Evasion of cell senescence in SHH medulloblastoma

The mechanisms leading to brain tumor formation are poorly understood. Using Ptch1^+/? mice as a medulloblastoma model, sequential mutations were found to shape tumor evolution. Initially, medulloblastoma preneoplastic lesions display loss of heterozygosity of the Ptch1 wild-type allele, an event associated with cell senescence in preneoplasia. Subsequently, p53 mutations lead to senescence evasion and progression from preneoplasia to medulloblastoma. These findings are consistent with a model where high levels of Hedgehog signaling caused by the loss of the tumor suppressor Ptch1 lead to oncogene-induced senescence and drive p53 mutations. Thus, cell senescence is an important characteristic of a subset of SHH medulloblastoma and might explain the acquisition of somatic TP53 mutations in human medulloblastoma. This mode of medulloblastoma formation contrasts with the one characterizing Li-Fraumeni patients with medulloblastoma, where TP53 germ-line mutations cause chromothriptic genomic instability and lead to mutations in Hedgehog signaling genes, which drive medulloblastoma growth. Here we discuss in detail these 2 alternative mechanisms leading to medulloblastoma tumorigenesis.     

Rescue of Holoprosencephaly in Fetal Alcohol-Exposed Cdon Mutant Mice by Reduced Gene Dosage of Ptch1

Holoprosencephaly (HPE) is a commonly occurring developmental defect in which midline patterning of the forebrain and midface is disrupted. Sonic hedgehog (SHH) signaling is required during multiple stages of rostroventral midline development, and heterozygous mutations in SHH pathway components are associated with HPE. However, clinical presentation of HPE is highly variable, and carriers of heterozygous mutations often lack apparent defects. It is therefore thought that such mutations must interact with more common modifiers, genetic and/or environmental. We have modeled this scenario in mice. Cdon mutant mice have a largely subthreshold defect in SHH signaling, rendering them sensitive to a wide spectrum of HPE phenotypes by additional hits that are themselves insufficient to produce HPE, including transient in utero exposure to ethanol. These variable HPE phenotypes may arise in embryos that fail to reach a threshold level of SHH signaling at a specific developmental stage. To provide evidence for this possibility, here we tested the effect of removing one copy of the negative regulator Ptch1 from Cdon^−/− embryos and compared their response to ethanol with that of Cdon^−/−;Ptch1^+/+ embryos. Ptch1 heterozygosity decreased the penetrance of HPE in this system by >75%. The major effect of reduced Ptch1 gene dosage was on penetrance, as those Cdon^−/−;Ptch1^+/− embryos that displayed HPE did not show major differences in phenotype from Cdon^−/−;Ptch1^+/+ embryos with ethanol-induced HPE. Our findings are consistent with the notion that even in an etiologically complex model of HPE, the level of SHH pathway activity is rate-limiting. Furthermore, the clinical outcome of an individual carrying a SHH pathway mutation will likely reflect the sum effect of both deleterious and protective modifier alleles and their interaction with non-genetic risk factors like fetal alcohol exposure.     

Mutation of the LXCXE Binding Cleft of pRb Facilitates Transformation by ras In Vitro but Does Not Promote Tumorigenesis In Vivo

Background

The Retinoblastoma protein (pRB) is a key tumor suppressor that is functionally inactivated in most cancers. pRB regulates the cell division cycle and cell cycle exit through protein–protein interactions mediated by its multiple binding interfaces. The LXCXE binding cleft region of pRB mediates interactions with cellular proteins that have chromatin regulatory functions. Chromatin regulation mediated by pRB is required for a stress responsive cell cycle arrest, including oncogene induced senescence. The in vivo role of chromatin regulation by pRB during senescence, and its relevance to cancer is not clear.

Methodology/Principal Findings

Using gene-targeted mice, uniquely defective for pRB mediated chromatin regulation, we investigated its role during transformation and tumor progression in response to activation of oncogenic ras. We report that the pRB^∆L mutation confers susceptibility to escape from HrasV12 induced senescence and allows transformation in vitro, although these cells possess high levels of DNA damage. Intriguingly, LSL-Kras, Rb1 ^∆L/∆L mice show delayed lung tumor formation compared to controls. This is likely due to the increased apoptosis seen in the early hyperplastic lesions shortly following ras activation that inhibits tumor progression. Furthermore, DMBA treatment to induce sporadic ras mutations in other tissues also failed to reveal greater susceptibility to cancer in Rb1 ^∆L/∆L mice.

