Targeting FOXO3a Research Articles

IL-1β promotes esophageal squamous cell carcinoma growth and metastasis through FOXO3A by activating the PI3K/AKT pathway

Esophageal cancer is a common type of cancer that poses a significant threat to human health. While the pro-inflammatory cytokine IL-1β has been known to contribute to the development of various types of tumors, its role in regulating esophageal cancer progression has not been extensively studied. Our studies found that the expression of IL-1β and FOXO3A was increased in esophageal squamous cell carcinoma (ESCC). IL-1β not only increased the proliferation, migration, and invasion of two ESCC cell lines but also promoted tumor growth and metastasis in nude mice. We also observed that IL-1β and FOXO3A regulated the process of epithelial-mesenchymal transition (EMT) and autophagy. The PI3K/AKT pathway was found to be involved in the changes of FOXO3A with the expression level of IL-1β. The AKT agonist (SC79) reversed the reduction of FOXO3A expression caused by the knockdown of IL-1β, indicating that IL-1β plays a role through the PI3K/AKT/FOXO3A pathway. Furthermore, the knockdown of FOXO3A inhibited ESCC development and attenuated the pro-cancer effect of overexpressed IL-1β. Targeting IL-1β and FOXO3A may be potentially valuable for the diagnosis and treatment of ESCC.

The Role of microRNA-23a-3p in the Progression of Human Aging Process by Targeting FOXO3a

Aging results in deterioration of body functions and, ultimately, death. miRNAs contribute to the regulation of aging. The aim of this study was to explore the contribution of miRNAs to aging and senescence-related changes in gene expression. The expression changes of miRNAs in the blood of people and animal samples collected from different age subjects were examined using Affymetrix miRNA 4.0 microarray and qRT-PCR. MTT assay and flow cytometry were used to examine the effect of miR-23a on cell functions in WI-38 cells. The expression levels of 48 miRNAs, including miR-23a, miR-21, and miR-100, in the blood samples were higher in the middle-aged group than in the young or elderly group. Animal studies further suggested that the expression of miR-23a increased with age. In addition, upregulation of miR-23a dramatically suppressed the cell proliferation and arrested the WI-38 cell cycle in vitro. FOXO3a has been identified as a target gene of miR-23a. MiR-23a downregulated the expression of FOXO3a in WI-38 cells. MiRNAs have different expression levels in different age groups. miR-23a could suppress cell proliferation and arrest the cell cycle in WI-38 cells, which elucidated the mechanism through which miR-23a exerts pivotal role in WI-38 cells by targeting FOXO3a.

Overcoming adaptive resistance in AML by synergistically targeting FOXO3A-GNG7-mTOR axis with FOXO3A inhibitor Gardenoside and rapamycin

Therapeutic targeting FOXO3A (a forkhead transcription factor) represents a promising strategy to suppress acute myeloid leukemia (AML). However, the effective inhibitors that target FOXO3A are lacking and the adaptive response signaling weakens the cytotoxic effect of FOXO3A depletion on AML cells. Here, we show that FOXO3A deficiency induces a compensatory response involved in the reactive activation of mTOR that leads to signaling rebound and adaptive resistance. Mitochondrial metabolism acts downstream of mTOR to provoke activation of JNK/c-JUN via reactive oxygen species (ROS). At the molecular level, FOXO3A directly binds to the promoter of G protein gamma subunit 7 (GNG7) and preserves its expression, while GNG7 interacts with mTOR and restricts phosphorylated activation of mTOR. Consequently, combinatorial inhibition of FOXO3A and mTOR show a synergistic cytotoxic effect on AML cells and prolongs survival in a mouse model of AML. Through a structure-based virtual screening, we report one potent small-molecule FOXO3A inhibitor (Gardenoside) that exhibits a strong effect of anti-FOXO3A DNA binding. Gardenoside synergizes with rapamycin to substantially reduce tumor burden and extend survival in AML patient-derived xenograft model. These results demonstrate that mTOR can mediate adaptive resistance to FOXO3A inhibition and validate a combinatorial approach for treating AML.

