FOXO3a Transcription Research Articles

FoxO3a transcription factor differentially modulates the metastatic potential of ER+ and ER− breast tumors

FoxO3a (F3a) transcription factor has been reported to suppress estrogen(E2)‐dependent breast cancer cell growth and tumorigenesis through its functional interaction with the estrogen receptor α (ER). However, the role of this interplay in tumor metastasis is poorly defined and is the aim of the present study. To this purpose, F3a and its active form AAA, were over‐expressed both in ER+ and in ER− breast cancer cell lines. Our results show decreased migration and invasion of F3a/ER+ cells compared to control, paralleled by a strong upregulation of Caveolin‐1 (Cav‐1), the main constituent of caveolae that seems to function as a negative regulator of cell transformation and invasion in breast cancer, and a significant reduction of MMP‐13 mRNA. F3a mediated effects were more evident in AAA expressing cells and were emphasized by E2. On the other hand, in ER− cells, F3a and AAA increased cell migration and invasion with a concomitant induction of MMP‐13 transcripts, while no difference was observed in Cav‐1 levels. Since both Cav‐1 and MMP‐13 promoters bear Forkhead canonical motifs next to ER binding sequences, our hypothesis is that F3a exerts a protective role in ER+ breast cancers through F3a/ER mediated regulation of Cav‐1 and MMP‐13, while it could represent a bad prognostic factor in ER− breast tumors. Financial support has been provided by AIRC (Associazione Italiana Ricerca Cancro).

Retinoic Acid/Alpha-Interferon Combination Inhibits Growth and Promotes Apoptosis in Mantle Cell Lymphoma through Akt-Dependent Modulation of Critical Targets

Mantle cell lymphoma (MCL) is characterized by a profound deregulation of the mechanisms controlling cell-cycle progression and survival. We herein show that the combination of 9-cis-retinoic acid (RA) and IFN-α induces marked antiproliferative and proapoptotic effects in MCL cells through the modulation of critical targets. Particularly, IFN-α enhances RA-mediated G(0)-G(1) cell accumulation by downregulating cyclin D1 and increasing p27(Kip1) and p21(WAF1/Cip1) protein levels. Furthermore, RA/IFN-α combination also induces apoptosis by triggering both caspases-8 and -9 resulting in Bax and Bak activation. In particular, RA/IFN-α treatment downregulates the antiapoptotic Bcl-xL and Bfl-1 proteins and upregulates the proapoptotic BH3-only Noxa protein. Sequestration of Mcl-1 and Bfl-1 by upregulated Noxa results in the activation of Bid, and the consequent induction of apoptosis is inhibited by Noxa silencing. Noxa upregulation is associated with nuclear translocation of the FOXO3a transcription factor as consequence of RA/IFN-α-induced Akt inhibition. Pharmacologic suppression of Akt, but not of TORC1, increases Noxa protein levels and downregulates Bfl-1 protein supporting the conclusion that the inhibition of the Akt pathway, the resulting FOXO3a activation and Noxa upregulation are critical molecular mechanisms underlying RA/IFN-α-dependent MCL cell apoptosis. These results support the potential therapeutic value of RA/IFN-α combination in MCL management.

Foxo3a transcription factor is a negative regulator of Skp2 and Skp2 SCF complex

Skp2 (S-phase kinase-associated protein-2) SCF complex displays E3 ligase activity and oncogenic activity by regulating protein ubiquitination and degradation, in turn regulating cell cycle entry, senescence and tumorigenesis. The maintenance of the integrity of Skp2 SCF complex is critical for its E3 ligase activity. The Skp2 F-box protein is a rate-limiting step and key factor in this complex, which binds to its protein substrates and triggers ubiquitination and degradation of its substrates. Skp2 is found to be overexpressed in numerous human cancers, which has an important role in tumorigenesis. The molecular mechanism by which the function of Skp2 and Skp2 SCF complex is regulated remains largely unknown. Here we show that Foxo3a transcription factor is a novel and negative regulator of Skp2 SCF complex. Foxo3a is found to be a transcriptional repressor of Skp2 gene expression by directly binding to the Skp2 promoter, thereby inhibiting Skp2 protein expression. Surprisingly, we found for the first time that Foxo3a also displays a transcription-independent activity by directly interacting with Skp2 and disrupting Skp2 SCF complex formation, in turn inhibiting Skp2 SCF E3 ligase activity and promoting p27 stability. Finally, we show that the oncogenic activity of Skp2 is repressed by Foxo3a overexpression. Our results not only reveal novel insights into how Skp2 SCF complex is regulated, but also establish a new role for Foxo3a in tumor suppression through a transcription-dependent and independent manner.

