Nuclear Localization Of FoxO3a Research Articles

Regorafenib induces Bim-mediated intrinsic apoptosis by blocking AKT-mediated FOXO3a nuclear export

Regorafenib (REGO) is a synthetic oral multi-kinase inhibitor with potent antitumor activity. In this study, we investigate the molecular mechanisms by which REGO induces apoptosis. REGO induced cytotoxicity, inhibited the proliferation and migration ability of cells, and induced nuclear condensation, and reactive oxygen species (ROS)-dependent apoptosis in cancer cells. REGO downregulated PI3K and p-AKT level, and prevented FOXO3a nuclear export. Most importantly, AKT agonist (SC79) not only inhibited REGO-induced FOXO3a nuclear localization and apoptosis but also restored the proliferation and migration ability of cancer cells, further demonstrating that REGO prevented FOXO3a nuclear export by deactivating PI3K/AKT. REGO treatment promotes Bim expression via the FOXO3a nuclear localization pathway following PI3K/AKT inactivation. REGO induced Bim upregulation and translocation into mitochondria as well as Bim-mediated Bax translocation into mitochondria. Fluorescence resonance energy transfer (FRET) analysis showed that REGO enhanced the binding of Bim to Bak/Bax. Knockdown of Bim, Bak and Bax respectively almost completely inhibited REGO-induced apoptosis, demonstrating the key role of Bim by directly activating Bax/Bak. Knockdown of Bax but not Bak inhibited REGO-induced Drp1 oligomerization in mitochondria. In conclusion, our data demonstrate that REGO promotes apoptosis via the PI3K/AKT/FOXO3a/Bim-mediated intrinsic pathway.

Overcoming Basal Autophagy, Kangai Injection Enhances Cisplatin Cytotoxicity by Regulating FOXO3a-Dependent Autophagic Cell Death and Apoptosis in Human Lung Adenocarcinoma A549/DDP Cells.

Cisplatin resistance is one of the major obstacles in the treatment of nonsmall cell lung cancer (NSCLC). Kangai injection (KAI), a Chinese herbal medicine, has been used in tumors as adjuvant treatment, but its exact antitumor mechanism is still unclear. In this study, we first demonstrated that cisplatin-resistant A549/DDP cells showed a higher level of basal autophagy in response to cisplatin treatment with increasing autophagic protein expression levels of Beclin 1, p62, and LC3 compared to cisplatin-sensitive A549/DDP cells; then, we assessed the antitumor effect of KAI in cisplatin-resistant lung adenocarcinoma A549/DDP cells. Our results showed that KAI exhibited direct cytotoxic and chemosensitizing effects in A549/DDP cells. Combining KAI with cisplatin promoted A549/DDP cell apoptosis, which was confirmed by cell cycle arrest, condensed nuclear chromatin, annexin V fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining, and apoptosis-related protein expression. In addition, combining KAI with cisplatin induced autophagic cell death in A549/DDP cells with a high level of basal autophagy, as indicated by an increase in LC3 spot count, an accumulation of Beclin 1 and LC3 II, and reduced p62 protein expression. We also found that the apoptosis and autophagic cell death induced by cotreatment of KAI and cisplatin in A549/DDP cells were FOXO3a-dependent as indicated by decreased p-FOXO3a expression and increased FOXO3a nuclear localization, respectively. Furthermore, the FOXO3a gene knockdown assay further confirmed that KAI enhanced cisplatin cytotoxicity in A549/DDP cells with a high level of basal autophagy by inducing apoptosis and autophagic cell death in a FOXO3a-dependent manner. These findings suggest that the combination of KAI and cisplatin might support the potential clinical treatment as a novel strategy to overcome cisplatin resistance.

TCRP1 activated by mutant p53 promotes NSCLC proliferation via inhibiting FOXO3a

Previously, our lab explored that tongue cancer resistance-associated protein (TCRP1) plays a central role in cancer chemo-resistance and progression. Absolutely, TCRP1 was significantly increased in lung cancer. But the mechanism is far from elucidated. Here, we found that TCRP1 was increased in p53-mutant non-small-cell lung cancer (NSCLC), comparing to that in NSCLC with wild type p53. Further study showed that mutant p53 couldn’t bind to the promoter of TCRP1 to inhibit its expression. While the wild type p53 did so. Next, loss-and gain-of-function assays demonstrated that TCRP1 promoted cell proliferation and tumor growth in NSCLC. Regarding the mechanism, TCRP1 encouraged AKT phosphorylation and blocked FOXO3a nuclear localization through favoring FOXO3a ubiquitination in cytoplasm, thus, promoted cell cycle progression. Conclusionly, TCRP1 was upregulated in NSCLC cells with mutant p53. TCRP1 promoted NSCLC progression via regulating cell cycle.

