body Myositis (sIBM). In Polymyositis (PM), inflammation is more pronounced and atrophy is symmetric and proximal. IBM and PM share various inflammatory markers. We found that forkhead family transcription factor Foxo3A is directed to the nucleus and Atrogin-1 transcript is increased in both conditions. Expression of Aβ in transgenic mice and differentiated C2C12 myotubes was sufficient to upregulate Atrogin-1 mRNA and cause atrophy. Aβi reduces levels of p-Akt and downstream p-Foxo3A, resulting in Foxo3A translocation and Atrogin-1 induction. In a mouse model of autoimmune myositis, cellular inflammation alone was associated with similar Foxo3A and Atrogin changes. Thus, either Aβi accumulation or cellular immune stimulation may independently drive muscle atrophy in sIBM and PM, respectively, through pathways converging on Foxo and Atrogin-1. In sIBM it is additionally possible that both mechanisms synergize.

Phosphorylation of FOXO3a on Ser-7 by p38 Promotes Its Nuclear Localization in Response to Doxorubicin

FOXO3a is a forkhead transcription factor that regulates a multitude of important cellular processes, including proliferation, apoptosis, differentiation, and metabolism. Doxorubicin treatment of MCF-7 breast carcinoma cells results in FOXO3a nuclear relocation and the induction of the stress-activated kinase p38 MAPK. Here, we studied the potential regulation of FOXO3a by p38 in response to doxorubicin. Co-immunoprecipitation studies in MCF-7 cells demonstrated a direct interaction between p38 and FOXO3a. We also showed that p38 can bind and phosphorylate a recombinant FOXO3a directly in vitro. HPLC-coupled phosphopeptide mapping and mass spectrometric analyses identified serine 7 as a major site for p38 phosphorylation. Using a phosphorylated Ser-7 FOXO3a antibody, we demonstrated that FOXO3a is phosphorylated on Ser-7 in response to doxorubicin. Immunofluorescence staining studies showed that upon doxorubicin treatment, the wild-type FOXO3a relocalized to the nucleus, whereas the phosphorylation-defective FOXO3a (Ala-7) mutant remained largely in the cytoplasm. Treatment with SB202190 also inhibits the doxorubicin-induced FOXO3a Ser-7 phosphorylation and nuclear accumulation in MCF-7 cells. In addition, doxorubicin caused the nuclear translocation of FOXO3a in wild-type but not p38-depleted mouse fibroblasts. Together, our results suggest that p38 phosphorylation of FOXO3a on Ser-7 is essential for its nuclear relocalization in response to doxorubicin.