Number 3 March 2011

VolumeVolume 15 1 -- NumberNumber 31 May March- Sept ember2011 1997

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Scope

The Atlas of Genetics and Cytogenetics in Oncology and Haematology is a peer reviewed on-line journal in open access, devoted to genes, cytogenetics, and clinical entities in cancer, and cancer-prone diseases. It presents structured review articles ("cards") on genes, leukaemias, solid tumours, cancer-prone diseases, more traditional review articles on these and also on surrounding topics ("deep insights"), case reports in hematology, and educational items in the various related topics for students in Medicine and in Sciences.

Editorial correspondance

Jean-Loup Huret Genetics, Department of Medical Information, University Hospital F-86021 Poitiers, France tel +33 5 49 44 45 46 or +33 5 49 45 47 67 [email protected] or [email protected]

Staff Mohammad Ahmad, Mélanie Arsaban, Houa Delabrousse, Marie-Christine Jacquemot-Perbal, Maureen Labarussias, Vanessa Le Berre, Anne Malo, Catherine Morel-Pair, Laurent Rassinoux, Sylvie Yau Chun Wan - Senon, Alain Zasadzinski. Philippe Dessen is the Database Director, and Alain Bernheim the Chairman of the on-line version (Gustave Roussy Institute – Villejuif – France).

The Atlas of Genetics and Cytogenetics in Oncology and Haematology (ISSN 1768-3262) is published 12 times a year by ARMGHM, a non profit organisation, and by the INstitute for Scientific and Technical Information of the French National Center for Scientific Research (INIST-CNRS) since 2008.

The Atlas is hosted by INIST-CNRS (http://www.inist.fr)

http://AtlasGeneticsOncology.org

The PDF version of the Atlas of Genetics and Cytogenetics in Oncology and Haematology is a reissue of the original articles published in collaboration with the Institute for Scientific and Technical Information (INstitut de l’Information Scientifique et Technique - INIST) of the French National Center for Scientific Research (CNRS) on its electronic publishing platform I-Revues. Online and PDF versions of the Atlas of Genetics and Cytogenetics in Oncology and Haematology are hosted by INIST-CNRS.

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Editor

Jean-Loup Huret (Poitiers, France) Editorial Board

Sreeparna Banerjee (Ankara, Turkey) Solid Tumours Section Alessandro Beghini (Milan, Italy) Genes Section Anne von Bergh (Rotterdam, The Netherlands) Genes / Leukaemia Sections Judith Bovée (Leiden, The Netherlands) Solid Tumours Section Vasantha Brito-Babapulle (London, UK) Leukaemia Section Charles Buys (Groningen, The Netherlands) Deep Insights Section Anne Marie Capodano (Marseille, France) Solid Tumours Section Fei Chen (Morgantown, West Virginia) Genes / Deep Insights Sections Antonio Cuneo (Ferrara, Italy) Leukaemia Section Paola Dal Cin (Boston, Massachussetts) Genes / Solid Tumours Section Louis Dallaire (Montreal, Canada) Education Section Brigitte Debuire (Villejuif, France) Deep Insights Section François Desangles (Paris, France) Leukaemia / Solid Tumours Sections Enric Domingo-Villanueva (London, UK) Solid Tumours Section Ayse Erson (Ankara, Turkey) Solid Tumours Section Richard Gatti (Los Angeles, California) Cancer-Prone Diseases / Deep Insights Sections Ad Geurts van Kessel (Nijmegen, The Netherlands) Cancer-Prone Diseases Section Oskar Haas (Vienna, Austria) Genes / Leukaemia Sections Anne Hagemeijer (Leuven, Belgium) Deep Insights Section Nyla Heerema (Colombus, Ohio) Leukaemia Section Jim Heighway (Liverpool, UK) Genes / Deep Insights Sections Sakari Knuutila (Helsinki, Finland) Deep Insights Section Lidia Larizza (Milano, Italy) Solid Tumours Section Lisa Lee-Jones (Newcastle, UK) Solid Tumours Section Edmond Ma (Hong Kong, China) Leukaemia Section Roderick McLeod (Braunschweig, Germany) Deep Insights / Education Sections Cristina Mecucci (Perugia, Italy) Genes / Leukaemia Sections Yasmin Mehraein (Homburg, Germany) Cancer-Prone Diseases Section Fredrik Mertens (Lund, Sweden) Solid Tumours Section Konstantin Miller (Hannover, Germany) Education Section Felix Mitelman (Lund, Sweden) Deep Insights Section Hossain Mossafa (Cergy Pontoise, France) Leukaemia Section Stefan Nagel (Braunschweig, Germany) Deep Insights / Education Sections Florence Pedeutour (Nice, France) Genes / Solid Tumours Sections Elizabeth Petty (Ann Harbor, Michigan) Deep Insights Section Susana Raimondi (Memphis, Tennesse) Genes / Leukaemia Section Mariano Rocchi (Bari, Italy) Genes Section Alain Sarasin (Villejuif, France) Cancer-Prone Diseases Section Albert Schinzel (Schwerzenbach, Switzerland) Education Section Clelia Storlazzi (Bari, Italy) Genes Section Sabine Strehl (Vienna, Austria) Genes / Leukaemia Sections Nancy Uhrhammer (Clermont Ferrand, France) Genes / Cancer-Prone Diseases Sections Dan Van Dyke (Rochester, Minnesota) Education Section Roberta Vanni (Montserrato, Italy) Solid Tumours Section Franck Viguié (Paris, France) Leukaemia Section José Luis Vizmanos (Pamplona, Spain) Leukaemia Section Thomas Wan (Hong Kong, China) Genes / Leukaemia Sections

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3) Atlas of Genetics and Cytogenetics in Oncology and Haematology

OPEN ACCESS JOURNAL AT INIST-CNRS

Volume 15, Number 3, March 2011

Table of contents

Gene Section

AGER (advanced glycosylation end product-specific receptor) 239 Geetha Srikrishna, Barry Hudson ANG (angiogenin, ribonuclease, RNase A family, 5) 244 Shouji Shimoyama ATF5 (activating transcription factor 5) 252 Arthur KK Ching, Nathalie Wong BRE (brain and reproductive organ-expressed (TNFRSF1A modulator)) 255 Yiu-Loon Chui, Kenneth Ka-Ho Lee, John Yeuk-Hon Chan DDX1 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 1) 259 Takahiko Hara, Kiyoko Tanaka DIO2 (deiodinase, iodothyronine, type II) 262 Ana Luiza Maia, Simone Magagnin Wajner, Leonardo B Leiria GFI1B (growth factor independent 1B transcription repressor) 266 Lothar Vassen, Tarik Möröy LRP5 (low density lipoprotein receptor-related protein 5) 270 Zhendong Alex Zhong, Bart O Williams MIF (macrophage migration inhibitory factor (glycosylation-inhibiting factor)) 276 Jan-Philipp Bach, Michael Bacher, Richard Dodel NEU3 (sialidase 3 (membrane sialidase)) 280 Kazunori Yamaguchi, Taeko Miyagi NPY1R (neuropeptide Y receptor Y1) 283 Massimiliano Ruscica, Elena Dozio, Luca Passafaro, Paolo Magni REPS2 (RALBP1 associated Eps domain containing 2) 288 Salvatore Corallino, Luisa Castagnoli XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6) 293 Sabina Pucci, Maria Josè Zonetti

Leukaemia Section t(6;22)(p21;q11) 297 Jean-Loup Huret

Solid Tumour Section t(11;22)(q24;q12) in giant cell tumour of bone 299 Jean-Loup Huret

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3) Atlast(11;14)(q 13;q32)of Genetics in multiple myeloma and Cytogenetics Huret JL, Laï JL in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

t(11;22)(q24;q12) in rhabdomyosarcomas (RMS) 300 Jean-Loup Huret t(11;22)(q24;q12) in solid pseudopapillary tumour of the pancreas 302 Jean-Loup Huret

Deep Insight Section

MTA1 of the MTA (metastasis-associated) gene family and its encoded proteins: molecular and regulatory functions and role in human cancer progression 304 Yasushi Toh, Garth L Nicolson Role of p38α in apoptosis: implication in cancer development and therapy 317 Almudena Porras, Carmen Guerrero

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3)