AMPK promotes skeletal muscle autophagy through activation of forkhead FoxO3a and interaction with Ulk1

In skeletal muscle, protein levels are determined by relative rates of protein synthesis and breakdown. The balance between synthesis and degradation of intracellular components determines the overall muscle fiber size. AMP-activated protein kinase (AMPK), a sensor of cellular energy status, was recently shown to increase myofibrillar protein degradation through the expression of MAFbx and MuRF1. In the present study, the effect of AMPK activation by AICAR on autophagy was investigated in muscle cells. Our results show that FoxO3a transcription factor activation by AMPK induces the expression of the autophagy-related proteins LC3B-II, Gabarapl1, and Beclin1 in primary mouse skeletal muscle myotubes and in the Tibialis anterior (TA) muscle. Time course studies reveal that AMPK activation by AICAR leads to a transient nuclear relocalization of FoxO3a followed by an increase of its cytosolic level. Moreover, AMPK activation leads to the inhibition of mTORC1 and its subsequent dissociation of Ulk1, Atg13, and FIP200 complex. Interestingly, we identify Ulk1 as a new interacting partner of AMPK in muscle cells and we show that Ulk1 is associated with AMPK under normal conditions and dissociates from AMPK during autophagy process. Moreover, we find that AMPK phosphorylates FoxO3a and Ulk1. In conclusion, our data show that AMPK activation stimulates autophagy in skeletal muscle cells through its effects on the transcriptional function of FoxO3a and takes part in the initiation of autophagosome formation by interacting with Ulk1. Here, we present new evidences that AMPK plays a crucial role in the fine tuning of protein expression programs that control skeletal muscle mass.

JNK-Mediated FOXO Expression Plays a Critical Role in EGFR Tyrosine Kinase Inhibitor-Induced BIM Expression and Apoptosis

BIM, a key proapoptotic member of the BCL-2 family of proteins, is essential for apoptosis triggered by tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR). However, the precise molecular mechanism by which EGFR-TKIs induce BIM expression has remained unclear. A previous study of ours showed that the activetion of c-Jun NH2-terminal kinase (JNK) is critical for the TKI-induced apoptosis in PC-9 cells, a gefitinib-sensitive human NSCLC cell line. In this study, we therefore examined the effect of JNK activation on BIM expression and further investigated the mechanism responsible for TKI-induced apoptosis in PC-9 cells. Northern blotting analysis revealed that the TKI AG1478 induced a substantial increase in the level of BIM mRNA. However, this TKI-induced increase was not observed in dominant-negative JNK overexpressing cell line J12A5 or in the TKI-resistant cell line HP-5R, in which JNK is not activated in response to AG1478. Therefore, JNK activation was correlated with the up-regulation of BIM expression. BIM is known to be a downstream target of forkhead box protein O (FOXO) transcription factors. Immunoblot analysis indicated that the levels of FOXO1, FOXO3a, and FOXO4 transcription factors increased after AG1478 treatment of PC-9 cells but that they were not increased in either J12A5 or HP-5R cells, indicating that FOXO was increased in PC-9 cells through JNK activation. FOXO1 knockdown in PC-9 cells decreased EGFR-TKI-induced BIM expression and apoptosis. These findings provide evidence that JNK activation and subsequent increased FOXO expression play a critical role in EGFR-TKI-induced BIM expression and apoptosis.

Neocytolysis Is Associated with Changes in Increase of Mitochondrial Content and Impaired Protection From Reactive Oxygen Species

