Overexpression Of FOXO3a Research Articles

Elucidating the role of the FoxO3a transcription factor in the IGF-1-induced migration and invasion of uveal melanoma cancer cells.

Uveal melanoma (UM) is the most common primary intraocular malignant tumor of adults. It has high mortality rate due to liver metastasis. However, the epidemiology and pathogenesis of liver metastasis in UM are not elucidated and there is no effective therapy available for preventing the development of this disease. IGF-1 is a growth factor involved in cell proliferation, malignant transformation and inhibition of apoptosis. In previous report, IGF-1 receptor was found to be highly expressed in UM and this was related to tumor prognosis. FoxO3a is a Forkhead box O (FOXO) transcription factor and a downstream target of the IGF-1R/PI3K/Akt pathway involved in a number of physiological and pathological processes including cancer. However, the role of FoxO3a in UM is unknown. In the present study, we investigated fundamental mechanisms in the growth, migration and invasion of UM and the involvement of FoxO3a. IGF-1 increased the cell viability, invasion, migration and S-G2/M cell cycle phase accumulation of UM cells. Western blot analysis showed that IGF-1 led to activation of Akt and concomitant phosphorylation of FoxO3a. FoxO3a phosphorylation was associated with its translocation into the cytoplasm from the nucleus and its functional inhibition led to the inhibition of expression of Bim and p27, but an increase in the expression of Cyclin D1. The effects of IGF-1 on UM cells were reversed by LY294002 (a PI3K inhibitor) or Akt siRNA, and the overexpression of FoxO3a also attenuated basal invasion and migration of UM. Taken all together, these results suggest that inhibition of FoxO3a by IGF-1 via the PI3K/Akt pathway has an important role in IGF-1 induced proliferation and invasion of UM cells. These findings also support FoxO3a and IGF signaling may represent a valid target for investigating the development of new strategies for the treatment and prevention of the pathology of UM.

FOXO3a orchestrates glioma cell responses to starvation conditions and promotes hypoxia-induced cell death.

Forkhead boxO (FOXO) transcription factors are homeostatic regulators adjusting diverse cellular processes crucial for metabolism and survival. In gliomas, FOXOs have been shown to modulate cell death, proliferation and differentiation. Here, we investigated the role of FOXO3a in human malignant gliomas with special regard to starvation conditions. Expression of FOXO3a increased with WHO grade and was accentuated in the perinecrotic niche, colocalizing with hypoxia‑inducible factor1α (HIF1α) expression. FOXO3a was upregulated in hypoxia and translocation of FOXO3a from the cytoplasm to the nucleus was induced by serum starvation, pharmacological inhibition of protein kinaseB(PKB) and hypoxia. Overexpression of FOXO3a induced tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) expression and resulted in spontaneous cell death. Knockdown of FOXO3a(shFOXO3a), on the one hand, enhanced the sensitivity of glioma cells towards H2O2 under normoxia. On the other hand, it decreased consumption of glucose and oxygen, resulting in improved survival during glucose and oxygen deprivation. Mechanistically, in shFOXO3a cells, hypoxia‑response element reporter activity, as well as the expression of common HIF1α target genes, was increased, suggesting disinhibited HIF1α signaling. However, glucose transporter1 (GLUT1) expression was inversely regulated, and this may have been caused by an upregulation of TP53 in shFOXO3a cells. These data reveal a novel role of FOXO3a‑dependent gene regulation in the complex adaptive responses of gliomas towards starvation signals. Strategies that target FOXO3a function may directly or indirectly alter glioma cell behavior and viability in the hypoxic niche.

NF-κB–driven suppression of FOXO3a contributes to EGFR mutation-independent gefitinib resistance

Therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs, such as gefitinib or erlotinib) significantly prolongs survival time for patients with tumors harboring an activated mutation on EGFR; however, up to 40% of lung cancer patients exhibit acquired resistance to EGFR-TKIs with an unknown mechanism. FOXO3a, a transcription factor of the forkhead family, triggers apoptosis, but the mechanistic details involved in EGFR-TKI resistance and cancer stemness remain largely unclear. Here, we observed that a high level of FOXO3a was correlated with EGFR mutation-independent EGFR-TKI sensitivity, the suppression of cancer stemness, and better progression-free survival in lung cancer patients. The suppression of FOXO3a obviously increased gefitinib resistance and enhanced the stem-like properties of lung cancer cells; consistent overexpression of FOXO3a in gefitinib-resistant lung cancer cells reduced these effects. Moreover, we identified that miR-155 targeted the 3'UTR of FOXO3a and was transcriptionally regulated by NF-κB, leading to repressed FOXO3a expression and increased gefitinib resistance, as well as enhanced cancer stemness of lung cancer in vitro and in vivo. Our findings indicate that FOXO3a is a significant factor in EGFR mutation-independent gefitinib resistance and the stemness of lung cancer, and suggest that targeting the NF-κB/miR-155/FOXO3a pathway has potential therapeutic value in lung cancer with the acquisition of resistance to EGFR-TKIs.

Juglanthraquinone C Induces Intracellular ROS Increase and Apoptosis by Activating the Akt/Foxo Signal Pathway in HCC Cells.

Juglanthraquinone C (JC), a naturally occurring anthraquinone extracted from Juglans mandshurica, could induce apoptosis of cancer cells. This study aims to investigate the detailed cytotoxicity mechanism of JC in HepG2 and BEL-7402 cells. The Affymetrix HG-U133 Plus 2.0 arrays were first used to analyze the mRNA expression exposed to JC or DMSO in HepG2 cells. Consistent with the previous results, the data indicated that JC could induce apoptosis and hyperactivated Akt. The Western blot analysis further revealed that Akt, a well-known survival protein, was strongly activated in HepG2 and BEL-7402 cells. Furthermore, an obvious inhibitory effect on JC-induced apoptosis was observed when the Akt levels were decreased, while the overexpression of constitutively active mutant Akt greatly accelerated JC-induced apoptosis. The subsequent results suggested that JC treatment suppressed nuclear localization and increased phosphorylated levels of Foxo3a, and the overexpression of Foxo3a abrogated JC-induced apoptosis. Most importantly, the inactivation of Foxo3a induced by JC further led to an increase of intracellular ROS levels by suppressing ROS scavenging enzymes, and the antioxidant N-acetyl-L-cysteine and catalase successfully decreased JC-induced apoptosis. Collectively, this study demonstrated that JC induced the apoptosis of hepatocellular carcinoma (HCC) cells by activating Akt/Foxo signaling pathway and increasing intracellular ROS levels.

Reduced FoxO3a expression causes low autophagy in idiopathic pulmonary fibrosis fibroblasts on collagen matrices.

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease, and fibroblasts derived from patients with IPF are resistant to type I collagen matrix-induced cell death. The alteration of the PTEN-Akt axis permits IPF fibroblasts to maintain a pathological phenotype on collagen by suppressing autophagy. However, the precise underlying mechanism by which the Akt downstream molecule suppresses autophagic activity remains elusive. FoxO3a is a direct target of Akt and is implicated with the transcriptional activation of autophagy. Therefore, we investigated whether reduced FoxO3a expression causes abnormally low autophagy in IPF fibroblasts on collagen. We found that FoxO3a mRNA and protein levels are low in IPF fibroblasts, which subsequently suppresses the autophagosomal marker LC3B expression on collagen matrix. In contrast, the majority of control fibroblasts showed an increase in FoxO3a and LC3B expression at both the mRNA and protein levels. The luciferase assay confirmed that FoxO3a binds to the promoter region of LC3B and transcriptionally activates LC3B. The overexpression of wild-type FoxO3a increased LC3B mRNA and protein expression in IPF fibroblasts, whereas the dominant negative FoxO3a decreased the LC3B level in control fibroblasts. The inhibition of autophagic activity sensitized control fibroblasts to collagen matrix-induced cell death. In contrast, enhanced viability was found when autophagic function was inhibited in IPF fibroblasts. Our study showed that aberrantly low FoxO3a expression participates in reducing autophagic activity via transcriptional suppression of LC3B in IPF fibroblasts on collagen. This suggests that low autophagic activity by the alteration of FoxO3a may contribute to IPF progression.

8‑bromo‑7‑methoxychrysin induces apoptosis by regulating Akt/FOXO3a pathway in cisplatin‑sensitive and resistant ovarian cancer cells.

