FOXO Target Research Articles

CGMP Signaling Increases Antioxidant Gene Expression by Activating Forkhead Box O3A in the Colon Epithelium

Signaling through cGMP has therapeutic potential in the colon, where it has been implicated in the suppression of colitis and colon cancer. In this study, we tested the ability of cGMP and type 2 cGMP-dependent protein kinase (PKG2) to activate forkhead box O (FoxO) in colon cancer cells and in the colon epithelium of mice. We show that activation of PKG2 in colon cancer cells inhibited cell proliferation, inhibited AKT, and activated FoxO. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and protein levels, and reduced epithelial reduction-oxidation (redox) stress. FoxO3a was the most prominent isoform in the distal colon epithelium, with prominent luminal staining. FoxO3a levels were reduced in Prkg2-/- animals, and FoxO target genes were unaffected by 8Br-cGMP challenge invitro. Treatment of mice with the phosphodiesterase-5 inhibitor vardenafil (Levitra) mobilized FoxO3a to the nucleus of luminal epithelial cells, which corresponded to increased FoxO target gene expression, reduced redox stress, and increased epithelial barrier integrity. Treatment of human colonic biopsy specimens with 8Br-cGMP also activated catalase and manganese superoxide dismutase expression, indicating that this pathway is conserved in humans. Taken together, these results identify a novel signaling pathway in the colon epithelium, where FoxO tumor suppressors could provide protection from redox stress. Moreover, this pathway is regulated by endogenous cGMP/PKG2 signaling, and can be targeted using phosphodiesterase-5 inhibitors.

MicroRNA mir-34 provides robustness to environmental stress response via the DAF-16 network in C. elegans.

Diverse stresses and aging alter expression levels of microRNAs, suggesting a role for these posttranscriptional regulators of gene expression in stress modulation and longevity. Earlier studies demonstrated a central role for the miR-34 family in promoting cell cycle arrest and cell death following stress in human cells. However, the biological significance of this response was unclear. Here we show that in C. elegans mir-34 upregulation is necessary for developmental arrest, correct morphogenesis, and adaptation to a lower metabolic state to protect animals against stress-related damage. Either deletion or overexpression of mir-34 lead to an impaired stress response, which can largely be explained by perturbations in DAF-16/FOXO target gene expression. We demonstrate that mir-34 expression is regulated by the insulin signaling pathway via a negative feedback loop between miR-34 and DAF-16/FOXO. We propose that mir-34 provides robustness to stress response programs by controlling noise in the DAF-16/FOXO-regulated gene network.

Allium Roseum L. Extract Exerts Potent Suppressive Activities on Chronic Myeloid Leukemia K562 Cell Viability Through the Inhibition of BCR-ABL, PI3K/Akt, and ERK1/2 Pathways and the Abrogation of VEGF Secretion

ABSTRACTUse of plant extracts, alone or combined to the current chemotherapy as chemosensitizers, has emerged as a promising strategy to overcome tumor drug resistance. Here, we investigated the anticancer activity of Allium roseum L. extracts, a wild edible species in North Africa, on human Chronic Myeloid Leukemia (CML) K562 cells. The dehydrated aqueous extract (DAE) disturbed the cell cycle progression and induced the apoptosis of K562 cells. Chemical analysis of DAE showed a diversity of organosulfur compounds S-alk(en)yl-cysteine sulfoxides (RCSO) and high amount of allicin, suggesting that such molecule may be behind its antitumor effect. DAE was efficient in inhibiting K562 cell viability. DAE inhibitory effect was associated with the dephosphorylation of the BCR-ABL kinase and interfered with ERK1/2, Akt, and STAT5 pathways. Furthermore, we found that DAE-induced inactivation of Akt kinase led to the activation of its target FOXO3 transcription factor, enhancing the expression of FOXO3-regulated proapoptotic effectors, Bim and Bax, and cell cycle inhibitor p27. Finally, we found that DAE reduced the secretion of vascular endothelial growth factor. Overall, our data suggest that A. roseum extract has great potential as a nontoxic cheap and effective alternative to conventional chemotherapy.