Abstract 1029Red blood cells (RBCs) continuously transport large amount of oxygen over their life time and require precise mechanism to protect themselves from oxidative stress. RBCs cannot respond to rapid oxygen changes by synthesizing enzymes and other proteins. Chronic hypoxia enhances erythropoiesis with ensuing polycythemia. With return to normoxia, red cell mass is reduced by neocytolysis, characterized by selective hemolysis of the young RBCs. Neocytolysis was described in astronauts, in those descending from high-altitude, and in newborn babies leaving hypoxic environment of uterus. While it has been suggested that neocytolysis is caused by very low erythropoietin levels, its molecular basis remains obscure. However, we argue against this postulate since RBCs lack pathway for erythropoietin signaling. We hypothesize that rapid changes of hypoxia-regulated hypoxia-inducible transcription factors (HIFs) regulated genes (other than erythropoietin) may be responsible, one such a gene (BNIP3L/NIX) regulates mitochondrial autophagy. Upon normoxic return young RBCs generated in hypoxia cannot cope because of decreased levels of oxidant protecting defenses regulated by HIF-dependent FOXO3a transcription factor.In order to test this hypothesis, we created an animal model depicted in Figure 1. We placed C57/BL6 mice in a hypoxia chamber at 12 % O2, (equivalent to 4500 m of altitude) for 10 days and then returned them to a normoxic condition. We measured hematocrit levels and reticulocyte count before and after hypoxia treatment. [Display omitted] Legend: BH: Before Hypoxia, PH: Post Hypoxia *; P value ≤ 0.05, **; P value ≤0.01, ***; P value ≤0.001, P value calculated using student T test comparing values before hypoxia.Epo levels increased 1.6 fold during hypoxia and then reduced up to undetectable level at PH day 4. Then Epo gradually increased to ∼3 fold during PH day 10∼28. During PH day 10∼21, the mice became anemic, even though Epo and reticulocytes remained high. These results suggest that neocytolysis occurs after several days of exposure to normoxia and it is not caused by Epo mechanism.To investigate the molecular basis of the observed neocytolysis in this mouse model, we measured the mitochondrial content in reticulocytes, anti-oxidative enzyme activities (glutathione peroxidase and reductase, catalase, and superoxide dismutase) that scavenge reactive oxygen species in RBCs, possibly coexistent with up-regulation of mitochondrial content upon normoxic return. Reticulocytes at returning normoxia generated more mitochondria several days after normoxic return, In contrast catalase activity was reduced during hypoxia and at PH day 4, but by PH day 10 its activity increased, and the catalase activity decrease coincided with a decrease in hematocrit.To investigate whether hypoxia drives neocytolysis under our conditions, we tested 2 known HIF target genes, Bnip3L (also called Nix), a pro-apoptotic protein that causes mitochondrial autophagy. Bnip3L mRNA was induced 9x during hypoxia and reduced 2x at PH day 6, compared to before hypoxia. We also analyzed Foxo3a, a transcription factor, in sorted reticulocytes (CD71+/TER119+/Mitochondria+) which regulates cellular stress responses such as catalase and superoxide dismutase (SOD). Foxo3a was slightly increased during hypoxia and reduced 4x at PH day 6 from levels before hypoxia.In conclusion, we developed mouse model to study neocytolysis. Our data suggest that increased mitochondria retained by Bnip3L repression leads to an accumulation of reactive oxygen species (whether in reticulocytes, platelets or leukocytes), and that young RBCs formed in hypoxia with insufficient antioxidant enzyme activity cannot survive because of excessive reactive oxygen species, with ensuing hemolysis. Studies of the role of other blood cells, as well as human studies of mountain climbers upon their return to sea level, are in progress. Disclosures:No relevant conflicts of interest to declare.

Abstract 3779: ARHI-induced autophagy is mediated through activation of FoxO3a and induction of its target genes to promote the formation of autophagic complexes

Abstract The role of autophagy in oncogenesis remains ambiguous and the mechanisms that induce autophagy and regulate its outcome in human cancer are poorly understood. ARHI (DIRAS3), a Ras-related tumor suppressor, is downregulated in >60% of ovarian cancers. Re-expression of ARHI in ovarian cancer cells induces autophagy in culture and in xenografts, by blocking Ras activation and inhibiting PI3 kinase activity. ARHI also participates directly in the formation of autophagosomes by upregulating Atg4 and co-localizing with processed LC-3 II. Here we show that inhibition of PI3 kinase/Akt signaling and Ras activation by ARHI is mediated, in part, by down regulation of EGFR expression and EGFR phosphorylation. Inhibition of these signaling pathways reduced phosphorylation of FoxO3a and enhanced nuclear accumulation of FoxO3a transcription factor, resulting in increased transcription of Atg4, MAP-LC-3, and PUMA. Knockdown of FoxO3a by siRNA reduced ARHI- and rapamycin-induced autophagy. Whereas ARHI inhibits Class I PI3K activity, its expression induces Class III PI3K activity and contributes to the enhanced autophagy. Thus, ARHI-induced autophagy is through activation of the transcription factor FoxO3a, which increases the expression of several autophagy-related genes that contribute to the formation of the autophagic complexes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3779. doi:10.1158/1538-7445.AM2011-3779

Feedback upregulation of HER3 (ErbB3) expression and activity attenuates antitumor effect of PI3K inhibitors