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

AGER (advanced glycosylation end product- specific receptor) Geetha Srikrishna, Barry Hudson Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, USA (GS), Columbia University Medical Center, 630 West 168th St. New York, NY 10032, USA (BH)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/AGERID594ch6p21.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI AGERID594ch6p21.txt

signal peptide (aa 1-22), followed by three Identity immunoglobulin-like domains, a V-type domain, (aa 23-116) and two C type domains (C1: aa 124- Other names: MGC22357; RAGE 221 and C2: 227-317), a single transmembrane HGNC (Hugo): AGER domain (aa 343-363), and a short cytoplasmic Location: 6p21.32 domain (aa 364-404) necessary for signaling. The prevalent isoforms of RAGE are full length RAGE, RAGE_v1 or endogenous secretory (es RAGE) which lacks the cytosolic and transmembrane domains and therefore can be secreted into the extracellular space, and N-terminal truncated Figure 1. Schematic of human chromosome 6. RAGE (RAGE_v2) which lacks N-terminal V domain and therefore cannot bind ligands. DNA/RNA RAGE_v2 does not form mature protein. Through Description its ability to scavenge RAGE ligands, soluble RAGE isoforms (sRAGE) are believed to act a The human AGER (RAGE) gene lies within the decoy receptor by regulating signaling mediated by major histocompatibility complex class III region activation of full length RAGE. Expression of on chromosome 6, which contains genes involved isoforms is tissue specific, suggesting tight tissue- in immune responses, such as TNFalpha, specific regulation of expression. sRAGE can also lymphotoxin, complement components and be formed by ectodomain cleavage by homeobox gene HOX12. It comprises 11 exons and ADAM10/MMP9. 10 introns, and a 5' flanking region that regulates its A number of NF-kappaB sites have been identified transcription. The resulting transcribed mRNA of in the RAGE 5' regulatory region. In addition, ~1.4 kb with a short 3'UTR is alternatively spliced, transcription is also controlled by other pro- and nearly twenty isoforms have been identified in inflammatory transcription factors such as SP-1 and different tissues such as lung, liver, kidney, smooth AP-2. muscle, endothelial cells and brain. The different At least 30 polymorphisms are known, most of RAGE gene splice variants have been named which are single nucleotide polymorphisms (SNP). RAGE, RAGE_v1 to RAGE_v19 according to the A Gly to Ser change at an N-glycosylation sequon Human Gene Nomenclature Committee. RAGE is at position 82, and two 5' flanking polymorphisms composed of a number of distinct protein domains; at position -374 and -429 lead to altered function an extracellular region (aa 1-342) composed of a and expression of RAGE.

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AGER (advanced glycosylation end product-specific receptor) Srikrishna G, Hudson B

Protein Description The V and C1 domains in the extracellular region of RAGE form an integrated structural unit, while C2 is fully independent, attached to VC1 through a flexible linker. RAGE was originally identified as a receptor for advanced glycation end products, but it also interacts with other structurally unrelated ligands including HMGB1, several members of the S100 family, amyloid-beta peptide, transthyretin and beta2 integrin Mac-1. By virtue of its multi- domain structure and ability to recognize different classes of ligands, RAGE behaves as a pattern recognition receptor (PRR) akin to innate immune receptors such as Toll-like receptors (TLRs) in orchestrating immune responses. However, unlike other PRRs that predominantly bind exogenous ligands, RAGE binds endogenous ligands, especially those considered to be damage associated molecular pattern molecules (DAMPs). AGEs, HMGB1, Abeta peptides, S100B, S100A1, S100A2 and S100A5 bind to the V domain, S100A12 binds to V-C1 domains, and S100A6 interacts with V-C2 domain. Studies on S100 protein-RAGE interactions also suggest that multimerization of ligand and receptor occurs and that formation of these higher ordered complexes may be essential for signal transduction. In addition to contribution by protein interaction domains, post-translational Figure 2. Schematic of RAGE protein and its domains. modifications such as glycosylation of the RAGE is a multi-ligand receptor consisting of three Ig- domains (V, C1 and C2), a transmembrane domain and a receptors, or acetylation or phosphorylation of cytosolic tail required for RAGE-mediated intracellular ligands could also play important roles in defining signaling. The V and C1 domains in the extracellular region specificity of interactions, multimerization and of RAGE form an integrated structural unit, while C2 is fully downstream signaling. RAGE has two N- independent, attached to VC1 through a flexible linker. Many ligands bind to the V domain, while some also glycosylation sites on the V-domain and both sites interact with the V-C1 or V-C2 domains. The V domain has are occupied by complex and hybrid or high N-glycosylation sites both of which are modified. Ligand mannose N-glycans. A subpopulation is modified binding activates multiple signaling pathways and regulates by carboxylated glycans, which promote interaction gene expression through the transcription factors NF- kappaB, CREB and SP1 (From Rauvala H, Rouhiainen A. with HMGB1, S100A8/S100A9 and S100A12. In Biochim Biophys Acta. 2010 Jan-Feb;1799(1-2):164-70. addition, the quaternary structure of RAGE might Reproduced with permission from publishers). also account for the diversity of ligand recognition. Function Though the cytoplasmic domain lacks endogenous kinase activity or any other known signaling motif, Normal physiological functions of RAGE include studies indicate that the cytoplasmic domain is embryonal neuronal growth, myogenesis, essential for intracellular signaling. mobilization of dendritic cells, activation and differentiation of T cells, stem cell migration and Expression osteoclast maturation. HMGB1 interaction of RAGE is highly expressed during embryonic RAGE results in stimulation of myogenesis. RAGE development, especially in the brain, but levels mediates trophic and toxic effects of S100B on decrease in adult tissues. RAGE is found at low embryonal neurons, and promotes neurite levels in neurons, endothelial cells, mononuclear outgrowth and neuronal regeneration promoted by phagocytes, smooth muscle cells, and constitutively HMGB1. RAGE also plays an important role in the expressed at high levels in the lung. regulation of osteoclast maturation and function, Localisation and bone remodeling. Ligand interaction promotes activation of - Full length: membrane: single pass type I intracellular signaling pathways including the membrane protein. MAPK pathway, RAC-1 and CDC42, NADPH - Isoforms: secreted. oxidase, PI3 kinase and JAK/STAT pathway, and

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activation of NF-kappaB. RAGE expression is Common bile duct cancer induced in inflammatory settings, since its Note transcription is controlled by several transcription RAGE is expressed on human biliary cancer cells, factors as mentioned above. Thus a positive feed- and expression correlates with their invasive ability. forward loop evolves in ligand rich inflammatory settings, perpetuating the pathology. sRAGE is Glioma believed to regulate signaling mediated by Note activation of full length RAGE. Binding of RAGE HMGB1/RAGE signaling pathways promote the to HMGB1 induces RAGE shedding by ADAM10 growth and migration of human glioblastoma cells. metalloprotease, thus possibly representing another Inhibition of RAGE-HMGB1 interactions decreases pathway for negatively regulating RAGE mediated growth and metastases of gliomas in mice. cellular activation. Skin cancer Implicated in Note RAGE is expressed in human melanoma cells and Gastric cancer promotes ligand-dependent growth and invasion. Note RAGE null mice are resistant to the onset of RAGE is constitutively expressed in human gastric inflammation mediated skin tumors in mice. carcinoma cell lines, and poorly differentiated Lung cancer human gastric carcinomas preferentially express RAGE. Strong RAGE expression is seen in cells at Note the invasive edge of tumors and correlates with RAGE, as well as its ligands, is highly expressed in invasion and lymph node metastasis. Studies in normal lung, but unlike other cancers, RAGE is Chinese population show that Gly82Ser markedly reduced in human lung carcinomas. polymorphism on RAGE is associated with Down-regulation correlates with advanced tumor increased risk for gastric cancer. stages, suggesting that RAGE may have tumor suppressive functions in lung cancer. Colon cancer Tumor microenvironment Note RAGE expression is increased in advanced colon Note tumors. Co-expression of RAGE and its ligands Many RAGE ligands are expressed and secreted not HMGB1 and S100P is strongly associated with only by cancer cells but also by cells within the invasion and metastasis of human colorectal cancer. tumor microenvironment, including myeloid RAGE appears to be at the interface of derived cells and vascular cells. These ligands inflammation and colon cancer, since RAGE interact with the receptor in both autocrine and deficient mice are resistant to the onset of colitis paracrine manners, promoting tumor growth, associated colon cancer. invasion, angiogenesis and metastasis. Pancreatic cancer Inflammation and immune responses Note Note Expression of RAGE is strongest in pancreatic RAGE and its ligands are highly enriched in cancer cells with high metastatic ability, and RAGE immune and inflammatory foci and their interaction may play an important role in the viability of promotes upregulation of inflammatory cytokines, pancreatic tumor cells against stress-induced adhesion molecules and matrix metalloproteinases. apoptosis. RAGE ligand S100P is overexpressed in They are therefore implicated in many pancreatic cancer. inflammatory conditions including colitis and arthritis. RAGE is upregulated in synovial tissue Prostate cancer macrophages and its ligands are abundant in Note inflamed synovial tissue. Activation leads to RAGE and ligands are highly expressed on prostate increased stimulation of chondrocytes and cancer cell lines, untreated prostate cancer tissue synoviocytes, promoting ongoing inflammation and and hormone-refractory prostate cancer tissue, and autoimmunity in arthritis. RAGE mediates HMGB1 RAGE promotes growth and invasion of prostate activation of dendritic cells in response to DNA cancer cells in response to ligand activation. containing immune complexes, contributing to Oral squamous cell cancer autoimmune pathogenesis. Blockade of RAGE interactions suppresses myelin basic protein Note induced experimental autoimmune RAGE expression closely associates with histologic encephalomyelitis. Inhibition of RAGE-ligand differentiation, invasiveness, angiogenesis and interactions or RAGE deletion protects mice from recurrence of oral squamous cell carcinoma. septic shock induced by caecal ligation and