Combined Therapy of Yishen Zhuanggu Decoction and Caltrate D600 Alleviates Postmenopausal Osteoporosis by Targeting FoxO3a and Activating the Wnt/β-Catenin Pathway

Background Postmenopausal osteoporosis (PMO) is the most prevalent metabolic bone disease in women. Yishen Zhuanggu (YSZG) decoction and Caltrate D600 reportedly affects bone formation. This study aimed to investigate the efficacy and mechanism of YSZG decoction combined with Caltrate D600 in PMO treatment. Methods Ovariectomy-induced PMO rat model was treated with YSZG or/and Caltrate D600 for 12 weeks. Femur bone mineral density (BMD), osteoporosis-related protein expression, and serum parameters were measured. Pathological features of femur bone tissues were observed using hematoxylin and eosin staining. Serum levels of oxidative stress parameters were measured using corresponding commercial kits. The mRNA and protein expression of FoxO3a, Wnt, and β-catenin was detected using qRT-PCR and western blotting. Results The BMD and ultimate load of PMO rats were increased after treatment with YSZG. YSZG treatment promoted the bone trabeculae formation of PMO rats. YSZG treatment also induced bone differentiation and suppress oxidative stress in PMO rats, evidenced by the increased BALP, Runx2, OPG, SOD, and CAT levels, as well as the decreased TRACP 5b, RANKL, ROS, and MDA levels. Additionally, YSZG treatment downregulated the FoxO3a expression and upregulated the levels of Wnt and β-catenin in PMO rats. Caltrate D600 addition showed an auxiliary effect for YSZG. Conclusion YSZG decoction exerts the antiosteoporotic effect on PMO by restraining the FoxO3a expression and activating the Wnt/β-catenin pathway, which has an impressive synergistic effect with Caltrate D600.

Identification of the key miRNA; hsa-miR-1269a targeting TP53, Caspase-9 and FOXO3a in breast cancer cells under hypoxia by integrated bioinformatics analysis

BackgroundHypoxia microenvironment allied too many vital events relevant to tumorigenesis such as; proliferation, speedy angiogenesis, metastasis, poor prognosis and resistance to several anticancer therapies. miRNAs are established as key players during cellular adaptations in metabolism, proliferation, DNA repair, and apoptosis in various cancer including breast cancer under hypoxia. However, the results have been unsatisfactory so far supporting the need of identifying novel players in hypoxia signaling that can be an ideal candidate for breast cancer therapy. ObjectiveThe current study aimed to find out the DE miRNAs and their target mRNAs/genes related to breast cancer progression and metastasis under hypoxia via in silico analysis. MethodsBased on microarray analysis of DE miRNAs, a string of computational approaches was used to select the most prominent hypoximiRs and their targets. Targets of these DE miRNAs were identified by using miRDB and TargetScan v7.2. Simultaneously miRNA enrichment and GO analysis were utilized to select their associated biological pathways. Further sequence alignment, molecular docking and semiquantitative RT-PCR were performed to select the most suitable targets of candidate miRNA. ResultsThe combined study of GO, KEGG, and miRNAs-mRNAs network analysis pragmatically demonstrated that hsa-miR-1269a and its selected target genes (TP53, Caspase-9, and FOXO3a) show maximum interaction with other miRNAs and shared common interaction pathways. Sequence alignment, molecular docking and RT-PCR further confirmed the targets of hsa-miR-1269a and revealed that it may inhibit cell growth by promoting apoptosis. ConclusionOur findings concluded that, TP53, Caspase-9 and FOXO3a could be the probable targets of hsa-miR-1269a that ultimately promotes breast cancer cells survival and proliferation under hypoxia. In conclusion, this computational study eventually suggest that, hsa-miR-1269a could be considered as a valuable prognostic marker for breast cancer therapeutics.

FOXO3a regulates lipid accumulation and adipocyte inflammation in adipocytes through autophagy : Role of FOXO3a in obesity.