We examined the effects of an inhibitor of PI3K, XL147, against human breast cancer cell lines with constitutive PI3K activation. Treatment with XL147 resulted in dose-dependent inhibition of cell growth and levels of pAKT and pS6, signal transducers in the PI3K/AKT/TOR pathway. In HER2-overexpressing cells, inhibition of PI3K was followed by up-regulation of expression and phosphorylation of multiple receptor tyrosine kinases, including HER3. Knockdown of FoxO1 and FoxO3a transcription factors suppressed the induction of HER3, InsR, IGF1R, and FGFR2 mRNAs upon inhibition of PI3K. In HER2(+) cells, knockdown of HER3 with siRNA or cotreatment with the HER2 inhibitors trastuzumab or lapatinib enhanced XL147-induced cell death and inhibition of pAKT and pS6. Trastuzumab and lapatinib each synergized with XL147 for inhibition of pAKT and growth of established BT474 xenografts. These data suggest that PI3K antagonists will inhibit AKT and relieve suppression of receptor tyrosine kinase expression and their activity. Relief of this feedback limits the sustained inhibition of the PI3K/AKT pathway and attenuates the response to these agents. As a result, PI3K pathway inhibitors may have limited clinical activity overall if used as single agents. In patients with HER2-overexpressing breast cancer, PI3K inhibitors should be used in combination with HER2/HER3 antagonists.

TEAD1-dependent expression of the FoxO3a gene in mouse skeletal muscle

BackgroundTEAD1 (TEA domain family member 1) is constitutively expressed in cardiac and skeletal muscles. It acts as a key molecule of muscle development, and trans-activates multiple target genes involved in cell proliferation and differentiation pathways. However, its target genes in skeletal muscles, regulatory mechanisms and networks are unknown.ResultsIn this paper, we have identified 136 target genes regulated directly by TEAD1 in skeletal muscle using integrated analyses of ChIP-on-chip. Most of the targets take part in the cell process, physiology process, biological regulation metabolism and development process. The targets also play an important role in MAPK, mTOR, T cell receptor, JAK-STAT, calcineurin and insulin signaling pathways. TEAD1 regulates foxo3a transcription through binding to the M-CAT element in foxo3a promoter, demonstrated with independent ChIP-PCR, EMSA and luciferase reporter system assay. In addition, results of over-expression and inhibition experiments suggest that foxo3a is positively regulated by TEAD1.ConclusionsOur present data suggests that TEAD1 plays an important role in the regulation of gene expression and different signaling pathways may co-operate with each other mediated by TEAD1. We have preliminarily concluded that TEAD1 may regulate FoxO3a expression through calcineurin/MEF2/NFAT and IGF-1/PI3K/AKT signaling pathways in skeletal muscles. These findings provide important clues for further analysis of the role of FoxO3a gene in the formation and transformation of skeletal muscle fiber types.

NF-κB/Rel: agonist and antagonist roles in HIV-1 latency

To discuss recent advances in our understanding of the diverse roles of NF-κB/Rel family members in HIV-1 latency. Various NF-κB/Rel family members can reinforce maintenance of HIV-1 latency. For example, p50 recruits histone deacetylase 1 to the HIV-1 long terminal repeat promoting chromatin condensation and reduced RNA Pol II recruitment. Low-level NF-κB activation during homeostatic proliferation of memory CD4 T cells induced by IL-7 and TCR signaling or OX40 action promotes expression of antiapoptotic gene targets such as BCL2 and BCLXL. Additionally, the IκB kinase phosphorylates FOXO3a transcription factor, blocking its induction of proapoptotic genes. These combined effects promote memory CD4 T-cell survival, thus maintaining the latent reservoir. Conversely, when the nontumorigenic phorbol ester prostratin is combined with histone deacetylase inhibitors, potent synergistic activation of latent HIV-1 occurs involving nuclear expression of NF-κB. These recent findings highlight both the antagonistic and agonistic effects of the NF-κB signaling pathway on HIV-1 latency. Synergistic inducers might be useful for flushing of latent virus from reservoirs in infected patients. The ultimate, albeit lofty, goal is to achieve full viral eradication. However, a more reasonable goal might be a functional cure where patients experience a drug-free remission.