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puncture. RAGE null mice are also resistant to skin Kientsch-Engel R, Schmidt AM, Stremmel W, Stern DM, and colon inflammation and inflammation-based Katus HA, Nawroth PP, Bierhaus A. Posttranslationally modified proteins as mediators of sustained intestinal tumorigenesis. inflammation. Am J Pathol. 2006 Oct;169(4):1223-37 Diabetes Bierhaus A, Stern DM, Nawroth PP. RAGE in Note inflammation: a new therapeutic target? Curr Opin Investig Drugs. 2006 Nov;7(11):985-91 RAGE, as a receptor for advanced glycation end products and other pro-inflammatory ligands, Zhou Z, Immel D, Xi CX, Bierhaus A, Feng X, Mei L, Nawroth P, Stern DM, Xiong WC. Regulation of osteoclast contributes to micro and macrovascular changes in function and bone mass by RAGE. J Exp Med. 2006 Apr diabetes. RAGE over-expression in transgenic mice 17;203(4):1067-80 is associated with increased vascular injury, Dattilo BM, Fritz G, Leclerc E, Kooi CW, Heizmann CW, diabetic nephropathy and neuropathy, while RAGE Chazin WJ. The extracellular region of the receptor for deletion confers partial protection from these advanced glycation end products is composed of two diabetes-associated changes. independent structural units. Biochemistry. 2007 Jun 12;46(23):6957-70 Atherosclerosis and ischemia Donato R. RAGE: a single receptor for several ligands and Note different cellular responses: the case of certain S100 Increased RAGE expression is found in endothelial proteins. Curr Mol Med. 2007 Dec;7(8):711-24 cells in non-diabetic patients with peripheral Leclerc E, Fritz G, Weibel M, Heizmann CW, Galichet A. occlusive vascular disease. sRAGE reduces S100B and S100A6 differentially modulate cell survival by atherosclerotic lesions and inflammation in interacting with distinct RAGE (receptor for advanced glycation end products) immunoglobulin domains. J Biol normoglycemic Apo E null mice, and reduced Chem. 2007 Oct 26;282(43):31317-31 neointima expansion in wild type mice following Logsdon CD, Fuentes MK, Huang EH, Arumugam T. femoral artery injury. Studies on ischemia- RAGE and RAGE ligands in cancer. Curr Mol Med. 2007 reperfusion injury of the heart in wild type and Dec;7(8):777-89 RAGE null mice show that infarct size and severity Ostendorp T, Leclerc E, Galichet A, Koch M, Demling N, of tissue damage is dependent on HMGB1-RAGE Weigle B, Heizmann CW, Kroneck PM, Fritz G. Structural interactions following necrotic cell death. and functional insights into RAGE activation by multimeric Neuronal degeneration S100B. EMBO J. 2007 Aug 22;26(16):3868-78 Xie J, Burz DS, He W, Bronstein IB, Lednev I, Shekhtman Note A. Hexameric calgranulin C (S100A12) binds to the RAGE is expressed on neurons, microglia and receptor for advanced glycated end products (RAGE) endothelial cells in the brain, and binds the using symmetric hydrophobic target-binding patches. J Biol multimeric form of amyloid-beta peptide. Binding Chem. 2007 Feb 9;282(6):4218-31 leads to activation of NADPH oxidase, generation Gebhardt C, Riehl A, Durchdewald M, Németh J, of reactive oxygen species, activation of NF- Fürstenberger G, Müller-Decker K, Enk A, Arnold B, Bierhaus A, Nawroth PP, Hess J, Angel P. RAGE signaling kappaB and CREB, and upregulation of cytokines sustains inflammation and promotes tumor development. J and chemokines, thus promoting Exp Med. 2008 Feb 18;205(2):275-85 neuroinflammation. The associated up-regulation Hudson BI, Carter AM, Harja E, Kalea AZ, Arriero M, Yang and release of other RAGE ligands such as HMGB1 H, Grant PJ, Schmidt AM. Identification, classification, and and S100 proteins further amplifies this cascade, expression of RAGE gene splice variants. FASEB J. 2008 leading to neuronal degeneration. sRAGE has been May;22(5):1572-80 shown to be beneficial in animal models of Turovskaya O, Foell D, Sinha P, Vogl T, Newlin R, Nayak Alzheimer's disease. RAGE null mice are also J, Nguyen M, Olsson A, Nawroth PP, Bierhaus A, Varki N, partially protected from diabetes-induced loss of Kronenberg M, Freeze HH, Srikrishna G. RAGE, carboxylated glycans and S100A8/A9 play essential roles neuronal function. in colitis-associated carcinogenesis. Carcinogenesis. 2008 Oct;29(10):2035-43 References Xie J, Reverdatto S, Frolov A, Hoffmann R, Burz DS, Bierhaus A, Humpert PM, Morcos M, Wendt T, Chavakis Shekhtman A. Structural basis for pattern recognition by T, Arnold B, Stern DM, Nawroth PP. Understanding RAGE, the receptor for advanced glycation end products (RAGE). the receptor for advanced glycation end products. J Mol J Biol Chem. 2008 Oct 3;283(40):27255-69 Med. 2005 Nov;83(11):876-86 Bierhaus A, Nawroth PP. Multiple levels of regulation Bierhaus A, Humpert PM, Stern DM, Arnold B, Nawroth determine the role of the receptor for AGE (RAGE) as PP. Advanced glycation end product receptor-mediated common soil in inflammation, immune responses and cellular dysfunction. Ann N Y Acad Sci. 2005 diabetes mellitus and its complications. Diabetologia. 2009 Jun;1043:676-80 Nov;52(11):2251-63 Ding Q, Keller JN. Splice variants of the receptor for Kalea AZ, Schmidt AM, Hudson BI. RAGE: a novel advanced glycosylation end products (RAGE) in human biological and genetic marker for vascular disease. Clin Sci brain. Neurosci Lett. 2005 Jan 3;373(1):67-72 (Lond). 2009 Apr;116(8):621-37 Andrassy M, Igwe J, Autschbach F, Volz C, Remppis A, Leclerc E, Fritz G, Vetter SW, Heizmann CW. Binding of Neurath MF, Schleicher E, Humpert PM, Wendt T, S100 proteins to RAGE: an update. Biochim Biophys Acta. Liliensiek B, Morcos M, Schiekofer S, Thiele K, Chen J, 2009 Jun;1793(6):993-1007

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Lin L, Park S, Lakatta EG. RAGE signaling in inflammation cardiovascular disease. Expert Rev Mol Med. 2009 Mar and arterial aging. Front Biosci. 2009 Jan 1;14:1403-13 12;11:e9 Maillard-Lefebvre H, Boulanger E, Daroux M, Gaxatte C, Yan SF, Yan SD, Ramasamy R, Schmidt AM. Tempering Hudson BI, Lambert M. Soluble receptor for advanced the wrath of RAGE: an emerging therapeutic strategy glycation end products: a new biomarker in diagnosis and against diabetic complications, neurodegeneration, and prognosis of chronic inflammatory diseases. inflammation. Ann Med. 2009;41(6):408-22 Rheumatology (Oxford). 2009 Oct;48(10):1190-6 Zhang L, Postina R, Wang Y. Ectodomain shedding of the Ramasamy R, Yan SF, Schmidt AM. RAGE: therapeutic receptor for advanced glycation end products: a novel target and biomarker of the inflammatory response--the therapeutic target for Alzheimer's disease. Cell Mol Life evidence mounts. J Leukoc Biol. 2009 Sep;86(3):505-12 Sci. 2009 Dec;66(24):3923-35 Riehl A, Németh J, Angel P, Hess J. The receptor RAGE: Rauvala H, Rouhiainen A. Physiological and Bridging inflammation and cancer. Cell Commun Signal. pathophysiological outcomes of the interactions of HMGB1 2009 May 8;7:12 with cell surface receptors. Biochim Biophys Acta. 2010 Jan-Feb;1799(1-2):164-70 Schmidt AM, Sahagan B, Nelson RB, Selmer J, Rothlein R, Bell JM. The role of RAGE in amyloid-beta peptide- Rojas A, Figueroa H, Morales E. Fueling inflammation at mediated pathology in Alzheimer's disease. Curr Opin tumor microenvironment: the role of multiligand/RAGE Investig Drugs. 2009 Jul;10(7):672-80 axis. Carcinogenesis. 2010 Mar;31(3):334-41 Sparvero LJ, Asafu-Adjei D, Kang R, Tang D, Amin N, Im Sims GP, Rowe DC, Rietdijk ST, Herbst R, Coyle AJ. J, Rutledge R, Lin B, Amoscato AA, Zeh HJ, Lotze MT. HMGB1 and RAGE in inflammation and cancer. Annu Rev RAGE (Receptor for Advanced Glycation Endproducts), Immunol. 2010 Mar;28:367-88 RAGE ligands, and their role in cancer and inflammation. J Transl Med. 2009 Mar 17;7:17 Srikrishna G, Nayak J, Weigle B, Temme A, Foell D, Hazelwood L, Olsson A, Volkmann N, Hanein D, Freeze Srikrishna G, Freeze HH. Endogenous damage-associated HH. Carboxylated N-glycans on RAGE promote S100A12 molecular pattern molecules at the crossroads of binding and signaling. J Cell Biochem. 2010 Jun inflammation and cancer. Neoplasia. 2009 Jul;11(7):615- 1;110(3):645-59 28 Yan SF, Ramasamy R, Schmidt AM. Soluble RAGE: Yan SD, Bierhaus A, Nawroth PP, Stern DM. RAGE and therapy and biomarker in unraveling the RAGE axis in Alzheimer's disease: a progression factor for amyloid-beta- chronic disease and aging. Biochem Pharmacol. 2010 May induced cellular perturbation? J Alzheimers Dis. 15;79(10):1379-86 2009;16(4):833-43 This article should be referenced as such: Yan SF, Ramasamy R, Schmidt AM. Receptor for AGE (RAGE) and its ligands-cast into leading roles in diabetes Srikrishna G, Hudson B. AGER (advanced glycosylation and the inflammatory response. J Mol Med. 2009 end product-specific receptor). Atlas Genet Cytogenet Mar;87(3):235-47 Oncol Haematol. 2011; 15(3):239-243. Yan SF, Ramasamy R, Schmidt AM. The receptor for advanced glycation endproducts (RAGE) and

