Genome-wide DNA methylation analysis in blood cells from patients with Werner syndrome

Werner syndrome is a progeroid disorder characterized by premature age-related phenotypes. Although it is well established that autosomal recessive mutations in the WRN gene is responsible for Werner syndrome, the molecular alterations that lead to disease phenotype remain still unidentified

Unregulated miR-96 Induces Cell Proliferation in Human Breast Cancer by Downregulating Transcriptional Factor FOXO3a

FOXO transcription factors are key tumor suppressors in mammalian cells. Until now, suppression of FOXOs in cancer cells was thought to be mainly due to activation of multiple onco-kinases by a phosphorylation-ubiquitylation-mediated cascade. Therefore, it was speculated that inhibition of FOXO proteins would naturally occur through a multiple step post-translational process. However, whether cancer cells may downregulate FOXO protein via an alternative regulatory mechanism is unclear. In the current study, we report that expression of miR-96 was markedly upregulated in breast cancer cells and breast cancer tissues compared with normal breast epithelial cells (NBEC) and normal breast tissues. Ectopic expression of miR-96 induced the proliferation and anchorage-independent growth of breast cancer cells, while inhibition of miR-96 reduced this effect. Furthermore, upregulation of miR-96 in breast cancer cells resulted in modulation of their entry into the G1/S transitional phase, which was caused by downregulation of cyclin-dependent kinase (CDK) inhibitors, p27Kip1 and p21Cip1, and upregulation of the cell-cycle regulator cyclin D1. Moreover, we demonstrated that miR-96 downregulated FOXO3a expression by directly targeting the FOXO3a 3′-untranslated region. Taken together, our results suggest that miR-96 may play an important role in promoting proliferation of human breast cancer cells and present a novel mechanism of miRNA-mediated direct suppression of FOXO3a expression in cancer cells

Two step activation of FOXO3 by AMPK generates a coherent feed-forward loop determining excitotoxic cell fate

Cerebral ischemia and excitotoxic injury induce transient or permanent bioenergetic failure, and may result in neuronal apoptosis or necrosis. We have previously shown that ATP depletion and activation of AMP-activated protein kinase (AMPK) during excitotoxic injury induces neuronal apoptosis by transcription of the proapoptotic BH3 only protein, Bim. AMPK, however, also exerts pro-survival functions in neurons. The molecular switches that determine these differential outcomes are not well understood. Using an approach combining biochemistry, single cell imaging and computational modeling, we here demonstrate that excitotoxic injury activated the bim promoter in a FOXO3-dependent manner. The activation of AMPK reduced AKT activation, and led to dephosphorylation and nuclear translocation of FOXO3. Subsequent mutation studies indicated that bim gene activation during excitotoxic injury required direct FOXO3 phosphorylation by AMPK in the nucleus as a second activation step. Inhibition of this phosphorylation prevented Bim expression and protected neurons against excitotoxic and oxygen/glucose deprivation-induced injury. Systems analysis and computational modeling revealed that these two activation steps defined a coherent feedforward loop; a network motif capable of filtering any effects of short-term AMPK activation on bim gene induction. This may prevent unwanted AMPK-mediated Bim expression and apoptosis during transient or physiological bioenergetic stress

Gene Expression of the Tumour Suppressor LKB1 Is Mediated by Sp1, NF-Y and FOXO Transcription Factors

The serine/threonine kinase LKB1 is a tumour suppressor that regulates multiple biological pathways, including cell cycle control, cell polarity and energy metabolism by direct phosphorylation of 14 different AMP-activated protein kinase (AMPK) family members. Although many downstream targets have been described, the regulation of LKB1 gene expression is still poorly understood. In this study, we performed a functional analysis of the human LKB1 upstream regulatory region. We used 200 base pair deletion constructs of the 5′-flanking region fused to a luciferase reporter to identify the core promoter. It encompasses nucleotides −345 to +52 relative to the transcription start site and coincides with a DNase I hypersensitive site. Based on extensive deletion and substitution mutant analysis of the LKB1 promoter, we identified four cis-acting elements which are critical for transcriptional activation. Using electrophoretic mobility shift assays as well as chromatin immunoprecipitations, we demonstrate that the transcription factors Sp1, NF-Y and two forkhead box O (FOXO) family members FOXO3 and FOXO4 bind to these elements. Overexpression of these factors significantly increased the LKB1 promoter activity. Conversely, small interfering RNAs directed against NF-Y alpha and the two FOXO proteins greatly reduced endogenous LKB1 expression and phosphorylation of LKB1's main substrate AMPK in three different cell lines. Taken together, these results demonstrate that Sp1, NF-Y and FOXO transcription factors are involved in the regulation of LKB1 transcription