Highlights of This Issue

Effective treatments for pancreatic adenocarcinoma that are less invasive and less toxic are needed. In this study, Glazer and colleagues used targeted delivery of gold nanoparticles to treat pancreatic adenocarcinoma cells in vitro and in vivo. They demonstrated that the nanoparticles released heat upon exposure to a noninvasive focused radiofrequency field. Animals treated with the targeted gold nanoparticles and the radiofrequency field suffered no toxicities and did not have evidence of nonmalignant tissue abnormalities, while the malignant cells showed evidence of thermally induced apoptosis and necrosis. This experimental approach has potential for the noninvasive treatment of cancer.IL-6 production is increased in drugresistant ovarian cancer cells and in the serum and ascites of ovarian cancer patients. Siltuximab is a chimeric murine anti-human IL-6 monoclonal antibody that has been studied previously in human cancers. Here, Guo and colleagues found that siltuximab specifically suppressed IL-6-induced Stat3 phosphorylation and Stat3 nuclear translocation in ovarian cancer cells. Treatment with siltuximab significantly decreased Stat3 downstream protein levels, including MCL-1, Bcl-XL and survivin. Siltuximab also increased the cytotoxic effects of paclitaxel in vitro. These results suggest that inhibition of IL-6 signaling may be effective for ovarian cancer treatmentEpithelial-mesenchymal transition (EMT) plays a critical role in cancer progression. Although the Foxo3a transcription factor is known to regulate the expression of cell cycle- and apoptosis-related genes, its role in EMT needs to be explored further. Here, Shiota and colleagues examined Foxo3a expression in urothelial cancer specimens. They found that Foxo3a expression levels were decreased in invasive urothelial cancer and that Foxo3a was an independent prognostic factor. Further, Foxo3a regulated the motility of urothelial cancer cells through the regulation of EMT-associated proteins (Twist1, YB-1 and E-cadherin). These results implicate Foxo3a in cancer progression and the regulation of EMT.Snail, which contributes to epithelialmesenchymal transition (EMT), has been implicated in cancer progression. Here, Kosaka and colleagues investigated the biological significance of Snail in upper urinary tract urothelial carcinoma (UTUC). They observed strong Snail staining in invasive UTUC, and found that Snail was an independent prognostic factor. Further, targeting Snail by siRNA resulted in decreased invasion activity (and an accompanying reduction in mesenchymal markers and matrix metalloproteinases) in urothelial carcinoma cells. These results suggest that Snail-induced EMT represents an attractive target for the treatment of UTUC.

OSI-027, a Dual TORC1/TORC2 Inhibitor, Induces Bim- and Puma-Mediated Apoptosis In Lymphoid Malignancy

AbstractAbstract 970The mammalian target of rapamycin, mTOR, is a highly conserved serine/threonine kinase known to play a role in regulating mRNA translation, cell cycle progression, cell proliferation and apoptosis. As a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, mTOR is a component of two distinct complexes, TORC1 and TORC2. While TORC1 facilitates cell cycle progression from G1 into S phase by phosphorylating p70S6 kinase and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), TORC2 catalyzes the activating phosphorylation of Akt on Ser473, providing a feedback loop for further activation of mTOR. Phase II trials have shown activity of the TORC1-selective inhibitor rapamycin and its analogs in a wide range of lymphoma subtypes. The purpose of this study was to evaluate the anti-proliferative and pro-apoptotic effects of the dual TORC1/TORC2 inhibitor OSI-027 in human neoplastic lymphoid cells in vitro. MTS assays demonstrated that OSI-027 inhibited proliferation in a wide range of lymphoid lines, including SeAx (Sezary syndrome), DoHH2 (large cell lymphoma), RL (follicular lymphoma) and Jurkat (T cell ALL), as well as clinical lymphoma and T cell ALL samples, with IC50 values ranging from 0.078 to 10 μM. Propidium iodide staining followed by flow cytometry for subdiploid cells revealed induction of apoptosis within 48 h of treatment with OSI-027 (but not rapamycin) in SeAx, DoHH2, and Jurkat cells. Examination of Jurkat variants with alterations in key proteins involved in the death receptor versus mitochondrial pathway revealed diminished apoptotic responses to OSI-027 when Bcl-2 was overexpressed or caspase 9 was silenced, indicating involvement of the mitochondrial pathway. Immunoblotting for Bcl-2 family members revealed upregulation of Bim and Puma after a 48-hour exposure to OSI-027 but not rapamycin. This upregulation was also seen at the mRNA level, with a 12- to 20-fold increase in Puma mRNA and 4- to 12-fold induction of Bim mRNA. Small interfering RNA (siRNA)-mediated knockdown of Bim and Puma significantly diminished the apoptotic response to OSI-027. Because the Foxo3a transcription factor has been implicated in Bim and Puma expression and is known to be activated when Akt is inhibited, we next examined whether Bim and Puma induction was Foxo3a-dependent. Luciferase reporter assays showed that OSI-027 activated the full-length Puma and Bim promoters and that this activation was diminished when the Foxo3a binding sites were deleted or mutated. In addition, OSI-027 induced nuclear translocation of Foxo3a, while Foxo3a siRNA diminished OSI-027-induced apoptosis in Jurkat cells. Collectively, these results indicate that OSI-027 inhibits proliferation and induces apoptosis in a wide range of neoplastic lymphoid cells through a process that involves Foxo3a-mediated upregulation of Bim and Puma. These results also suggest that dual inhibition of TORC1 and TORC2 may be an effective treatment strategy in lymphoid malignancy. Disclosures:Barr:OSI Pharmaceuticals: Employment. Witzig:Novartis and Celgene: Patents & Royalties, Research Funding, Served on advisory boards with Novartis and Celgene – both uncompensated with compensation to Mayo Clinic.

Molecular Mechanism Regulating Foxo In Leukemia Initiating Cells of Chronic Myeloid Leukemia.