Conclusions/Significance

Our data suggests that chromatin regulation by pRB can function to limit proliferation, but its loss fails to contribute to cancer susceptibility in ras driven tumor models because of elevated levels of DNA damage and apoptosis.     

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-α) secretion by macrophages was essential for robust SHH activation, as TNF-α^−/− macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-α or blockade of TNF-α receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.     

In Vitro Comparison of Hypericin and 5-Aminolevulinic Acid-Derived Protoporphyrin IX for Photodynamic Inactivation of Medulloblastoma Cells

Background

Hypericin (HYP) is a naturally occurring photosensitizer. Cellular uptake and photodynamic inactivation after incubation with this photosensitizer have neither been examined in medulloblastoma cells in vitro, nor compared with 5-aminolevulinic acid-derived protoporphyrin IX (5-ALA-derived PpIX).

Methods

In 3 medulloblastoma cell lines (D283 Med, Daoy, and D341 Med) the time- and concentration-dependent intracellular accumulation of HYP and 5-ALA-derived PpIX was analyzed by fluorescence microscopy (FM) and FACS. Photocytotoxicity was measured after illumination at 595 nm (HYP) and 635 nm (5-ALA-derived PpIX) in D283 Med cells and compared to U373 MG glioma cells.

Results

All medulloblastoma cell lines exhibited concentration- and time-dependent uptake of HYP. Incubation with HYP up to 10 µM resulted in a rapid increase in fluorescence intensity, which peaked between 2 and 4 hours. 5-ALA-derived PpIX accumulation increased in D283 Med cells by 22% over baseline after 5-ALA incubation up to 1.2 mM. Photocytotoxicity of 5-ALA-derived PpIX was higher in D283 Med medulloblastoma compared to U373MG glioma. The [lethal dose (light dose that is required to reduce cell survival to 50% of control)] of 5-ALA-derived PpIX was 3.8 J/cm² in D283 Med cells versus 5.7 J/cm2 in U373MG glioma cells. Photocytotoxicity of HYP in D283 Med cells was determined at 2.5 µM after an incubation time of 2 h and an illumination wavelength of 595 nm. The value was 0.47 J/cm².

Conclusion

By its 5-fold increase in fluorescence over autofluorescence levels HYP has excellent properties for tumor visualization in medulloblastomas. The high photocytotoxicity of HYP, compared to 5-ALA-derived PpIX, is convincingly demonstrated by its 8- to 13-fold lower . Therefore HYP might be a promising molecule for intraoperative visualization and photodynamic treatment of medulloblastomas.     

Epigenetic Changes in Basal Cell Carcinoma Affect SHH and WNT Signaling Components

Background

The genetic background of Basal Cell Carcinoma (BCC) has been studied extensively, while its epigenetic makeup has received comparatively little attention. Epigenetic alterations such as promoter hypermethylation silence tumor suppressor genes (TSG) in several malignancies.

Objective

We sought to analyze the promoter methylation status of ten putative (tumor suppressor) genes that are associated with Sonic Hedgehog (SHH), WNT signaling and (hair follicle) tumors in a large series of 112 BCC and 124 healthy control samples by methylation-specific PCR.

Results

Gene promoters of SHH (P = 0.016), adenomatous polyposis coli (APC) (P = 0.003), secreted frizzled-related protein 5 (SFRP5) (P = 0.004) and Ras association domain family 1A (RASSF1A) (P = 0.023) showed significantly more methylation in BCC versus normal skin. mRNA levels of these four genes were reduced for APC and SFRP5 in BCC (n = 6) vs normal skin (n = 6). Down regulation of SHH, APC and RASSF1A could be confirmed on protein level as well (P<0.001 for all genes) by immunohistochemical staining. Increased canonical WNT activity was visualized by β-catenin staining, showing nuclear β-catenin in only 28/101 (27.7%) of BCC. Absence of nuclear β-catenin in some samples may be due to high levels of membranous E-cadherin (in 94.1% of the samples).

Conclusions

We provide evidence that promoter hypermethylation of key players within the SHH and WNT pathways is frequent in BCC, consistent with their known constitutive activation in BCC. Epigenetic gene silencing putatively contributes to BCC tumorigenesis, indicating new venues for treatment.     