8-bromo-7-methoxychrysin (BrMC), a novel chrysin analog, was reported to have anti-cancer activities. The aim of the present study was to investigate the molecular mechanism of 8-bromo-7-methoxychrysin (BrMC)-induced apoptosis via the Akt/forkhead box O3a (FOXO3a) pathway in cisplatin (DDP)-sensitive and -resistant ovarian cancer cells. The human ovarian cancer cell lines A2780 and A2780/DDP were cultured in vitro. Various molecular techniques were used to assess the expression of FOXO3a and B cell lymphoma 2 (Bcl-2)-interacting mediator of cell death (Bim) in cisplatin-sensitive and -resistant ovarian cancer cells. Different concentrations of BrMC induced apoptosis in cisplatin-sensitive and -resistant ovarian cancer cells. BrMC-induced apoptotic cell death occurred mainly by the activation of Akt, which was accompanied by the overexpression of transcription factor FOXO3a, with a concomitant increase in the expression levels of Bim. Silencing Bim expression by using small interfering RNA, attenuated the induction of apoptosis by BrMC treatment. The results indicated that BrMC-induced apoptosis in cisplatin-sensitive and -resistant ovarian cancer cells may occur via the regulation of Akt/FOXO3a, leading to Bim transcription.

SIRT1 and FOXO Mediate Contractile Differentiation of Vascular Smooth Muscle Cells under Cyclic Stretch

Background/Aims: Physiological mechanical stretch in vivo helps to maintain the quiescent contractile differentiation of vascular smooth muscle cells (VSMCs), but the underlying mechanisms are still unclear. Here, we investigated the effects of SIRT1 in VSMC differentiation in response to mechanical cyclic stretch. Methods and Results: Rat VSMCs were subjected to 10%-1.25Hz-cyclic stretch in vitro using a FX-4000T system. The data indicated that the expression of contractile markers, including α-actin, calponin and SM22α, was significantly enhanced in VSMCs that were subjected to cyclic stretch compared to the static controls. The expression of SIRT1 and FOXO3a was increased by the stretch, but the expression of FOXO4 was decreased. Decreasing SIRT1 by siRNA transfection attenuated the stretch-induced expression of contractile VSMC markers and FOXO3a. Furthermore, increasing SIRT1 by either treatment with activator resveratrol or transfection with a plasmid to induce overexpression increased the expression of FOXO3a and contractile markers, and decreased the expression of FOXO4 in VSMCs. Similar trends were observed in VSMCs of SIRT1 (+/-) knockout mice. The overexpression of FOXO3a promoted the expression of contractile markers in VSMCs, while the overexpression of FOXO4 demonstrated the opposite effect. Conclusion: Our results indicated that physiological cyclic stretch promotes the contractile differentiation of VSMCs via the SIRT1/FOXO pathways and thus contributes to maintaining vascular homeostasis.

Peroxisome proliferator-activated receptor γ coactivator 1α and FoxO3A mediate chondroprotection by AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) inhibits chondrocyte procatabolic responses to inflammation and biomechanical injury. This study was undertaken to test the hypothesis that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and FoxO3A, 2 major AMPK downstream targets, mediate the chondroprotective effect of AMPK activation. We assessed the activity of AMPKα (threonine 172 phosphorylation) and the expression of PGC-1α and FoxO3A in human chondrocytes and AMPKα1- or AMPKα2-knockout mouse chondrocytes by Western blotting, and in mouse knee cartilage by immunohistochemistry. We also knocked down or overexpressed PGC-1α and FoxO3A by small interfering RNA or plasmid DNA transfection, respectively. We assessed mitochondrial superoxide generation using MitoSOX Red. Expression of PGC-1α and FoxO3A was enhanced by pharmacologic AMPK activator A-769662 but impaired in AMPKα1(-/-) or AMPKα2(-/-) mouse chondrocytes. Reduced expression of PGC-1α and FoxO3A was observed in mouse knee instability-induced osteoarthritis (OA) cartilage and in aged C57BL/6 mouse knee cartilage. Knockdown of PGC-1α and FoxO3A enhanced, but limited the ability of A-769662 to inhibit, phosphorylation of p65 NF-κB (Ser(536) ) and procatabolic responses induced by inflammatory cytokines. Forced expression of PGC-1α and FoxO3A induced increased expression of superoxide dismutase 2 (SOD2) and catalase, but A-769662 failed to increase the expression of SOD2 and catalase in either PGC-1α- or FoxO3A-knockdown chondrocytes. Last, menadione-induced superoxide generation was inhibited by AMPK pharmacologic activators and by overexpression of PGC-1α or FoxO3A. PGC-1α and FoxO3A limit oxidative stress and at least partially mediate the capacity of AMPK activity to block procatabolic responses in chondrocytes, and therefore have the potential to inhibit the progression of cartilage damage in OA.

