Forkhead Transcription Factor Research Articles

Abstract B70: Differential expression of regulatory T cells and Th17 cells are indicative of tumor recurrence in pN0 stage I lung cancer patients

Abstract Background: Up to 30% of lung cancer (Stage I) with the most favorable outcome recur within 5 years after surgery. Predicting relapse in patients treated curatively with surgical resection for stage I non-small cell lung cancer (NSCLC) remains difficult. The sentinel lymph nodes (SLNs) are the first lymph nodes reached by metastatic cancer cells from a primary tumor. In a recent immunologic study, immunologic changes were detected in these lymph nodes prior to pathologic invasion. This study evaluated the pattern of failure after surgical resection in pN0 lung cancers and determines whether regulatory T (Treg) cells and Th17 cells were prognostic indicator for recurrence. Methods: Patients over 18 years of age who were clinically diagnosed with stage I lung cancer were enrolled. Patients receiving prior lung cancer treatment or thoracic radiation were excluded from enrollment. During surgery, the sentinel lymph nodes were isolated using a handheld gamma probe, followed by the dissection of the visible mediastinal, hilar, and peribronchial lymph nodes. Samples from normal lung tissue, the tumor mass, and all dissected lymph nodes were sectioned and conventionally examined using H&E staining and immunohistochemistry. In addition, the forkhead transcription factor3 (Foxp3) and transcription factor retinoic acid-related orphan receptor (ROR)-γ, recently described to be essential for differentiation of Tregs and Th17 cells, were evaluated by real time RT-PCR. Results: Between October 2009 and October 2010, 27 patients with clinical stage I NSCLC were enrolled consecutively. Among them, twenty patients (thirteen men, seven women, mean age 67.6 years) pathologically staged as N0 were analyzed. Eight patients had squamous cell carcinoma and twelve patients had adenocarcinoma. The expression of Foxp3 in the tumor mass was increased in adenocarcinoma compared to squamous cell carcinoma (P=0.05). The Foxp3 expression and Treg cells in the tumor mass were not significantly associated with recurrence. However, Foxp3 expression and Treg cells’ presence in SLNs were significantly increased in patients with recurring disease compared to nonrecurring disease (P=0.03 and P=0.01, respectively). The number of Th17 cells in tumor mass was decreased in patients with recurring pN0 disease compared to those with non-recurring disease (P=0.04). ROR-γ expression and Th17 cells presence in SLNs were not related with recurrence. Conclusion: The results of this study suggest that the increased number of Treg cells in SLNs and decreased number of Th17 cells in tumor mass might be a promising indicator of recurrence. Citation Format: Chan Kwon Park, Seung Joon Kim. Differential expression of regulatory T cells and Th17 cells are indicative of tumor recurrence in pN0 stage I lung cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B70.

A Hot Water Extract of Sideritis scardica Prolongs Life Span and Enhances Heat Shock Resistance in Caenorhabditis elegans

Sideritis scardica is a Lamiaceae plant that is endemic to the alpine zone of the Balkan Peninsula. The tea of S. scardica has been handed down as a “tea of longevity” in the Rhodope region of Bulgaria for an unknown amount of time. In this study, we prepared a hot water extract of S. scardica (SHWE) and examined its effects on both life span and stress response in living tissue using Caenorhabditis elegans and its transgenic mutants. The life span of wild-type N2 worms was prolonged by approximately 15% at the SHWE concentration of 5 µg/mL and approximately 22% at the SHWE concentration of 50 µg/mL, as compared with the control group. The effect of SHWE on the expression of heat shock protein 16.2 (HSP-16.2) under heat stress was investigated using TJ375 worms, a transgenic mutant of C. elegans. In the TJ375 worms pretreated with SHWE, the fluorescence intensity of green fluorescent protein fluorescence, which indicates the expression of HSP-16.2, was significantly increased. In the assay using TJ356 worms, the worms pretreated with SHWE did not show the translocation of DAF-16, a forkhead transcription factor class O homolog, from the cytoplasm to nucleus under heat stress. Additionally, under heat stress, the pretreatment of SHWE improved the survival rate of GR1307 worms, a knockout mutant of daf-16. These results indicate that SHWE enhances HSP-16.2 expression through a stress-response pathway (eg, HSF-1 pathway) other than the DAF-16 pathway, resulting in a prolonged life span of C. elegans under heat stress.