Intestinal-Specific TNFα Overexpression Induces Crohn’s-Like Ileitis in Mice

Background and Aim

Human and animal studies have clearly established tumor necrosis factor (TNF)α as an important mediator of Crohn’s disease pathogenesis. However, whether systemic or only local TNFα overproduction is required for the development of chronic intestinal inflammation and Crohn’s disease remains unclear. The aim of this study was to assess the contribution of intestinal epithelial-derived TNFα to the development of murine Crohn’s-like ileitis.

Methods

We adapted the well-established TNF^∆ARE/+ mouse model of Crohn’s disease (which systemically overexpresses TNFα) to generate a homozygous mutant strain that overexpress TNFα only within the intestinal epithelium. Intestinal-specific TNF^{i∆ARE/i∆ARE} mice were examined for histopathological signs of gut inflammation and extraintestinal manifestations of Crohn’s disease. The mucosal immune phenotype was characterized, and the contribution of specific lymphocyte populations to the pathogenesis of TNF^{i∆ARE/i∆ARE} ileitis was assessed.

Results

TNF^{i∆ARE/i∆ARE} mice had increased mucosal and systemic TNFα levels compared to wild-type controls (P<0.001), as well as severe chronic ileitis with increased neutrophil infiltration and villous distortion, but no extraintestinal manifestations (P<0.001 vs. wild-type controls). The gut mucosal lymphocytic compartment was also expanded in TNF^{i∆ARE/i∆ARE} mice (P<0.05), consisting of activated CD69⁺ and CD4⁺CD62L^- lymphocytes (P<0.05). FasL expression was significantly elevated in the mesenteric lymph nodes of TNF^{i∆ARE/i∆ARE} mice (P<0.05). Adoptive transfer of mucosal TNF^{i∆ARE/i∆ARE} lymphocytes resulted in ileitis in immunologically naïve severe combined immunodeficiency recipients (P<0.05 vs. wild-type controls), indicating an effector phenotype that was associated with increased production of both Th1 (IFNγ) and Th2 (IL-5, IL-13) cytokines.

Conclusion

Intestinal epithelial-derived TNFα is sufficient for the induction of Crohn’s-like ileitis, but not for the occurrence of extraintestinal manifestations, in TNF^{i∆ARE/i∆ARE} mice. These effects were associated with generation of effector lymphocytes within the intestinal mucosa and dysregulated apoptosis. Thus, targeted intestinal blockade of TNFα may provide an effective means to neutralize gut-derived TNFα with reduced side effects.     

Serum level of adiponectin is a surrogate independent biomarker of radiographic disease progression in early rheumatoid arthritis: results from the ESPOIR cohort

Introduction

Adipokines such as adiponectin, leptin, and visfatin/nicotinamide phosphoribosyltransferase (NAMPT) have recently emerged as pro-inflammatory mediators involved in the pathophysiology of rheumatoid arthritis (RA). We aimed to determine whether serum adipokine levels independently predicted early radiographic disease progression in early RA.

Methods

In total, 791 patients were included from the prospective Etude et Suivi des POlyarthrites Indifférenciées Récentes (ESPOIR) cohort who met the American College of Rheumatology-European League Against Rheumatism criteria for RA (n = 632) or had undifferentiated arthritis (UA) (n = 159). Enzyme-linked immunosorbent assay (ELISA) was used to assess baseline serum levels of adiponectin, leptin, and visfatin/NAMPT. In the RA group, we tested the association of serum adipokine levels and (a) baseline radiographic damage and (b) radiographic disease progression, defined as a change >0 or ≥5 in total Sharp-van der Heijde Score (∆SHS) between inclusion and 1 year (∆SHS ≥1 or rapid radiographic progression: ∆SHS ≥5), adjusting for confounders (age, sex, body-mass index, insulin resistance, C-reactive protein level, Disease Activity Score in 28 joints, Health Assessment Questionnaire score, autoantibody status, steroid use, and radiographic evidence of RA damage at inclusion).