FOXO3a is a widely studied transcription factor and plays an important role in a variety of biology. The purpose of this study was to explore the role and potential mechanism of FOXO3a on lipid accumulation and adipocyte inflammation in adipocytes through regulation of autophagy. The obese mouse model was successfully induced by high-fat diet. SiRNA targeting FOXO3a was transfected into differentiation of 3T3-L1 adipocytes to reduce the expression of FOXO3a. The culture medium of RAW264.7 cells was added to the differentiated 3T3-L1 adipocytes to form a co-culture system. Subsequently, ELISA or AdipoRed assay was performed to measure the expression of triglyceride (TG) and cholesterol (TC) in mouse adipose tissue or differentiation of 3T3-L1 adipocytes. Adipocyte differentiation was detected by Oil Red O-staining. Ad-mCherry-GFP-LC3II was used to detect the level of autophagy in differentiation of 3T3-L1 adipocytes. Western blotting or qRT-PCR was used to detect the expression of FOXO3a, autophagy-related proteins (beclin 1, CEBPβ, PPARγ, ACC1 and KLF4), inflammatory cytokines (TNF-α, IL-1β, IL-6 and MCP1), NF-κB signal pathway-related proteins or adipokines (Adiponectin, AdipoR1 and resistin) in differentiated 3T3-L1 or RAW264.7 cells. The expression of FOXO3a and autophagy levels were significantly increased in visceral adipose tissue of obese mice and differentiation of 3T3-L1 adipocytes. Downregulation of FOXO3a significantly inhibited the autophagy and lipid accumulation in differentiation of 3T3-L1 adipocytes. In addition, FOXO3a knockdown significantly reduced Lipopolysaccharide (LPS)-induced inflammation and adipokines release in RAW264.7 cells treated with the culture medium of 3T3-L1 adipocytes. These above activity changes could be reversed by autophagy inducer rapamycin. FOXO3a could promote lipid accumulation and inflammation in differentiated 3T3-L1 adipocytes by targeting autophagy. Our results provide a new theoretical basis for FOXO3a to regulate obesity.

MiR-29b-3p protects cardiomyocytes against endotoxin-induced apoptosis and inflammatory response through targeting FOXO3A

Cardiac dysfunction represents a main component of death induced by sepsis in critical care units. And microRNAs (miRNAs) have been reported as important modulators or biomarkers of sepsis. However, the molecular detail of miRNAs involved in septic cardiac dysfunction remains unclear. Here we showed that endotoxin (lipopolysaccharide, LPS) significantly down-regulated expression of miR-29b-3p in heart. Increased expression of miR-29b-3p by lentivirus improved cardiac function and attenuated damage of cardiac induced by LPS in mice. Furthermore, overexpression or knockdown of miR-29b-3p showed its crucial roles on regulation of apoptosis and production of pro-inflammatory cytokines in NRCMs through directly targeting FOXO3A. miR-29b-3p ameliorates inflammatory damage likely via reducing activation of MAPKs and nuclear-translocation of NF-κB to block LPS-activated NF-κB signaling. Notably, miR-29b is also down-regulated in septic patients' plasma compared with normal subjects, indicating a potential clinical relevance of miR-29b. Taken together, our findings demonstrate that upregulation of miR-29b-3p can attenuate myocardial injury induced by sepsis via regulating FOXO3A, which provide a potential therapy target for interference of septic cardiac dysfunction.

AURKA Enhances Autophagy of Adipose Derived Stem Cells to Promote Diabetic Wound Repair via Targeting FOXO3a

AURKA regulates apoptosis and autophagy in a diverse range of diseases and exhibits promising clinical efficacy; however, the role of AURKA in regulating adipose-derived stem cells (ADSCs) and repairing diabetic wound remains unclear. Here, we showed that ADSCs subjected to high glucose stress displayed an obvious induction of AURKA and FOXO3a, and a significant increase in autophagy and apoptosis. AURKA was confirmed to regulate autophagy through FOXO3a. AURKA-mediated autophagy inhibited high-glucose-induced apoptosis of ADSCs. Furthermore, co-immunoprecipitation and chromatin immunoprecipitation assays were employed to investigate the interaction of AURKA and FOXO3a. FOXO3a bound to its own promoter and transactivated its own expression. AURKA was found to interact with FOXO3a to regulate FOXO3a activity. In diabetic mice, ADSCs overexpressing AURKA led to a decrease of apoptosis of ADSCs and promoted wound healing in the skin. Taken together, our data suggest that transcriptional regulation of FOXO3a by high-glucose-mediated AURKA is necessary for ADSCs autophagy. Our data reveal a potential therapeutic strategy for targeting AURKA involved in high-glucose-induced anti-apoptotic autophagy in ADSCs.