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Atlas of Genetics and Cytogenetics

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Gene Section Review

ANG (angiogenin, ribonuclease, RNase A family, 5) Shouji Shimoyama Gastrointestinal Unit, Settlement Clinic, 4-20-7, Towa, Adachi-ku, Tokyo, 120-0003, Japan (SS)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/ANGID635ch14q11.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI ANGID635ch14q11.txt

35% identical and 68% homologous to the Identity pancreatic RNAse A sequence. The overall crystal Other names: ALS9; HEL168; MGC22466; structure of ANG shows a similarity to, but the MGC71966; RNASE4; RNASE5 biological actions of ANG differ distinctly from HGNC (Hugo): ANG those of RNAse A. ANG possesses two distinct regions: a ribonucleolytic and a noncatalytic site, Location: 14q11.2 both being critical for angiogenic activity. Besides the ribonucleolytic activity, ANG differs from RNAse A in noncatalytic activities such as interactions with endothelial and smooth muscle cells and subsequent cellular responses in the events of neovascularization, including basement membrane degradation, signal transduction, and DNA/RNA nuclear translocation. Expression ANG mRNA is expressed in a wide spectrum of cells including neoplastic cells as well as normal epithelial cells, fibroblasts, peripheral blood cells, Starts at 2152336 and ends at 2162345 in NCBI reference and vascular endothelial cells. sequence NT_026437.12. Gene map locus is available at NCBI Nucleotide. Total length of ANG DNA is 10010 Localisation nucleotides. The coding region starts at 2162723 and ends Strikingly, ANG localizes freely in the circulation, at 2162166 including stop codon TAA. and is translocated into the nucleus. Nuclear translocation of ANG triggers subsequent cell Protein proliferation. However, the precise mechanisms for Description why serum ANG is inactive and continuous angiogenesis does not take place remain unknown. The amino acid sequence is available at NCBI protein locus AAA51678. It consists of a signal Function peptide from amino acid 1 to 24 and a mature I. Ribonuclease activity peptide from amino acid 25 to 147. The catalytic activity of ANG is several orders of ANG is a basic, single chain potent blood-vessel magnitude weaker than that of RNAse A, this being inducing protein with a molecular weight of 14 kDa partly due to the partial occupation of the which was originally discovered in conditioned pyrimidine-binding pocket of RNAse A by media of a human colon carcinoma cell line HT-29. glutamine-117 residue so that the substrate binding ANG belongs to the RNAse superfamily, being is compromised. Key amino acids for the ribo-

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The secondary structure elements of human ANG are depicted by purple boxes. The numbers on the upper and lower sides of each element indicate respectively the beginning and end amino acid residue positions (Acharya et al., 1994). The whole amino acid sequences are shown below and the signal peptide sequences (1-24) are enclosed by box. The H elements form helix structure (Acharya et al., 1994). Key amino acids for the ribonucleolytic activity of ANG (His13, Lys40, and His114) are indicated by plus (+) signs, and residues necessary for angiogenesis (60-68 and 109) are underlined. nucleolytic activity of ANG are His13, Lys40, or several secondary message cascades such as His114 of ANG, a catalytic triad, but mutations of extracellular signal related kinase 1/2 (ERK1 and these amino acids also reduce ANG induced ERK2), protein kinase B/Akt, and stress-associated angiogenesis, suggesting that the ribonucleolytic protein kinase/c-Jun N-terminal kinase activity of ANG, although weak, is necessary for (SAPK/JNK). the angiogenic activity of ANG. Furthermore, 3) Nuclear translocation several arginines are essential for ribonucleolytic The nuclear mechanisms underlying the function of and angiogenic activities. ANG remain elusive. Internalization could involve II. Angiogenic activity cell surface ANG binding to proteins as well as to In addition to the catalytic activity, cell binding other molecules such as proteoglycans, followed by sites which encompass residues 60-68 of the endocytosis. In this event, ANG interacts directly surface loop as well as asparagine-109 are with intracellular protein alpha-actinin-2 followed necessary for angiogenesis. The variants by translocation into the nucleus through the undergoing alterations of these residues lack any nuclear pore in a passive manner. After nuclear angiogenic activity while the enzymatic activity retention, ANG binds to carrier proteins through a remains intact. Inversely, replacing the surface loop sequence 29-IMRRRGL-35 (nuclear localization in RNAse A (residues 59-73) with the signal) of ANG and to the ANG-binding element of corresponding region of ANG (residue 57-70) ribosomal DNA (CTCT repeats) and subsequently, bestows a neovascularization activity to the RNAse stimulates ribosomal RNA transcription. Nuclear A. translocation is essential for cell proliferation since 1) Basement membrane degradation it is considered a third messenger and promotes Amino acid residues from Lys60 to Asn68 of the gene activation and transcription events, and ANG constitute a cell surface receptor binding site. inhibition of the nuclear translocation of angiogenin Accordingly, a 42 kDa endothelial cell surface abolishes ANG-induced angiogenesis. Interestingly, protein was identified as an ANG binding protein, the expression of cell surface receptors responsible which was later found to be a smooth muscle type for internalization as well as for the nuclear alpha-actin. The ANG-actin complex dissociates translocation of ANG also depends on the cell from the cell surface and activates a tissue type density. plasminogen activator, thus accelerating III. Roles of ANG in physiological angiogenesis degradation of the basement membrane and The above biological events, which are distinct extracellular matrix that allows endothelial cells to from those of RNAse A, are regulated tightly by the penetrate or migrate through the extracellular cell density-dependent expression of ANG matrix more easily, an initial step of receptors. The discovery of the uniquely regulated neovascularization. Furthermore, fibulin-1, an expression of ANG receptors provides us with the important molecule for stabilization of the blood following conceivable mechanisms for ANG related vessel wall, binds to ANG, suggesting that the angiogenesis. In the region where ANG-fibulin-1 complex modulates new blood neovascularization is required, ANG binds to the vessel formation and stabilization. endothelial surface 42 kDa receptor, and the ANG- 2) Signal transduction 42 kDa receptor complex dissociates from the cell Besides the 42 kDa ANG receptor, a 170 kDa surface and stimulates proteolytic activity, thus molecule later found on the endothelial surface is facilitating the penetration of endothelial cells responsible for signal transduction, an important through the extracellular matrix. After the leading process leading to cell proliferation. ANG activates cells migrate away, the endothelial cell density in

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the vicinity of migrating cells might be sparse, and with a mortality rate of 133 per million. Breast such cell sparsity triggers the endothelial cancer incidence rates have increased in most proliferation machinery that includes signal geographic regions. transduction, ANG internalization, and nuclear Prognosis translocation. A 170 kDa receptor is one of the The ANG level in sera and the roles of ANG in receptors responsible for this orchestrated process. breast cancer patients seem to be conflicting. Some Once the microenvironment is filled up with the studies found significantly increased serum ANG in sufficient amount of endothelial cells and the breast cancer patients in comparison with normal vascular network is established, such cell controls while other studies failed to find such a proliferating events diminish. Therefore, the above difference. The serum ANG level is significantly cell density dependent biological events are decreased after breast cancer resection, suggesting intelligent mechanisms where the proliferation that the source of ANG is at least in part the breast machinery and subsequent angiogenic switch are on cancer cells. However, there is conflicting evidence when neovascularization is needed while they are concerning the role of ANG. The correlation off to prevent unwanted angiogenesis. between ANG expression in tissue or in sera and Homology patient survival was inverse, neutral, or even Of the 123 amino acids of human ANG, 43 (35%) positive. The absence of any increase in serum and 25 are respectively identical to human ANG levels in early stage breast cancer patients pancreatic RNAse or to other RNAse, and 16 are suggests that ANG may have clinical implications conservative replacements, constituting an overall when breast cancer progresses to the advanced homology of 68%. stage. Pancreas cancer Implicated in Disease Various cancers The pancreas is composed of exocrine (acinar glands and pancreas duct) and endocrine (islets of Note Langerhans) components. Both can give rise to There is growing evidence that increased ANG malignant neoplasms, but adenocarcinoma arising expression in tissue and/or in sera is correlated with from the pancreatic ducts is representative of all tumor aggressiveness. These facts are explained at pancreatic cancers. least in part by the hypothesis that ANG in Prognosis malignancy plays roles in the proliferation and migration of malignant cells, mimicking endothelial Pancreatic cancer is one of the most aggressive cell behavior during physiological angiogenesis. As diseases with most cancers already in later stages at described earlier, ANG could activate proteolytic presentation, the 5-year survival rate being around activity, so that ANG-expressing malignant cells 5% both in USA and in Europe. Investigations are allowed to invade through the extracellular concerning ANG expression in pancreatic cancer matrix and enter into the bloodstream. In addition, are scarce. ANG in sera is elevated in pancreatic the continuous translocation of ANG to the nucleus cancer patients as compared with healthy of HeLa cells in a cell density-independent manner volunteers, and increased ANG mRNA in tissue or suggests that cancer cells are also targets for ANG, increased ANG in sera has been correlated with and that ANG per se is a contributing factor for cancer aggressiveness. In addition, the involvement sustained cell growth and the constant supply of of ANG in the cancer microenvironment has been ribosomes, a characteristic of malignant cells. suggested by findings of the ANG expression in Several ANG antagonists have been introduced and chronic pancreatitis adjacent to pancreatic cancer some have proved to be effective inhibitors for the but not in pure chronic pancreatitis. Cancer derived establishment or metastasis of human tumors in fibroblasts also express ANG. athymic mice. These compounds include a Gastric cancer monoclonal antibody, antisense oligonucleotides Disease complementary to the AUG translational start site Gastric cancer, the third most common cancer and region of ANG, translocation blocker, enzymatic the second leading cause of cancer death among inhibitor targeting ANG enzymatic active site, men, arises in an estimated one million new cases ANG binding polypeptide complementary to the in both sexes worldwide. Gastric cancer is receptor binding site of ANG, and internalization anatomically classified as noncardia and cardia pathway blocker. cancers, and the former incidence has declined Female breast cancer while the latter incidence has increased. Disease Helicobacter pylori infection is one of the risk Female breast cancer is globally the most common factors for noncardia cancer. Adenocarcinomas cancer with an annual incidence of 1,15 million account for a large majority of gastric cancer worldwide. It is also the leading cause of death, histologic diagnoses.