Propofol Postconditioning Ameliorates Hypoxia/Reoxygenation induced H9c2 Cells Apoptosis and Autophagy via upregulating Forkhead Transcription Factors under Hyperglycemia

Administration of propofol, an intravenous anesthetic with antioxidant property, immediately at the onset of post‐ischemic reperfusion (propofol postconditioning, P‐PostC) has been shown to confer cardioprotection against ischemia‐reperfusion injury , while the underlying mechanism remains incompletely understood. Also, whether or not P‐PostC can confer cardioprotection in diabetes or under hyperglycemic condition has yet to be explored. The FoxO transcription factors were reported to play critical roles in promoting cardiomyocyte survival during conditions of oxidative stress through induction of antioxidants and cell survival pathways (J Biol Chem. 2011;286(9):7468–78) and was also involved in hypoxic postconditioning mediated neuroprotection. However, the role of Foxo in postconditioning mediated protection in the heart and in particular in high glucose condition is unknown. We hypothesized that P‐PostC may attenuate hypoxia/reoxygenation (H/R) injury in cardiac origin H9c2 cells by attenuating apoptosis and autophagy through upregulating FoxO1 and FoxO3a under hyperglycemia. Rat heart‐derived H9c2 cells were exposed to high glucose (HG) for 48 hours, then subjected to H/R (composed of 8 hours of hypoxia followed by 12 hours of reoxygenation) in the absence or presence of postconditioning with various concentrations of propofol at the onset of reoxygenation. Our data showed that HG with or without H/R decreased cell viability, increased lactate dehydrogenase (LDH) leakage and the production of reactive oxygen species (ROS) in H9c2 cells, all of which were significantly reversed by propofol (P‐PostC), especially at the concentration of 25μM. Moreover, we found that propofol (P25) decreased H9c2 cells apoptosis and autophagy that were associated with increased FoxO1 and FoxO3a expression. The protective effects of propofol (P25) against H/R injury were reversed by silencing FoxO1 or FoxO3a. Taken together, these results indicated that propofol postconditioning attenuated H9c2 cardiac cells apoptosis and autophagy induced by H/R injury through upregulating FoxO1 and FoxO3a under hyperglycemia.Support or Funding InformationSupported by National Natural Science Foundation of China (NSFC) grant (81970247)

Value of human mitochondrial transcription termination factor 3 and forkhead box protein 3 in predicting the prognosis of non-small cell lung cancer

Objective To explore the prognostic value of human mitochondrial transcription termination factor 3 (hMTERF3) and forkhead box protein 3 (Foxp3) in non-small cell lung cancer (NSCLC). Methods The clinical data of 88 patients with NSCLC who were admitted to the Third Medical Center of PLA General Hospital from March 2017 to March 2018 were retrospectively analyzed. All patients were diagnosed by pathological puncture. The patients were followed-up by telephone for 12 months, and according to the prognosis, the patients were divided into good prognosis group and poor prognosis group. The pathological tissues were taken from all patients, and the expressions of hMTERF3 and Foxp3 proteins were detected by immunohistochemistry. The expressions of hMTERF3 and Foxp3 in the good prognosis group and the poor prognosis group were compared. Logistic regression model was used to analyze the risk factors of poor prognosis in patients with NSCLC. Results Of 88 patients, 61 patients (69.3%) had good prognosis and 27 patients (30.7%) had poor prognosis. The positive expression rate of hMTERF3 in the good prognosis group was 57.4% (35/61), which was significantly lower than that in the poor prognosis group (81.5%, 22/27) (χ 2= 4.766, P= 0.029). The positive expression rate of Foxp3 in the good prognosis group was 55.7% (34/61), which was significantly lower than that in the poor prognosis group (85.2%, 23/27) (χ 2= 7.113, P= 0.008). The proportions of patients with medium and high differentiation or stage Ⅰ- Ⅱ in the good prognosis group were 82.0% (50/61) and 68.8% (42/61), respectively, which were significantly higher than those in the poor prognosis group [48.15% (13/27) and 25.93% (7/27)] (both P < 0.05). Logistic regression analysis showed that the poor differentiation, stage Ⅲ-Ⅳ, hMTERF3-positive and Foxp3-positive were the risk factors for poor prognosis in NSCLC patients (all P < 0.05). Conclusions The positive expression rates of hMTERF3 and Foxp3 in patients with good prognosis are lower. The hMTERF3-positive and Foxp3-positive are risk factors for poor prognosis in NSCLC patients. Key words: Carcinoma, non-small-cell lung; Mitochondrial proteins; Forkhead transcription factors; Human mitochondrial transcription termination factor 3; Forkhead box protein 3; Prognosis