Inhibition of FOXO3 Tumor Suppressor Function by βTrCP1 through Ubiquitin-Mediated Degradation in a Tumor Mouse Model

The ubiquitin-proteasome system is the primary proteolysis machine for controlling protein stability of the majority of regulatory proteins including those that are critical for cancer development. The forkhead box transcription factor FOXO3 plays a key role in regulating tumor suppression; however, the control of FOXO3 protein stability remains to be established. It is crucial to elucidate the molecular mechanisms underlying the ubiquitin-mediated degradation of FOXO3 tumor suppressor.Here we show that betaTrCP1 oncogenic ubiquitin E3-ligase interacts with FOXO3 and induces its ubiquitin-dependent degradation in an IkappaB kinase-beta phosphorylation dependent manner. Silencing betaTrCP1 augments FOXO3 protein level, resulting in promoting cellular apoptosis in cancer cells. In animal models, increasing FOXO3 protein level by silencing betaTrCP1 suppresses tumorigenesis, whereas decreasing FOXO3 by over-expressing betaTrCP1 promotes tumorigenesis and tumor growth in vivo.This is a unique demonstration that the betaTrCP1-mediated FOXO3 degradation plays a crucial role in tumorigenesis. These findings significantly contribute to understanding of the control of FOXO3 stability in cancer cells and may provide opportunities for developing innovative anticancer therapeutic modalities

FoxO and Stress Responses in the Cnidarian Hydra vulgaris

Background: In the face of changing environmental conditions, the mechanisms underlying stress responses in diverse organisms are of increasing interest. In vertebrates, Drosophila, and Caenorhabditis elegans, FoxO transcription factors mediate cellular responses to stress, including oxidative stress and dietary restriction. Although FoxO genes have been identified in early-arising animal lineages including sponges and cnidarians, little is known about their roles in these organisms. Methods/Principal Findings: We have examined the regulation of FoxO activity in members of the well-studied cnidarian genus Hydra. We find that Hydra FoxO is expressed at high levels in cells of the interstitial lineage, a cell lineage that includes multipotent stem cells that give rise to neurons, stinging cells, secretory cells and gametes. Using transgenic Hydra that express a FoxO-GFP fusion protein in cells of the interstitial lineage, we have determined that heat shock causes localization of the fusion protein to the nucleus. Our results also provide evidence that, as in bilaterian animals, Hydra FoxO activity is regulated by both Akt and JNK kinases. Conclusions: These findings imply that basic mechanisms of FoxO regulation arose before the evolution of bilaterians an

Longevity in mice: is stress resistance a common factor?

A positive relationship between stress resistance and longevity has been reported in a multitude of studies in organisms ranging from yeast to mice. Several mouse lines have been discovered or developed that exhibit extended longevities when compared with normal, wild-type mice of the same genetic background. These long-living lines include the Ames dwarf, Snell dwarf, growth hormone receptor knockout (Laron dwarf), IGF-1 receptor heterozygote, Little, α-MUPA knockout, p66shc knockout, FIRKO, mClk-1 heterozygote, thioredoxin transgenic, and most recently the Klotho transgenic mouse. These mice are described in terms of the reported extended lifespans and studies involving resistance to stress. In addition, caloric restriction (CR) and stress resistance are briefly addressed for comparison with genetically altered mice. Although many of the long-living mice have GH/IGF-1/insulin signaling-related alterations and enhanced stress resistance, there are some that exhibit life extension without an obvious link to this hormone pathway. Resistance to oxidative stress is by far the most common system studied in long-living mice, but there is evidence of enhancement of resistance in other systems as well. The differences in stress resistance between long-living mutant and normal mice result from complex interrelationships among pathways that appear to coordinate signals of growth and metabolism, and subsequently result in differences in lifespan

Co-Depletion of Cathepsin B and uPAR Induces G0/G1 Arrest in Glioma via FOXO3a Mediated p27Kip1 Upregulation

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined.Cathepsin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27(Kip1) and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by alphaVbeta3/PI3K/AKT/FOXO pathway as observed by the decreased alphaVbeta3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27(Kip1) and FOXO3a when treated with Ly294002 (10 microM) and increased luciferase expression with the siRNA and Ly294002 treatments when the FOXO binding promoter region of p27(Kip1) was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly.Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27(Kip1) accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells