FoxO1 Activity Research Articles

Vascular endothelial growth factor encoded by Parapoxviruses can regulate metabolism and survival of triple negative breast cancer cells

Dysbiotic microbiomes are linked to many pathological outcomes including different metabolic disorders like diabetes, atherosclerosis and even cancer. Breast cancer is the second leading cause of cancer associated death in women, and triple negative breast cancer (TNBC) is the most aggressive type with major challenges for intervention. Previous reports suggested that Parapoxvirus signatures are one of the predominant dysbiotic viral signatures in TNBC. These viruses encode several genes that are homologs of human genes. In this study, we show that the VEGF homolog encoded by Parapoxviruses, can induce cell proliferation, and alter metabolism of breast cancer and normal breast cells, through alteration of MAPK-ERK and PI3K-AKT signaling. In addition, the activity of the transcription factor FoxO1 was altered by viral-encoded VEGF through activation of the PI3K-AKT pathway, leading to reprogramming of cellular metabolic gene expression. Therefore, this study provides new insights into the function of viral-encoded VEGFs, which promoted the growth of the breast cancer cells and imparted proliferative phenotype with altered metabolism in normal breast cells.

Longevity effects of hispidol in Caenorhabditis elegans.

In this study, we investigated the longevity effects of hispidol, a 6,4'-dihydroxyaurone, using the Caenorhabditis elegans model system. Our lifespan assay data revealed that hispidol could prolong the lifespan of wild-type worms under normal culture condition. Moreover, hispidol increased the survival rate of the worms against a heat stress condition through up-regulated expressions of HSP-16.2. Similarly, hispidol protected worms from paraquat-induced oxidative stress. We also found that the hispidol elevated the activities of antioxidant enzymes, thereby attenuating the generation of intracellular reactive oxygen species. These results suggest that the enhancement of lifespan and stress resistance by the hispidol treatment might be attributed to its strong in vivo antioxidant capacity and regulation of stress proteins. Further tests on the aging-related factors revealed that hispidol could regulate the speed of pharyngeal pumping, indicating the association of dietary restriction with the hispidol-mediated longevity. However, there were no significant alterations in the body length of the worms between the groups. We then investigated the effects of hispidol on body movement and lipofuscin accumulation in aged worms. Interestingly, these healthspan parameters were strongly improved by the hispidol treatment. Our genetic studies showed no significant change in the lifespan of the daf-16 null mutants by hispidol supplementation. In addition, enhanced nuclear translocation of DAF-16 was observed in the hispidol-fed DAF-16::GFP fused transgenic mutants, suggesting the requirement of DAF-16/FOXO activation for the longevity effect of hispidol.

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

SummaryCoactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.

Up-regulation of FoxO1 contributes to adverse vascular remodelling in type 1 diabetic rats.

Vascular complications from diabetes often result in poor outcomes for patients, even after optimized interventions. Forkhead box protein O1 (FoxO1) is a key regulator of cellular metabolism and plays an important role in vessel formation and maturation. Alterations of FoxO1 occur in the cardiovascular system in diabetes, yet the role of FoxO1 in diabetic vascular complications is poorly understood. In Streptozotocin (STZ)‐induced type 1 diabetic rats, FoxO1 expression was up‐regulated in carotid arteries at 8 weeks of diabetes that was accompanied with adverse vascular remodelling characterized as increased wall thickness, carotid medial cross‐sectional area, media‐to‐lumen ratio and decreased carotid artery lumen area. This adverse vascular remodelling induced by hyperglycaemia in diabetic rats required FoxO1 activation as pharmacological inhibition of FoxO1 with 50mg/kg AS1842856 (AS) reversed vascular remodelling in type 1 diabetic rats. The adverse vascular remodelling in type 1 diabetes mellitus (T1DM) occurred concomitantly with increases in pro‐inflammatory factors, adhesion factors, apoptosis, NOD‐like receptor family protein‐3 inflammasome activation and the phenotypic switch of arterial smooth muscle cells, which were all reversed by AS. In addition, FoxO1 inhibition counteracted the down‐regulation of its upstream mediator PDK1 in T1DM. PDK1 activator reduced FoxO1 nuclear translocation, which serves as the basis for subsequent transcriptional regulation during hyperglycaemia. Taken together, our data suggest that FoxO1 is a critical trigger for type 1 diabetes‐induced vascular remodelling in rats, and inhibition of FoxO1 thus offers a potential therapeutic option for diabetes‐associated cardiovascular diseases.

