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author:("arsan, Aly")
1.  Cell Fate Decisions in Malignant Hematopoiesis: Leukemia Phenotype Is Determined by Distinct Functional Domains of the MN1 Oncogene 
PLoS ONE  2014;9(11):e112671.
Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia (T-ALL), share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.
doi:10.1371/journal.pone.0112671
PMCID: PMC4234417  PMID: 25401736
3.  Barnacle: detecting and characterizing tandem duplications and fusions in transcriptome assemblies 
BMC Genomics  2013;14:550.
Background
Chimeric transcripts, including partial and internal tandem duplications (PTDs, ITDs) and gene fusions, are important in the detection, prognosis, and treatment of human cancers.
Results
We describe Barnacle, a production-grade analysis tool that detects such chimeras in de novo assemblies of RNA-seq data, and supports prioritizing them for review and validation by reporting the relative coverage of co-occurring chimeric and wild-type transcripts. We demonstrate applications in large-scale disease studies, by identifying PTDs in MLL, ITDs in FLT3, and reciprocal fusions between PML and RARA, in two deeply sequenced acute myeloid leukemia (AML) RNA-seq datasets.
Conclusions
Our analyses of real and simulated data sets show that, with appropriate filter settings, Barnacle makes highly specific predictions for three types of chimeric transcripts that are important in a range of cancers: PTDs, ITDs, and fusions. High specificity makes manual review and validation efficient, which is necessary in large-scale disease studies. Characterizing an extended range of chimera types will help generate insights into progression, treatment, and outcomes for complex diseases.
doi:10.1186/1471-2164-14-550
PMCID: PMC3751903  PMID: 23941359
Transcriptome assembly; Chimeric transcripts; Fusion; Partial tandem duplication; PTD; Internal tandem duplication; ITD; RNA-seq; Transcriptome
4.  Concurrent CIC mutations, IDH mutations and 1p/19q loss distinguish oligodendrogliomas from other cancers 
The Journal of pathology  2011;226(1):7-16.
Oligodendroglioma is characterized by unique clinical, pathological, and genetic features. Recurrent losses of chromosomes 1p and 19q are strongly associated with this brain cancer but knowledge of the identity and function of the genes affected by these alterations is limited. We performed exome sequencing on a discovery set of 16 oligodendrogliomas with 1p/19q co-deletion to identify new molecular features at base-pair resolution. As anticipated, there was a high rate of IDH mutations: all cases had mutations in either IDH1 (14/16) or IDH2 (2/16). In addition, we discovered somatic mutations and insertions/deletions in the CIC gene on chromosome 19q13.2 in 13/16 tumours. These discovery set mutations were validated by deep sequencing of 13 additional tumours, which revealed 7 others with CIC mutations, thus bringing the overall mutation rate in oligodendrogliomas in this study to 20/29 (69%). In contrast, deep sequencing of astrocytomas and oligoastrocytomas without 1p/19q loss revealed that CIC alterations were otherwise rare (1/60; 2%). Of the 21 non-synonymous somatic mutations in 20 CIC-mutant oligodendrogliomas, 9 were in exon 5 within an annotated DNA interacting domain and 3 were in exon 20 within an annotated protein interacting domain. The remaining 9 were found in other exons and frequently included truncations. CIC mutations were highly associated with oligodendroglioma histology, 1p/19q co-deletion and IDH1/2 mutation (p<0.001). Although we observed no differences in the clinical outcomes of CIC mutant versus wild-type tumors, in a background of 1p/19q co-deletion, hemizygous CIC mutations are likely important. We hypothesize that the mutant CIC on the single retained 19q allele is linked to the pathogenesis of oligodendrogliomas with IDH mutation. Our detailed study of genetic aberrations in oligodendroglioma suggests a functional interaction between CIC mutation, IDH1/2 mutation and 1p/19q co-deletion.
doi:10.1002/path.2995
PMCID: PMC3246739  PMID: 22072542
Glioma; Oligodendroglioma; Next Generation Sequencing; Capicua; IDH1
5.  Heterogeneity of breast cancer stem cells as evidenced with Notch-dependent and Notch-independent populations 
Cancer Medicine  2012;1(2):105-113.