MiR-155 targeting FoxO3a regulates oral cancer cell proliferation, apoptosis, and DDP resistance through targeting FoxO3a.

FoxO3a is a well-defined tumor suppressor gene in the forkhead transcription factor O subfamily (FoxO), and its reduction is related to the occurrence of various tumors. It was found that the expression of miR-155 is abnormally elevated in oral cancer tissues, suggesting that miR-155 may be a tumor-promoting gene for oral cancer. Bioinformatics analysis revealed a targeted complementary binding site between miR-155 and FoxO3a. In this study, we established a cisplatin-resistant cell line of oral cancer to compare the expression of miR-155 and FoxO3a in parental oral cancer cells, and explore whether miR-155 regulates the expression of FoxO3a and affects the proliferation and apoptosis of oral cancer cells. Dual luciferase reporter gene assay validated the targeted regulatory role between miR-155 and FoxO3a. The DDP-resistant oral cancer cell line KB/DDP was established, and the expressions of miR-155 and FoxO3a were compared. The CCK-8 assay was used to calculate the resistance index (RI). KB/DDP cells were divided into miR-NC group and miR-155 inhibitor group. Cell apoptosis was detected by flow cytometry. Cell proliferation ability was detected by EdU staining. There is a targeted regulatory relationship between miR-155 and FoxO3a mRNA. The expression of miR-155 was significantly higher, while the expression of Foxo3a mRNA and protein was obviously lower in KB/DDP cells than that in KB cells. CCK-8 experiments showed that the same concentration of DDP exhibited apparently lower proliferation inhibition in KB/DDP cells than KB cells. Compared with the miR-NC group, transfection of miR-155 inhibitor markedly increased the expression of Foxo3a in KB/DDP cells, which significantly attenuated cell proliferation and enhanced apoptosis in DDP-treated cells. Increased expression of miR-155 and decreased expression of Foxo3a were associated with DDP resistance in oral cancer cells. Inhibition of miR-155 expression upregulated Foxo3a expression, restrained oral cancer cell proliferation, promoted cell apoptosis, and enhanced DDP sensitivity in oral cancer cells.

Carbon black nanoparticles induce pulmonary fibrosis through NLRP3 inflammasome pathway modulated by miR-96 targeted FOXO3a.

Carbon black nanoparticle (CBNP) is a core constituent of air pollutants like fine particulate matter (PM2.5) as well as a common manufactural material. It was proved to pose adverse effects on lung function and even provoke pulmonary fibrosis. However, the underlying mechanisms of CBNPs-induced pulmonary fibrosis remain unclear. The present study aimed to investigate the mechanism of fibrotic effects caused by CBNPs in rat lung and human bronchial epithelial (16HBE) cells. Forty-nine male rats were randomly subjected to 7 groups, means the 14-day exposure group (30 mg/m3), the 28-day exposure groups (5 mg/m3 and 30 mg/m3), the 90-day exposure group (30 mg/m3) and their respective controls. Rats were nose-only-inhaled CBNPs. 16HBE cells were treated with 0, 50, 100 and 200 μg/mL CBNPs respectively for 24 h. Besides, Forkhead transcription factor class O (FOXO)3a and miR-96 overexpression or suppression 16HBE cells were established to reveal relative mechanisms. Our results suggested CBNPs induced pulmonary fibrosis in time- and dose-dependent manners. CBNPs induced persisting inflammation in rat lung as observed by histopathology and cytology analyses in whole lung lavage fluid (WLL). Both invivo and invitro, CBNPs exposure significantly increased the expression of NLRP3 inflammasome, accompanied by the increased reactive oxygen species (ROS), decreased miR-96 and increased FOXO3a expressions dose -and time-dependently. MiR-96 overexpression or FOXO3a suppression could partially rescue the fibrotic effects through inhibiting NLRP3 inflammasome. Conclusively, our research show that CBNPs-induced pulmonary fibrosis was at least partially depended on activation of NLRP3 inflammasome which modulated by miR-96 targeting FOXO3a.