S100A11 Promotes Liver Steatosis via FOXO1-Mediated Autophagy and Lipogenesis.

Background & AimsNonalcoholic fatty liver disease (NAFLD) is becoming a severe liver disorder worldwide. Autophagy plays a critical role in liver steatosis. However, the role of autophagy in NAFLD remains exclusive and under debate. In this study, we investigated the role of S100 calcium binding protein A11 (S100A11) in the pathogenesis of hepatic steatosis.MethodsWe performed liver proteomics in a well-established tree shrew model of NAFLD. The expression of S100A11 in different models of NAFLD was detected by Western blot and/or quantitative polymerase chain reaction. Liver S100A11 overexpression mice were generated by injecting a recombinant adenovirus gene transfer vector through the tail vein and then induced by a high-fat and high-cholesterol diet. Cell lines with S100a11 stable overexpression were established with a recombinant lentiviral vector. The lipid content was measured with either Bodipy staining, Oil Red O staining, gas chromatography, or a triglyceride kit. The autophagy and lipogenesis were detected in vitro and in vivo by Western blot and quantitative polymerase chain reaction. The functions of Sirtuin 1, histone deacetylase 6 (HDAC6), and FOXO1 were inhibited by specific inhibitors. The interactions between related proteins were analyzed by a co-immunoprecipitation assay and immunofluorescence analysis.ResultsThe expression of S100A11 was up-regulated significantly in a time-dependent manner in the tree shrew model of NAFLD. S100A11 expression was induced consistently in oleic acid–treated liver cells as well as the livers of mice fed a high-fat diet and NAFLD patients. Both in vitro and in vivo overexpression of S100A11 could induce hepatic lipid accumulation. Mechanistically, overexpression of S100A11 activated an autophagy and lipogenesis process through up-regulation and acetylation of the transcriptional factor FOXO1, consequently promoting lipogenesis and lipid accumulation in vitro and in vivo. Inhibition of HDAC6, a deacetylase of FOXO1, showed similar phenotypes to S100A11 overexpression in Hepa 1–6 cells. S100A11 interacted with HDAC6 to inhibit its activity, leading to the release and activation of FOXO1. Under S100A11 overexpression, the inhibition of FOXO1 and autophagy could alleviate the activated autophagy as well as up-regulated lipogenic genes. Both FOXO1 and autophagy inhibition and Dgat2 deletion could reduce liver cell lipid accumulation significantly.ConclusionsA high-fat diet promotes liver S100A11 expression, which may interact with HDAC6 to block its binding to FOXO1, releasing or increasing the acetylation of FOXO1, thus activating autophagy and lipogenesis, and accelerating lipid accumulation and liver steatosis. These findings indicate a completely novel S100A11-HDAC6-FOXO1 axis in the regulation of autophagy and liver steatosis, providing potential possibilities for the treatment of NAFLD.

The ERG-Regulated LINC00920 Promotes Prostate Cancer Cell Survival via the 14-3-3ε-FOXO Pathway.

Numerous noncoding transcripts have been reported to correlate with cancer development and progression. Nevertheless, there remains a paucity of long noncoding RNAs (lncRNA) with well-elucidated functional roles. Here, we leverage the International Cancer Genome Consortium-Early Onset Prostate Cancer transcriptome and identify the previously uncharacterized lncRNA LINC00920 to be upregulated in prostate tumors. Phenotypic characterization of LINC00920 revealed its positive impact on cellular proliferation, colony formation, and migration. We demonstrate that LINC00920 transcription is directly activated by ERG, an oncogenic transcription factor overexpressed in 50% of prostate cancers. Chromatin isolation by RNA purification-mass spectrometry revealed the interaction of LINC00920 with the 14-3-3ε protein, leading to enhanced sequestration of tumor suppressive FOXO1. Altogether, our results provide a rationale on how ERG overexpression, partly by driving LINC00920 transcription, could confer survival advantage to prostate cancer cells and potentially prime PTEN-intact prostate cells for cellular transformation through FOXO inactivation. IMPLICATIONS: The study describes a novel lncRNA-mediated mechanism of regulating the FOXO signaling pathway and provides additional insight into the role of ERG in prostate cancer cells.