Pdcd2l Promotes Palmitate-Induced Pancreatic Beta-Cell Apoptosis as a FoxO1 Target Gene.

Transcription factor FoxO1 is a key regulator of the insulin-signaling pathway, and is reported to play important roles in pancreatic β cell differentiation, proliferation, apoptosis and stress resistance. The multifunctional nature of FoxO1 is due to its regulation of various downstream targets. Previous studies in our lab identified potential FoxO1 target genes using the ChIP-DSL technique and one of those genes, Pdcd2l, was selected for further study. We found that the expression of Pdcd2l was increased with palmitate treatment; the luciferase assay result revealed that enhanced Pdcd2l promoter activity was responsible for the elevation of Pdcd2l expression. ChIP-PCR was performed to confirm the combination of FoxO1 to Pdcd2l promoter, result showing that FoxO1 could bind to Pdcd2l promoter and this binding was further enhanced after palmitate treatment. Overexpression of FoxO1 significantly induced Pdcd2l promoter activity, leading to increased mRNA level; consistently, interference of FoxO1 abolished the increment of Pdcd2l gene expression triggered by palmitate treatment. In addition, overexpression of Pdcd2l could further increase the percentage of apoptotic cells induced by palmitate incubation, whilst interference of Pdcd2l partially reversed the palmitate-induced apoptosis together with activated Caspase-3, indicating that the latter may play a part in this process. Therefore, in this study, we confirmed the binding of FoxO1 to the Pdcd2l gene promoter and studied the role of Pdcd2l in β cells for the first time. Our results suggested that FoxO1 may exert its activity partially through the regulation of Pdcd2l in palmitate-induced β cell apoptosis and could help to clarify the molecular mechanisms of β cell failure in type 2 diabetes.

The BANK1 SLE-risk variants are associated with alterations in peripheral B cell signaling and development in humans

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the development of autoantibodies that drive disease pathogenesis. Genetic studies have associated nonsynonymous variants in the BANK1 B cell scaffolding gene with susceptibility to SLE and autoantibodies in lupus. To determine how the BANK1 SLE-risk variants contribute to the dysregulated B cell program in lupus, we performed genotype/phenotype studies in human B cells. Targeted phospho-proteomics were used to evaluate BCR/CD40 signaling in human B cell lines engineered to express the BANK1 risk or non-risk variant proteins. We found that phosphorylation of proximal BCR signaling molecules was reduced in B cells expressing the BANK1 risk protein compared to the non-risk protein. Similar to these findings, we observed decreased B cell signaling in primary B cells from genotyped healthy control subjects carrying the BANK1 risk haplotype, including blunted BCR- and CD40-dependent AKT activation. Consistent with decreased AKT activation, we found that BANK1 risk B cells expressed increased basal levels of FOXO1 protein and increased expression of FOXO1 target genes upon stimulation compared to non-risk B cells. Healthy subjects carrying the BANK1 risk haplotype were also characterized by an expansion of memory B cells. Taken together, our results suggest that the SLE susceptibility variants in the BANK1 gene may contribute to lupus by altering B cell signaling, increasing FOXO1 levels, and enhancing memory B cell development.

Hepatic Notch1 deletion predisposes to diabetes and steatosis via glucose-6-phosphatase and perilipin-5 upregulation

Notch signaling pathways have recently been implicated in the pathogenesis of metabolic diseases. However, the role of hepatic Notch signaling in glucose and lipid metabolism remains unclear and needs further investigation as it might be a candidate therapeutic target in metabolic diseases such as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). We used hepatocyte-specific Notch1 knockout (KO) mice and liver biopsies from NASH and NAFLD patients to analyze the role of Notch1 in glucose and lipid metabolism. Hepatocyte-specific Notch1 KO mice were fed with a high fat diet (HFD) or a regular diet (RD). We assessed the metabolic phenotype, glucose and insulin tolerance tests, and liver histology. Hepatic mRNA expression was profiled by Affymetrix Mouse Gene arrays and validated by quantitative reverse transcription PCR (qPCR). Akt phosphorylation was visualized by immunoblotting. Gene expression was analyzed in liver biopsies from NASH, NAFLD, and control patients by qPCR. We found that Notch1 KO mice had elevated fasting glucose. Gene expression analysis showed an upregulation of glucose-6-phosphatase, involved in the final step of gluconeogenesis and glucose release from glycogenolysis, and perilipin-5, a regulator of hepatic lipid accumulation. When fed with an HFD KO mice developed overt diabetes and hepatic steatosis. Akt was highly phosphorylated in KO animals and the Foxo1 target gene expression was altered. Accordingly, a reduction in Notch1 and increase in glucose-6-phosphatase and perilipin-5 expression was observed in liver biopsies from NAFLD/NASH compared with controls. Notch1 is a regulator of hepatic glucose and lipid homeostasis. Hepatic impairment of Notch1 expression may be involved in the pathogenesis of human NAFLD/NASH.