GW25-e0862 Overexpression or Silencing of FOXO3a Affects Proliferation of Endothelial Progenitor Cells and Expression of Cell Cycle Regulatory Proteins

GW25-e0862 Overexpression or Silencing of FOXO3a Affects Proliferation of Endothelial Progenitor Cells and Expression of Cell Cycle Regulatory Proteins

The antiproliferative activity of kinase inhibitors in chronic myeloid leukemia cells is mediated by FOXO transcription factors.

Chronic myeloid leukemia (CML) is initiated and maintained by the tyrosine kinase BCR-ABL which activates a number of signal transduction pathways, including PI3K/AKT signaling and consequently inactivates FOXO transcription factors. ABL-specific tyrosine kinase inhibitors (TKIs) induce minimal apoptosis in CML progenitor cells, yet exert potent antiproliferative effects, through as yet poorly understood mechanisms. Here, we demonstrate that in CD34+ CML cells, FOXO1 and 3a are inactivated and relocalized to the cytoplasm by BCR-ABL activity. TKIs caused a decrease in phosphorylation of FOXOs, leading to their relocalization from cytoplasm (inactive) to nucleus (active), where they modulated the expression of key FOXO target genes, such as Cyclin D1, ATM, CDKN1C, and BCL6 and induced G1 arrest. Activation of FOXO1 and 3a and a decreased expression of their target gene Cyclin D1 were also observed after 6 days of in vivo treatment with dasatinib in a CML transgenic mouse model. The over-expression of FOXO3a in CML cells combined with TKIs to reduce proliferation, with similar results seen for inhibitors of PI3K/AKT/mTOR signaling. While stable expression of an active FOXO3a mutant induced a similar level of quiescence to TKIs alone, shRNA-mediated knockdown of FOXO3a drove CML cells into cell cycle and potentiated TKI-induced apoptosis. These data demonstrate that TKI-induced G1 arrest in CML cells is mediated through inhibition of the PI3K/AKT pathway and reactivation of FOXOs. This enhanced understanding of TKI activity and induced progenitor cell quiescence suggests that new therapeutic strategies for CML should focus on manipulation of this signaling network. Stem Cells 2014;32:2324–2337

Overexpression or silencing of FOXO3a affects proliferation of endothelial progenitor cells and expression of cell cycle regulatory proteins.

Endothelial dysfunction is involved in the pathogenesis of many cardiovascular diseases such as atherosclerosis. Endothelial progenitor cells (EPCs) have been considered to be of great significance in therapeutic angiogenesis. Furthermore, the Forkhead box O (FOXO) transcription factors are known to be important regulators of cell cycle. Therefore, we investigated the effects of changes in FOXO3a activity on cell proliferation and cell cycle regulatory proteins in EPCs. The constructed recombinant adenovirus vectors Ad-TM (triple mutant)-FOXO3a, Ad-shRNA-FOXO3a and the control Ad-GFP were transfected into EPCs derived from human umbilical cord blood. Assessment of transfection efficiency using an inverted fluorescence microscope and flow cytometry indicated a successful transfection. Additionally, the expression of FOXO3a was markedly increased in the Ad-TM-FOXO3a group but was inhibited in the Ad-shRNA-FOXO3a group as seen by western blotting. Overexpression of FOXO3a suppressed EPC proliferation and modulated expression of the cell cycle regulatory proteins including upregulation of the cell cycle inhibitor p27kip1 and downregulation of cyclin-dependent kinase 2 (CDK2), cyclin D1 and proliferating cell nuclear antigen (PCNA). In the Ad-shRNA-FOXO3a group, the results were counter-productive. Furthermore, flow cytometry for cell cycle analysis suggested that the active mutant of FOXO3a caused a noticeable increase in G1- and S-phase frequencies, while a decrease was observed after FOXO3a silencing. In conclusion, these data demonstrated that FOXO3a could possibly inhibit EPC proliferation via cell cycle arrest involving upregulation of p27kip1 and downregulation of CDK2, cyclin D1 and PCNA.