Ghrelin reverses ductular reaction and hepatic fibrosis in a rodent model of cholestasis

The orexigenic peptide ghrelin (Ghr) stimulates hunger signals in the hypothalamus via growth hormone secretagogue receptor (GHS-R1a). Gastric Ghr is synthetized as a preprohormone which is proteolytically cleaved, and acylated by a membrane-bound acyl transferase (MBOAT). Circulating Ghr is reduced in cholestatic injuries, however Ghr’s role in cholestasis is poorly understood. We investigated Ghr’s effects on biliary hyperplasia and hepatic fibrosis in Mdr2-knockout (Mdr2KO) mice, a recognized model of cholestasis. Serum, stomach and liver were collected from Mdr2KO and FVBN control mice treated with Ghr, des-octanoyl-ghrelin (DG) or vehicle. Mdr2KO mice had lower expression of Ghr and MBOAT in the stomach, and lower levels of circulating Ghr compared to WT-controls. Treatment of Mdr2KO mice with Ghr improved plasma transaminases, reduced biliary and fibrosis markers. In the liver, GHS-R1a mRNA was expressed predominantly in cholangiocytes. Ghr but not DG, decreased cell proliferation via AMPK activation in cholangiocytes in vitro. AMPK inhibitors prevented Ghr-induced FOXO1 nuclear translocation and negative regulation of cell proliferation. Ghr treatment reduced ductular reaction and hepatic fibrosis in Mdr2KO mice, regulating cholangiocyte proliferation via GHS-R1a, a G-protein coupled receptor which causes increased intracellular Ca2+ and activation of AMPK and FOXO1, maintaining a low rate of cholangiocyte proliferation.

Hepatic insulin-degrading enzyme regulates glucose and insulin homeostasis in diet-induced obese mice

The insulin-degrading enzyme (IDE) is a metalloendopeptidase with a high affinity for insulin. Human genetic polymorphisms in Ide have been linked to increased risk for T2DM. In mice, hepatic Ide ablation causes glucose intolerance and insulin resistance when mice are fed a regular diet. ObjectiveThese studies were undertaken to further investigate its regulatory role in glucose homeostasis and insulin sensitivity in diet-induced obesity. MethodsTo this end, we have compared the metabolic effects of loss versus gain of IDE function in mice fed a high-fat diet (HFD). ResultsWe demonstrate that loss of IDE function in liver (L-IDE-KO mouse) exacerbates hyperinsulinemia and insulin resistance without changes in insulin clearance but in parallel to an increase in pancreatic β-cell function. Insulin resistance was associated with increased FoxO1 activation and a ~2-fold increase of GLUT2 protein levels in the liver of HFD-fed mice in response to an intraperitoneal injection of insulin. Conversely, gain of IDE function (adenoviral delivery) improves glucose tolerance and insulin sensitivity, in parallel to a reciprocal ~2-fold reduction in hepatic GLUT2 protein levels. Furthermore, in response to insulin, IDE co-immunoprecipitates with the insulin receptor in liver lysates of mice with adenoviral-mediated liver overexpression of IDE. ConclusionsWe conclude that IDE regulates hepatic insulin action and whole-body glucose metabolism in diet-induced obesity via insulin receptor levels.

Abstract P114: Foxo4 Regulates Vascular Smooth Muscle Soluble Guanylyl Cyclase Expression Expression

Soluble guanylyl cyclase (sGC) modulator drugs may offer a new approach to attenuate hypertension by enhancing vasorelaxation through production of cGMP. Loss of sGC leads to impaired vasorelaxation and hypertension, however the mechanisms that regulate sGC transcription are not fully understood. We recently published that Forkhead box subclass O (FoxO) transcription factor inhibition led to decreased sGC mRNA and protein expression and subsequent loss of downstream enzymatic sGC function and vasoreactivity in the aorta. We conducted luciferase experiments in HEK cells which identified several regions which were sensitive to FoxO inhibition and may be responsible for the regulation of sGCβ protein expression (n=4).The objective of this study was to identify which FoxO transcription factor(s) regulates sGC transcription in vascular aortic smooth muscle cells (SMCs) and at what location on the promoter. FoxO1 shRNA produced a 3-fold increase in sGCα and sGCβ mRNA expression (n=3) and a 27% elevation in sGCβ protein expression (n=6). FoxO3a shRNA produced a similar 2-fold increase in sGCα and sGCβ mRNA (n=3) and a 41% increase in sGCβ protein expression (n=6). FoxO4 shRNA, however, produced approximately 55% decrease in sGCα and sGCβ mRNA (n=3) and a similar 52% decrease in sGCβ protein expression (n=6). FoxO1 and FoxO3a shRNA treatment caused no significant change in pVASP expression at baseline or following stimulation with the nitric oxide donor molecule DEA NONOate (n=3). FoxO4 shRNA treatment resulted in a 73% decrease in pVASP expression at baseline and blunted the DEA NONOate-mediated increase in phosphorylation by 33% (n=3). Preliminary ChIP experiments in human aortic SMCs and have identified 4 sites of enriched Foxo4 binding on the human sGCβ promoter consistent with locations previously identified as sensitive to FoxO inhibitor. These data indicate FoxO4 plays a critical role in the maintenance of sGC transcription, protein expression, and downstream enzymatic function of sGC in vascular smooth muscle. Additionally, our data demonstrate that human sGCβ promoter expression requires functional FoxO activity and FoxO4 promoter binding occurs in promoter regions of human smooth muscle found to be important for promoter-luciferase expression.