Studies have suggested the potential importance of Notch signaling to the cancer stem cell population in some tumors, but it is not known whether all cells in the cancer stem cell fraction require Notch activity. To address this issue, we blocked Notch activity in MCF-7 cells by expressing a dominant-negative MAML-GFP (dnMAML) construct, which inhibits signaling through all Notch receptors, and quantified the effect on tumor-initiating activity. Inhibition of Notch signaling reduced primary tumor sphere formation and side population. Functional quantification of tumor-initiating cell numbers in vivo showed a significant decrease, but not a complete abrogation, of these cells in dnMAML-expressing cells. Interestingly, when assessed in secondary assays in vitro or in vivo, there was no difference in tumor-initiating activity between the dnMAML-expressing cells and control cells. The fact that a subpopulation of dnMAML-expressing cells was capable of forming primary and secondary tumors indicates that there are Notch-independent tumor-initiating cells in the breast cancer cell line MCF-7. Our findings thus provide direct evidence for a heterogeneous cancer stem cell pool, which will require combination therapies against multiple oncogenic pathways to eliminate the tumor-initiating cell population.
doi:10.1002/cam4.18
PMCID: PMC3544441  PMID: 23342261
Breast cancer; cancer stem cells; dominant-negative MAML; MCF-7; Notch
6.  Twist1 Transcriptional Targets in the Developing Atrio-Ventricular Canal of the Mouse 
PLoS ONE  2012;7(7):e40815.
Malformations of the cardiovascular system are the most common type of birth defect in humans, frequently affecting the formation of valves and septa. During heart valve and septa formation, cells from the atrio-ventricular canal (AVC) and outflow tract (OFT) regions of the heart undergo an epithelial-to-mesenchymal transformation (EMT) and invade the underlying extracellular matrix to give rise to endocardial cushions. Subsequent maturation of newly formed mesenchyme cells leads to thin stress-resistant leaflets. TWIST1 is a basic helix-loop-helix transcription factor expressed in newly formed mesenchyme cells of the AVC and OFT that has been shown to play roles in cell survival, cell proliferation and differentiation. However, the downstream targets of TWIST1 during heart valve formation remain unclear. To identify genes important for heart valve development downstream of TWIST1, we performed global gene expression profiling of AVC, OFT, atria and ventricles of the embryonic day 10.5 mouse heart by tag-sequencing (Tag-seq). Using this resource we identified a novel set of 939 genes, including 123 regulators of transcription, enriched in the valve forming regions of the heart. We compared these genes to a Tag-seq library from the Twist1 null developing valves revealing significant gene expression changes. These changes were consistent with a role of TWIST1 in controlling differentiation of mesenchymal cells following their transformation from endothelium in the mouse. To study the role of TWIST1 at the DNA level we performed chromatin immunoprecipitation and identified novel direct targets of TWIST1 in the developing heart valves. Our findings support a role for TWIST1 in the differentiation of AVC mesenchyme post-EMT in the mouse, and suggest that TWIST1 can exert its function by direct DNA binding to activate valve specific gene expression.
doi:10.1371/journal.pone.0040815
PMCID: PMC3397961  PMID: 22815831
7.  TRAF6 is an amplified oncogene bridging the RAS and NF-κB pathways in human lung cancer  
The Journal of Clinical Investigation  2011;121(10):4095-4105.
Somatic mutations and copy number alterations (as a result of deletion or amplification of large portions of a chromosome) are major drivers of human lung cancers. Detailed analysis of lung cancer–associated chromosomal amplifications could identify novel oncogenes. By performing an integrative cytogenetic and gene expression analysis of non–small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) cell lines and tumors, we report here the identification of a frequently recurring amplification at chromosome 11 band p13. Within this region, only TNF receptor–associated factor 6 (TRAF6) exhibited concomitant mRNA overexpression and gene amplification in lung cancers. Inhibition of TRAF6 in human lung cancer cell lines suppressed NF-κB activation, anchorage-independent growth, and tumor formation. In these lung cancer cell lines, RAS required TRAF6 for its oncogenic capabilities. Furthermore, TRAF6 overexpression in NIH3T3 cells resulted in NF-κB activation, anchorage-independent growth, and tumor formation. Our findings show that TRAF6 is an oncogene that is important for RAS-mediated oncogenesis and provide a mechanistic explanation for the previously apparent importance of constitutive NF-κB activation in RAS-driven lung cancers.
doi:10.1172/JCI58818
PMCID: PMC3195480  PMID: 21911935
8.  Constitutively overexpressed 21 kDa protein in Hodgkin lymphoma and aggressive non-Hodgkin lymphomas identified as cytochrome B5b (CYB5B) 
Molecular Cancer  2010;9:14.