Effect of electroacupuncture on silent information regulator 1, fork head transcription factor O1 and proopiomelanocortin in the hypothalamus of rats with obesity induced by high-fat diet

To investigate the effect of electroacupuncture (EA) on silent information regulator 1 (SIRT1), forkhead transcription factor O1 (FoxO1), and proopiomelanocortin (POMC) in the hypothalamus of rats with high-fat diet-induced obesity (DIO), as well as the mechanism of EA in regulating central appetite peptides to help lose weight. Male Wistar rats were randomly divided into normal group, model group, EA group, EA＋inhibitor group, inhibitor group, and sham-operation group, with 10 rats in each group. High-fat diet was used to establish a rat model of DIO. The rats in the EA group and EA＋inhibitor group were given EA at "Fenglong" (ST40), "Zhongwan "(CV12)，"Guanyuan "(CV4), and"Zusanli" (ST36) with continuous wave at a frequency of 2 Hz and an intensity of 1 mA, for 10 minutes each time. The rats in the EA＋inhibitor group and inhibitor group were given tube placement in the third ventricle and injection of the specific SIRT1 antagonist EX-527. The rats in the sham-operation group were given tube placement in the third ventricle and injection of artificial cerebrospinal fluid. The above treatment was given 3 times a week for 8 weeks in total. Body weight, food intake, and Lee's index were observed before and after treatment. An automatic biochemical analyzer was used to measure the serum levels of total cholesterol (TC), triglyceride (TG), and free fatty acid (FFA), and Western blot was used to measure the protein expression of SIRT1, FoxO1, acetylated FoxO1(AC-FoxO1), and POMC in the hypothalamus. Before treatment, the model group, the EA group, the EA＋inhibitor group, the inhibitor group, and the sham-operation group had significantly higher body weight and food intake than the normal group (P<0.01), and the model group and the sham-operation group had a significantly higher Lee's index than the normal group (P<0.05). Compared with the model group after treatment, the EA group and the EA＋inhibitor group had significant reductions in body weight, food intake, TC, and the protein expression of AC-FoxO1 (P<0.01, P<0.05) and significant increases in the protein expression of SIRT1, FoxO1 and POMC (P<0.01, P<0.05); the EA group had significant reductions in Lee's index and the levels of TG, FFA(P<0.05，P<0.01)；the inhibitor group had significant increases in food intake, the serum levels of TC, TG, FFA(P<0.01) and significant reductions in the protein expression of SIRT1, FoxO1 and POMC (P<0.01，P<0.05). Compared with the EA group, the EA＋inhibitor group and the inhibitor group had significant increases in body weight, food intake, Lee's index, the levels of TG, FFA and the protein expression of AC-FoxO1 (P<0.01, P<0.05) and significant reductions in the protein expression of SIRT1, FoxO1 and POMC (P<0.01); the inhibitor group had significant increases in the serum levels of TC (P<0.01). Compared with the EA＋inhibitor group, the inhibitor group had significant increases in body weight, food intake, the serum levels of TC, TG, FFA, and the protein expression of AC-FoxO1 (P<0.01), as well as significant reductions in the protein expression of SIRT1, FoxO1 and POMC (P<0.01). In rats with DIO, EA can effectively up-regulate the expression of SIRT1 in the hypothalamus, exert a deacetylation effect on FoxO1, and promote the expression of the downstream appetite-inhibiting peptide POMC, which may be one of the mechanisms of EA to help lose weight by regulating central appetite peptides in the obesity model.

Macrophage-Derived Exosomal Mir-155 Regulating Cardiomyocyte Pyroptosis and Hypertrophy in Uremic Cardiomyopathy

miR-155 was synthesized and loaded into exosomes in increased infiltration of macrophages in a uremic heart.The released exosomal fusion with the plasma membrane leads to the release of miR-155 into the cytosoland translational repression of forkhead transcription factors of the O class (FoxO3a) in cardiomyocytes. Finally, macrophage-derived miR-155-containing exosomes promoted cardiomyocyte pyroptosis and uremic cardiomyopathy changes (cardiac hypertrophy and fibrosis) by directly targeting FoxO3a in uremic mice.