FAM65B controls the proliferation of transformed and primary T cells.

Cell quiescence is controlled by regulated genome-encoded programs that actively express genes which are often down-regulated or inactivated in transformed cells. Among them is FoxO1, a transcription factor that imposes quiescence in several cell types, including T lymphocytes. In these cells, the FAM65B encoding gene is a major target of FOXO1. Here, we show that forced expression of FAM65B in transformed cells blocks their mitosis because of a defect of the mitotic spindle, leading to G2 cell cycle arrest and apoptosis. Upon cell proliferation arrest, FAM65B is engaged in a complex containing two proteins well known to be involved in cell proliferation i.e. the HDAC6 deacetylase and the 14.3.3 scaffolding protein. In primary T cells, FAM65B is down-regulated upon T cell receptor engagement, and maintaining its expression blocks their proliferation, establishing that the decrease of FAM65B expression is required for proliferation. Conversely, inhibiting FAM65B expression in naive T lymphocytes decreases their activation threshold. These results identify FAM65B as a potential new target for controlling proliferation of both transformed and normal cells.

Abstract 2009: Role of FOXO1 in oncogenic program of B cell precursor acute lymphoblastic leukemia (BCP-ALL)

Abstract Although treatment of B cell precursor acute lymphoblastic leukemia (BCP-ALL) in children has shown eminent progress over the past decades, about 15% of patients can still not be cured, however. Therefore, new therapeutic strategies are warranted. FOXO1 together with other transcription factors including EBF1, PAX5 and TCF3 plays a central role in differentiation of pre-B-cells. In cooperation with PAX5 and other B-cell transcription factors, FOXO1 induces expression of SYK, BLNK, RAG1, RAG2, AICDA and IL7RA genes. Of note, SYK and IL7RA signaling contribute to the oncogenic program of BCP-ALL, which utilize pre-B cell survival and proliferation program. In this study, we demonstrate that FOXO1 is highly expressed in most of the BCP-ALL cell lines. By using immunofluorescence assays we show that FOXO1 is localized in the nucleus of BCP-ALL cells. Overexpression of constitutively active AKT1 (inactivating FOXO1) negatively regulates the growth of BCP-ALL cells. Inhibition of FOXO1 by shRNA results in cell death in the RS4;11 BCP-ALL cell line. BCP-ALL cells are more sensitive to growth arrest and apoptosis induced by a small molecular FOXO1 inhibitor AS1842856 than other classical Hodgkin lymphoma and non-Hodgkin lymphoma cell lines. Gene expression profiling of BCP-ALL cells after treatment with the FOXO1 inhibitor for 24h shows the significantly down-regulation of FOXO target genes including VPREB3, IL1B, KLHL24 and BNIP3L. Interestingly, at the same time, some FOXO targets like NOXA, RUNX2 and PRDM1 were found upregulated, indicating a complex effect of the FOXO1 inhibitor. We conclude that FOXO1 expression is critical for the BCP-ALL oncogenic program and its targeting represents a novel therapeutic approach. Citation Format: Fan Wang, Alexey Ushmorov, Thomas Wirth. Role of FOXO1 in oncogenic program of B cell precursor acute lymphoblastic leukemia (BCP-ALL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2009.