Results

Adiponectin level was independently associated with baseline total SHS (adjusted β = 0.12; P = 0.006). It was also associated with ∆SHS ≥1 (adjusted odds ratio (aOR) = 1.84 (1.25 to 2.72)) involving erosive as well as narrowing disease progression (aOR = 1.73 (1.17 to 2.55) and 1.93 (1.04 to 3.57), respectively). Serum adiponectin level predicted ∆SHS ≥5 (aOR = 2.0 (1.14 to 3.52)). Serum leptin level was independently associated only with ∆SHS >0 (aOR = 1.59 (1.05 to 2.42)). Conversely, serum visfatin/NAMPT level and radiographic disease progression were unrelated. Considering the receiver-operated characteristic curves, the best adiponectin cut-offs were 4.14 μg/ml for ∆SHS ≥1 and 6.04 μg/ml for ∆SHS ≥5, with a good specificity (58% and 75% for ∆SHS ≥1 and ∆SHS ≥5, respectively) and high negative predictive values (75% and 92% for ∆SHS ≥1 or ∆SHS ≥5, respectively).

Conclusion

Serum adiponectin level is a simple useful biomarker associated with early radiographic disease progression in early RA, independent of RA-confounding factors and metabolic status.     

The RAVE complex is an isoform-specific V-ATPase assembly factor in yeast

The regulator of ATPase of vacuoles and endosomes (RAVE) complex is implicated in vacuolar H⁺-translocating ATPase (V-ATPase) assembly and activity. In yeast, rav1∆ mutants exhibit a Vma⁻ growth phenotype characteristic of loss of V-ATPase activity only at high temperature. Synthetic genetic analysis identified mutations that exhibit a full, temperature-independent Vma⁻ growth defect when combined with the rav1∆ mutation. These include class E vps mutations, which compromise endosomal sorting. The synthetic Vma⁻ growth defect could not be attributed to loss of vacuolar acidification in the double mutants, as there was no vacuolar acidification in the rav1∆ mutant. The yeast V-ATPase a subunit is present as two isoforms, Stv1p in Golgi and endosomes and Vph1p in vacuoles. Rav1p interacts directly with the N-terminal domain of Vph1p. STV1 overexpression suppressed the growth defects of both rav1∆ and rav1∆vph1∆, and allowed RAVE-independent assembly of active Stv1p-containing V-ATPases in vacuoles. Mutations causing synthetic genetic defects in combination with rav1∆ perturbed the normal localization of Stv1–green fluorescent protein. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vma⁻ phenotypes arise from defects in Vph1p-containing complexes caused by rav1∆, combined with defects in Stv1p-containing V-ATPases caused by the second mutation. Thus RAVE is the first isoform-specific V-ATPase assembly factor.     

Functions of Ceramide Synthase Paralogs YPR114w and YJR116w of Saccharomyces cerevisiae

Ceramide is synthesized in yeast by two redundant acyl-CoA dependent synthases, Lag1 and Lac1. In lag1∆ lac1∆ cells, free fatty acids and sphingoid bases are elevated, and ceramides are produced through the redundant alkaline ceramidases Ypc1 and Ydc1, working backwards. Even with all four of these genes deleted, cells are surviving and continue to contain small amounts of complex sphingolipids. Here we show that these residual sphingolipids are not synthesized by YPR114w or YJR116w, proteins of unknown function showing a high degree of homology to Lag1 and Lac1. Indeed, the hextuple lag1∆ lac1∆ ypc1∆ ydc1∆ ypr114w∆ yjr116w∆ mutant still contains ceramides and complex sphingolipids. Yjr116w∆ exhibit an oxygen-dependent hypersensitivity to Cu²⁺ due to an increased mitochondrial production of reactive oxygen species (ROS) and a mitochondrially orchestrated programmed cell death in presence of copper, but also a general copper hypersensitivity that cannot be counteracted by the antioxidant N-acetyl-cysteine (NAC). Myriocin efficiently represses the synthesis of sphingoid bases of ypr114w∆, but not its growth. Both yjr116w∆ and ypr114w∆ have fragmented vacuoles and produce less ROS than wild type, before and after diauxic shift. Ypr114w∆/ypr114w∆ have an increased chronological life span. Thus, Yjr116w and Ypr114w are related, but not functionally redundant.     

Exercise and obesity in fibromyalgia: beneficial roles of IGF-1 and resistin?