Mmu_circ_0000790 Is Involved in Pulmonary Vascular Remodeling in Mice with HPH via MicroRNA-374c-Mediated FOXC1

Recently, the identification of several circular RNAs (circRNAs) as vital regulators of microRNAs (miRNAs) underlines the increasing complexity of non-coding RNA (ncRNA)-mediated regulatory networks. This study aimed to explore the effects of mmu_circ_0000790 on the biological behaviors of pulmonary artery smooth muscle cells (PASMCs) in hypoxic pulmonary hypertension (HPH). The HPH mouse model and hypoxia-induced PASMC model were initially established, and the expression of mmu_circ_0000790 in the pulmonary vascular tissues and hypoxic PASMCs was determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). A series of in vitro experiments such as dual-luciferase, RNA pull-down, and RNA-binding protein immunoprecipitation (RIP) assays were conducted to evaluate the interactions among mmu_circ_0000790, microRNA-374c (miR-374c), and forkhead transcription factor 1 (FOXC1). The potential physiological functions of mmu_circ_0000790, miR-374c, and FOXC1 in hypoxic PASMCs were investigated through gain- and loss-of function approaches. Upregulated mmu_circ_0000790 was found in both the HPH-pulmonary vascular tissues and hypoxic PASMCs. Additionally, mmu_circ_0000790 could competitively bind to miR-374c and consequently upregulate the target gene of miR-374c, FOXC1. It was also observed that mmu_circ_0000790 induced proliferation and inhibited apoptosis of hypoxic PASMCs, which further promoted the pulmonary vascular remodeling in mice with HPH. Therefore, we speculate that mmu_circ_0000790 may serve as a prospective target for the treatment of patients with HPH.

Low expression and hypermethylation of FOXP3 in regulatory T cells are associated with asthma in children.

The aim of the present study was to determine the expression and methylation levels of forkhead transcription factor P3 (FOXP3) in peripheral blood CD4+CD25+ regulatory T cells (Tregs) harvested from children with asthma, and to explore the pathogenesis of asthma. The percentages of CD4+CD25+FOXP3+ Tregs in CD4+ T lymphocytes from 15 children with asthma and 15 healthy controls were measured by flow cytometry, and FOXP3 mRNA expression in the CD4+CD25+ Tregs was measured by reverse transcriptase-quantitative PCR. In addition, the forced expiratory volume in one second (FEV1) was measured to determine lung function. The methylation statuses of 16 CpG sites in two regions of the FOXP3 gene's exon and intron were analysed with bisulfite-specific PCR and pyrophosphate sequencing. The differences in methylation levels between the asthma and control groups were compared. The percentage of CD4+CD25+FOXP3+ Tregs in CD4+ T lymphocytes and FOXP3 mRNA expression were significantly lower in children with asthma than in control children (P<0.05). The FOXP3 mRNA levels in children with asthma were positively correlated with FEV1 (P<0.001; r=0.895). The methylation levels in 12 of the 16 studied CpG loci of the FOXP3 gene, and of the 6th CpG locus in the exon regions, were significantly higher in the asthma group compared with the control group (P<0.05). In summary, low expression and hypermethylation of the FOXP3 gene in the peripheral blood were associated with the pathogenesis of asthma in children. Thus, the FOXP3 mRNA expression level can be used to predict the severity of asthma in children.

The Critical Role of SIRT1 in Parkinson's Disease: Mechanism and Therapeutic Considerations.

Silence information regulator 1 (SIRT1), a member of the sirtuin family, targets histones and many non-histone proteins and participates in various physiological functions. The enzymatic activity of SIRT1 is decreased in patients with Parkinson’s disease (PD), which may reduce their ability to resist neuronal damage caused by various neurotoxins. As far as we know, SIRT1 can induce autophagy by regulating autophagy related proteins such as AMP-activated protein kinase, light chain 3, mammalian target of rapamycin, and forkhead transcription factor 1. Furthermore, SIRT1 can regulate mitochondrial function and inhibit oxidative stress mainly by maintaining peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in a deacetylated state and thus maintaining a constant level of PGC-1α. Other studies have demonstrated that SIRT1 may play a role in the pathophysiology of PD by regulating neuroinflammation. SIRT1 deacetylases nuclear factor-kappa B and thus reduces its transcriptional activity, inhibits inducible nitric oxide synthase expression, and decreases tumor necrosis factor-alpha and interleukin-6 levels. SIRT1 can also upregulate heat shock protein 70 by deacetylating heat shock factor 1 to increase the degradation of α-synuclein oligomers. Few studies have focused on the relationship between SIRT1 single nucleotide polymorphisms and PD risk, so this topic requires further research. Based on the neuroprotective effects of SIRT1 on PD, many in vitro and in vivo experiments have demonstrated that some SIRT1 activators, notably resveratrol, have potential neuroprotective effects against dopaminergic neuronal damage caused by various neurotoxins. Thus, SIRT1 plays a critical role in PD development and might be a potential target for PD therapy.