Abstract 1017: Pro-inflammatory cytokine secretion and gene networks associate with pancreatic cancer induced cachexia

Abstract Introduction: Pancreatic cancer (PC) is associated with a high rate of cachexia, which specifically diminishes quality of life, prohibits effective therapies, and subsequently contributes to morbidity and mortality. Unfortunately, mechanisms underlying this cancer-induced muscle wasting in the human disease remain incompletely described, in part due to limited translational models. Therefore, we hypothesize that the development of more representative models of PC cachexia will allow for development of therapeutic targets for cachexia. To test our hypothesis, we propose to 1) establish the first patient-derived xenograft (PDX) cancer cachexia models to identify the muscle associated with PC induced cachexia and 2) support these results by examining the corresponding skeletal muscle of PC patients whom contributed to the PDX models. Methods: Rectus abdominis muscle was biopsied from surgically resected PC patients and matched non-cancer controls. After surgical harvest of PC specimen, PDX models were derived in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice and skeletal muscle was subsequently harvested for histologic investigations on ultrastructural disorganization and qRT-PCR for atrophy-related transcription factors differentially regulated in PC patients. Systemic cytokine expression profiles were analyzed with luminex technology and confirmed by ELISA. Results: Rectus biopsies from patients with resected PC displayed marked muscle fiber atrophy, increased extracellular space, greater variation in fiber size and shape and more centralized nuclei compared to controls. These architectural abnormalities were also present in mice bearing xenografts from corresponding PC patients. Despite the absence of an adaptive immune system, PDX mice demonstrated high levels of systemic TNFα, IL-1β, IL6 and KC (IL8) with a concomitant decrease in anti-inflammatory cytokine IL10 when compared to matched controls. Further, skeletal muscle from both patients with PC and mice bearing PDX tumors demonstrated increased expression of the Forkhead boxO1 (FoxO1) transcription factor and FoxO target gene and E3 ubiquitin ligase, MuRF1, both of which have been directly implicated in the regulation of muscle mass. Conclusions: Preoperative muscle wasting in PC is associated with characteristic architectural abnormalities and elevated FoxO1-MuRF1 levels. Mice bearing PDX demonstrate comparable elevations in circulating pro-inflammatory cytokines, muscle pathology and FoxO1-MuRF1 levels. These results provide a valid translational model of cachexia which suggests a central role for FoxO1 and MuRF1 in PC-associated muscle wasting. Citation Format: Daniel Delitto, Sarah M. Judge, Rachel L. Nosacka, Andrea Knowlton, Fernanda G. Rocha, Kevin E. Behrns, Steven J. Hughes, Shannon M. Wallet, Andrew R. Judge, Jose G. Treviño. Pro-inflammatory cytokine secretion and gene networks associate with pancreatic cancer induced cachexia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1017.

Abstract 4661: Loss of LKB1 in NSCLC confers sensitivity to MEK inhibition by regulating activation of AKT-FOXO3 pathway

Abstract The use of MEK inhibitors for non-small cell lung cancer (NSCLC) has shown little efficacy in clinical trials, even in the case of tumors with mutant KRAS where one might predict good outcomes. From these data, it is clear that the success of MEK inhibitors is going to rely on finding a biomarker that predicts sensitivity to this type of therapy. Our area of interest is finding a way to treat LKB1 mutant tumors and, surprisingly, an in silico screen of drug sensitivity data for NSCLC cell lines determined that four MEK inhibitors were among the top drugs that were significantly associated with LKB1 loss. We confirmed the effects MEK inhibition by evaluating 23 lung cancer cell lines with known LKB1 status. The results of the study showed that MEK inhibition with trametinib led to a significant reduction in cell viability in LKB1 mutant cell lines when compared to cell lines with wild type LKB1. In addition, we investigated MEK sensitivity by restoring wild-type (Wt) LKB1 in lung cancer cell lines with LKB1 loss, or by silencing LKB1 in lung cancer cell LKB1-Wt lines. Transduction of LKB1 resulted in significant MEK resistance in three of the five LKB1 add-back lines, while silencing LKB1 induced MEK sensitivity in all five LKB1-Wt lines tested. The mechanism behind these observed results appears to be through phosphorylation of AKT and its downstream target FOXO3, which are important determinants of the apoptotic response to MEK inhibition. With LKB1 transduction into mutant cell lines we see an increase in the activating phosphorylation of AKT, a protein involved in survival mechanisms, and an increase in the deactivating phosphorylation of FOXO3, a transcription factor implicated in increased levels of apoptosis. Our data suggest that the identification of LKB1 activity may be promising biomarker for the sensitivity to MEK inhibition by regulating activation of AKT-FOXO3 pathway in NSCLC. Citation Format: Tadaaki Yamada, Jacob M. Kaufman, Joseph M. Amann, Seiji Yano, David P. Carbone. Loss of LKB1 in NSCLC confers sensitivity to MEK inhibition by regulating activation of AKT-FOXO3 pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4661.