Extracellular signal-regulated kinase signaling-mediated induction and interaction of FOXO3a and p53 contribute to the inhibition of nasopharyngeal carcinoma cell growth by curcumin.

Curcumin, one of the main bioactive components extracted from a traditional Chinese medicinal herb, exhibits potent anticancer activity against many types of cancer cells including nasopharyngeal carcinoma (NPC). However, the detailed molecular mechanism underlying this is not clearly understood. In this study, we showed that curcumin significantly inhibited the growth of NPC cells in a dose-and time-dependent manner as determined by MTT assays, while increasing apoptosis was also observed as measured by flow cytometry for the FITC-Annexin V and propidium iodide (PI) label and Hoechst 33258 staining. To further explore the potential mechanism, we showed that curcumin increased the phosphorylation of ERK1/2 but not p38 MAPK in a time-dependent manner, and induced protein expression of the tumor suppressors FOXO3a and p53 in a dose-dependent manner, which were not observed in the presence of PD98059, an inhibitor of ERK1/2. Furthermore, silencing of FOXO3a and p53 genes by siRNAs overcame the inhibitory effect of curcumin on cell proliferation. Silencing or blockade of p53 using siRNA or chemical inhibitor abrogated the effect of curcumin on expression of FOXO3a protein; silencing or overexpression of FOXO3a had no further effect on curcumin-induced p53 protein expression. Furthermore, blockade of ERK1/2 and exogenous expression of FOXO3a restored the effect of curcumin on growth of cells. Together, our studies show that curcumin inhibits growth and induces apoptosis of NPC cells through ERK1/2-mediated increase in the protein expression and interaction of p53 and FOXO3a. p53 is upstream of FOXO3a, which form a regulatory loop that mediates the effect of curcumin. This study unveils a new mechanism by which curcumin inhibits the proliferation and induces apoptosis of human NPC cells.

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.

Semaphorin 3A upregulates FOXO 3a-dependent MelCAM expression leading to attenuation of breast tumor growth and angiogenesis.

Semaphorin 3A (Sema 3A), a member of semaphorin family, serves as a guidance clue during embryonic development and is known as a candidate tumor suppressor that attenuates breast tumor progression by binding with its co-receptor, neuropilin-1 (NRP-1). However, the underlying mechanism by which Sema 3A suppresses breast tumor growth is still unexplored. In this study, we report that Sema 3A regulates phosphorylation and nuclear translocation of phosphatase and tensin homolog (PTEN) and FOXO 3a. Moreover, Sema 3A controls NRP-1-mediated PTEN-dependent FOXO 3a activation. Overexpression of PTEN and FOXO 3a enhances Sema 3A-induced attenuation of breast cancer cell migration. Chromatin immunoprecipitation and electrophoretic mobility shift assay data revealed that FOXO 3a regulates MelCAM at the transcriptional level. Furthermore, Sema 3A induces NRP-1-mediated MelCAM expression through PTEN and FOXO 3a. The data also showed that vascular endothelial growth factor-induced angiogenesis is inhibited by Sema 3A. Loss of or gain in function study revealed that Sema 3A modulates phosphorylation of PTEN and FOXO 3a and expression of MelCAM, leading to suppression of tumor growth and angiogenesis using in vivo mice model. Clinical specimen analysis revealed that reduced expression of Sema 3A and p-PTEN are correlated with enhanced breast cancer progression, further strengthening our in vitro and in vivo findings. Correlation of relapse-free survival of breast cancer patients (n=2878) with expression levels of Sema 3A, NRP-1, FOXO 3a and MelCAM were studied by Kaplan-Meier analysis. Statistical analysis revealed a close association between reduced expression of Sema 3A and MelCAM with that of poor patient's survival. Our study demonstrated a novel mechanism of regulation of tumor suppression by Sema 3A in coordination with a chain of tumor-suppressor genes, which in turn inhibits breast cancer cell migration, tumor growth and angiogenesis.