Detoxification-related gene expression accompanies anhydrobiosis in the foliar nematode (Aphelenchoides fragariae)

The foliar nematode (Aphelenchoides fragariae) is a quarantined pest that infects a broad range of herbaceous and woody plants. Previous work has demonstrated its remarkable ability to survive rapid and extreme desiccation, although the specific molecular mechanisms underlying its anhydrobiotic response have not been characterized. The authors used RNA sequencing and de novo transcriptome assembly to compare patterns of gene expression between hydrated and 24-hr desiccated nematodes. In total, 2,083 and 953 genes were significantly up- and downregulated, respectively, in desiccated nematodes. Of the 100 annotated genes with the largest positive fold-changes, more than one third encoded putative detoxification-related proteins. Genes encoding enzymes of Phase I and Phase II detoxification systems were among the most strongly upregulated in the transcriptome, including 35 cytochrome p450s, 23 short chain dehydrogenase/reductases, 5 glutathione-S-transferases, and 22 UDP-glucuronosyltransferases. Genes encoding heat shock proteins, unfolded protein response enzymes, and intrinsically disordered proteins were also upregulated. Anhydrobiosis in A. fragariae appears to involve both strategies to minimize protein misfolding and aggregation, and wholesale induction of the cellular detoxification machinery. These processes may be controlled in part through the activity of forkhead transcription factors similar to Caenorhabditis elegans’ daf-16, a number of which were differentially expressed under desiccation.

A novel analysis of gene array data: yeast cell cycle.

Many gene array studies of the yeast cell cycle have been performed (Cho RJ, Campbell MJ, Winzeler EA et al. A genome-wide transcriptional analysis of the mitotic cell cycle. Mol Cell 1998;2:65–73; Orlando DA, Lin CY, Bernard A et al. Global control of cell-cycle transcription by coupled CDK and network oscillators. Nature 2008;453:944–7; Pramila T, Wu W, Miles S et al. The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle. Genes Dev 2006;20:2266–78; Spellman PT, Sherlock G, Zhang MQ et al. Comprehensive identification of cell cycle–regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. MBoC 1998;9:3273–97). Largely, these studies contain elements drawn from laboratory experiments. The present investigation determines cell division cycle (CDC) genes solely from the data (Orlando DA, Lin CY, Bernard A et al. Global control of cell-cycle transcription by coupled CDK and network oscillators. Nature 2008;453:944–7). It is shown by simple reasoning that the dynamics of the approximately 6000 yeast genes are described by an approximately six-dimensional space. This leads a precisely determined cell-cycle period, along with the quality and timing of the identified CDC genes. Convincing evidence for the role of the identified genes is obtained. While these show good agreement with standard CDC gene representatives (Orlando DA, Lin CY, Bernard A et al. Global control of cell-cycle transcription by coupled CDK and network oscillators. Nature 2008;453:944–7; Spellman PT, Sherlock G, Zhang MQ et al. Comprehensive identification of cell cycle–regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. MBoC 1998;9:3273–97; de Lichtenberg U, Jensen LJ, Fausbøll A et al. Comparison of computational methods for the identification of cell cycle-regulated genes. Bioinformatics 2005;21:1164–71) several hundred newly revealed CDC genes appear, which merit attention. The present approach employs an adaptation of a method introduced to study turbulent flows (Schmid PJ. Dynamic mode decomposition of numerical and experimental data. J Fluid Mech 2010;656:5–28), “dynamic mode decomposition” (DMD). From this, one can infer that singular value decomposition, analysis of the data entangles the underlying (gene) dynamics implicit in the data; and that DMD produces the disentangling transformation. It is the assertion of this study that a new tool now exists for the analysis of the gene array signals, and in particular for investigating the yeast cell cycle.