Forkhead box O member FOXO1 regulates the majority of follicle-stimulating hormone responsive genes in ovarian granulosa cells

FSH promotes maturation of ovarian follicles. One pathway activated by FSH in granulosa cells (GCs) is phosphatidylinositol-3 kinase/AKT. The AKT target FOXO1 is reported to function primarily as a repressor of FSH genes, including Ccnd2 and Inha. Based on its broad functions in other tissues, we hypothesized that FOXO1 may regulate many more GC genes. We transduced GCs with empty adenovirus or constitutively active FOXO1 followed by treatment with FSH for 24 h, and conducted RNA deep sequencing. Results show that FSH regulates 3772 genes ≥2.0-fold; 60% of these genes are activated or repressed by FOXO1. Pathway Studio Analysis revealed enrichment of genes repressed by FOXO1 in metabolism, signaling, transport, development, and activated by FOXO1 in signaling, cytoskeletal functions, and apoptosis. Gene regulation was verified by q-PCR (eight genes) and ChIP analysis (two genes). We conclude that FOXO1 regulates the majority of FSH target genes in GCs.

Skin expression of mammalian target of rapamycin and forkhead box transcription factor O1, and serum insulin-like growth factor-1 in patients with acne vulgaris and their relationship with diet.

Acne vulgaris is a multifactorial disorder of the pilosebaceous units. Several studies have reported that insulin-like growth factor (IGF)-1, forkhead box transcription factor (Fox)O1 and mammalian target of rapamycin (mTOR) interactions may be the key to understanding the links between genetic and environmental factors in acne vulgaris. To evaluate the immunohistochemical detection of mTOR and FoxO1 in the skin, and the serum level of IGF-1 in patients with acne vulgaris. This study was carried out on 60 participants, including 40 patients with acne and 20 controls. A diet questionnaire was administered to the patients and controls. Serum levels of IGF-1 were measured using enzyme-linked immunosorbent assay, and skin biopsies were taken from lesions on the backs of the patients and controls. FoxO1 and mTOR expression was detected using immunohistochemistry. A significantly higher serum IGF-1 level was found in the patients with acne than in the controls. The cytoplasmic expression of FoxO1 was found to be significantly greater in the acne group, whereas in the control subjects this expression was likely to be nuclear. Both the cytoplasmic expression and the nuclear expression of mTOR were significantly more intense in the patients with acne than in the controls. Excess consumption of a high-glycaemic-load diet was significantly associated with higher serum levels of IGF-1 and cytoplasmic expression of FoxO1 and mTOR. These results suggest that FoxO1, mTOR, serum IGF-1 and a high-glycaemic-load diet may play a role in acne pathogenesis.

Characterization of the direct targets of FOXO transcription factors throughout evolution.

Summary FOXO transcription factors (FOXOs) are central regulators of lifespan across species, yet they also have cell‐specific functions, including adult stem cell homeostasis and immune function. Direct targets of FOXOs have been identified genome‐wide in several species and cell types. However, whether FOXO targets are specific to cell types and species or conserved across cell types and throughout evolution remains uncharacterized. Here, we perform a meta‐analysis of direct FOXO targets across tissues and organisms, using data from mammals as well as Caenorhabditis elegans and Drosophila. We show that FOXOs bind cell type‐specific targets, which have functions related to that particular cell. Interestingly, FOXOs also share targets across different tissues in mammals, and the function and even the identity of these shared mammalian targets are conserved in invertebrates. Evolutionarily conserved targets show enrichment for growth factor signaling, metabolism, stress resistance, and proteostasis, suggesting an ancestral, conserved role in the regulation of these processes. We also identify candidate cofactors at conserved FOXO targets that change in expression with age, including CREB and ETS family factors. This meta‐analysis provides insight into the evolution of the FOXO network and highlights downstream genes and cofactors that may be particularly important for FOXO's conserved function in adult homeostasis and longevity.