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Prognosis Liver cancer The 5-year survival rate of gastric cancer in Japan is Disease double that of the United States and Europe. The The global incidence and mortality rate of liver better treatment outcomes are ascribed partly to the cancer is ranked among top 5 (in men) and top 10 social screening program that attempts to capitalize (in women) cancer types. The hepatitis B virus and on the benefit of early detection, and partly to hepatitis C virus are the most important risk factors systematic lymph node dissection. ANG in sera is for liver cancer. Histologic classification separates increased in gastric cancer patients and is decreased hepatocellular carcinoma (HCC) (liver cell origin) by resection, suggesting gastric cancer to be a from cholangiocarcinoma, which arises from source of ANG. Increased mRNA expression in intrahepatic bile ducts. HCC is the most common gastric cancer tissues or increased serum ANG histology of liver cancer which is characterized by levels is correlated with cancer progression, vigorous neovascularization. proliferation ability, and poor patient prognosis. Prognosis Colorectal cancer The serum ANG is increased in hypervascular Disease hepatocellular carcinoma. The serum ANG level Colon cancer is the fourth most commonly decreases after therapy but again increases at diagnosed cancer and fourth most frequent cause of recurrence, suggesting the usefulness of serum cancer death among men. Colon cancer incidence ANG measurement for monitoring the disease and rates have increased in most parts of the world. prediction of patient survival. However, other Typically, there is a pathologic evolution from investigators found a neutral correlation between benign adenomas to cancer (adenoma-carcinoma serum ANG and survival. The ANG sequence), so that colorectal cancer screening aims immunoreactivity is correlated with poorer to detect lesions at the adenoma stage and interrupt histological differentiation. the adenoma carcinoma sequence, ultimately Cytogenetics reducing colorectal cancer incidence and mortality. 14q11.2 is found to be highly amplified in Prognosis hepatoblastoma. Colorectal cancer exhibits increased serum ANG Prostate cancer concentration, and the degree of elevation is correlated with cancer progression. ANG message Disease expression in colorectal cancer tissue has also been Prostate cancer is the second most frequently correlated with poor patient survival. diagnosed cancer among men. Prognosis is excellent for early stage disease while it is poor for Cytogenetics those diagnosed with advanced cases, pointing to Overexpression of the 14q11.1-14q11.2 product the benefit of earlier diagnosis. Measurement of was observed in a colon adenocarcinoma cell line. prostate specific antigen helps to detect biologically Lung cancer indolent prostate cancer. Disease Prognosis Lung cancer is the most frequently diagnosed The immunoreactivity of ANG is more evident cancer among men. The mortality rate is the highest according to prostate epithelial cells evolution from among men and the second highest among women a benign to an invasive phenotype. In vitro analyses worldwide. Cigarette smoking is the most important using prostate cancer cell line have elucidated that risk factor for lung cancer. The main histologic ANG is one of the elements responsible for types of lung cancer are adenocarcinoma, squamous tumorigenicity and tumor growth. Furthermore, cell carcinoma, large cell carcinoma, and small cell serum ANG is more increased in hormone- carcinoma. The stage of the disease is a strong refractory patients than in healthy controls. predictor of survival, suggesting that early detection Leukemia is needed for improvement in treatment outcomes. Disease Prognosis Leukemias are malignancies that affect blood- Immunoreactivity in lung cancer tissue is correlated forming stem cells in the bone marrow. Leukemia, with tumor size and positive nodal involvement. a heterogeneous group of malignancies, is classified Recently, the detection of ANG in exhaled breath into several subtypes according to the major cell condensate has been achieved, and breath based type such as acute lymphoblastic leukemia (ALL), ANG may help in the early detection of lung chronic lymphoblastic leukemia (CLL), acute cancer. myeloid leukemia (AML), chronic myeloid Cytogenetics leukemia (CML), etc. Acute types refer to cancers DNA damage in 14q11.2 was found in asbestos- arising in immature stem cells while chronic types exposed lung cancer patients. refer to cancers arising in mature stem cells.

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Prognosis message in urothelial cancer correlates with poor The ANG level in sera was increased in AML and patient survival or cancer progression. Recently, CML, although other investigators failed to find ANG in the urine has been found to be increased in such correlation. In sharp contrast to the clinical bladder cancer patients. significance of serum ANG in other solid tumors, Melanoma elevated serum ANG in AML and CML is correlated with better patient survival. Disease Melanoma is placed as the leading cause of skin Cytogenetics cancer death and its incidence has dramatically 14q11.2 is one of the risk foci for ALL. increased over the last ten years. It is characterized Hodgkin and non-hodgkin as having a notorious resistance to currently lymphomas available therapies so that early detection and intervention is needed. Disease Lymphomas, malignancies of the lymphoid cells, Prognosis are divided on the basis of their pathologic features Survival clearly worsens with increasing tumor into Hodgkin lymphoma (HL) and non-Hodgkin thickness. Thin lesions exhibit excellent survival lymphoma (NHL). HL almost always develops in a outcomes while the MST of patients with advance lymph node or other lymphoid structure and spread cases is around 8 months. The ANG in sera is to nearby nodes. HL is characterized by the increased in melanoma patients, and increased presence of Hodgkin Reed Sternberg cells. It is one serum ANG correlates with malignancy potential, of the most common cancers diagnosed in younger accordingly, ANG contributes directly to A375 persons. The proportions of patients being melanoma cell proliferation. However, other diagnosed below 50 years old accounts for 60%. investigators failed to find such a correlation. NHL occurs in more elderly patients in the context Gynecological cancers of HIV-related immunosuppression. NHL with HIV Disease shows extensively poorer survivals than those Cancers of the uterus and ovary are respectively the without HIV. second and the sixth most frequently diagnosed Prognosis cancer among women. Cancer of the uterus is In sharp contrast to the other solid malignancies, further classified as cancer of the cervix and corpus, serum ANG concentrations in patients with HL or and cancer of the cervix uteri shows the highest NHL are less than or the same as those in healthy incidence among the three gynecological controls. Increased serum ANG renders no or an malignancies. Cancer of the cervix uteri can be inverse impact on survival in NHL patients. attributed to persistent infection with carcinogenic Cytogenetics genotypes of human papilloma virus. The three One subtype of NHL experiences multiple most common histological types of cancer of the translocations at 14q11.2. cervix uteri are squamous, adenosquamous, and adenocarcinoma. Adenocarcinoma is the most Kidney and bladder cancer common histology of cancer of the corpus uteri and Disease ovary. Women with ovarian cancer have poorer Kidney and bladder cancers are placed among the survival rates than those with other gynecological top ten cancer types in both sexes. Cancer of the cancers. urinary bladder most commonly originates in the Prognosis urothelium, the epithelium that lines the bladder. Serum ANG is significantly increased in ovarian Bladder cancer incidence is significantly higher in cancer patients, while other studies failed to find males than in females. There are three major such a difference. Increased serum ANG histologic types of bladder cancer: transitional cell concentration is correlated with cancer progression carcinoma, squamous cell carcinoma, and in ovary and cervix uteri. adenocarcinoma, the former being overwhelmingly the most common. The majority of cancers of the Cytogenetics kidney are renal cell carcinomas, which arise from Gains on 14q11.2 are associated with renal tubules. On the other hand, cancer of the renal chemoresistant ovarian cancer. pelvis designated as transitional cell carcinoma comprises the minority. References Prognosis Kurachi K, Davie EW, Strydom DJ, Riordan JF, Vallee BL. The serum level of ANG is increased in renal cell Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor. Biochemistry. 1985 Sep carcinoma and bladder cancer; however, the 24;24(20):5494-9 increase in serum ANG level does not correlate Strydom DJ, Fett JW, Lobb RR, Alderman EM, Bethune with patient survival for renal cell carcinoma. On JL, Riordan JF, Vallee BL. Amino acid sequence of human the other hand, increased serum ANG or ANG