Metformin Suppresses Expression of the Selenoprotein P Gene via an AMP-activated Kinase (AMPK)/FoxO3a Pathway in H4IIEC3 Hepatocytes

Selenoprotein P (SeP; encoded by SEPP1 in humans) is a liver-derived secretory protein that induces insulin resistance in type 2 diabetes. Suppression of SeP might provide a novel therapeutic approach to treating type 2 diabetes, but few drugs that inhibit SEPP1 expression in hepatocytes have been identified to date. The present findings demonstrate that metformin suppresses SEPP1 expression by activating AMP-activated kinase (AMPK) and subsequently inactivating FoxO3a in H4IIEC3 hepatocytes. Treatment with metformin reduced SEPP1 promoter activity in a concentration- and time-dependent manner; this effect was cancelled by co-administration of an AMPK inhibitor. Metformin also suppressed Sepp1 gene expression in the liver of mice. Computational analysis of transcription factor binding sites conserved among the species resulted in identification of the FoxO-binding site in the metformin-response element of the SEPP1 promoter. A luciferase reporter assay showed that metformin suppresses Forkhead-response element activity, and a ChIP assay revealed that metformin decreases binding of FoxO3a, a direct target of AMPK, to the SEPP1 promoter. Transfection with siRNAs for Foxo3a, but not for Foxo1, cancelled metformin-induced luciferase activity suppression of the metformin-response element of the SEPP1 promoter. The overexpression of FoxO3a stimulated SEPP1 promoter activity and rescued the suppressive effect of metformin. Metformin did not affect FoxO3a expression, but it increased its phosphorylation and decreased its nuclear localization. These data provide a novel mechanism of action for metformin involving improvement of systemic insulin sensitivity through the regulation of SeP production and suggest an additional approach to the development of anti-diabetic drugs.

FoxO3a Contributes to the Reprogramming Process and the Differentiation of Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells, which are morphologically and functionally similar with embryonic stem (ES) cells, have been successfully generated from somatic cells through defined reprogramming transcription factors. Forkhead class O3a (FoxO3a) has been recently reported to play an important role in the homeostasis and maintenance of certain types of stem cells; however, the role of FoxO3a in the reprogramming process and differentiation of iPS cells remains unclear. In this study, we investigate the function of FoxO3a during the reprogramming process and characterize the properties of iPS cells from FoxO3a-wild type and -null mouse embryonic fibroblasts (MEFs). Our results show that the FoxO3a-null iPS cells are similar to the wild-type iPS cells in the levels of ES cell markers, alkaline phosphatase activity, and formation of teratoma in vivo. The reprogramming process is delayed in the FoxO3a-null MEFs compared to the wild-type MEFs; whereas the overexpression of FoxO3a partially recovers the impaired reprogramming efficiency in the null group. More importantly, FoxO3a deficiency impairs the neuronal lineage differentiation potential of iPS cells in vitro. These results suggest that FoxO3a affects the reprogramming kinetics and the neuronal lineage differentiation potential of the resulting iPS cells. Therefore, this study demonstrates a novel function of FoxO3a in cell reprogramming, which will help the development of alternative strategies for generating iPS cells.

Hydrogen peroxide induced impairment of endothelial progenitor cell viability is mediated through a FoxO3a dependant mechanism

ObjectivesIncreased oxidative stress has been suggested to contribute to the functional impairment of endothelial progenitor cells (EPCs). The Forkhead box O transcription factors (FoxOs) are critical regulators involved in various cellular processes including cell apoptosis. Here, we investigated whether FoxOs are required in oxidative stress induced EPC apoptosis. Methods and resultsEPCs were cultured from cord blood derived mononuclear cells and treated with hydrogen peroxide (H2O2) for induction of oxidative stress. Incubation with H2O2 dose dependently reduced viability and increased apoptosis in EPCs. Western blotting showed that EPCs predominantly expressed FoxO3a and the expression was markedly increased upon H2O2 treatment. Transduction with adenoviral vectors expressing either a wide-type or a non-phosphorylatable, constitutively active mutant of FoxO3a led to further increased apoptosis of EPCs after H2O2 treatment. Conversely, FoxO3a silencing rescued EPCs from these H2O2 induced deleterious effects. Overexpression of FoxO3a also increased the level of the pro-apoptotic protein Bim, whereas FoxO3a silencing downregulated H2O2 induced Bim expression. Furthermore, Matrigel assay demonstrated that FoxO3a overexpression significantly impaired the tube forming ability of EPCs, whereas its silencing completely protected EPCs from H2O2 induced decrease of capillary formation. ConclusionsThese data suggest that oxidative stress induced impairment of EPC survival is mediated through a FoxO3a dependant mechanism, possibly by transcriptional regulation of Bim. Our data indicate FoxO3a as a potential therapeutic target for improvement of EPC number and function in patients with ischemic heart disease.