FoxO1 regulates apoptosis induced by asbestos in the MT-2 human T-cell line

Asbestos is known to cause malignant mesothelioma and lung cancer. Recent studies implicate tumor immunity in the development of various tumors, including malignant mesothelioma. In order to establish an in vitro T-cell model to clarify the effects of long-term exposure of asbestos on tumor immunity, in this study, human T-cell line MT-2 cells were cultured with asbestos for longer than 8 months and the resultant cells (MT-2Rst) were assessed for the expression of forkhead transcription factor FoxO1. Gene expression analysis revealed that the amount of FoxO1 mRNA decreased after long-term exposure of the MT-2 cells to asbestos. In accordance with this reduction in FoxO1, pro-apoptotic Foxo1 target genes Puma, Fas ligand and Bim were also seen to be down-regulated in MT-2Rst cells. Furthermore, shRNA-mediated knock-down of FoxO1 reduced the number of apoptotic parental MT-2 cells after treatment with asbestos. On the other hand, over-expression of FoxO1 did not affect asbestos-induced apoptosis in MT-2Rst cells. These results suggested that FoxO1 played an important role in regulating asbestos-induced apoptosis and confirmed the presence of multiple pathways regulating resistance to asbestos in MT-2Rst cells.

GSK3β Modulates Excess Nutrient‐Induced Autophagy Impairment in Human Aortic Endothelial Cells via FOXO1

Dysregulated autophagy, the process by which damaged proteins and organelles are turned over, and decreased AMP‐activated protein kinase (AMPK) activity are each associated with type 2 diabetes and atherogenesis. In a human aortic endothelial cell (HAEC) model of poorly‐controlled type 2 diabetes, we observed that the same concentrations of excess nutrients that increased inflammation and apoptosis also decreased AMPK activity and autophagy. Our current objective was to determine whether glycogen synthase kinase 3β (GSK3β), an enzyme whose hyperactivity has been associated with diabetes, atherogenesis and inhibition of AMPK activity, attenuates autophagy and AMPK activity in nutrient‐laden HAECs.AMPK activity was measured by protein levels of p‐AMPKThr172 and the SAMS peptide assay. GSK3β activity was measured by protein levels of p‐GSK3βSer9. GSK3β activity was increased by treating cells with Akt inhibitor VIII (Akti) and GSK3β activity was suppressed by infecting HAECs with lentivirus knocking down GSK3β (shGSK3β) or expressing the kinase‐dead (K85A) mutant. Changes in Lysotracker staining reflected changes in lysosome acidity. Autophagosome formation was evaluated by measuring bafilomycin‐induced changes in protein levels of LC3‐II, by both Western blot and immunofluorescence. P‐FOXO1Thr24 and mRNA levels of PI3KC3 and SOD2, FOXO1 target genes, were used to evaluate FOXO1 signaling.A combination of 25 mM glucose and 0.4 mM palmitate (excess nutrient conditions) inhibited basal autophagy and AMPK activity and also increased GSK3β activity. Treatment of HAECs with Akti decreased Lysotracker staining under both control and excess nutrient conditions. Suppression of GSK3β activity attenuated excess nutrient‐induced losses in Lysotracker staining. Under both control and excess nutrient conditions, suppression of GSK3β activity increased LC3‐II protein levels and knockdown of GSK3β increased FOXO1 signaling.These data suggest that increases in GSK3β activity may impair autophagy in our HAEC model of poorly‐controlled type 2 diabetes by decreasing lysosome acidity and autophagosome formation. The mechanism by which GSK3β affects each of these aspects of autophagy remains to be clarified, but may involve modulation of FOXO1 signaling.Support or Funding InformationThis research was supported by NIH T32 Multidisciplinary Training in Cardiovascular Research HL007224.