Background
We have previously reported a novel constitutively overexpressed 21 kDa protein in Hodgkin Lymphoma (HL) and aggressive Non-Hodgkin Lymphomas (NHL). The objective of the current study was to 1) identify this protein using two independent methods, 2) study the expression of the protein and its encoding mRNA in reactive lymph nodes, normal lymphocytes and CD34+ bone marrow precursor cells, 3) analyse patterns of expression of the protein in tissue microarrays assembled from a large number of diagnostic clinical biopsies from patients with HL, and 4) determine the copy number variation and mutation status of the encoding gene in HL cell lines.
Results
Peptide sequencing by LC-MS/MS and protein identification by protein array screening identified a single protein, CYB5B. No mutations were detected in the CYB5B gene in HL cell lines. Quantitative PCR showed CYB5B gene expression was increased in HL and NHL cell lines. Array CGH using a submegabase resolution tiling array revealed gains in the CYB5B locus in HL cell lines KMH2 and L428. Membrane expression was seen in Reed-Sternberg cells in clinical biopsies from patients with HL but not in reactive lymph nodes. Bone marrow CD34+ precursor cells were CYB5B negative on the cell surface. RT-PCR assays of RNA extracted from T and B cell enriched fractions obtained from normal peripheral blood mononuclear cells, reactive lymph nodes, tonsils and normal bone marrow samples showed no evidence of increased mRNA levels of CYB5B in comparison to housekeeping gene GAPDH.
Conclusions
The 21 kDa protein overexpressed in HL and aggressive NHL is identical to CYB5B. CYB5B gene expression is increased in a subset of HL and NHL cell lines tested. This is associated with CYB5B gene amplification in HL cell lines KMH2 and L428. CYB5B may be a potential target for antibody-based therapy of HL and aggressive NHL as although cytoplasmic expression is present in reactive lymphocytes, it is not expressed on the cell surface of non-neoplastic lymphocytes or bone marrow precursor cells.
doi:10.1186/1476-4598-9-14
PMCID: PMC2829491  PMID: 20100355
9.  Slug is a direct Notch target required for initiation of cardiac cushion cellularization 
The Journal of Cell Biology  2008;182(2):315-325.
Snail family proteins are key regulators of epithelial-mesenchymal transition, but their role in endothelial-to-mesenchymal transition (EMT) is less well studied. We show that Slug, a Snail family member, is expressed by a subset of endothelial cells as well as mesenchymal cells of the atrioventricular canal and outflow tract during cardiac cushion morphogenesis. Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)–9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT. We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells. In contrast, transforming growth factor β (TGF-β) induces Snail but not Slug. Interestingly, activation of Notch in the context of TGF-β stimulation results in synergistic up-regulation of Snail in endothelial cells. Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.
doi:10.1083/jcb.200710067
PMCID: PMC2483533  PMID: 18663143
10.  Jagged1-mediated Notch activation induces epithelial-to-mesenchymal transition through Slug-induced repression of E-cadherin 
The Journal of Experimental Medicine  2007;204(12):2935-2948.
Aberrant expression of Jagged1 and Notch1 are associated with poor outcome in breast cancer. However, the reason that Jagged1 and/or Notch overexpression portends a poor prognosis is unknown. We identify Slug, a transcriptional repressor, as a novel Notch target and show that elevated levels of Slug correlate with increased expression of Jagged1 in various human cancers. Slug was essential for Notch-mediated repression of E-cadherin, which resulted in β-catenin activation and resistance to anoikis. Inhibition of ligand-induced Notch signaling in xenografted Slug-positive/E-cadherin–negative breast tumors promoted apoptosis and inhibited tumor growth and metastasis. This response was associated with down-regulated Slug expression, reexpression of E-cadherin, and suppression of active β-catenin. Our findings suggest that ligand-induced Notch activation, through the induction of Slug, promotes tumor growth and metastasis characterized by epithelial-to-mesenchymal transition and inhibition of anoikis.
doi:10.1084/jem.20071082
PMCID: PMC2118507  PMID: 17984306
11.  FADD Negatively Regulates Lipopolysaccharide Signaling by Impairing Interleukin-1 Receptor-Associated Kinase 1-MyD88 Interaction▿  
Molecular and Cellular Biology  2007;27(21):7394-7404.