Abstract 4365: Estrogen receptor β agonists reverses letrozole therapy resistance of breast cancer through FOXO3 and FOXM1 mediated actions

Abstract Introduction: Breast cancer (BC) is the most common cancer in women worldwide and leading cause of cancer death. More than 70% of cancers are estrogen receptor alpha (ERα) positive. Despite effective therapeutic strategies for treating hormone receptor-positive (HR+) breast cancer, resistance to endocrine therapy that is either de novo or acquired still occurs. Understanding the molecular pathways that contribute to BC resistance will enable to develop new treatments for improving the efficacy of endocrine therapy. Studies have shown that estrogen receptor beta (ERβ) and its agonists inhibits the different types of cancers including BC. Herein, we investigated mechanism(s) for therapeutic efficacies of ERβ agonists in breast cancer. Methods: To investigate the mechanisms of ERβ agonists (S-Equol and LY500307), in the treatment of breast cancer, we used therapy sensitive (MCF7-Aro) and letrozole resistant (MCF7-Aro-LTLT) cells and ERβ knock down cell lines, generated by CRISPR-Cas9 technology. The wild type and ERβ knock down cells with or without treatment of ERβ agonists were analyzed, for cell proliferation, protein (Western), Gene expression array analysis, RNA-seq analysis, data-independent acquisition (DIA) mass spectrometry analyses and RNA (RT-qPCR) analysis. Xenograft and patient derived BC explants (PDEX) were used to test the antiproliferative activity of ERβ agonists. Results: Our results demonstrated that treatment with ERβ agonists, S-Equol and LY500307 inhibited the growth of both endocrine therapy sensitive and resistant breast cancer cells. Gene expression array analysis of LY500307 treated breast cancer cells showed the modulation of signaling molecules involved in cell death and cell cycle pathways. RNA-seq analysis showed that treatment with S-equol modulated the ERβ target genes involved in tumor progression and resistance to hormone therapies. Mass spectrometry based DIA analysis and Reactome pathway analysis revealed that S-equol treatment modulated the key proteins involved in DNA replication and cell cycle. Both ERβ agonists S-Equol and LY500307 inhibited the cell proliferation of parental cells whereas they could not elicits the inhibitory effect on the knock out cells. By using ERα positive PDX tumors, we have demonstrated that both the ERβ agonists inhibited the proliferation of human BC tumors. Our mechanistic studies confirmed that increased FOXO3 activation by S-equol in LTLT cells can reverse letrozole resistance. Our results showed that inhibiting FOXM1 by ERβ agonists significantly reduce the proliferation of letrozole-resistant cells. These changes were not observed in ERβ knockdown cells confirming the anti-proliferative role of ERβ in breast cancer progression. Conclusions: Collectively, our results suggest that ERβ agonists may offer a new targeted therapy for endocrine therapy resistant breast tumors. (Supported by BCRP 151884/DOD grant W81XWH-16-1-0294 and P30 CA-54174 NCI-Mays cancer center grant). Citation Format: Kumaraguruparan Ramasamy, Suryavathi Viswanadhapalli, M.A. Sureshkumar, Susan Weintraub, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. Estrogen receptor β agonists reverses letrozole therapy resistance of breast cancer through FOXO3 and FOXM1 mediated actions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4365.

CAMP-dependent protein kinase inhibits FoxO activity and regulates skeletal muscle plasticity in mice.