AbstractAbstract 3391Chronic myeloid leukemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. Although the development of imatinib, a small molecule inhibitor of ABL, represents a breakthrough in the treatment of CML, major part of patients treated in chronic phase CML are not off therapy due to resistance or intolerance. Recent studies have suggested that imatinib is a potent inhibitor against differentiated leukemia cells, but does not deplete leukemia-initiating cells (LICs) responsible for recurrence of CML. To date, therapeutics that can eradicate CML LICs, however, have remained under investigation. To overcome these clinical problems, here we studied the molecular mechanisms regulating maintenance of imatinib-resistant CML LICs by forkhead transcription factor Foxo3a.We first generated a mouse CML model by using retroviral induction of BCR-ABL-ires-GFP gene into mouse immature hematopoietic cells, and the cells were subsequently transplanted into irradiated recipient mice. These experiments showed that CML LICs were highly enriched in c-Kit+Lin−Sca-1+ (KLS+) population in BCR-ABL+ CML cells. Serial transplantation experiments for CML LICs originated from Foxo3a-deficient mice and littermate wild-type mice indicated that Foxo3a-deficiency reduced lethality of recipient mice at third transplantation. Although recipients that transplanted with wild-type LICs developed CML and acute lymphocytic leukemia (ALL) at third transplantation, we did not observe development of ALL or CML in recipients of Foxo3a deficient LICs after 45 days post-third transplantation, suggesting that the Foxo3a deficient LICs lose their potential to generate malignancies. In addition, a combination of Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo, indicating that Foxo3a plays an essential role for the maintenance of imatinib-resistant CML LICs (Naka et al., Nature 463, 676–680, 2010).Interestingly, when we examined sub-cellular localization of Foxo3a transcription factor in the CML LICs, we found two CML LIC populations; one population was the cells with nuclear localization of Foxo3a (Foxo3a transcription factor is active) and the other population was the cells with cytoplasmic localization of Foxo3a (Foxo3a is inactive). To understand the molecular mechanisms regulating Foxo3a in CML LICs, we next evaluated the activity of upstream BCR-ABL, PI3K, PDK1, and Akt signaling pathway by fluorescence immunohistochemistry. BCR-ABL activity that was determined by phosphorylation levels of CrkL, a down-stream target of BCR-ABL, was detected in almost all of the CML LICs. However, unexpectedly, phosphorylation levels of Akt in the CML LICs with nuclear localization of Foxo3a appeared to be lower than that in the CML LICs with cytoplasmic localization of Foxo3a, despite it is widely believed that BCR-ABL induces activation of Akt signal. Consistent with Akt phosphorylation status, we detected low levels phosphorylation of PDK1 and PI3K p85, upstream regulators for Akt, in the CML LICs with nuclear localization of Foxo3a. Interestingly, expression levels of the cell proliferation antigen Ki67 were lower in the CML LICs with nuclear Foxo3a than that in the CML LICs with cytoplasmic Foxo3a. These results suggest that Foxo3a responsible for maintenance of imatinib-resistant CML LICs may be regulated by molecular mechanisms that are involved in dormancy in CML LICs. Disclosures:No relevant conflicts of interest to declare.

Comparison of intracellular signalling by insulin and the hypermitogenic AspB10 analogue in MCF‐7 breast adenocarcinoma cells

We compared mitogenicity and intracellular signalling by human insulin and the AspB10 (X-10) human insulin analogue in MCF-7 human mammary adenocarcinoma cells. By flow analysis of phosphorylated histone H3 or cell cycle distributions, insulin and X-10 were mitogenic at physiologically relevant concentrations (2 nm to 74 pm range), with X-10 being approximately 3-fold more mitogenic than insulin. By western blotting with phospho-specific antibodies, insulin induced phosphorylation of IRS-1, Akt, p70S6K, S6 ribosomal protein, 4E-BP1, FoxO3a, FoxO1, p44/42 MAPK and the EGFR. Blocking with wortmannin, rapamycin and U0126 showed that these signalling events conformed to the canonical PI3K pathway. IRS-1 (Ser302) phosphorylation was abolished by wortmannin and rapamycin, suggesting a feedback from the PI3K pathway on insulin signalling. Compared with equimolar insulin, X-10 caused up to 2-fold higher phosphorylation of all proteins examined in this study. The phosphorylation sites that responded most strongly to insulin were not generally the same as those responding most strongly to X-10. In the PI3K pathway, the most X-10-sensitive protein localized to the translation-regulating arm (p70S6K), with FoxO3a and FoxO1 transcription factors showing a more comparable response to insulin and X-10. Using flow analysis, we confirmed the correlation between insulin-triggered translational activation in G0/G1 (S6 phosphorylation) and S-phase entry by MCF-7 cells. In summary, our findings implicate asymmetrical PI3K pathway activation and specifically stimulation of protein translation in the hypermitogenic effect of insulin analogues such as X-10. It remains to be shown whether these findings are relevant to other human mammary cancer cell types.