ING4 suppresses hepatocellular carcinoma via a NF-κB/miR-155/FOXO3a signaling axis.

The tumor suppressor ING4 has been shown to be reduced in human HCC. The alteration of ING4 contributes to HCC progression. However, its effect in HCC and the potential mechanism is largely unclear. Herein, we found that downregulation of ING4 in HCC tumor tissues was closely associated with cancer staging, tumor size and vascular invasion. Lentivirus-mediated ING4 overexpression significantly inhibited proliferation, migration and invasion, and induced cell cycle G1 phase arrest and apoptosis in MHCC97H human HCC cells. Moreover, overexpression of ING4 dramatically suppressed MHCC97H tumor cell growth and metastasis to lung in vivo in athymic BALB/c nude mice. Mechanistic studies revealed that overexpression of ING4 markedly increased expression of FOXO3a both at the mRNA and protein level as well as enhanced nuclear level and transcriptional activity of FOXO3a in MHCC97H tumor cells. In addition, ING4 repressed transcriptional activity of NF-κB and expression of miR-155 targeting FOXO3a. Knockdown of ING4 exhibited opposing effects in MHCC97L human HCC cells. Interestingly, knockdown of FOXO3a attenuated not only ING4-elicited tumor suppression but also ING4-mediated regulatory effect on FOXO3a downstream targets, confirming that FOXO3a is involved in ING4-directed tumor-inhibitory effect in HCC. Overexpression of miR-155 attenuated ING4-induced upregulation of FOXO3a, whereas inhibition of miR-155 blunted ING4 knockdown-induced reduction of FOXO3a. Furthermore, inhibition of NF-κB markedly impaired ING4 knockdown-induced upregulation of miR-155 and downregulation of FOXO3a. Taken together, our study provided the first compelling evidence that ING4 can suppress human HCC growth and metastasis to a great extent via a NF-κB/miR-155/FOXO3a pathway.

Arsenic Trioxide Suppressed Migration and Angiogenesis by Targeting FOXO3a in Gastric Cancer Cells.

Arsenic trioxide (As2O3), a traditional remedy in Chinese medicine, has been used in acute promyelocytic leukemia (APL) research and clinical treatment. Previous studies have shown that As2O3 exerts its potent antitumor effects in solid tumors by regulating cell proliferation and survival. The aim of this study was to investigate whether As2O3 inhibited gastric cancer cell migration and angiogenesis by regulating FOXO3a expression. We found that As2O3 reduced gastric cancer cell viability in a dose-dependent manner and also inhibited cell migration and angiogenesis in vitro. Western blotting and immunofluorescence showed that As2O3 downregulated the levels of p-AKT, upregulated FOXO3a expression in the nucleus, and attenuated downstream Vascular endothelial growth factor (VEGF) and Matrix metallopeptidase 9 (MMP9) expression. Moreover, we demonstrated that knockdown of FOXO3a significantly reversed the inhibition of As2O3 and promoted cell migration and angiogenesis in vitro. Further, As2O3 significantly inhibited xenograft tumor growth and angiogenesis by upregulating FOXO3a expression in vivo. However, knockdown of FOXO3a attenuated the inhibitory effect of As2O3 in xenograft tumors, and increased microvessel density (MVD) and VEGF expression. Our results demonstrated that As2O3 inhibited migration and angiogenesis of gastric cancer cells by enhancing FOXO3a expression.