Although we have shown that catecholamines suppress the activity of the Ubiquitin-Proteasome System (UPS) and atrophy-related genes expression through a cAMP-dependent manner in skeletal muscle from rodents, the underlying mechanisms remain unclear. Here, we report that a single injection of norepinephrine (NE; 1mgkg-1 ; s.c) attenuated the fasting-induced up-regulation of FoxO-target genes in tibialis anterior (TA) muscles by the stimulation of PKA/CREB and Akt/FoxO1 signaling pathways. In addition, muscle-specific activation of PKA by the overexpression of PKA catalytic subunit (PKAcat) suppressed FoxO reporter activity induced by (1) a wild-type; (2) a non-phosphorylatable; (3) a non-phosphorylatable and non-acetylatable forms of FoxO1 and FoxO3; (4) downregulation of FoxO protein content, and probably by (5) PGC-1α up-regulation. Consistently, the overexpression of the PKAcat inhibitor (PKI) up-regulated FoxO activity and the content of Atrogin-1 and MuRF1, as well as induced muscle fiber atrophy, the latter effect being prevented by the overexpression of a dominant negative (d. n.) form of FoxO (d.n.FoxO). The sustained overexpression of PKAcat induced fiber-type transition toward a smaller, slower, and more oxidative phenotype and improved muscle resistance to fatigue. Taken together, our data provide the first evidence that endogenous PKA activity is required to restrain the basal activity of FoxO and physiologically important to maintain skeletal muscle mass.

Hepatic activation of FOXO3 triggers positive feedback-loop for mTORC2-Akt and enhances oxidative damage-associated hepatocellular carcinogenesis

Hepatic activation of FOXO3 triggers positive feedback-loop for mTORC2-Akt and enhances oxidative damage-associated hepatocellular carcinogenesis

MicroRNA-124 Overexpression in Schwann Cells Promotes Schwann Cell-Astrocyte Integration and Inhibits Glial Scar Formation Ability.

Schwann cell (SC) transplantation is a promising approach for the treatment of spinal cord injury (SCI); however, SC grafts show a low migratory capacity within the astrocytic environment, which inevitably hampers their therapeutic efficacy. The purpose of this study was to explore mechanisms to modify the characteristics of SCs and astrocytes (ASs), as well as to adjust the SC-AS interface to break the SC-AS boundary, thus improving the benefits of SCI treatment. We observed that the expression levels of miR-124 in SCs and ASs were significantly lower than those in the normal spinal cord. Furthermore, overexpressing miR-124 in SCs (miR-124-SCs) significantly inhibited gene and protein expression levels of SC-specific markers, such as GFAP and Krox20. The expression of neurotrophic factors, Bdnf and Nt-3, was up-regulated in miR-124-SCs without affecting their proliferation. Further, the boundary assay showed an increased number of miR-124-SCs that had actively migrated and entered the astrocytic region to intermingle with ASs, compared with normal SCs. In addition, although Krox20 protein expression was down-regulated in miR-124-SCs, the luciferase assay showed that Krox20 is not a direct target of miR-124. RNA sequencing of miR-124-SCs revealed seven upregulated and eleven downregulated genes involved in cell migration and motility. Based on KEGG pathway and KOG functional analyses, changes in these genes corresponded to the activation of Hippo, FoxO, and TGF-beta signaling pathways, cytokine-cytokine receptor interactions, and the cell cycle. Finally, co-culturing of miR-124-SCs and ASs in a transwell system revealed that GFAP and p-STAT3 protein expression in ASs was significantly reduced. Collectively, these results show that overexpression of miR-124 in SCs promotes SC-AS integration in vitro and may attenuate the capacity of ASs to form glial scars. Thus, this study provides novel insights into modifying SCs by overexpressing miR-124 to improve their therapeutic potential in SCI.

Molecular regulation and function of FoxO3 in chronic kidney disease.

FOXOs are transcription factors that regulate downstream target genes to counteract to cell stress. Here we review the function and regulation of FOXO transcription factors, the mechanism of FOXO3 activation in the kidney, and the role of FOXO3 in delaying the development of chronic kidney disease (CKD). Progressive renal hypoxia from vascular dropout and metabolic perturbation is a pathogenic factor for the initiation and development of CKD. Hypoxia and low levels of α-ketoglutarate generated from the TCA cycle inhibit prolyl hydroxylase domain (PHD)-mediated prolyl hydroxylation of FoxO3, thus reducing FoxO3 protein degradation via the ubiquitin proteasomal pathway, similar to HIF stabilization under hypoxic conditions. FoxO3 accumulation and nuclear translocation activate two key cellular defense mechanisms, autophagy and antioxidative response in renal tubular cells, to reduce cell injury and promote cell survival. FoxO3 directly activates the expression of Atg proteins, which replenishes core components of the autophagic machinery to allow sustained autophagy in the chronically hypoxic kidney. FoxO3 protects mitochondria by stimulating the expression of superoxide dismutase 2 (SOD2), as tubular deletion of FoxO3 in mice results in reduced SOD2 levels and profound mitochondrial damage. Knowledge gained from animal studies may help understand the function of stress responsive transcription factors that could be targeted to prevent or treat CKD.