Lipopolysaccharide (LPS) engages Toll-like receptor 4 (TLR4) on various cells to initiate inflammatory and angiogenic pathways. FADD is an adaptor protein involved in death receptor-mediated apoptosis. Here we report a role for FADD in regulation of TLR4 signals in endothelial cells. FADD specifically attenuates LPS-induced activation of c-Jun NH2-terminal kinase and phosphatidylinositol 3′-kinase in a death domain-dependent manner. In contrast, FADD-null cells show hyperactivation of these kinases. Examining physical associations of endogenous proteins, we show that FADD interacts with interleukin-1 receptor-associated kinase 1 (IRAK1) and MyD88. LPS stimulation increases IRAK1-FADD interaction and recruitment of the IRAK1-FADD complex to activated MyD88. IRAK1 is required for FADD-MyD88 interaction, as FADD does not associate with MyD88 in IRAK1-null cells. By shuttling FADD to MyD88, IRAK1 provides a mechanism for controlled and limited activation of the TLR4 signaling pathway. Functionally, enforced FADD expression inhibited LPS- but not vascular endothelial growth factor-induced endothelial cell sprouting, while FADD deficiency led to enhanced production of proinflammatory cytokines induced by stimulation of TLR4 and TLR2, but not TLR3. Reconstitution of FADD reversed the enhanced production of proinflammatory cytokines. Thus, FADD is a physiological negative regulator of IRAK1/MyD88-dependent responses in innate immune signaling.
doi:10.1128/MCB.00600-07
PMCID: PMC2169044  PMID: 17785432
12.  Notch Activation Induces Endothelial Cell Cycle Arrest and Participates in Contact Inhibition: Role of p21Cip1 Repression†  
Molecular and Cellular Biology  2004;24(20):8813-8822.
Although previous studies demonstrate that appropriate Notch signaling is required during angiogenesis and in vascular homeostasis, the mechanisms by which Notch regulates vascular function remain to be elucidated. Here, we show that activation of the Notch pathway by the ligand Jagged1 reduces the proliferation of endothelial cells. Notch activation inhibits proliferation of endothelial cells in a cell-autonomous manner by inhibiting phosphorylation of the retinoblastoma protein (Rb). During cell cycle entry, p21Cip1 is upregulated in endothelial cells. Activated Notch inhibits mitogen-induced upregulation of p21Cip1 and delays cyclin D-cdk4-mediated Rb phosphorylation. Notch-dependent repression of p21Cip1 prevents nuclear localization of cyclin D and cdk4. The necessity of p21Cip1 for nuclear translocation of cyclin D-cdk4 and S-phase entry in endothelial cells was demonstrated by targeted downregulation of p21Cip1 by using RNA interference. We further demonstrate that when endothelial cells reach confluence, Notch is activated and p21Cip1 is downregulated. Inhibition of the Notch pathway at confluence prevents p21Cip1 downregulation and induces Rb phosphorylation. We suggest that Notch activation contributes to contact inhibition of endothelial cells, in part through repression of p21Cip1 expression.
doi:10.1128/MCB.24.20.8813-8822.2004
PMCID: PMC517869  PMID: 15456857
13.  Activated Notch4 Inhibits Angiogenesis: Role of β1-Integrin Activation 
Molecular and Cellular Biology  2002;22(8):2830-2841.
Notch4 is a member of the Notch family of transmembrane receptors that is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions. The sprouting of endothelial cells from microvessels, or angiogenesis, involves the modulation of the endothelial cell phenotype. Based on the function of other Notch family members and the expression pattern of Notch4, we postulated that Notch4 activation would modulate angiogenesis. Using an in vitro endothelial-sprouting assay, we show that expression of constitutively active Notch4 in human dermal microvascular endothelial cells (HMEC-1) inhibits endothelial sprouting. We also show that activated Notch4 inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane in vivo. Activated Notch4 does not inhibit HMEC-1 proliferation or migration through fibrinogen. However, migration through collagen is inhibited. Our data show that Notch4 cells exhibit increased β1-integrin-mediated adhesion to collagen. HMEC-1 expressing activated Notch4 do not have increased surface expression of β1-integrins. Rather, we demonstrate that Notch4-expressing cells display β1-integrin in an active, high-affinity conformation. Furthermore, using function-activating β1-integrin antibodies, we demonstrate that activation of β1-integrins is sufficient to inhibit VEGF-induced endothelial sprouting in vitro and angiogenesis in vivo. Our findings suggest that constitutive Notch4 activation in endothelial cells inhibits angiogenesis in part by promoting β1-integrin-mediated adhesion to the underlying matrix.
doi:10.1128/MCB.22.8.2830-2841.2002
PMCID: PMC133705  PMID: 11909975

Results 1-13 (13)