Blunting Autoantigen-induced FOXO3a Protein Phosphorylation and Degradation Is a Novel Pathway of Glucocorticoids for the Treatment of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease affecting multiple organs. Glucocorticoids (GCs), the potent anti-inflammatory drugs, remain as a cornerstone in the treatment for SLE; nevertheless, their clinical efficacy is compromised by the side effects of long term treatment and resistance. To improve the therapeutic efficacy of GCs in SLE, it is important to further decipher the molecular mechanisms of how GCs exert their anti-inflammatory effects. In this investigation, FOXO3a was identified as a molecule that was down-regulated in the course of SLE. Of interest, GC treatment was found to rescue FOXO3a expression both in SLE mice and in SLE patients. Gain- and loss-of-function studies demonstrated that FOXO3a played a crucial role in GC treatment of SLE via inhibiting inflammatory responses. Further studies showed that the up-regulation of FOXO3a by GCs relied on the suppression of pI3K/AKT-mediated FOXO3a phosphorylation and the arrest of FOXO3a in the nucleus. Finally, our data revealed that FOXO3a was critical for GC-mediated inhibition of NF-κB activity, which might involve its interaction with NF-κB p65 protein. Collectively, these data indicated that FOXO3a played an important role in GC treatment of SLE by suppressing pro-inflammatory response, and targeting FOXO3a might provide a novel therapeutic strategy against SLE.

FOXO3a and Posttranslational Modifications Mediate Glucocorticoid Sensitivity in B-ALL.

Glucocorticoids are widely used to treat B acute lymphoblastic leukemia (B-ALL); however, the molecular mechanism underlying glucocorticoid response and resistance is unclear. In this study, the role and regulation of FOXO3a in mediating the dexamethasone response in B-ALL were investigated. The results show that FOXO3a mediates the cytotoxic function of dexamethasone. In response to dexamethasone, it was found that FOXO3a translocates into the nucleus, where it induces the expression of downstream targets, including p27Kip1 and Bim, important for proliferative arrest and cell death in the sensitive RS4;11 and SUP-B15 B-ALL cells. FOXO3a activation by dexamethasone is mediated partially through the suppression of the PI3K/Akt signaling cascade. Furthermore, two posttranslational modifications were uncovered, phosphorylation on Ser-7 and acetylation on Lys-242/5, that associated with FOXO3a activation by dexamethasone. Immunoblot analysis showed that the phosphorylation on Ser-7 of FOXO3a is associated with p38/JNK activation, whereas the acetylation on Lys-242/5 is correlated with the downregulation of SIRT1/2/6 and the induction of the acetyltransferase CBP/p300. Collectively, these results indicate that FOXO3a is essential for dexamethasone response in B-ALL cells, and its nuclear translocation and activation is associated with its phosphorylation on Ser-7 and acetylation on Lys-242/245. These posttranslational events can be exploited as biomarkers for B-ALL diagnosis and as drug targets for B-ALL treatment, particularly for overcoming the glucocorticoid resistance. FOXO3a and its posttranslational regulation are essential for dexamethasone response, and targeting FOXO3a and sirtuins may enhance the dexamethasone-induced cytotoxicity in B-ALL cells.

Simvastatin prevents triple-negative breast cancer metastasis in pre-clinical models through regulation of FOXO3a.

We previously reported using statins was correlated with improved metastasis-free survival in aggressive breast cancer. The purpose of this study was to examine the effect of statins on metastatic colonization by triple-negative breast cancer (TNBC) cells. TNBC cell lines were treated with simvastatin and then studied for cell cycle progression and proliferation in vitro, and metastasis formation in vivo, following injection of statin-treated cells. Reverse-phase protein assay (RPPA) analysis was performed on statin-treated and control breast cancer cells. RNA interference targeting FOXO3a was used to measure the impact of simvastatin on FOXO3a-expressing cells. The prognostic value of FOXO3a mRNA expression was examined in eight public breast cancer gene expression datasets including 1479 patients. Simvastatin increased G1/S-phase arrest of the cell cycle and inhibited both proliferation and migration of TNBC cells in vitro. In vitro pre-treatment and in vivo treatment with simvastatin reduced metastases. Phosphorylated FOXO3a was downregulated after simvastatin treatment in (RPPA) analysis. Ectopic expression of FOXO3a enhanced mammosphere formation and migratory capacity in vitro. Knockdown of FOXO3a attenuated the effect of simvastatin on mammosphere formation and migration. Analysis of public gene expression data demonstrates FOXO3a mRNA downregulation was independently associated with shorter metastasis-free survival in all breast cancers, as well as in TNBC breast cancers. Simvastatin inhibits in vitro endpoints associated with metastasis through a FOXO3a mechanism and reduced metastasis formation in vivo. FOXO3a expression is prognostic for metastasis formation in patient data. Further investigation of simvastatin as a cancer therapy is warranted.