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tumor derived angiogenin. Biochemistry. 1985 Sep Olson KA, French TC, Vallee BL, Fett JW. A monoclonal 24;24(20):5486-94 antibody to human angiogenin suppresses tumor growth in athymic mice. Cancer Res. 1994 Sep 1;54(17):4576-9 Rybak SM, Fett JW, Yao QZ, Vallee BL. Angiogenin mRNA in human tumor and normal cells. Biochem Biophys Russo N, Shapiro R, Acharya KR, Riordan JF, Vallee BL. Res Commun. 1987 Aug 14;146(3):1240-8 Role of glutamine-117 in the ribonucleolytic activity of human angiogenin. Proc Natl Acad Sci U S A. 1994 Apr Shapiro R, Strydom DJ, Olson KA, Vallee BL. Isolation of 12;91(8):2920-4 angiogenin from normal human plasma. Biochemistry. 1987 Aug 11;26(16):5141-6 Raines RT, Toscano MP, Nierengarten DM, Ha JH, Auerbach R. Replacing a surface loop endows Riordan JF, Vallee BL. Human angiogenin, an organogenic ribonuclease A with angiogenic activity. J Biol Chem. 1995 protein. Br J Cancer. 1988 Jun;57(6):587-90 Jul 21;270(29):17180-4 Shapiro R, Fox EA, Riordan JF. 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Am J Hum Genet. 1990 Dec;47(6):973- epithelial ovarian cancer. Clin Cancer Res. 1997 81 Sep;3(9):1579-86 Hallahan TW, Shapiro R, Vallee BL. Dual site model for Chopra V, Dinh TV, Hannigan EV. Serum levels of the organogenic activity of angiogenin. Proc Natl Acad Sci interleukins, growth factors and angiogenin in patients with U S A. 1991 Mar 15;88(6):2222-6 endometrial cancer. J Cancer Res Clin Oncol. Hu GF, Chang SI, Riordan JF, Vallee BL. An angiogenin- 1997;123(3):167-72 binding protein from endothelial cells. Proc Natl Acad Sci U Gho YS, Chae CB. Anti-angiogenin activity of the peptides S A. 1991 Mar 15;88(6):2227-31 complementary to the receptor-binding site of angiogenin. Hallahan TW, Shapiro R, Strydom DJ, Vallee BL. J Biol Chem. 1997 Sep 26;272(39):24294-9 Importance of asparagine-61 and asparagine-109 to the Hu GF, Riordan JF, Vallee BL. A putative angiogenin angiogenic activity of human angiogenin. 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Shimoyama S, Yamasaki K, Kawahara M, Kaminishi M. Brunner B, Gunsilius E, Schumacher P, Zwierzina H, Gastl Increased serum angiogenin concentration in colorectal G, Stauder R. Blood levels of angiogenin and vascular cancer is correlated with cancer progression. Clin Cancer endothelial growth factor are elevated in myelodysplastic Res. 1999 May;5(5):1125-30 syndromes and in acute myeloid leukemia. J Hematother Stem Cell Res. 2002 Feb;11(1):119-25 Wechsel HW, Bichler KH, Feil G, Loeser W, Lahme S, Petri E. Renal cell carcinoma: relevance of angiogenetic Gho YS, Yoon WH, Chae CB. Antiplasmin activity of a factors. Anticancer Res. 1999 Mar-Apr;19(2C):1537-40 peptide that binds to the receptor-binding site of angiogenin. J Biol Chem. 2002 Mar 22;277(12):9690-4 Etoh T, Shibuta K, Barnard GF, Kitano S, Mori M. Angiogenin expression in human colorectal cancer: the Glenjen N, Mosevoll KA, Bruserud Ø. Serum levels of role of focal macrophage infiltration. 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J Cell Biochem. 2000 Jan;76(3):452-62 Ni X, Ma Y, Cheng H, Jiang M, Guo L, Ji C, Gu S, Cao Y, Xie Y, Mao Y. Molecular cloning and characterization of a Sheen-Chen SM, Eng HL, Chen WJ, Chou FF, Chen HS. novel human Rab ( Rab2B) gene. J Hum Genet. Serum level of angiogenin in breast cancer. Anticancer 2002;47(10):548-51 Res. 2000 Nov-Dec;20(6C):4769-71 Olson KA, Byers HR, Key ME, Fett JW. Inhibition of Shimoyama S, Kaminishi M. Increased angiogenin prostate carcinoma establishment and metastatic growth in expression in gastric cancer correlated with cancer mice by an antiangiogenin monoclonal antibody. Int J progression. J Cancer Res Clin Oncol. 2000 Cancer. 2002 Apr 20;98(6):923-9 Aug;126(8):468-74 Xu ZP, Tsuji T, Riordan JF, Hu GF. The nuclear function of Bodner-Adler B, Hefler L, Bodner K, Leodolter S, et al. angiogenin in endothelial cells is related to rRNA Serum levels of angiogenin (ANG) in invasive cervical production. Biochem Biophys Res Commun. 2002 Jun cancer and in cervical intraepithelial neoplasia (CIN). 7;294(2):287-92 Anticancer Res. 2001 Jan-Feb;21(1B):809-12 Chao Y, Li CP, Chau GY, Chen CP, King KL, Lui WY, Yen Liu S, Yu D, Xu ZP, Riordan JF, Hu GF. Angiogenin SH, Chang FY, Chan WK, Lee SD. Prognostic significance activates Erk1/2 in human umbilical vein endothelial cells. of vascular endothelial growth factor, basic fibroblast Biochem Biophys Res Commun. 2001 Sep 14;287(1):305- growth factor, and angiogenin in patients with resectable 10 hepatocellular carcinoma after surgery. Ann Surg Oncol. Lixin R, Efthymiadis A, Henderson B, Jans DA. Novel 2003 May;10(4):355-62 properties of the nucleolar targeting signal of human Hisai H, Kato J, Kobune M, Murakami T, Miyanishi K, et al. angiogenin. Biochem Biophys Res Commun. 2001 Jun Increased expression of angiogenin in hepatocellular 1;284(1):185-93 carcinoma in correlation with tumor vascularity. Clin Olson KA, Byers HR, Key ME, Fett JW. Prevention of Cancer Res. 2003 Oct 15;9(13):4852-9 human prostate tumor metastasis in athymic mice by Majumder PK, Yeh JJ, George DJ, Febbo PG, Kum J, et antisense targeting of human angiogenin. Clin Cancer al. Prostate intraepithelial neoplasia induced by prostate Res. 2001 Nov;7(11):3598-605 restricted Akt activation: the MPAKT model. Proc Natl Pilch H, Schlenger K, Steiner E, Brockerhoff P, Knapstein Acad Sci U S A. 2003 Jun 24;100(13):7841-6 P, Vaupel P. Hypoxia-stimulated expression of angiogenic Shimoyama S, Kaminishi M. Angiogenin in sera as an growth factors in cervical cancer cells and cervical cancer- independent prognostic factor in gastric cancer. J Cancer derived fibroblasts. Int J Gynecol Cancer. 2001 Mar- Res Clin Oncol. 2003 Apr;129(4):239-44 Apr;11(2):137-42 Xu ZP, Tsuji T, Riordan JF, Hu GF. Identification and Sun W, Schuchter LM. Metastatic melanoma. Curr Treat characterization of an angiogenin-binding DNA sequence Options Oncol. 2001 Jun;2(3):193-202 that stimulates luciferase reporter gene expression. Ugurel S, Rappl G, Tilgen W, Reinhold U. Increased serum Biochemistry. 2003 Jan 14;42(1):121-8 concentration of angiogenic factors in malignant melanoma Giles FJ, Vose JM, Do KA, Johnson MM, Manshouri T, et patients correlates with tumor progression and survival. J al. Clinical relevance of circulating angiogenic factors in Clin Oncol. 2001 Jan 15;19(2):577-83 patients with non-Hodgkin's lymphoma or Hodgkin's Verstovsek S, Kantarjian H, Aguayo A, Manshouri T, et al. lymphoma. Leuk Res. 2004 Jun;28(6):595-604 Significance of angiogenin plasma concentrations in Molica S, Vitelli G, Levato D, Giannarelli D, Vacca A, patients with acute myeloid leukaemia and advanced Cuneo A, Ribatti D, Digiesi G. Serum angiogenin is not myelodysplastic syndrome. Br J Haematol. 2001 elevated in patients with early B-cell chronic lymphocytic Aug;114(2):290-5 leukemia but is prognostic factor for disease progression. Xu Z, Monti DM, Hu G. Angiogenin activates human Eur J Haematol. 2004 Jul;73(1):36-42 umbilical artery smooth muscle cells. Biochem Biophys Musolino C, Alonci A, Bellomo G, Loteta B, Quartarone E, Res Commun. 2001 Jul 27;285(4):909-14 Gangemi D, Massara E, Calabrò L. Levels of soluble Bevona C, Sober AJ. Melanoma incidence trends. angiogenin in chronic myeloid malignancies: clinical Dermatol Clin. 2002 Oct;20(4):589-95, vii implications. Eur J Haematol. 2004 Jun;72(6):416-9