Reciprocal regulation between GCN2 (eIF2AK4) and PERK (eIF2AK3) through the JNK-FOXO3 axis to modulate cancer drug resistance and clonal survival

Pharmaceutical inhibitors of the endoplasmic reticulum (ER)-stress modulator PERK (eIF2AK3) have demonstrated anticancer activities in combination therapies, but their effectiveness as a single agent is limited, suggesting the existence of possible compensatory cellular responses. To explore the potential mechanisms involved, we performed time-course drug treatment experiments on the parental MCF-7 and drug resistant MCF-7EpiR and MCF-7TaxR breast cancer cells and identified GCN2 (eIF2AK4) as a molecule that can potentially cooperate with PERK to regulate FOXO3 via JNK and AKT to modulate drug response. Consistently, GCN2 knockdown severely impaired the clonal survival of parental and resistant MCF-7 cells and sensitised them to epirubicin and paclitaxel treatment. Western blot, RT-qPCR and ChIP analyses also confirmed that GCN2 inactivation causes an induction of JNK and thereby FOXO3 activity, culminating in an increase in PERK activity and expression at the transcription level. Conversely, PERK-inactivation using GSK2606414-induces an induction in GCN2 expression and activity also associated with JNK. In agreement, we also showed that the perk−/− MEFs, expressing elevated levels of P-JNK, JNK, GCN2 and reduced levels of P-AKT and P-FOXO3, have lower clonogenicity and are more sensitive to epirubicin compared to wild-type MEFs. Similarly, gcn2−/− MEFs expressing augmented levels of P-JNK, JNK, P-PERK, PERK and lower levels of P-AKT and P-FOXO3 also had lower clonogenicity and were more sensitive to epirubicin and PERK-inhibition. In addition, JNK1/2 deletion in MEFs resulted in reduced levels of GCN2, FOXO3, PERK, P-PERK expression as well as FOXO3 activity and enhanced clonal survival and resistance to PERK-inhibition. Together these results demonstrate that GCN2 cooperates with PERK through the JNK-FOXO3 axis in a reciprocal negative feedback loop to mediate cancer chemotherapeutic drug response and clonal survival, advocating the potential of targeting GCN2 as a therapeutic strategy for treating cancer and for overcoming drug resistance.

Serine-threonine kinase ROCK2 regulates germinal center B cell positioning and cholesterol biosynthesis.

Germinal center (GC) responses require B cells to respond to a dynamic set of intercellular and microenvironmental signals that instruct B cell positioning, differentiation, and metabolic reprogramming. RHO-associated coiled-coil-containing protein kinase 2 (ROCK2), a serine-threonine kinase that can be therapeutically targeted by ROCK inhibitors or statins, is a key downstream effector of RHOA GTPases. Although RHOA-mediated pathways are emerging as critical regulators of GC responses, the role of ROCK2 in B cells is unknown. Here, we found that ROCK2 was activated in response to key T cell signals like CD40 and IL-21 and that it regulated GC formation and maintenance. RNA-Seq analyses revealed that ROCK2 controlled a unique transcriptional program in GC B cells that promoted optimal GC polarization and cholesterol biosynthesis. ROCK2 regulated this program by restraining AKT activation and subsequently enhancing FOXO1 activity. ATAC-Seq (assay for transposase-accessible chromatin with high-throughput sequencing) and biochemical analyses revealed that the effects of ROCK2 on cholesterol biosynthesis were instead mediated via a novel mechanism. ROCK2 directly phosphorylated interferon regulatory factor 8 (IRF8), a crucial mediator of GC responses, and promoted its interaction with sterol regulatory element-binding transcription factor 2 (SREBP2) at key regulatory regions controlling the expression of cholesterol biosynthetic enzymes, resulting in optimal recruitment of SREBP2 at these sites. These findings thus uncover ROCK2 as a multifaceted and therapeutically targetable regulator of GC responses.