Abstract 27: The role of the mTORC2-dependent regulation of FOXO3a in UVB-induced apoptosis

Abstract The greatest environmental risk factor for developing non-melanoma skin cancer (NMSC) is ultraviolet B (UVB) radiation. Both complexes of the mammalian target of rapamycin (mTOR) signaling pathway, the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-resistant mTOR complex 2 (mTORC2), are stimulated in response to UVB. Cell survival in UVB-induced keratinocyte carcinogenesis has been shown to be mTORC2-depedent. The tumor suppressor FOXO3a, a member of the Forkhead box family of proteins, is an important transcription factor involved in regulating cell survival. FOXO3a activity is attenuated as a result of AKT activation, which is downstream of mTORC2. UVB has been shown to activate AKT through the mTORC2 pathway; however, the role of FOXO3a in UVB-induced apoptosis in keratinocytes has yet to be studied. To investigate the role of mTORC2 and FOXO3a in UVB-induced apoptosis, we utilized both human keratinocytes (HaCaT cells) as well as inducible Rictor-deficient mouse embryonic fibroblasts (iRictKO cells) following treatment with 4-hydroxy-tamoxifen (4OHT). We also employed the use of inhibitors and shRNA targeting Rictor or mSIN1. The Rictor and mSIN1 proteins are key structural components unique to mTORC2 and their removal dramatically lowers mTORC2 activity. Finally, we compared the effects of shRNA targeting FOXO3a with the expression of a constitutively active mutant FOXO3a. For all the experiments described, cells were exposed to an apoptotic dose of UVB and allowed to incubate for defined experimentally established intervals. Our results show that there is an mTORC2-dependent regulation of FOXO3a expression, localization, and activity upon UVB-irradiation. Through western analysis we demonstrate that UVB exposure promotes the phosphorylation of FOXO3a by AKT in cells with intact mTORC2 signaling, which is repressed when mTORC2 is inhibited. This phosphorylation has been shown in other systems to reduce FOXO3a activity by causing a cytoplasmic localization of the protein. In agreement with this, we observed a rapid cytoplasmic localization of FOXO3a and decreased FOXO3a promoter activation in response to UVB, which is rescued following disruption of mTORC2 signaling. Additionally, we find that FOXO3a protein expression is reduced upon UVB-irradiation when mTORC2-signaling is reduced. These data support previous findings that suggest the mTORC2-driven FOXO3a cytoplasmic sequestration protects FOXO3a from dephosphorylation and degradation. Taken together, these studies provide strong evidence that mTORC2 plays a critical role in regulating FOXO3a expression, localization and activity following UVB-irradiation. In addition, our results support the hypothesis that the mTORC2-dependent UVB-induced apoptosis is due at least in part to FOXO3a. Supported by ES19242 (LMS) Citation Format: Robert P. Feehan, Lisa M. Shantz. The role of the mTORC2-dependent regulation of FOXO3a in UVB-induced apoptosis. [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 27. doi:10.1158/1538-7445.AM2015-27

Salinomycin decreases doxorubicin resistance in hepatocellular carcinoma cells by inhibiting the β-catenin/TCF complex association via FOXO3a activation