Introduction

Severe fatigue is a major health problem in fibromyalgia (FM). Obesity is common in FM, but the influence of adipokines and growth factors is not clear. The aim was to examine effects of exercise on fatigue, in lean, overweight and obese FM patients.

Methods

In a longitudinal study, 48 FM patients (median 52 years) exercised for 15 weeks. Nine patients were lean (body mass index, BMI 18.5 to 24.9), 26 overweight (BMI 25 to 29.9) and 13 obese. Fatigue was rated on a 0 to 100 mm scale (fibromyalgia impact questionnaire [FIQ] fatigue) and multidimensional fatigue inventory (MFI-20) general fatigue (MFIGF). Higher levels in FIQ fatigue and MFIGF indicate greater degree of fatigue. Free and total IGF-1, neuropeptides, adipokines were determined in serum and cerebrospinal fluid (CSF).

Results

Baseline FIQ fatigue correlated negatively with serum leptin (r = -0.345; P = 0.016) and nerve growth factor (NGF; r = -0.412; P = 0.037). In lean patients, baseline MFIGF associated negatively with serum resistin (r = -0.694; P = 0.038). FIQ Fatigue associated negatively with CSF resistin (r = -0.365; P = 0.073). Similarly, FIQ fatigue (r = -0.444; P = 0.026) and MFIGF correlated negatively with CSF adiponectin (r = -0.508; P = 0.01). In lean patients, FIQ fatigue (P = 0.046) decreased after 15 weeks. After 30 weeks, MFIGF decreased significantly in lean (MFIGF: P = 0.017), overweight (MFIGF: P = 0.001), and obese patients (MFIGF: P = 0.016). After 15 weeks, total IGF-1 increased in lean (P = 0.043) patients. ∆Total IGF-1 differed significantly between lean and obese patients (P = 0.010). ∆Total IGF-1 related negatively with ∆MFIGF after 15 weeks (r = -0.329; P = 0.050). After 30 weeks, ∆FIQ fatigue negatively correlated with ∆NGF (r = -0.463; P = 0.034) and positively with ∆neuropeptide Y (NPY) (r = 0.469; P = 0.032). Resistin increased after 30 weeks (P = 0.034). ∆MFIGF correlated negatively with ∆resistin (r = -0.346; P = 0.031), being strongest in obese patients (r = -0.815; P = 0.007). In obese patients, ∆FIQ fatigue after 30 weeks correlated negatively with ∆free IGF-1 (r = -0.711; P = 0.032).

Conclusions

Exercise reduced fatigue in all FM patients, this effect was achieved earlier in lean patients. Baseline levels of resistin in both serum and CSF associated negatively with fatigue. Resistin was increased after the exercise period which correlated with decreased fatigue. Changes in IGF-1 indicate similar long-term effects in obese patients. This study shows reduced fatigue after moderate exercise in FM and indicates the involvement of IGF-1 and resistin in these beneficial effects.

Trial registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT00643006","term_id":"NCT00643006"}}NCT00643006     

MicroRNA Biogenesis and Hedgehog-Patched Signaling Cooperate to Regulate an Important Developmental Transition in Granule Cell Development

The Dicer1, Dcr-1 homolog (Drosophila) gene encodes a type III ribonuclease required for the canonical maturation and functioning of microRNAs (miRNAs). Subsets of miRNAs are known to regulate normal cerebellar granule cell development, in addition to the growth and progression of medulloblastoma, a neoplasm that often originates from granule cell precursors. Multiple independent studies have also demonstrated that deregulation of Sonic Hedgehog (Shh)-Patched (Ptch) signaling, through miRNAs, is causative of granule cell pathologies. In the present study, we investigated the genetic interplay between miRNA biogenesis and Shh-Ptch signaling in granule cells of the cerebellum by way of the Cre/lox recombination system in genetically engineered models of Mus musculus (mouse). We demonstrate that, although the miRNA biogenesis and Shh-Ptch-signaling pathways, respectively, regulate the opposing growth processes of cerebellar hypoplasia and hyperplasia leading to medulloblastoma, their concurrent deregulation was nonadditive and did not bring the growth phenotypes toward an expected equilibrium. Instead, mice developed either hypoplasia or medulloblastoma, but of a greater severity. Furthermore, some genotypes were bistable, whereby subsets of mice developed hypoplasia or medulloblastoma. This implies that miRNAs and Shh-Ptch signaling regulate an important developmental transition in granule cells of the cerebellum. We also conclusively show that the Dicer1 gene encodes a haploinsufficient tumor suppressor gene for Ptch1-induced medulloblastoma, with the monoallielic loss of Dicer1 more severe than biallelic loss. These findings exemplify how genetic interplay between pathways may produce nonadditive effects with a substantial and unpredictable impact on biology. Furthermore, these findings suggest that the functional dosage of Dicer1 may nonadditively influence a wide range of Shh-Ptch-dependent pathologies.     