AKT inhibitor AZD5363 suppresses stemness and promotes anti-cancer activity of 3,3′-diindolylmethane in human breast cancer cells

3,3′-diindolylmethane (DIM) is a dimer compound converted from Indoly-3-carbinol that had been studied as promising chemo-preventive agent against breast cancer. In this study, we observed that proportion of CD133+Nanog+ subpopulation in MCF-7 cells was significantly increased after DIM administration with up-regulated AKT activity by using CyTOF assay. SPADE analysis revealed this stem-like subpopulation exhibited apoptosis-resistance property against DIM treatment. By combining with AKT inhibitor AZD5363, DIM induced CD133 expression could be suppressed. In addition, a combination treatment of MCF-7 and MDA-MB-231 breast cancer cells with DIM and AZD5363 showed synergistic decreases in cell proliferation and induced apoptosis. Furthermore, results from imaging flow cytometry suggested that FoxO3a nuclear localization and PUMA expression could be improved by combination of AZD5363 with DIM. Taken together, the above observations suggested that the combination of AZD5363 with DIM could be developed as potential therapy for breast cancer.

Purple corn extract (PCE) alleviates cigarette smoke (CS)-induced DNA damage in rodent blood cells by activation of AMPK/Foxo3a/MnSOD pathway

ABSTRACT Purple corn extract (PCE) is a nutraceutical, an activator of AMPK, and it has antioxidants and anticancer properties. Therefore, PCE could be a candidate for alleviating cigarette smoke (CS)-induced oxidative DNA damage. This study examined whether PCE can have a protective effect on blood cells in an animal model of cigarette smoke (CS)-induced DNA damage. PCE was orally administered to CS-inhaled Spraque-Dawley (SD) rats, followed by the target cells being examined for markers of DNA damage. The study also sought to elucidate the mechanism of PCE action in the PCE treated animals. SD rat inhalation of CS was for once a day for 30 min, repeated for 7 days. PCE was administered orally before CS inhalation. Pretreatment of the animals with oral PCE kept the numbers of white blood cells (WBC) as well as neutrophils (NE), lymphocytes (LY), monocytes (Mo), eosinophils (EO), abd jasophils (BA) from increasing as those were increased in the CS-inhaling SD rats. The amount of phosphorylated γ-H2AX, a DNA damage marker, was assayed in the circulating blood cells collected from the animals and western blot analysis with anti-Foxo3a, p-Foxo3a, p-AMPK, MnSOD antibodies were performed on those cells. PCE protected the circulating blood cells from CS inhalation-induced DNA damage by 44% as assayed by increases in γ-H2AX. PCE also increased the nuclear localization of Foxo3a by 52% over control cells. Mechanistically, PCE appears to efficiently protect various blood cell types from CS-induced DNA damage through removal of ROS via activation of the AMPK/Foxo3a/MnSOD pathway.

FoxO3a suppresses neuropeptide W expression in neuronal cells and in rat hypothalamus and its implication in hypothalamic-pituitary-adrenal (HPA) axis.

FoxO3a, a forkhead family member of transcription factors, is involved in the regulation of cell metabolism, proliferation, differentiation and apoptosis. However, whether FoxO3a participates in the regulation of glucocorticoids induced-hypothalamic-pituitary-adrenal (HPA) dysfunction is still unknown. Our present results indicate that dexamethasone(DEX) increased FoxO3a expression in PC12 and hypothalamic neuronal cultures in correlation to reduced expression of NPW, a process that could be blocked by GR2 antagonist. DEX restrained the phosphorylation of Akt and FoxO3a, but not ERK1/2 phosphorylation, resulting with FoxO3a nuclear localization. Overexpression of FoxO3a inhibited NPW expression, while FoxO3a knockdown by siRNA had the opposite effect. The regulatory region of NPW promoter contains multiple FoxO3a binding sites, and FoxO3a bonding to these sites inhibited its transcriptional activity. In a rat model, chronic administration of corticosterone reduced animals' body weight and sucrose consumption and caused stress- depression like behavior. Corticosterone treatment induced a marked increase in FoxO3a levels, while decreased the expression of NPW protein in the hypothalamus. Immunofluorescent double labeling demonstrated that FoxO3a and NPW were collocated in the hypothalamus. Taken together, these data indicate that NPW is a new direct downstream target gene of FoxO3a. FoxO3a suppressed the transcription of NPW and modulated glucocorticoids-induced HPA dysfunction by directly regulating the expression of NPW. Thus, present findings suggest that FoxO3a and NPW may be potential therapeutic targets for endocrine and psychiatric disorders.