Doxorubicin is a conventional and effective chemotherapy drug against hepatocellular carcinoma (HCC). However, during long-term doxorubicin monotherapy, HCC cells may eventually develop acquired-resistance to doxorubicin which results in recurrence and a poor prognosis. Salinomycin, an ionophore antibiotic, was recently reported to selectively kill human cancer stem cells (CSCs) which were response for chemoresistance. In this study, salinomycin was found to exert synergistic cytotoxicity with doxorubicin in HCC cells and be capable of inhibiting doxorubicin-induced epithelial-mesenchymal transition (EMT), an important cellular process involved in the acquired chemoresistance of tumors. Further experiments revealed that FOXO3a, a multifunctional transcription factor that can be activated by salinomycin, was vital in mediating doxorubicin-induced EMT. In addition, activated FOXO3a disturbed the interaction between β-catenin and TCF and inhibited the expression of β-catenin/TCF target genes (ZEB1, c-Myc and CyclinD1), which played important roles in doxorubicin-induced EMT in HCC cells. Finally, the enhanced curative efficacy of combination treatment of doxorubicin and salinomycin for HCC was confirmed in established xenograft models. In summary, the present study identifies a new doxorubicin-based chemotherapy for advanced HCC and provides a potential anti-cancer strategy targeting FOXO3a and related cell pathway molecules.

MicroRNA-155 Is Involved in the Pathogenesis of Ulcerative Colitis by Targeting FOXO3a

MicroRNAs (miRNAs) are important posttranscriptional regulators of gene expression. The precise role of miRNAs in ulcerative colitis (UC) is not completely understood. The purpose of this study was to identify miRNAs that are induced in patients with active UC and to assess the effect of miR-155 on improving intestinal inflammation. The miRNA profiles in patients with active UC (n = 20) and healthy subjects (n = 16) were examined using miRNA microarrays. miR-155 upregulation was confirmed by quantitative RT-PCR. Regulation of the target gene FOXO3a expression by miR-155 was assessed using luciferase reporter construct assays and miR-155 mimic or inhibitor transfections. The effects of FOXO3a or miR-155 on IκBα or IL-8 were detected by Western blot or enzyme-linked immunosorbent assay in HT29 cells, respectively. We identified 68 miRNAs that were differentially expressed (33 upregulated and 35 downregulated) in patients with active UC compared with healthy controls. One of the upregulated miRNAs in the UC tissue was miR-155 (1.22-fold, P < 0.03), which plays a key role in the regulation of inflammatory pathways. In patients with active UC, miR-155 was significantly upregulated, and the expression of FOXO3a dramatically decreased. Luciferase reporter assays demonstrated that miR-155 directly targets FOXO3a and affects the protein expression of FOXO3a in HT29 cells. Moreover, silenced FOXO3a and the overexpression of miR-155 increased the levels of IL-8 in TNF-α-treated HT29 cells by suppressing the inhibitory IκBα. miR-155 appears to play a role in the intestinal inflammation of patients with active UC by downregulating the expression of FOXO3a. This process may activate the nuclear factor kappa B signaling pathway.

Nuclear Localization Marker of FOXO3a: Can it be Used to Predict Doxorubicin Response?

Nuclear Localization Marker of FOXO3a: Can it be Used to Predict Doxorubicin Response?

Abstract P5-06-02: The Role of Breast Tumor Kinase (Brk) in Cell Cycle Control

Abstract Brk promotes cell proliferation and has been associated with multiple mitogenic pathways. However, the role of Brk in the cell cycle is still elusive. Here, we present a mechanism of Brk-induced cell cycle progression. Brk downregulates the cell cycle inhibitor p27 by altering its transcription factor FoxO3a subcellular localization. p27 is a key component of the G1/S cell cycle checkpoint and downregulation of p27 promotes S phase entry. The progression of the cell from G1 to S phase, induced by Brk, is independent of growth factors suggesting a role for Brk in malignant transformation. FoxO3a appears to be a major component in p27 down-regulation by Brk as the effect of Brk on p27 expression is significantly abrogated in cells transfected with siRNA targeting FoxO3a. The induction of FoxO3a nuclear exclusion by Brk is inhibited by LY294002 indicating that the mechanism is mediated by the PI3-kinase/Akt pathway. The mechanism underlying PI3-kinase mediated Brk function is still under investigation with evidence suggesting the involvement of the translocation of epidermal growth factor receptor. These data suggest that perturbation of p27 expression induced by Brk causes S phase entrance. Deregulation of the cell cycle is a key event in neoplasia, and thus, the mechanism presented here likely contributes to breast cancer development. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-06-02.