Downregulation of Manganese Superoxide Dismutase by Angiotensin II in Cardiac Fibroblasts of Rats: Association With Oxidative Stress in Myocardium

The aim of this study was to determine whether angiotensin II (ANG II) affects the protein and mRNA expressions of the mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD) in cardiac fibroblasts of rats through inducing the phosphorylation of the proteins Akt and FOXO3a, thereby contributing to the oxidative stress in the myocardium. Cardiac fibroblasts (passage 2) from normal male adult rats were cultured to confluency and incubated in serum-free Dulbecco's modified Eagle's medium for 24 h. The cells were then preincubated with/without the tested inhibitors for 1 h and then further incubated with/without ANG II (1 µmol/l) for 24 h. ANG II increased the production of superoxide ions in the cardiac fibroblasts, and decreased the activity levels of both Mn-SOD and CuZn-SOD, but not the activity levels of catalase and glutathione peroxidase. ANG II also decreased the mRNA and protein expressions of Mn-SOD, but not those of CuZn-SOD, catalase, and glutathione peroxidase. The likely mechanism through which ANG II produces the effect of reducing Mn-SOD activity is by reducing the extent of binding of FOXO3a to the Mn-SOD promoter. In control fibroblasts, inhibition of FOXO3a transcription with small-interfering RNA (siRNA) led to a reduction in the binding of FOXO3a to the Mn-SOD promoter, and a concomitant reduction in Mn-SOD gene expression. Our data suggest that when Akt is phosphorylated by ANG II, P-Akt is translocated from the cytoplasm to the nucleus; subsequently, nuclear phosphorylation of FOXO3a by P-Akt leads to relocalization of FOXO3a from the nucleus to the cytosol, resulting in a decrease in its transcriptional activity, and consequently in Mn-SOD expression. The likelihood of such a mechanism of action is further strengthened by the fact that inhibition of phosphoinositide 3-kinase with wortmannin or LY 294002, and Akt inhibition, were shown to lead to a decrease in P-AKT and to a consequent increase in Mn-SOD mRNA expression. Our data indicate that ANG II inactivates FOXO3a by activating Akt, leading to a reduction in the expression of the antioxidant Mn-SOD, and thereby potentially contributing to oxidative stress in the myocardium.

Western diet changes cardiac acyl-CoA composition in obese rats: a potential role for hepatic lipogenesis

The "lipotoxic footprint" of cardiac maladaptation in diet-induced obesity is poorly defined. We investigated how manipulation of dietary lipid and carbohydrate influenced potential lipotoxic species in the failing heart. In Wistar rats, contractile dysfunction develops at 48 weeks on a high-fat/high-carbohydrate "Western" diet, but not on low-fat/high-carbohydrate or high-fat diets. Cardiac content of the lipotoxic candidates--diacylglycerol, ceramide, lipid peroxide, and long-chain acyl-CoA species--was measured at different time points by high-performance liquid chromatography and biochemical assays, as was lipogenic capacity in the heart and liver by qRT-PCR and radiometric assays. Changes in membranes fluidity were also monitored using fluorescence polarization. We report that Western feeding induced a 40% decrease in myocardial palmitoleoyl-CoA content and a similar decrease in the unsaturated-to-saturated fatty acid ratio. These changes were associated with impaired cardiac mitochondrial membrane fluidity. At the same time, hepatic lipogenic capacity was increased in animals fed Western diet (+270% fatty acid elongase activity compared with high-fat diet), while fatty acid desaturase activity decreased over time. Our findings suggest that dysregulation of lipogenesis is a significant component of heart failure in diet-induced obesity.

Role for the Proapoptotic Factor BIM in Mediating Imatinib-induced Apoptosis in a c-KIT-dependent Gastrointestinal Stromal Tumor Cell Line

The c-KIT receptor tyrosine kinase is constitutively activated and oncogenic in the majority of gastrointestinal stromal tumors. The identification of selective inhibitors of c-KIT, such as imatinib, has provided a novel therapeutic approach in the treatment of this chemotherapy refractory tumor. However, despite the clinical importance of these findings and the potential it provides as a model system for understanding targeted therapy, this approach has not yielded curative outcomes in most patients, and the biochemical pathways connecting c-KIT inhibition to cell death are not completely understood. Here, we show that inhibition of c-KIT with imatinib in gastrointestinal stromal tumors (GISTs) triggered the up-regulation of the proapoptotic protein BIM via both transcriptional and post-translational mechanisms. The inhibition of c-KIT by imatinib increased levels of the dephosphorylated and deubiquitinated form of BIM as well as triggered the accumulation of the transcription factor FOXO3a on the BIM promoter to activate transcription of BIM mRNA. Furthermore, using RNA interference directed against BIM, we demonstrated that BIM knockdown attenuated the effects of imatinib, suggesting that BIM functionally contributes to imatinib-induced apoptosis in GIST. The identification and characterization of the pathways that mediate imatinib-induced cell death in GIST provide for a better understanding of targeted therapy and may facilitate the development of new therapeutic approaches to further exploit these pathways.