Increased FoxO3a expression prevents osteoblast differentiation and matrix calcification

Forkhead Box O transcription factors play important roles in bone metabolism by defending against oxidative stress and apoptosis. FoxO3a is of special interest as it is the predominant isoform expressed in bone. In osteoblasts, the administration of 1,25 dihydroxyvitamin D3 (1,25D3) increases FoxO3a expression, and alters calcium handling. We therefore queried whether FoxO3a participates in vitamin D-mediated regulation of calcium transport pathways or matrix calcification, independent of reactive oxygen species (ROS) formation. To examine this possibility, we differentiated MC3T3-E1 cells into mature osteoblast-like cells over 7 days. This coincided with an increased ability to mineralize extracellular matrix. FoxO3a expression increased throughout differentiation. 1,25D3 enhanced both FoxO3a mRNA and protein expression. Immunofluorescence microscopy found increased FoxO3a nuclear localization with differentiation and after treatment with 1,25D3. Live cell ratiometric imaging with Fura-2AM identified significant L-type calcium channel mediated calcium uptake that was enhanced by 1,25D3. We observed expression of both Cav1.2 and Cav1.3, although expression decreased throughout differentiation and was not altered by 1,25D3 treatment. FoxO3a overexpression reduced calcium uptake and calcium deposition. FoxO3a overexpression also prevented alterations in calcium channel expression and the cell differentiation associated decrease in expression of Runx2 and increased expression of osteocalcin, findings consistent with a failure for the cells to differentiate. Based on both our expression and functional data, we suggest that high levels of FoxO3a prevent osteoblast differentiation and matrix calcification.

Matrine inhibits the proliferation of pituitary tumor cells by decreasing Foxo3a phosphorylation and promoting Foxo3a nuclear localization.

Pituitary tumors account for 10% of intracranial cancer, and are difficult to treat with chemotherapy. The aim of the present study was to explore the role of matrine on the proliferation of pituitary cancer cells, as well as the molecular mechanism of matrine in progression and development of pituitary tumors. Matrine significantly suppressed the proliferation of pituitary cancer cells in a dose- and time-dependent manner. Western blotting results showed that the phosphorylation levels of AKT serine/threonine kinase (Akt) and forkhead box O3A (Foxo3a) decreased as the concentration of matrine increased. Matrine increased the nuclear localization of Foxo3a and the expression of proapoptotic genes, such as BCL2 like 11 and BCL2 associated X apoptosis regulator, and inhibited the levels of cytoplasmic Foxo3a. In conclusion, matrine promoted cell death of pituitary cancer cells and was involved in Akt/Foxo3a signaling pathway.

Suppression of Endothelial-to-Mesenchymal Transition by SIRT (Sirtuin) 3 Alleviated the Development of Hypertensive Renal Injury.

Endothelial-to-mesenchymal transition (EndoMT) has recently emerged as a potentially important contributor in promoting fibrosis in chronic kidney disease. However, little is known about the role and molecular basis of its involvement in hypertensive renal injury. Here, we aim to determine the role of SIRT (sirtuin) 3 on EndoMT in hypertensive renal injury and to explore its underlying mechanisms. We found that SIRT3 expression was significantly reduced in Ang II (angiotensin II)-induced hypertensive model, accompanied with induction of EndoMT and increased reactive oxygen species and renal fibrosis. In SIRT3-/- (SIRT3 knockout) mice subjected to Ang II infusion, renal dysfunction was aggravated with an increased EndoMT and reactive oxygen species level, whereas in SIRT3-TgEC (SIRT3 endothelial cell-specific transgenic) mice, the Ang II-induced renal fibrosis and EndoMT and oxidative stress were ameliorated. With primary mouse glomerular endothelial cells, we confirmed that Ang II treatment initiated EndoMT and decreased catalase expression, which were suppressed by SIRT3 overexpression. Using immunoprecipitation, luciferase, and chromatin immunoprecipitation assay, we demonstrated that SIRT3-mediated deacetylation and nuclear localization of Foxo3a (forkhead box O3a) resulted in activated Foxo3a-dependent catalase expression. Moreover, Foxo3a knockdown abolished SIRT3-mediated suppression of EndoMT. In conclusion, these results established the SIRT3-Foxo3a-catalase pathway as a critical factor in the maintenance of endothelial homeostasis and point to an important role of EndoMT in the vascular pathology of renal fibrosis, which may provide a new therapeutic target to impede the progression of hypertensive renal injury.

Oligonol, a Low-Molecular Weight Polyphenol Derived from Lychee, Alleviates Muscle Loss in Diabetes by Suppressing Atrogin-1 and MuRF1.

Stimulation of the ubiquitin-proteasome pathway—especially E3 ubiquitin ligases Atrogin-1 and MuRF1—is associated with muscle loss in diabetes. Elevated lipid metabolites impair myogenesis. Oligonol, a low molecular weight polyphenol derived from lychee, exhibited anti-diabetic and anti-obesity properties, suggesting it could be a proper supplement for attenuating muscle loss. Dietary (10 weeks) oligonol supplementation (20 or 200 mg/kg diet) on the skeletal muscle loss was investigated in diabetic db/db mice. Transcription factors NF-κB and FoxO3a involved in regulation of Atrogin-1 and MuRF1 were also investigated. Attenuation of muscle loss by oligonol (both doses) was associated with down-regulation of Atrogin-1 and MuRF1 gene expression. Oligonol supplementation decreased NF-κB expression in the nuclear fraction compared with db/db mice without oligonol supplement. Upregulation of sirtuin1 (SIRT1) expression prevented FoxO3a nuclear localization in db/db mice supplemented with oligonol. Marked increases in AMPKα activity and Ppara mRNA expression leading to lower lipid accumulation by oligonol provided additional benefits for attenuating muscle loss. Oligonol limited palmitate-induced senescent phenotype and cell cycle arrest and suppressed Atrogin-1 and MuRF1 mRNA expression in palmitate-treated C2C12 muscle cells, thus contributing to improving the impaired myotube formation. In conclusion, oligonol-mediated downregulation of Atrogin-1 and MuRF1 gene expression alleviates muscle loss and improves the impaired myotube formation, indicating that oligonol supplementation may be useful for the attenuation of myotube loss.