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.

Dynamic Interaction of USP14 with the Chaperone HSC70 Mediates Crosstalk between the Proteasome, ER Signaling, and Autophagy.

SummaryUSP14 is a deubiquitinating enzyme associated with the proteasome important for protein degradation. Here we show that upon proteasome inhibition or expression of the mutant W58A-USP14, association of USP14 with the 19S regulatory particle is disrupted. MS-based interactomics revealed an interaction of USP14 with the chaperone, HSC70, in neuroblastoma cells. Proteasome inhibition enhanced binding of USP14 to HSC70, and to XBP1u and IRE1α proteins, demonstrating a role in the unfolded protein response. Striatal neurons expressing mutant huntingtin exhibited reduced USP14 and HSC70 levels, whereas inhibition of HSC70 downregulated USP14. Furthermore, proteasome inhibition or use of the mutant W58A-USP14 facilitated the interaction of USP14 with the autophagy protein, GABARAP. Functionally, overexpression of W58A-USP14 increased GABARAP positive autophagosomes in striatal neurons, and this was abrogated using the HSC70 inhibitor, VER-155008. Modulation of the USP14-HSC70 axis may represent a potential therapeutic target in HD to beneficially influence multiple proteostasis pathways.

Abstract C49: Knockdown of FOXN2 enhances adhesion and reduces migration in pancreatic cancer cells

Abstract FOXN2, a member of the forkhead domain binding protein family, was identified as a master regulator whose expression and activity are increased in the progression from pancreatic intraepithelial neoplasia (PanIN) to pancreatic ductal adenocarcinoma (PDA). Forkhead transcription factor proteins have been identified as essential components of both oncogenic and tumor suppressive pathways. Deregulation of FOX proteins has a direct role in cancer initiation, maintenance, progression, and drug resistance. In particular, other studies have linked FOXN2 expression to better outcomes in T-cell leukemia and glioblastoma multiforme. Additionally, recent studies indicate that FOXN2 impairs lung cancer cell proliferation in vivo and in vitro. Finally, recent studies in breast cancer cells indicate potential conflicting roles for the FOXN2 transcription factor in maintenance of cancer stem cell phenotypes and in migration and invasion. In the present study, we sought to understand the role of FOXN2 specifically in human pancreatic malignancy. Data from the TCGA database indicates that high levels of FOXN2 are associated with poor overall survival. While knockdown of FOXN2 does not significantly affect proliferation or colony formation of pancreatic cancer cells, we report that knockdown of FOXN2 in PANC-1 and BxPC-3 cells significantly enhances cellular adhesion. Additionally, FOXN2 knockdown reduces cellular invasion through collagen in transwell migration assays. Preliminary data suggest that FOXN2 may mediate some of its effects on adhesion and migration through regulation of Akt2 expression. Overall, our studies suggest that high FOXN2 expression and activity contribute to pancreatic malignancy. A better understanding of the regulation and function of the FOXN2 transcription factor should help us to uncover its potential as a therapeutic target, as well as a predictive marker for cancer. Citation Format: Blanca Santibanez, Sabrina Oliveira, Kelsie Duggan, Kate Lu, Kristen Johnson. Knockdown of FOXN2 enhances adhesion and reduces migration in pancreatic cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C49.

FOXO3a regulates rhinovirus-induced innate immune responses in airway epithelial cells