Brain Insulin Signaling Is Increased in Insulin-Resistant States and Decreases in FOXOs and PGC-1α and Increases in Aβ1-40/42 and Phospho-Tau May Abet Alzheimer Development.

Increased coexistence of Alzheimer disease (AD) and type 2 diabetes mellitus (T2DM) suggests that insulin resistance abets neurodegenerative processes, but linkage mechanisms are obscure. Here, we examined insulin signaling factors in brains of insulin-resistant high-fat–fed mice, ob/ob mice, mice with genetically impaired muscle glucose transport, and monkeys with diet-dependent long-standing obesity/T2DM. In each model, the resting/basal activities of insulin-regulated brain protein kinases, Akt and atypical protein kinase C (aPKC), were maximally increased. Moreover, Akt hyperactivation was accompanied by hyperphosphorylation of substrates glycogen synthase kinase-3β and mammalian target of rapamycin and FOXO proteins FOXO1, FOXO3A, and FOXO4 and decreased peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) expression. Akt hyperactivation was confirmed in individual neurons of anterocortical and hippocampal regions that house cognition/memory centers. Remarkably, β-amyloid (Aβ1–40/42) peptide levels were as follows: increased in the short term by insulin in normal mice, increased basally in insulin-resistant mice and monkeys, and accompanied by diminished amyloid precursor protein in monkeys. Phosphorylated tau levels were increased in ob/ob mice and T2DM monkeys. Importantly, with correction of hyperinsulinemia by inhibition of hepatic aPKC and improvement in systemic insulin resistance, brain insulin signaling normalized. As FOXOs and PGC-1α are essential for memory and long-term neuronal function and regeneration and as Aβ1–40/42 and phospho-tau may increase interneuronal plaques and intraneuronal tangles, presently observed aberrations in hyperinsulinemic states may participate in linking insulin resistance to AD.

Graded Foxo1 activity in Treg cells differentiates tumour immunity from spontaneous autoimmunity.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 have a pivotal role in maintaining immunological self-tolerance; yet, excessive Treg cell activities suppress anti-tumour immune responses. Compared to the resting Treg (rTreg) cell phenotype in secondary lymphoid organs, Treg cells in non-lymphoid tissues exhibit an activated Treg (aTreg) cell phenotype. However, the function of aTreg cells and whether their generation can be manipulated are largely unexplored. Here we show that the transcription factor Foxo1, previously demonstrated to promote Treg cell suppression of lymphoproliferative diseases, has an unexpected function in inhibiting aTreg-cell-mediated immune tolerance in mice. We find that aTreg cells turned over at a slower rate than rTreg cells, but were not locally maintained in tissues. aTreg cell differentiation was associated with repression of Foxo1-dependent gene transcription, concomitant with reduced Foxo1 expression, cytoplasmic localization and enhanced phosphorylation at the Akt sites. Treg-cell-specific expression of an Akt-insensitive Foxo1 mutant prevented downregulation of lymphoid organ homing molecules, and impeded Treg cell homing to non-lymphoid organs, causing CD8(+) T-cell-mediated autoimmune diseases. Compared to Treg cells from healthy tissues, tumour-infiltrating Treg cells downregulated Foxo1 target genes more substantially. Expression of the Foxo1 mutant at a lower dose was sufficient to deplete tumour-associated Treg cells, activate effector CD8(+) T cells, and inhibit tumour growth without inflicting autoimmunity. Thus, Foxo1 inactivation is essential for the migration of aTreg cells that have a crucial function in suppressing CD8(+) T-cell responses; and the Foxo signalling pathway in Treg cells can be titrated to break tumour immune tolerance preferentially.