Flavanol-Rich Lychee Fruit Extract Prevents Diabetes-induced Muscle Loss And Palmitate-induced Myotube Loss

Diabetes-induced muscle loss is associated with stimulated the muscle RING-finger protein 1 (MuRF1), a muscle specific E3 ubiquitin ligase. Elevated lipid metabolites impair myogenesis. Flavanol-rich lychee fruit extract (FRLFE) exhibited anti-diabetic and -obesity properties which would be an alternative approach for maintaining muscle homeostasis. PURPOSE: The underlying mechanisms of FRLFE on maintaining muscle homeostasis was studied in vivo and in vitro. METHODS: Dietary (10 wk) FRLFE supplementation (200 mg/kg diet) on the skeletal muscle size and transcription factors such as NF-κB and Foxo3a involved in regulation of MuRF1 were investigated in diabetic db/db mice (n=10/per group). The roles of FRLFE on cell cycle and senescent phenotype were investigated in palmitate-induced insulin resistance in C2C12 muscle cells. The statistical significance of the differences among the groups (P < 0.05) was determined by one-way ANOVA and following post hoc assessment by Student-Newman-Keuls Method. RESULTS: The average cross-sectional area was significantly increased by 1.6-fold after administration of FRLFE compared with non-treated db/db mice (420μm2). Prevention of muscle loss by FRLFE was associated with down regulation of MuRF1 mRNA expression (410% vs. 100%, p<0.05). Decreased NF-κB expression in the nuclear fraction was observed in FRLFE treated db/db mice compared with untreated mice (260% vs. 60%, p<0.05). At transcriptional level, FRLFE abated ceramide-induced MuRF1 promoter activity (200% vs. 100%, p<0.05). Restoration of SIRT1 expression (30% vs. 150%, p<0.05) prevented Foxo3a nuclear localization in FRLFE treated db/db mice. A strong β-galactosidase staining positive signal was observed in the palmitate-treated myoblasts. For cell cycle analysis, significantly increased proportion of cells in G2-phase by palmitate was observed compared with group without palmitate treatment (28.9% vs. 18.3%). Weak β-galactosidase staining positive signals in accordance with decreased proportion of cells in G2-phase (21.7%) were observed in FRLFE-treated myoblasts. CONCLUSION: The efficacy of FRLFE on preserving skeletal muscle mass and myogenic process indicates that FRLFE acts as an effective supplement for diabetes and/or obesity-induced muscle atrophy.

NOTCH1 Inhibits Activation of ATM by Impairing the Formation of an ATM-FOXO3a-KAT5/Tip60 Complex

SummaryThe DNA damage response (DDR) signal transduction pathway is responsible for sensing DNA damage and further relaying this signal into the cell. ATM is an apical DDR kinase that orchestrates the activation and the recruitment of downstream DDR factors to induce cell-cycle arrest and repair. We have previously shown that NOTCH1 inhibits ATM activation upon DNA damage, but the underlying mechanism remains unclear. Here, we show that NOTCH1 does not impair ATM recruitment to DNA double-strand breaks (DSBs). Rather, NOTCH1 prevents binding of FOXO3a and KAT5/Tip60 to ATM through a mechanism in which NOTCH1 competes with FOXO3a for ATM binding. Lack of FOXO3a binding to ATM leads to the loss of KAT5/Tip60 association with ATM. Moreover, expression of NOTCH1 or depletion of ATM impairs the formation of the FOXO3a-KAT5/Tip60 protein complex. Finally, we show that pharmacological induction of FOXO3a nuclear localization sensitizes NOTCH1-driven cancers to DNA-damage-induced cell death.

Shikonin induces apoptosis of lung cancer cells via activation of FOXO3a/EGR1/SIRT1 signaling antagonized by p300

Shikonin derivatives exert powerful cytotoxic effects including induction of apoptosis. Here, we demonstrate the cytotoxic efficacy of shikonin in vivo in xenograft models, which did not affect body weight as well as its reduction of cell viability in vitro using several non-small cell lung cancer (NSCLC) cell lines. We found that inhibition of AKT by shikonin activated the forkhead box (FOX)O3a/early growth response protein (EGR)1 signaling cascade and enhanced the expression of the target gene Bim, leading to apoptosis in lung cancer cells. Overexpression of wild-type or a constitutively active mutant of FOXO3a enhanced shikonin-induced Bim expression. The NAD+-dependent histone deacetylase sirtuin (SIRT)1 amplified the pro-apoptotic effect by deacetylating FOXO3a, which induced EGR1 binding to the Bim promoter and activated Bim expression. Meanwhile, PI3K/AKT activity was enhanced, whereas that of FOXO3a was reduced and p300 was upregulated by treatment with a sublethal dose of shikonin. FOXO3a acetylation was enhanced by p300 overexpression, while shikonin-induced Bim expression was suppressed by p300 overexpression, which promoted cell survival. FOXO3a acetylation was increased by p300 overexpression and treatment with SIRT1 inhibitor, improving cell survival. In addition, shikonin-induced FOXO3a nuclear localization was blocked by AKT activation and SIRT1 inhibition, which blocked Bim expression and conferred resistance to the cytotoxic effects of shikonin. The EGR1 increase induced by shikonin was restored by pretreatment with SIRT1 inhibitor. These results suggest that shikonin induces apoptosis in some lung cancer cells via activation of FOXO3a/EGR1/SIRT1 signaling, and that AKT and p300 negatively regulate this process via Bim upregulation.

Cdk5-Foxo3 axis: initially neuroprotective, eventually neurodegenerative in Alzheimer's disease models.

Deregulated Cdk5 causes neurotoxic amyloid beta peptide (Aβ) processing and cell death, two hallmarks of Alzheimer's disease, through the Foxo3 transcriptional factor in hippocampal cells, primary neurons and an Alzheimer's disease mouse model. Using an innovative chemical genetic screen, we identified Foxo3 as a direct substrate of Cdk5 in brain lysates. Cdk5 directly phosphorylates Foxo3, which increased its levels and nuclear translocation. Nuclear Foxo3 initially rescued cells from ensuing oxidative stress by upregulating MnSOD (also known as SOD2). However, following prolonged exposure, Foxo3 upregulated Bim (also known as BCL2L11) and FasL (also known as FASLG) causing cell death. Active Foxo3 also increased Aβ(1-42) levels in a phosphorylation-dependent manner. These events were completely inhibited either by expressing phosphorylation-resistant Foxo3 or by depleting Cdk5 or Foxo3, highlighting a key role for Cdk5 in regulating Foxo3. These results were confirmed in an Alzheimer's disease mouse model, which exhibited increased levels and nuclear localization of Foxo3 in hippocampal neurons, which preceded neurodegeneration and Aβ plaque formation, indicating this phenomenon is an early event in Alzheimer's disease pathogenesis. Collectively, these results show that Cdk5-mediated phospho-regulation of Foxo3 can activate several genes that promote neuronal death and aberrant Aβ processing, thereby contributing to the progression of neurodegenerative pathologies.

Abstract 2661: Bilateral blockade of MEK- and PI3K-driven pathways is effective in the treatment of KAS mutant mucinous colorectal cancer cells

Abstract Purpose. A significant proportion of sporadic colorectal cancers (CRCs) (10-15%) are of the mucinous subtype (&amp;gt;50% of the tumor). Mucinous colorectal adenocarcinomas (MCAs) are clinically, morphologically and molecularly distinct from nonmucinous CRCs as they show a different spectrum of genetic alterations (higher KRAS mutations, MSI-H, lower p53, high MUC2) and exhibit more aggressive behavior (more prone to peritoneal dissemination and lymph node involvement). MCAs are associated with poorer response to chemotherapy and have limited treatment options. The purpose of this study was to test the effectiveness of combinatorial targeting of two KRAS-associated parallel pathways in an attempt to counter adaptive resistance associated with single pathway inhibition. Methods. Two KRAS mutant MCA cell lines LS174T and RW7213 were treated with two small molecule inhibitors, GDC0973 and GDC0941, to block mitogen-activated extracellular regulated kinase kinase (MEK) and phosphoinositide 3-kinase (PI3K) respectively. Adaptive resistance of these two cell lines to the PI3K inhibitor (PI3Ki) was examined by protein phospho-receptor tyrosine kinase (RTK) arrays, western blots and immunocytochemistry. Results. Bioinformatic analysis of the colon cancer datasets in the cancer genome atlas (TCGA) showed increased mutation rates in mucinous over nonmucinous CRCs for effectors of RAS-RAF-MEK-ERK (80% versus 41.9%) and PI3K-AKT-mTOR (60% versus 21.8%) pathways. Both MCA cell lines were sensitive to MEK inhibitor (MEKi) treatment. In contrast, these cell lines, though initially sensitive to PI3Ki, later became resistant. Concomitant with the development of resistance were a) increase in phosphorylated RTKs including IR, IGF-1R, EGFR, ErbB2 b) increase in phosphorylated ERK and AKT, c) nuclear translocation of the transcription factor FOXO3A and d) a decrease in proapoptotic protein BIM. Combinatorial treatment of PI3Ki and MEKi synergistically reduced viability of MCA tumor cells with attendant decrease in phosphorylated ERK and AKT and increase in BIM. Conclusions. Our data suggest cellular dependence (‘addiction’) of KRAS-mutant mucinous CRC cells on hyperactivation of MEK and PI3K pathways. Interestingly, PI3K single agent inhibition initially triggers pathway downregulation and reduced tumor cell viability but later leads to development of adaptive resistance. Our studies suggest this resistance involves dynamic rewiring of signaling circuits mediated through FOXO3A nuclear localization, relief of RTK inhibition, MEK-ERK activation and BIM reduction. Adaptive resistance however, can be overcome by co-targeting of PI3K and MEK. Our studies thus provide a rational basis for MEKi and PI3Ki combination therapy for KRAS mutant CRCs. Citation Format: Murali R. Kuracha, Peter Thomas, Brian W. Loggie, Venkatesh Govindarajan. Bilateral blockade of MEK- and PI3K-driven pathways is effective in the treatment of KAS mutant mucinous colorectal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2661. doi:10.1158/1538-7445.AM2015-2661