Forkhead transcription factor class O (FOXO)3a, which plays a critical role in a wide variety of cellular processes, was also found to regulate cell-type-specific antiviral responses. Airway epithelial cells express FOXO3a and play an important role in clearing rhinovirus (RV) by mounting antiviral type I and type III interferon (IFN) responses. To elucidate the role of FOXO3a in regulating antiviral responses, we generated airway epithelial cell-specific Foxo3a knockout (Scga1b1-Foxo3a−/−) mice and a stable FOXO3a knockout human airway epithelial cell line. Compared to wild-type, Scga1b1-Foxo3a−/− mice show reduced IFN-α, IFN-β, IFN-λ2/3 in response to challenge with RV or double-stranded (ds)RNA mimic, Poly Inosinic-polycytidylic acid (Poly I:C) indicating defective dsRNA receptor signaling. RV-infected Scga1b1-Foxo3a−/− mice also show viral persistence, enhanced lung inflammation and elevated pro-inflammatory cytokine levels. FOXO3a K/O airway epithelial cells show attenuated IFN responses to RV infection and this was associated with conformational change in mitochondrial antiviral signaling protein (MAVS) but not with a reduction in the expression of dsRNA receptors under unstimulated conditions. Pretreatment with MitoTEMPO, a mitochondrial-specific antioxidant corrects MAVS conformation and restores antiviral IFN responses to subsequent RV infection in FOXO3a K/O cells. Inhibition of oxidative stress also reduces pro-inflammatory cytokine responses to RV in FOXO3a K/O cells. Together, our results indicate that FOXO3a plays a critical role in regulating antiviral responses as well as limiting pro-inflammatory cytokine expression. Based on these results, we conclude that FOXO3a contributes to optimal viral clearance and prevents excessive lung inflammation following RV infection.

The effect of FOXO gene family variants and global DNA metylation on RRMS disease

Multiple sclerosis is a chronic disease that usually occurs with exacerbations and remissions in young adults, affects the central nervous system white matter in multiple localization, and is thought to be the result of complex interactions of genetic and environmental factors, the most common form is relapsing-remitting MS. Forkhead transcription factors O class (FOXO) are responsible for the regulation of various cellular processes including cell cycle, apoptosis, DNA repair, cellular resistance and metabolism. DNA methylation is such an epigenetic change and has been shown to be associated with almost any biological process. The aim of our study to show the relation between the genetic variants of FOXO3a (rs2253310 rs4966936) and FOXO1 (rs3900833, rs4581585) and global DNA methylation in RRMS. We analyzed DNA obtained from 79 RRMS patients and 104 healthy individuals by PCR-RFLP method for the detection of genetic variants. For the determination of global DNA methylation, results were obtained using ELISA method. The data were evaluated statistically. As a result of our analysis; global DNA methylation is higher in RRMS patients compared to control individuals and it can be effective on the disease. In addition, it has been determined that variants of FOXO3a (rs2253310, rs4966936) and FOXO1 (rs3900833), which have been genotyped, may be effective in disease pathogenesis. These results suggest that DNAmethylation and FOXO gene variants may be effective in neuronal loss in RRMS.

Advanced Glycation End-Products and Hyperglycemia Increase Angiopoietin-2 Production by Impairing Angiopoietin-1-Tie-2 System.

The angiopoietin-Tie-2 system plays a crucial role in the maintenance of endothelial integrity. Hyperglycemia and advanced glycation end-products (AGEs) are involved in endothelial cell dysfunction responsible of the pathogenesis of microvascular complications of diabetes. Here, we investigated whether glycated serum (GS) or hyperglycemia (HG) affect the angiopoietin-Tie-2 system in the microvascular endothelial cells HMEC-1. We found that culture for 5 days in the presence of AGEs and HG (alone or in combination) decreased cell proliferation, increased reactive oxygen species (ROS) production, and reduced ratio between the oxidized and the reduced form of glutathione. Since angiopoietin-1 (Ang-1) signaling regulates angiopoietin-2 (Ang-2) expression through inactivation of the forkhead transcription factor FoxO1, we investigated intracellular signaling of Ang-1 and expression of Ang-2. HG and AGEs reduced phosphorylation of Akt and abrogated phosphorylation of FoxO1 induced by Ang-1 without affecting neither Tie-2 expression nor its activation. Furthermore, AGEs and/or HG induced nuclear translocation of FoxO1 and increased Ang-2 production. In conclusion, we demonstrated that both hyperglycemia and AGEs affect the angiopoietin-Tie-2 system by impairing Ang-1/Tie-2 signaling and by increasing Ang-2 expression. These results suggest that therapeutic strategies useful in preventing or delaying the onset of diabetic vascular complications should be aimed to preserve Ang-1 signaling.