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

Serotonin promotes the proliferation of serum-deprived hepatocellular carcinoma cells via upregulation of FOXO3a

BackgroundPeripheral serotonin is involved in tumorigenesis and induces a pro-proliferative effect in hepatocellular carcinoma (HCC) cells; however, the intracellular mechanisms by which serotonin exerts a mitogenic effect remain unclear. In this research, we examined whether FOXO3a, a transcription factor at the interface of crucial cellular processes, plays a role downstream of serotonin in HCC cells.ResultsThe cell viability and expression of FOXO3a was assessed in three HCC cell lines (Huh7, HepG2 and Hep3B) during serum deprivation in the presence or absence of serotonin. Serum free media significantly inhibited HCC proliferation and led to reduced expression and nuclear accumulation of FOXO3a. Knockdown of FOXO3a enhanced the ability of serum deprivation to inhibit HCC cells proliferation. And overexpression of non-phosphorylated FOXO3a in HCC cells reversed serum-deprivation-induced growth inhibition. Serotonin reversed the serum-deprivation-induced inhibition of cell proliferation and upregulated FOXO3a in Huh7 cells; however, serotonin had no effect on the proliferation of serum-deprived HepG2 or Hep3B cells. In addition to proliferation, serotonin also induced phosphorylation of AKT and FOXO3a in serum-deprived Huh7 cells but not in HepG2 and Hep3B cells. However, the phosphorylation of FOXO3a induced by serotonin did not export FOXO3a from nucleus to cytoplasm in serum-deprived Huh7 cells. Consequently, we demonstrated that serotonin promoted the proliferation of Huh7 cells by increasing the expression of FOXO3a. We also provide preliminary evidence that different expression levels of the 5-HT2B receptor (5-HT2BR) may contribute to the distinct effects of serotonin in different serum-deprived HCC cells.ConclusionsThis study demonstrates that FOXO3a functions as a growth factor in serum-deprived HCC cells and serotonin promotes the proliferation of serum-deprived HCC cells via upregulation of FOXO3a, in the presence of sufficient levels of the serotonin receptor 5-HT2BR. Drugs targeting the serotonin-5-HT2BR-FOXO3a pathway may provide a novel target for anticancer therapy.

FoxO3a Negatively Regulates Nerve Growth Factor-Induced Neuronal Differentiation Through Inhibiting the Expression of Neurochondrin in PC12 Cells

Forkhead box O3 (FoxO3a) is a forkhead family transcription factor playing important roles in non-neuronal differentiation, metabolism, proliferation, and survival, but its role in neuronal differentiation remains unclear. In the present study, we investigated the role of FoxO3a in neuronal differentiation and its underlying mechanisms. Our results showed that overexpression of FoxO3a inhibited neuronal differentiation of PC12 cells induced by nerve growth factor (NGF) while knockdown of FoxO3a by siRNA enhanced NGF-induced differentiation. DNA microarray analysis and quantitative reverse transcription PCR (RT-PCR) showed that the overexpression of FoxO3a significantly attenuated expression of neurochondrin (NCDN), a neurite outgrowth-related protein, in PC12 cells, while knocking down the expression of FoxO3a had the opposite effect. Bioinformatic studies found that the regulatory region of NCDN promoter contained multiple FoxO3a binding sites. Dual-luciferase reporter assay with report gene containing NCDN promoter showed that FoxO3a significantly decreased the transcription activity of NCDN promoter. These results indicate that NCDN is a direct downstream target of FoxO3a which negatively regulates the expression of NCDN. Interestingly, NGF-induced NCDN expression and cell differentiation was blocked by the inhibition of phosphatidylinositol-3-kinase (PI3K)-protein kinase B (PKB, Akt) signal pathway (activation of FoxO3a) and overexpression of FoxO3a. Moreover, knockdown of NCDN by siRNA blocked NGF-induced neuronal differentiation of PC12 cells while overexpression of NCDN significantly promoted neurite outgrowth. These results put together demonstrate that NCDN plays an important role in NGF-induced neuronal differentiation and suggest that FoxO3a inhibits NGF-induced neuronal differentiation, at least in part, by suppressing the expression of NCDN.