Enhanced Autophagy Promotes Survival of Peripheral Blast Cells from Murine Acute Myeloid Leukemia

Intensive chemotherapy for the treatment of leukemias inevitably provides cellular and metabolic stress to leukemic cells, leading to programmed cell death. In addition, anti-apoptotic property of leukemic cells could easily induce autophagy in response to anti-leukemic treatments and upregulation of autophagy is likely to contribute to the survival of leukemic cells by driving therapy resistance. Given that bone marrow (BM) supports leukemic cell proliferation by various types of stimuli from stromal cells or leukemic cells themselves, it is supposed that BM-occupying leukemic cells and circulating peripheral leukemic cells would have distinct difference in autophagic activity. However, comprehensive understanding of autophagic activity in leukemic cells has not been achieved so far. Here in this study, autophagic activities of leukemic cells in BM and peripheral blood (PB) from murine acute myeloid leukemia (AML) model driven by MLL-ENL fusion gene were evaluated. In the PB and BM from MLL-ENL AML mice, both mature (CD11b+c-Kit-) and immature (CD11b+c-Kit+) MLL-ENL+ leukemic cells showed no difference in apoptotic status by Annexin-V/DAPI staining regardless of c-Kit expression. By contrast, from cell cycle analysis, c-Kit- leukemic cells in the BM were found to have higher frequency of S/G2 phase than PB counterparts, indicating the proliferative potential of BM leukemic cells. When molecules of intracellular signaling pathways regarding proliferation and survival were assessed, components of MAPK and PI3K-mTOR signaling pathways such as Erk1/2, Akt, S6K, and S6 were highly phosphorylated in c-Kit- BM AML cells compared to c-Kit- PB AML cells, implying the importance of activated cytokine signaling in the BM of MLL-ENL AML. For the autophagic evaluation of PB and BM AML cells, MLL-ENL fusion gene was introduced into autophagy sensor mice, GFP-LC3 transgenic mice, enabling us to check autophagy by GFP. PB AML cells from these mice showed decreased GFP intensity compared to BM counterparts, which meant more activated degradation of autophagosome in peripheral AML cells. By western blotting analysis, PB AML cells had the enhanced conversion of LC3A-I to LC3A-II, indicating activation of autophagy. Furthermore, activation of stress responsive pathways such as nuclear localization of Foxo3a and enhanced phosphorylation of eIF2a was observed in c-Kit- PB AML cells. Interpretation of microarray data using public database (GSE9476 and GSE34577) comparing PB and BM cells from clinical AML samples revealed that PB AML cells have significantly higher expression of amino acid transporters. From these results, it is strongly suggested that peripheral AML cells have enhanced autophagic activity in vivo. To elucidate the functional role of activated autophagy in PB AML cells, MLL-ENL fusion gene was introduced into conditional Atg5 or Atg7 knockout mice (Atg5flox/flox or Atg7flox/flox), both of which are essential for autophagy. Interestingly, genetic deletion of Atg5 or Atg7 in irradiated recipients transplanted with Atg5flox/flox or Atg7flox/flox AML cells caused significant increase of apoptotic cells in peripheral leukemic cells and significant decrease of peripheral white blood cell (WBC) counts and donor cell engraftment in PB, whereas Atg5Δ/Δ or Atg7Δ/Δ MLL-ENL AML mice had neither prolonged survival nor reduced leukemia-initiating capacity in serial transplants. Similar to MLL-ENL AML model, genetic deletion of Atg7 in advanced phase of chronic myeloid leukemia (CML) induced by BCR-ABL plus NUP98-HOXA9 fusion genes caused decreased WBC counts and increased apoptosis in peripheral leukemic cells, indicating that circulating leukemic cells favor autophagy for their survival. It is of note that Atg7 deletion had no impact on apoptosis in normal CD11b+ myeloid fraction, implying that loss of Atg7 could spare normal myeloid cells. It remains to be seen what soluble factors could support the survival of AML cells in BM by suppressing autophagy, but our preclinical data provides the therapeutic potential of autophagy inhibition in the treatment of excessive peripheral leukocytosis. It is also highly expected that inhibition of autophagy could be more effective for BM AML cells when combined with chemotherapeutic agent to activate autophagy. DisclosuresSumitomo:Kyowa Hakko Kirin Co., Ltd.: Employment. Kurokawa:Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; SHIONOGI & CO., Ltd.: Consultancy; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Celgene K.K.: Consultancy, Speakers Bureau; Bristol-Myers Squibb Company: Consultancy, Research Funding, Speakers Bureau; Sanofi K.K.: Consultancy; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Astellas Pharma Inc., : Research Funding, Speakers Bureau; Dainippon Sumitomo Pharma Co.,Ltd.: Research Funding, Speakers Bureau; Asahi Kasei Co.: Research Funding, Speakers Bureau; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; MSD K.K.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Co.,Ltd.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; TAIHO PHARMACEUTICAL CO., LTD.: Research Funding; Teijin Pharma Limited: Research Funding; Alexion Pharmaceuticals K.K.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Mitsubishi Tanabe Pharma Corporation: Speakers Bureau; GlaxoSmithKline K.K.: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; Yakult Pharmaceutical Industry Co., Ltd.: Speakers Bureau; Ono Pharmaceutical Co.,Ltd.: Speakers Bureau; Miraca Holdings Inc.: Speakers Bureau; CSL Behring K.K.: Speakers Bureau.