1108-P: Resveratrol Inhibits Dapagliflozin-Induced Renal Gluconeogenesis through Modulating the PI3k/Akt/Foxo1 Pathway in HK-2 Cells

Aims: Dapagliflozin(Dapa) improves glycemia by increasing glucosuria, but it enhances renal gluconeogenesis, thus reducing its efficacy. Resveratrol (RSV) inhibits hepatic gluconeogenesis by increasing insulin sensitivity. Our study aims to determine if RSV can reduce dapa-induced renal gluconeogenesis via modulating the PI3K/Akt/FoxO1 pathway. Methods: Glucose produced via gluconeogenesis was tested by glucose assay kit. Changes in insulin signaling and gluconeogenic key enzymes were detected by RT-PCR and western blot. Results: Dapa induced glucose production and the expression of gluconeogenic enzymes (PEPCK, G6Pase) via increasing the expression of FoxO1 in HK-2 cells. RSV treatment reversed all these findings by up-regulating pFoxO1 expression. The PI3K inhibitor LY294002 restained the pFoxO1 expression and subsequently inhibited PEPCK and G6Pase expression via PI3K/Akt-mediated FoxO1 inactivation. Conclusion: Our data show that RSV ameliorates gluconeogenesis via modulating the PI3K-Akt-FoxO1-PEPCK/G6Pase pathway in dapa-treated HK-2 cells. This mechanism provides a basis for pharmaceutial intervention in order to achieve better hypoglycemic effect in future animal and human research. Disclosure X. Sun: None.

1699-P: Mechanisms and Roles of Foxo1-Akt Negative Feedback Pathway in Adipocytes

Objective: FoxO1 is a substrate of serine/threonine-kinase Akt. Although FoxO1 is phosphorylated by Akt and inactivated by insulin stimulation, constitutive activation of FoxO1 is reported to enhance Akt phosphorylation. Therefore, constitutive activation of FoxO1 may mimic insulin’s functions via Akt phosphorylation. Methods: Constitutively-active FoxO1 (CA-FoxO1) in 3T3-L1 adipocytes is overexpressed by adenovirus and Changes in insulin signaling and downstream actions was studied. Results: CA-FoxO1 induced Akt phosphorylation in differentiated 3T3-L1 adipocytes. Regarding downstream actions, CA-FoxO1 significantly enhanced glucose uptake (148.6% compared to controls, P =0.030), GLUT4 translocation (297.6%, P <0.01), lipogenesis (175.9%, P =0.030) and tended to enhance glycogen synthesis (128.7%, P =0.154), while in contrast, under insulin-stimulated condition, significantly suppressed protein synthesis (56.5%, P <0.01) and DNA synthesis (44.4%, P <0.01). Regarding insulin signaling, CA-FoxO1 enhanced phosphorylations of GSK-3, PRAS40, and p70S6K. The enhancement of PI 3-kinase activity by 3nM insulin is larger than that by CA-FoxO1, while Akt phosphorylation by 3nM insulin is smaller than that by CA-FoxO1, suggesting the mechanism of FoxO1-Akt feedback is downstream of PI 3-kinase. In contrast to previous papers, protein amounts of IR-β, p110α subunit of PI 3-kinase, or Rictor were not significantly changed by CA-FoxO1. Conclusion: Constitutive activation of FoxO1 induced Akt phosphorylation even in differentiated 3T3-L1 adipocytes, but FoxO1 activation mimicked only metabolic functions of glucose but not proliferative or oncogenic functions of insulin. This selective mimicking of insulin by FoxO1 activation in adipocytes may improve glucose metabolism with a low risk of tumorigenesis. The mechanisms of FoxO1-Akt feedback is suggested not to be the increase in IR-β or p110α subunit of PI 3-kinase as previously reported, but to be mainly downstream of PI 3-kinase. Disclosure T. Ohno: None. H. Ono: None. K. Yokote: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo, Eli Lilly Japan K.K., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Pfizer Japan Inc., Sanofi K.K., Shionogi & Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited. Speaker’s Bureau; Self; Abbott, Astellas Amgen, AstraZeneca K.K., Bayer Inc., FUJIFILM Pharmaceuticals U.S.A., Inc., Kaken Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho.

299-OR: Loss of Insulin and IGF1 Receptors in Muscle Impairs Complex-I Dependent Mitochondrial Bioenergetics and Supercomplex Formation via Foxo Transcription Factors