Soy Isoflavone Genistein-Mediated Downregulation of miR-155 Contributes to the Anticancer Effects of Genistein

ABSTRACTWe previously reported that dietary genistein inhibits mammary tumor growth and metastasis of the highly metastatic MDA-MB-435 cancer cells in immunocompromised mice. The purpose herein was to characterize the role of the novel oncogenic microRNA (miRNA) miR-155 in the anticancer effects of genistein in metastatic breast cancer. The effect of genistein was determined on breast cancer cell viability, apoptosis, and expression of miR-155 and its targets. At low physiologically relevant concentrations, genistein inhibits cell viability and induces apoptosis in metastatic MDA-MB-435 and Hs578t breast cancer cells, without affecting the viability of nonmetastatic MCF-7 breast cancer cells. In parallel with reduced cell viability, miR-155 is downregulated, whereas proapoptotic and anticell proliferative miR-155 targets FOXO3, PTEN, casein kinase, and p27 are upregulated in MDA-MB-435 and Hs578t cells in response to genistein treatment. However, miR-155 levels remain unchanged in response to genistein in the MCF-7 cells. Ectopic expression of miR-155 in MDA-MB-435 and Hs578t cells decreases the effects of genistein on cell viability and abrogates the effects of genistein on apoptosis and expression of proapoptotic genes. Therefore, genistein-mediated downregulation of miR-155 contributes to the anticancer effects of genistein in metastatic breast cancer.

Hepatic FOXO1 Target Genes Are Co-regulated by Thyroid Hormone via RICTOR Protein Deacetylation and MTORC2-AKT Protein Inhibition

MTORC2-AKT is a key regulator of carbohydrate metabolism and insulin signaling due to its effects on FOXO1 phosphorylation. Interestingly, both FOXO1 and thyroid hormone (TH) have similar effects on carbohydrate and energy metabolism as well as overlapping transcriptional regulation of many target genes. Currently, little is known about the regulation of MTORC2-AKT or FOXO1 by TH. Accordingly, we performed hepatic transcriptome profiling in mice after FOXO1 knockdown in the absence or presence of TH, and we compared these results with hepatic FOXO1 and THRB1 (TRβ1) ChIP-Seq data. We identified a subset of TH-stimulated FOXO1 target genes that required co-regulation by FOXO1 and TH. TH activation of FOXO1 was directly linked to an increase in SIRT1-MTORC2 interaction and RICTOR deacetylation. This, in turn, led to decreased AKT and FOXO1 phosphorylation. Moreover, TH increased FOXO1 nuclear localization, DNA binding, and target gene transcription by reducing AKT-dependent FOXO1 phosphorylation in a THRB1-dependent manner. These events were associated with TH-mediated oxidative phosphorylation and NAD(+) production and suggested that downstream metabolic effects by TH can post-translationally activate other transcription factors. Our results showed that RICTOR/MTORC2-AKT can integrate convergent hormonal and metabolic signals to provide coordinated and sensitive regulation of hepatic FOXO1-target gene expression.

Fatty acid induced metabolic memory involves alterations in renal histone H3K36me2 and H3K27me3

Accumulating evidence suggest that diabetic complications persist even after the maintenance of normal glucose levels. However, the molecular mechanisms involved are still unclear. In the present study, we have investigated the molecular mechanism behind the presence of insulin resistance (IR) condition even after normalization of circulating lipids levels both in vivo and in vitro. Persistent inhibition of insulin signalling in absence of elevated circulating lipids level confirms the presence of metabolic memory in our model of IR. IR in human urine derived podocyte-like epithelial cells (HUPECs) was developed by incubating cells with palmitate (750 μM) for 24 h and in SD rats by feeding high fat diet for 16 weeks. Inhibition of insulin induced FOXO1 (regulator of gluconeogenic genes) degradation persisted even after 48 h of palmitate removal from the culture media. Metabolic memory by palmitate was found to be associated with increased FOXO1 activity as evident from increased expression of FOXO1 target genes such as PDK4, p21, G6Pc and IGFBP1. To understand the reason for prolonged activation of FOXO1 and its target genes, chromatin immuno-precipitation (ChIP) was performed with histone H3K36me2 and H3K27me3 antibodies. ChIP assay shows persistent increase in abundance of histone H3K36me2 on promoter region of FOXO1. We also show decreased abundance of histone H3K27me3 on promoter region of FOXO1, in the kidneys of HFD fed rats, which persisted even after 8 weeks of diet reversal. Taken together, we provide first evidence that circulating lipids generate metabolic memory possibly by altering the abundance of histone H3K36me2 and H3K27me3 on FOXO1 promoter.