FoxO3a nuclear localization and its association with β-catenin and Smads in IFN-α-treated hepatocellular carcinoma cell lines.

Interferon-α2b (IFN-α2b) reduces proliferation and increases apoptosis in hepatocellular carcinoma cells by decreasing β-catenin/TCF4/Smads interaction. Forkhead box O-class 3a (FoxO3a) participates in proliferation and apoptosis and interacts with β-catenin and Smads. FoxO3a is inhibited by Akt, IκB kinase β (IKKβ), and extracellular-signal-regulated kinase (Erk), which promote FoxO3a sequestration in the cytosol, and accumulates in the nucleus upon phosphorylation by c-Jun N-terminal kinase (JNK) and p38 mitogen-activated kinase (p38 MAPK). We analyzed FoxO3a subcellular localization, the participating kinases, FoxO3a/β-catenin/Smads association, and FoxO3a target gene expression in IFN-α2b-stimulated HepG2/C3A and Huh7 cells. Total FoxO3a and Akt-phosphorylated FoxO3a levels decreased in the cytosol, whereas total FoxO3a levels increased in the nucleus upon IFN-α2b stimulus. IFN-α2b reduced Akt, IKKβ, and Erk activation, and increased JNK and p38 MAPK activation. p38 MAPK inhibition blocked IFN-α2b-induced FoxO3a nuclear localization. IFN-α2b enhanced FoxO3a association with β-catenin and Smad2/3/7. Two-step coimmunoprecipitation experiments suggest that these proteins coexist in the same complex. The expression of several FoxO3a target genes increased with IFN-α2b. FoxO3a knockdown prevented the induction of these genes, suggesting that FoxO3a acts as mediator of IFN-α2b action. Results suggest a β-catenin/Smads switch from TCF4 to FoxO3a. Such events would contribute to the IFN-α2b-mediated effects on cellular proliferation and apoptosis. These results demonstrate new mechanisms for IFN-α action, showing the importance of its application in antitumorigenic therapies.

ARHI (DIRAS3) induces autophagy in ovarian cancer cells by downregulating the epidermal growth factor receptor, inhibiting PI3K and Ras/MAP signaling and activating the FOXo3a-mediated induction of Rab7.

The process of autophagy has been described in detail at the molecular level in normal cells, but less is known of its regulation in cancer cells. Aplasia Ras homolog member I (ARHI; DIRAS3) is an imprinted tumor suppressor gene that is downregulated in multiple malignancies including ovarian cancer. Re-expression of ARHI slows proliferation, inhibits motility, induces autophagy and produces tumor dormancy. Our previous studies have implicated autophagy in the survival of dormant ovarian cancer cells and have shown that ARHI is required for autophagy induced by starvation or rapamycin treatment. Re-expression of ARHI in ovarian cancer cells blocks signaling through the PI3K and Ras/MAP pathways, which, in turn, downregulates mTOR and initiates autophagy. Here we show that ARHI is required for autophagy-meditated cancer cell arrest and ARHI inhibits signaling through PI3K/AKT and Ras/MAP by enhancing internalization and degradation of the epidermal growth factor receptor. ARHI-mediated downregulation of PI3K/AKT and Ras/ERK signaling also decreases phosphorylation of FOXo3a, which sequesters this transcription factor in the nucleus. Nuclear retention of FOXo3a induces ATG4 and MAP-LC3-I, required for maturation of autophagosomes, and also increases the expression of Rab7, required for fusion of autophagosomes with lysosomes. Following the knockdown of FOXo3a or Rab7, autophagolysosome formation was observed but was markedly inhibited, resulting in numerous enlarged autophagosomes. ARHI expression correlates with LC3 expression and FOXo3a nuclear localization in surgical specimens of ovarian cancer. Thus, ARHI contributes to the induction of autophagy through multiple mechanisms in ovarian cancer cells.