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Cho S, Beintema JJ, Zhang J. The ribonuclease A Lones MA, Heerema NA, Le Beau MM, Sposto R, et al. superfamily of mammals and birds: identifying new Chromosome abnormalities in advanced stage members and tracing evolutionary histories. Genomics. lymphoblastic lymphoma of children and adolescents: a 2005 Feb;85(2):208-20 report from CCG-E08. Cancer Genet Cytogenet. 2007 Jan 1;172(1):1-11 Hirukawa S, Olson KA, Tsuji T, Hu GF. Neamine inhibits xenografic human tumor growth and angiogenesis in Vihinen P, Kallioinen M, Vuoristo MS, Ivaska J, Syrjänen athymic mice. Clin Cancer Res. 2005 Dec 15;11(24 Pt KJ, Hahka-Kemppinen M, Kellokumpu-Lehtinen PL, 1):8745-52 Pyrhönen SO. Serum angiogenin levels predict treatment response in patients with stage IV melanoma. Clin Exp Hu H, Gao X, Sun Y, Zhou J, Yang M, Xu Z. Alpha-actinin- Metastasis. 2007;24(7):567-74 2, a cytoskeletal protein, binds to angiogenin. Biochem Biophys Res Commun. 2005 Apr 8;329(2):661-7 Passam FH, Sfiridaki A, Pappa C, Kyriakou D, Petreli E, et al. Angiogenesis-related growth factors and cytokines in Katona TM, Neubauer BL, Iversen PW, Zhang S, the serum of patients with B non-Hodgkin lymphoma; Baldridge LA, Cheng L. Elevated expression of angiogenin relation to clinical features and response to treatment. Int J in prostate cancer and its precursors. Clin Cancer Res. Lab Hematol. 2008 Feb;30(1):17-25 2005 Dec 1;11(23):8358-63 Suzuki M, Kato M, Yuyan C, Takita J, Sanada M, et al. Tsuji T, Sun Y, Kishimoto K, Olson KA, Liu S, Hirukawa S, Whole-genome profiling of chromosomal aberrations in Hu GF. Angiogenin is translocated to the nucleus of HeLa hepatoblastoma using high-density single-nucleotide cells and is involved in ribosomal RNA transcription and polymorphism genotyping microarrays. Cancer Sci. 2008 cell proliferation. Cancer Res. 2005 Feb 15;65(4):1352-60 Mar;99(3):564-70 Zhao H, Grossman HB, Delclos GL, Hwang LY, et al. Zhang H, Gao X, Weng C, Xu Z. Interaction between Increased plasma levels of angiogenin and the risk of angiogenin and fibulin 1: evidence and implication. Acta bladder carcinoma: from initiation ot recurrence. Cancer. Biochim Biophys Sin (Shanghai). 2008 May;40(5):375-80 2005 Jul 1;104(1):30-5 Chan HP, Lewis C, Thomas PS. Exhaled breath analysis: Chen Y, Zhang S, Chen YP, Lin JY. Increased expression novel approach for early detection of lung cancer. Lung of angiogenin in gastric carcinoma in correlation with tumor Cancer. 2009 Feb;63(2):164-8 angiogenesis and proliferation. World J Gastroenterol. 2006 Aug 28;12(32):5135-9 Duranyildiz D, Camlica H, Soydinc HO, Derin D, Yasasever V. Serum levels of angiogenic factors in early Kamangar F, Dores GM, Anderson WF. Patterns of cancer breast cancer remain close to normal. Breast. 2009 incidence, mortality, and prevalence across five continents: Feb;18(1):26-9 defining priorities to reduce cancer disparities in different geographic regions of the world. J Clin Oncol. 2006 May Eissa S, Swellam M, Labib RA, El-Zayat T, El Ahmady O. 10;24(14):2137-50 A panel of angiogenic factors for early bladder cancer detection: enzyme immunoassay and Western blot. J Urol. Nymark P, Wikman H, Ruosaari S, Hollmén J, et al. 2009 Mar;181(3):1353-60 Identification of specific gene copy number changes in asbestos-related lung cancer. Cancer Res. 2006 Jun Goon PK, Lip GY, Stonelake PS, Blann AD. Circulating 1;66(11):5737-43 endothelial cells and circulating progenitor cells in breast cancer: relationship to endothelial Song J, Wang J, Yang J, Jiang C, Shen W, Wang L. damage/dysfunction/apoptosis, clinicopathologic factors, Influence of angiogenin on the growth of A375 human and the Nottingham Prognostic Index. Neoplasia. 2009 melanoma cells and the expression of basic fibroblast Aug;11(8):771-9 growth factor. Melanoma Res. 2006 Apr;16(2):119-26 Ibaragi S, Yoshioka N, Li S, Hu MG, Hirukawa S, Sadow Tas F, Duranyildiz D, Oguz H, Camlica H, Yasasever V, PM, Hu GF. Neamine inhibits prostate cancer growth by Topuz E. Circulating serum levels of angiogenic factors suppressing angiogenin-mediated rRNA transcription. Clin and vascular endothelial growth factor receptors 1 and 2 in Cancer Res. 2009 Mar 15;15(6):1981-8 melanoma patients. Melanoma Res. 2006 Oct;16(5):405- 11 Jang SH, Song HD, Kang DK, Chang SI, Kim MK, Cho KH, Scheraga HA, Shin HC. Role of the surface loop on the Yoshioka N, Wang L, Kishimoto K, Tsuji T, Hu GF. A structure and biological activity of angiogenin. BMB Rep. therapeutic target for prostate cancer based on 2009 Dec 31;42(12):829-33 angiogenin-stimulated angiogenesis and cancer cell proliferation. Proc Natl Acad Sci U S A. 2006 Sep Papaemmanuil E, Hosking FJ, Vijayakrishnan J, et al. Loci 26;103(39):14519-24 on 7p12.2, 10q21.2 and 14q11.2 are associated with risk of childhood acute lymphoblastic leukemia. Nat Genet. Kawada M, Inoue H, Arakawa M, Takamoto K, Masuda T, 2009 Sep;41(9):1006-10 Ikeda D. Highly tumorigenic human androgen receptor- positive prostate cancer cells overexpress angiogenin. Yuan Y, Wang F, Liu XH, Gong DJ, Cheng HZ, Huang SD. Cancer Sci. 2007 Mar;98(3):350-6 Angiogenin is involved in lung adenocarcinoma cell proliferation and angiogenesis. Lung Cancer. 2009 Kim HM, Kang DK, Kim HY, Kang SS, Chang SI. Oct;66(1):28-36 Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear Gessner C, Rechner B, Hammerschmidt S, Kuhn H, et al. translocation of angiogenin in HUVE cells. Biochem Angiogenic markers in breath condensate identify non- Biophys Res Commun. 2007 Jan 12;352(2):509-13 small cell lung cancer. Lung Cancer. 2010 May;68(2):177- 84 Kim SW, Kim JW, Kim YT, Kim JH, Kim S, Yoon BS, Nam EJ, Kim HY. Analysis of chromosomal changes in serous This article should be referenced as such: ovarian carcinoma using high-resolution array comparative genomic hybridization: Potential predictive markers of Shimoyama S. ANG (angiogenin, ribonuclease, RNase A chemoresistant disease. Genes Chromosomes Cancer. family, 5). Atlas Genet Cytogenet Oncol Haematol. 2011; 2007 Jan;46(1):1-9 15(3):244-251.

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Gene Section Mini Review

ATF5 (activating transcription factor 5) Arthur KK Ching, Nathalie Wong Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong (AKKC, NW)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/ATF5ID50361ch19q13.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI ATF5ID50361ch19q13.txt

2 are non-coding exons and the size of open reading Identity frame is 849 bp. Other names: ATFX; FLJ34666; HMFN0395 HGNC (Hugo): ATF5 Protein Location: 19q13.33 Description Local order: Refer to mapping diagram. ATF5 consists of 282 amino acid with MW of 30.69 kDa (NCBI reference sequence DNA/RNA NP_036200.2). Description Expression The ATF5 gene spans a total genomic size of 5219 Northern blot analysis revealed ubiquitous bases and is composed of four exons. expression of ATF5, with highest levels in liver, lung, adipose tissue, heart, and skeletal muscle. Transcription Localisation The human ATF5 transcript is 2268 bp in size (NM_012068.4) and contains 4 exons. Exon 1 and Nucleus and cytoplasm.

Mapping diagram. Base on Human Mar. 2006 (NCBI36/hg18) Assembly.

DNA structure diagram. Relative size of the 4 exons of ATF5. Exon 1 and 2 are untranslated exons (NCBI reference sequence NM_012068.4). Blue area is non-coding region and pink is coding region.

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Protein structure diagram.