The activity of S. pombe LTR retrotransposons is activated in response to rapamycin

Long terminal repeat (LTR) retrotransposons are mobile genetic elements that are present in the genomes of most eukaryotes. They are closely related to retroviruses and mobilize through an RNA intermediate. The uncontrolled mobilization of retrotransposons is potentially harmful to the integrity of the genome and so the activity of these elements is subjected to strict host cell controls. We are using the fission yeast, Schizosaccharomyces pombe to study the signalling pathways that regulate the activity ofLTR retrotransposons. We have found that the expression and mobilization of the Tf2 LTR retrotransposons is activated in response to exposure to the immunosuppressant drug rapamycin. Rapamycin binds to the conserved FKBP12 protein (called Fkh1 in S. pombe) and the resulting FKBP12-rapamycin complex inhibits the kinase activity of the conserved the TORC1 complex. This suggests that Tf2 activity is under the control of the TORC1 signalling network which is a master regulator of cellular responses to nutrient and energy availability. However, the inhibition of TORC1 activity using a tor2 temperature sensitive allele or a direct chemical inhibitor (Torin) did not activate either the expression or mobilization of Tf2 elements. Therefore, rapamycin may be controlling Tf2 activity via a TORC1-independent pathway. We are currently defining this pathway and find that it is dependent upon the FKBP12 protein, Fkh1 and the Forkhead transcription factor, Fhl1.

Effect of lncRNA MEG3 on retinopathy in diabetic rats through regulating Fox01 expression.

The aim of this study was to investigate the effect of long non-coding ribonucleic acid (lncRNA) maternally expressed gene 3 (MEG3) on retinopathy in diabetic rats by regulating the expression of forkhead transcription factor 01 (Fox01). All rats were randomly divided into three groups, including the control group (n=10), diabetes mellitus (DM) group (n=10) and lncRNA MEG3 transfection group (n=10). The expressions of Fox01 and interleukin-1β (IL-1β) in the three groups were detected using immunohistochemical staining, quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting. Microscopic examinations showed that the retinal structure was clear and complete, the inner limiting membrane surface was smooth, and the cells were arranged orderly with uniform structure in the control group. In the DM group, the retinal ganglia were slightly thickened, the histiocytes were sparse and arranged disorderly, and the edema of the outer plexiform layer (OPL) was significant. Meanwhile, there was abnormal microvascular dilatation without neovascularization. In lncRNA MEG3 transfection group, the edema of retinal OPL was significantly alleviated when compared with the DM group, showing statistically significant differences (p<0.05). The results of immunohistochemical staining showed that the expressions of Fox01 and IL-1β in the inner plexiform layer and inner nuclear layer increased markedly in the DM group and lncRNA MEG3 transfection group when compared with those in the control group (p<0.05). However, they were both significantly declined in lncRNA MEG3 transfection group when compared with the DM group (p<0.05). Furthermore, Western blotting and qRT-PCR indicated that the protein and mRNA expressions of Fox01 and IL-1β in the retina of DM group and lncRNA MEG3 transfection group were remarkably higher than the control group (p<0.05). However, they were remarkably declined in lncRNA MEG3 transfection group when compared with the DM group (p<0.05). LncRNA MEG3 plays an important role in retinopathy in diabetic rats. In addition, it can ameliorate retinopathy in diabetic rats by inhibiting the expressions of IL-1β and Fox01.

Dietary Supplementation of Methyl Donor l-Methionine Alters Epigenetic Modification in Type 2 Diabetes.

The aim of the current study is to evaluate whether l-methionine supplementation (l-Met-S) improves type 2 diabetes-induced alterations in glucose and lipid metabolism by modulating one-carbon metabolism and methylation status. Diabetes is induced in male Sprague-Dawley rats using high-fat diet and low dose streptozotocin. At the end of study, various biochemical parameters, immunoblotting, qRT-PCR and ChIP-qPCR are performed. The first evidence that l-Met-S activates p-AMPK and SIRT1, very similar to "metformin," is provided. l-Met-S improves the altered key one-carbon metabolites in diabetic rats by modulating methionine adenosyl transferase 1A and cystathione β synthase expression. qRT-PCR shows that l-Met-S alleviates diabetes-induced increase in Forkhead transcription factor 1 expression and thereby regulating genes involved in glucose (G6pc, Pdk4, Pklr) and lipid metabolism (Fasn). Interestingly, l-Met-S inhibits the increased expression of DNMT1 and also prevents methylation of histone H3K36me2 under diabetic condition. ChIP assay shows that persistent increase in abundance of histone H3K36me2 on the promoter region of FOXO1 in diabetic rats and it is recovered by l-Met-S. The first evidence that dietary supplementation of l-Met prevents diabetes-induced epigenetic alterations and regulating methionine levels can be therapeutically exploited for the treatment of metabolic diseases is provided.