Foxo3a expression and acetylation regulate cancer cell growth and sensitivity to cisplatin

Many advanced cancers receive cisplatin-based chemotherapy. However, cisplatin resistance is a major obstacle for cancer chemotherapy. Foxo3a is a member of the Foxo transcription factor family, which modulates the expression of genes involved in DNA damage repair, apoptosis, and other cellular processes. In this study, we found that cisplatin-resistant cells were more sensitive to the anticancer agent mithramycin than their parental cells, and had a decreased level of Foxo3a expression. Foxo3a knockdown increased cell proliferation and resistance to cisplatin. On the other hand, mithramycin stimulated Foxo3a expression through reactive oxygen species production and sensitized cells to cisplatin, which was abolished by Foxo3a knockdown, while the acetylation status of Foxo3a was decreased in response to cisplatin treatment and was lower in cisplatin-resistant cells. Knockdown of Foxo3a-associated acetyltransferase p300 promoted cancer-cell growth and cisplatin resistance. In addition, non-acetylation-mimicking Foxo3a overexpression decreased cancer cell growth and sensitized cells to cisplatin less than wild-type Foxo3a overexpression. The current work may contribute to the evaluation of the therapeutic potential of inducing the Foxo3a pathway and acetylating the Foxo3a transcription factor, and lead to the reevaluation of cancer treatments based on mithramycin.

Blockade of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase and Murine Double Minute Synergistically Induces Apoptosis in Acute Myeloid Leukemia via BH3-Only Proteins Puma and Bim

Molecular aberrations of the Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK and/or Murine double minute (MDM2)/p53 signaling pathways have been reported in 80% and 50% of primary acute myeloid leukemia (AML) samples and confer poor outcome. In this study, antileukemic effects of combined MEK inhibition by AZD6244 and nongenotoxic p53 activation by MDM2 antagonist Nutlin-3a were investigated. Simultaneous blockade of MEK and MDM2 signaling by AZD6244 and Nutlin-3a triggered synergistic proapoptotic responses in AML cell lines [combination index (CI) = 0.06 +/- 0.03 and 0.43 +/- 0.03 in OCI/AML3 and MOLM13 cells, respectively] and in primary AML cells (CI = 0.52 +/- 0.01). Mechanistically, the combination upregulated levels of BH3-only proteins Puma and Bim, in part via transcriptional upregulation of the FOXO3a transcription factor. Suppression of Puma and Bim by short interfering RNA rescued OCI/AML3 cells from AZD/Nutlin-induced apoptosis. These results strongly indicate the therapeutic potential of combined MEK/MDM2 blockade in AML and implicate Puma and Bim as major regulators of AML cell survival.

Regulation of the Intracellular Localization of Foxo3a by Stress-Activated Protein Kinase Signaling Pathways in Skeletal Muscle Cells

Muscle atrophy is a debilitating process associated with many chronic wasting diseases, like cancer, diabetes, sepsis, and renal failure. Rapid loss of muscle mass occurs mainly through the activation of protein breakdown by the ubiquitin proteasome pathway. Foxo3a transcription factor is critical for muscle atrophy, since it activates the expression of ubiquitin ligase Atrogin-1. In several models of atrophy, inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway induces nuclear import of Foxo3a through an Akt-dependent process. This study aimed to identify signaling pathways involved in the control of Foxo3a nuclear translocation in muscle cells. We observed that after nuclear import of Foxo3a by PI3K/Akt pathway inhibition, activation of stress-activated protein kinase (SAPK) pathways induced nuclear export of Foxo3a through CRM1. This mechanism involved the c-Jun NH(2)-terminal kinase (JNK) signaling pathway and was independent of Akt. Likewise, we showed that inhibition of p38 induced a massive nuclear relocalization of Foxo3a. Our results thus suggest that SAPKs are involved in the control of Foxo3a nucleocytoplasmic translocation in C2C12 cells. Moreover, activation of SAPKs decreases the expression of Atrogin-1, and stable C2C12 myotubes, in which the p38 pathway is constitutively activated, present partial protection against atrophy.