Diabetes is associated with decreased muscle strength and mitochondrial metabolism which can lead to disability. Recently, we reported that insulin receptor (IR) and IGF-1 receptor (IGF1R) prevent muscle atrophy via suppression of FoxOs, but how FoxOs interact with mitochondrial function remains unclear. To test whether IR/IGF1R suppression of FoxOs coordinates muscle strength with mitochondrial function, we measured mitochondrial bioenergetics and RNA-Seq in mice lacking IR, IGF1R, and/or FoxO1/3/4 in muscle. Perinatal muscle-specific deletion of IR (MIRKO) showed a mild 20% decrease in pyruvate-driven respiratory capacity in soleus permeabilized fibers, whereas succinate/Rotenone respiration was not different. Deletion of both IR/IGF1R (MIGIRKO) showed a marked 60% decrease in pyruvate respiration and ATP production in soleus fibers. These mitochondrial abnormalities were reversed in muscle-specific IR/IGF1R and FoxO1/3/4 quintuple knockout (QKO), suggesting FoxO activation mediates the mitochondrial decline. FoxOs are known inducers of autophagy, but mitophagy quantification using MitoTIMER or mitoKeima biosensors was not altered in Day 21 Tamoxifen-inducible IR/IGF1R KO in adult muscle (IND-IGIRKO) or by acute in-vitro deletion of IR/IGF1R primary myotubes. Complex-I activity was decreased 50-70% in MIGIRKO and Day 21 IND-IGIRKO muscle, but other complex activities were unchanged. Furthermore, 14 complex-I subunits were decreased in MIGIRKO by RNA-Seq, and 13 of these were reversed in QKO. IND-IGIRKO also showed a decrease in pyruvate respiration, ATP production, complex-I activity, and complex-I/supercomplex content by blue-native gels 21 days after tamoxifen. Thus, loss of IR or both IR/IGF1R in muscle decreases complex-I driven mitochondrial respiration and supercomplex assemblies, at least in part by FoxO-mediated downregulation of mitochondrial genes. Disclosure G. Bhardwaj: None. C.M. Penniman: None. P.A. Suarez Beltran: None. J. Jena: None. C.M. Foster: None. K. Poro: None. T. Junck: None. A. Hinton: None. R. Souvenir: None. J. Fuqua: None. P.E. Morales: None. R. Bravo-Sagua: None. V.A. Lira: None. E.D. Abel: None. B.T. ONeill: None.

261-OR: CDK4-R24C Compensates for IRS2 Deficiency by Enhancing Beta-Cell Proliferation and Restoring Signaling through the AKT-Foxo1-PDX1 Pathway

Expanding functional beta cell mass remains a prime goal of diabetes research. We previously demonstrated that IRS2 deficient mice have reduced islet expression of cyclin D2, and that overexpressing cyclin D2 rescues proliferation in IRS2 deficient beta cells in vitro. Since cyclin D2 partners with CDK4 to drive cell cycle progression, we hypothesized that an activated form of CDK4, CDK4-R24C (resistant to inhibition by Ink4A cell cycle inhibitors) might rescue the in vivo proliferation defect in IRS2 deficient mice. Mice heterozygous for both whole body Irs2 deletion and whole body Cdk4-R24C knock-in were mated, yielding 6 genotypes of interest: Irs2+/+; Cdk4-WT, Irs2+/+; Cdk4-R24C/WT, Irs2+/+; Cdk4-R24C/R24C, Irs2-/-; Cdk4-WT, Irs2-/-; Cdk4-R24C/WT, Irs2-/-; Cdk4-R24C/R24C. Two copies, but not one, of Cdk4-R24C completely rescued fasting and non-fasting blood glucose, glucose intolerance, and beta cell mass and proliferation, without rescuing peripheral insulin resistance. Interestingly, Irs2-/-; Cdk4-WT islets contained dedifferentiated (reduced PDX1, nuclear FOXO1, ALDH1A3+) beta cells, and this was completely reversed in Irs2-/-; Cdk4-R24C/R24C islets. An in vitro primary beta cell model of increased nuclear FOXO1 activity (short-term starvation) resulted in mRNA changes consistent with nuclear FOXO1 activity (including reduced Pdx1) and mimicked IRS2 deficiency in vivo. Intriguingly, overexpressing cell cycle activators CDK4 and cyclin D2 partially rescued the mRNA changes indicative of nuclear FOXO1 activity, while expressing an inhibitor (CDKN2A/p16) did not. Overexpression of CDK4 increased FOXO1 phosphorylation. We conclude that CDK4 may play additional roles outside of the cell cycle in beta cells, by activating the insulin signaling pathway downstream of IRS2 and exerting antidiabetic action by maintaining beta cell differentiation. Disclosure R.E. Stamateris: None. R.B. Sharma: None. B. Gablaski: None. S.G. Rane: None. L.C. Alonso: Consultant; Self; Fairbanks Pharmaceuticals Inc. Research Support; Self; American Diabetes Association. Funding American Diabetes Association (1-18-IBS-233 to L.C.A.)