The PI3K/AKT Pathway and FOXO3a Transcription Factor Mediate High Glucose-Induced Apoptosis in Neonatal Rat Ventricular Myocytes.

Background:PI3K/AKT pathway plays major roles in regulating cardiomyocyte metabolism. The roles of PI3K/AKT pathway and FOXO3a in mediating high glucose-induced apoptosis in cardiomyocytes remain unclear.Objectives:In this experimental study, we investigated the mechanisms of the PI3K/AKT pathway and FOXO3a in mediating hyperglycemia-induced apoptosis in neonatal rat ventricular myocytes (NRVMs).Materials and Methods:NRVMs were adopted as the cell model to investigate the roles of PI3K/AKT and FOXO3a in mediating hyperglycemia-induced apoptosis in cardiomyocytes. Annexin-V-FITC staining and PI staining were used to evaluate the apoptosis in NRVMs under indicated conditions of serum starvation, high glucose exposure, and pharmacological or genetic manipulations on the expressions of PI3K/AKT and FOXO3a. Western blotting was conducted to evaluate the cytoplasmic/nuclear localization of FOXO3a in NRVMs exposed to high glucose. FOXO3a transcriptional activity was measured by luciferase reporter assay.Results:High glucose (30 mM) induced significant apoptosis in serum-starved NRVMs as compared with normal glucose (5 mM) control (12.01 ± 0.76% vs. 2.86 ± 0.55%; P < 0.001). Treatment with IGF1 attenuated hyperglycemia-induced apoptosis by 68% (3.23 ± 0.76% vs. 9.97 ± 1.29%; P < 0.001; n = 3) in comparison with the non-treated control. Treatment with PI3K inhibitor LY294002 enhanced hyperglycemia-induced apoptosis by 109% (20.83 ± 1.87% vs. 9.97 ± 1.29%; P < 0.001; n = 3) in comparison with the non-treated control. Over-expression of AKT by transduction with CA-AKT attenuated hyperglycemia-induced apoptosis by 47% (5.48 ± 0.35% vs.10.31 ± 0.94%; P < 0.001; n = 3) in comparison with the empty-vector control. Transduction with DN-AKT enhanced high glucose-induced apoptosis by 105% (21.13 ± 1.11% vs. 10.31 ± 0.94%; P < 0.001; n = 3) in comparison with the empty-vector control. Western blotting showed that high glucose induced a significant increase in FOXO3a nuclear localization. Luciferase reporter assay showed that high glucose induced a significant increase of 310% (P < 0.001; n = 3) in FOXO3a transcriptional activity against Fas ligand when NRVMs were transducted with TM-FOXO3a in comparison with the empty-vector control.Conclusions:The PI3K/AKT pathway mediated hyperglycemia-induced apoptosis of NRVMs through the translocation of FOXO3a to nuclei and the resultant enhanced transcriptional activity of FOXO3.

A RXR ligand 6-OH-11-O-hydroxyphenanthrene with antitumour properties enhances (-)-epigallocatechin-3-gallate activity in three human breast carcinoma cell lines.

(−)-Epigallocatechin-3-gallate (EGCG) and chemotherapeutic agents cotreatment can improve cytotoxicity against cancer cells. We showed that EGCG and the rexinoid 6-OH-11-O-hydroxyphenanthrene (IIF), given together, were cytotoxic toward MCF-7, MCF-7TAM, and MDA-MB-231, three breast carcinoma cell lines showing different molecular characteristics. Cell growth arrest and apoptosis were greater after EGCG and IIF cotreatment than after individual administration. Cytotoxicity was related to upregulation of 67-kDa laminin receptor (LR67), one of the principal molecular targets of EGCG, and activation of the nuclear retinoic X receptors (RXRs) pathway. Furthermore, the transcription factor Forkhead box O3 (Foxo3a), a protein able to trigger apoptosis through upregulation of genes necessary for cell death, was activated. EGCG and IIF cotreatment produced a significant nuclear import of Foxo3a from the cytoplasm in MCF-7, MCF-7TAM, and MDA-MB-231 cells. In MCF-7TAM cells only, Foxo3a nuclear localization was associated with p473AKT downregulation. For the first time we showed that when EGCG and IIF, two harmless molecules, were given together, they might increase cytotoxicity in three breast carcinoma cell lines, two of them being representative of poorly responsive breast carcinoma types.