Crosstalks between Cytokines and Sonic Hedgehog in Helicobacter pylori Infection: A Mathematical Model

Helicobacter pylori infection of gastric tissue results in an immune response dominated by Th1 cytokines and has also been linked with dysregulation of Sonic Hedgehog (SHH) signaling pathway in gastric tissue. However, since interactions between the cytokines and SHH during H. pylori infection are not well understood, any mechanistic understanding achieved through interpretation of the statistical analysis of experimental results in the context of currently known circuit must be carefully scrutinized. Here, we use mathematical modeling aided by restraints of experimental data to evaluate the consistency between experimental results and temporal behavior of H. pylori activated cytokine circuit model. Statistical analysis of qPCR data from uninfected and H. pylori infected wild-type and parietal cell-specific SHH knockout (PC-SHH^KO) mice for day 7 and 180 indicate significant changes that suggest role of SHH in cytokine regulation. The experimentally observed changes are further investigated using a mathematical model that examines dynamic crosstalks among pro-inflammatory (IL1β, IL-12, IFNγ, MIP-2) cytokines, anti-inflammatory (IL-10) cytokines and SHH during H. pylori infection. Response analysis of the resulting model demonstrates that circuitry, as currently known, is inadequate for explaining of the experimental observations; suggesting the need for additional specific regulatory interactions. A key advantage of a computational model is the ability to propose putative circuit models for in-silico experimentation. We use this approach to propose a parsimonious model that incorporates crosstalks between NFĸB, SHH, IL-1β and IL-10, resulting in a feedback loop capable of exhibiting cyclic behavior. Separately, we show that analysis of an independent time-series GEO microarray data for IL-1β, IFNγ and IL-10 in mock and H. pylori infected mice further supports the proposed hypothesis that these cytokines may follow a cyclic trend. Predictions from the in-silico model provide useful insights for generating new hypothesis and design of subsequent experimental studies.     

SUMO-targeted ubiquitin ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin

Centromeric histone H3, CENP-A^Cse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-A^Cse4 restricts its localization to centromeres. Mislocalization of CENP-A^Cse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1, since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization than either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.     

Protease Nexin-1 affects the migration and invasion of C6 glioma cells through the regulation of urokinase Plasminogen Activator and Matrix Metalloproteinase-9/2

Protease Nexin-1 (PN-1) or Serpine2 is a physiological regulator of extracellular proteases as thrombin and urokinase (uPA) in the brain. Besides, PN-1 is also implicated in some human cancers and further identified as a substrate for Matrix Metalloproteinase (MMP)-9, a key enzyme in tumor invasiveness. Our aim was to study the role of PN-1 in the migration and invasive potential of glioma cells, using the rat C6 glioma cell line as stable clones transfected with pAVU6 + 27 vector expressing PN-1 short-hairpin RNA. We find that PN-1 knockdown enhanced the in vitro migration and invasiveness of C6 cells which also showed a strong gelatinolytic activity by in situ zymography. PN-1 silencing did not alter prothrombin whereas increased uPA, MMP-9 and MMP-2 expression levels and gelatinolytic activity in a conditioned medium from stable C6 cells. Selective inhibitors for MMP-9 (Inhibitor I), MMP-2 (Inhibitor III) or exogenous recombinant PN-1 added to the culture medium of C6 silenced cells restored either the migration and invasive ability or gelatinolytic activity thus validating the specificity of PN-1 silencing strategy. Phosphorylation levels of extracellular signal-related kinases (Erk1/2 and p38 MAPK) involved in MMP-9 and MMP-2 signaling were increased in PN-1 silenced cells. This study shows that PN-1 affects glioma cell migration and invasiveness through the regulation of uPA and MMP-9/2 expression levels which contribute to the degradation of extracellular matrix during tumor invasion.