Function Homology ATF5 is a member of basic-region leucine zipper ATF5 gene is highly conserved in mammals. (bZIP) proteins family which binds the cAMP Protein identity percentage of human ATF5 response element (CRE) consensus sequence: compared with chimpanzee, cow, mouse, and rat is 5'GTGACGT(C/A)(G/A). This sequence is present 98.9, 88.0, 87.5 and 88.9 respectively. in many viral and cellular promoters. ATF within or between subgroups can form homo- or hetero-dimer Mutations through the bZIP domain and the dimer can then bind to the DNA through the basic-motif and Germinal function as a transcription factor. Recently, another Unknown. novel ATF5 consensus DNA binding sequence (CYTCTYCCTTW) was found in C6 glioma and Somatic MCF7 using a cyclic amplification and selection of Exon2 Leu141Phe, Exon2 Val257Met and Exon2 targets (CASTing) approach (Li et al., 2009). Arg275Trp. ATF5 is linked to many cellular function including cell cycle progression, metabolite homeostasis (Al Implicated in Sarraj et al., 2005; Watatani et al., 2007), cellular differentiation and apoptosis. It involves in the Various cancers proliferation and differentiation of neural cells Note (Angelastro et al., 2003; Angelastro et al., 2005; ATF5 is showed to be overexpressed in various Mason et al., 2005) and has been shown to take part cancers by TMA (tissue microarray) that includes in the skeletal development of mouse limb breast cancer, glioblastomas, adenocarcinomas, (Shinomura et al., 2006; Satake at al., 2009). Data transitional cell carcinomas, squamous cell from various groups also suggested that ATF5 can carcinomas and metastatic carcinomas of various function as anti-apoptotic factor (Devireddy et al., origin (Monaco et al., 2007). However, in 2001; Persengiev et al., 2002; Nishioka et al., hepatocellular carcinoma, ATF5 expression is 2009). down-regulated, suggesting that role of ATF5 in Coimmunoprecipitation and GST pull-down tumor is highly depending on the tumor type. analyses confirmed the association of the C- terminal bZIP motif of ATF5 with the PRL-1 Glioma PTPase domain and adjacent residues of PTP4A1 in Note vitro. SDS-PAGE analysis showed that PRL-1 ATF5 has been shown to be highly expressed in dephosphorylates ATF5 in vitro (Peters et al., perinecrotic palisades, the most aggressive forms of 2001). malignant gliomas. In a study of 28 tumors without ATF5 has been shown to interact with various perinecrotic palisades, the level of ATF5 expression proteins including Cyclin D3 (Liu et al., 2004), together with 4 other genes, negatively correlated GABAB receptors (White et al., 2000), HTLV-1 with time of patient survival. Interference of ATF5 viral protein Tax (Forgacs et al., 2005), E2 expression in glioma cell lines causes apoptosis but ubiguitin-conjugating enzyme Cdc34, PRL-1 and not in cultured astrocytes. These findings suggested DISC1 (Morris et al., 2003; Fujii et al., 2007; that ATF5 plays a role in maintaining cell survival Tomppo et al., 2009). It is a target of Cdc34- in glioma. dependent ubiquitin-mediated proteolysis (Pati et Cytogenetics al., 1999). Study indicates that during stress Unknown. condition, eIF2 is phosphorylated and subsequently direct ATF5 translation in cell (Watatani et al., Hybrid/Mutated gene 2008; Zhou et al., 2008). Recent study of the Unknown. promoter of ATF5 also suggested that its Abnormal protein transcription is regulated by EBF1 (Wei et al., Unknown. 2010).

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Hepatocellular carcinoma prognostic markers in human glioblastoma. J Neuropathol Exp Neurol. 2005 Nov;64(11):948-55 Note Forgacs E, Gupta SK, Kerry JA, Semmes OJ. The bZIP A study has showed that ATF5 is down-regulated in transcription factor ATFx binds human T-cell leukemia 60 out of 77 cases in HCC, as in contrast to adult virus type 1 (HTLV-1) Tax and represses HTLV-1 long normal liver where expression of ATF5 is terminal repeat-mediated transcription. J Virol. 2005 particularly high. Gene expression profiling was Jun;79(11):6932-9 also done by ectopic re-expression of ATF5 Mason JL, Angelastro JM, Ignatova TN, Kukekov VG, Lin suggesting cell cycle, actin skeleton regulation, G, Greene LA, Goldman JE. ATF5 regulates the proliferation and differentiation of oligodendrocytes. Mol MAPK signaling and focal adhesion are the Cell Neurosci. 2005 Jul;29(3):372-80 pathways modulated by ATF5. These findings suggested that ATF5 down regulation may Angelastro JM, Canoll PD, Kuo J, Weicker M, Costa A, Bruce JN, Greene LA. Selective destruction of contribute to the development of HCC. The glioblastoma cells by interference with the activity or inactivation mechanisms of ATF5 involve expression of ATF5. Oncogene. 2006 Feb 9;25(6):907-16 epigenetic silencing and chromosome copy number Chow LS, Lam CW, Chan SY, Tsao SW, To KF, Tong SF, loss. Hung WK, Dammann R, Huang DP, Lo KW. Identification of RASSF1A modulated genes in nasopharyngeal References carcinoma. Oncogene. 2006 Jan 12;25(2):310-6 Monaco SE, Angelastro JM, Szabolcs M, Greene LA. The Pati D, Meistrich ML, Plon SE. Human Cdc34 and Rad6B transcription factor ATF5 is widely expressed in ubiquitin-conjugating enzymes target repressors of cyclic carcinomas, and interference with its function selectively AMP-induced transcription for proteolysis. Mol Cell Biol. kills neoplastic, but not nontransformed, breast cell lines. 1999 Jul;19(7):5001-13 Int J Cancer. 2007 May 1;120(9):1883-90 White JH, McIllhinney RA, Wise A, Ciruela F, Chan WY, Watatani Y, Kimura N, Shimizu YI, Akiyama I, Tonaki D, et Emson PC, Billinton A, Marshall FH. The GABAB receptor al. Amino acid limitation induces expression of ATF5 interacts directly with the related transcription factors mRNA at the post-transcriptional level. Life Sci. 2007 Feb CREB2 and ATFx. Proc Natl Acad Sci U S A. 2000 Dec 6;80(9):879-85 5;97(25):13967-72 Gho JW, Ip WK, Chan KY, Law PT, Lai PB, Wong N. Re- Devireddy LR, Teodoro JG, Richard FA, Green MR. expression of transcription factor ATF5 in hepatocellular Induction of apoptosis by a secreted lipocalin that is carcinoma induces G2-M arrest. Cancer Res. 2008 Aug transcriptionally regulated by IL-3 deprivation. Science. 15;68(16):6743-51 2001 Aug 3;293(5531):829-34 Watatani Y, Ichikawa K, Nakanishi N, Fujimoto M, et al. Peters CS, Liang X, Li S, Kannan S, Peng Y, Taub R, Stress-induced translation of ATF5 mRNA is regulated by Diamond RH. ATF-7, a novel bZIP protein, interacts with the 5'-untranslated region. J Biol Chem. 2008 Feb the PRL-1 protein-tyrosine phosphatase. J Biol Chem. 1;283(5):2543-53 2001 Apr 27;276(17):13718-26 Zhou D, Palam LR, Jiang L, Narasimhan J, Staschke KA, Persengiev SP, Devireddy LR, Green MR. Inhibition of Wek RC. Phosphorylation of eIF2 directs ATF5 apoptosis by ATFx: a novel role for a member of the translational control in response to diverse stress ATF/CREB family of mammalian bZIP transcription factors. conditions. J Biol Chem. 2008 Mar 14;283(11):7064-73 Genes Dev. 2002 Jul 15;16(14):1806-14 Greene LA, Lee HY, Angelastro JM. The transcription Angelastro JM, Ignatova TN, Kukekov VG, Steindler DA, factor ATF5: role in neurodevelopment and neural tumors. Stengren GB, Mendelsohn C, Greene LA. Regulated J Neurochem. 2009 Jan;108(1):11-22 expression of ATF5 is required for the progression of neural progenitor cells to neurons. J Neurosci. 2003 Jun Li G, Li W, Angelastro JM, Greene LA, Liu DX. 1;23(11):4590-600 Identification of a novel DNA binding site and a transcriptional target for activating transcription factor 5 in Morris JA, Kandpal G, Ma L, Austin CP. DISC1 (Disrupted- c6 glioma and mcf-7 breast cancer cells. Mol Cancer Res. In-Schizophrenia 1) is a centrosome-associated protein 2009 Jun;7(6):933-43 that interacts with MAP1A, MIPT3, ATF4/5 and NUDEL: regulation and loss of interaction with mutation. Hum Mol Satake H, Ito K, Takahara M, Furukawa T, Takagi M, Genet. 2003 Jul 1;12(13):1591-608 Ogino T, Shinomura T. Spatio-temporal expression of activating transcription factor 5 in the skeletal development Liu W, Sun M, Jiang J, Shen X, Sun Q, Liu W, Shen H, Gu of mouse limb. Dev Growth Differ. 2009 Sep;51(7):669-76 J. Cyclin D3 interacts with human activating transcription factor 5 and potentiates its transcription activity. Biochem Tomppo L, Hennah W, Lahermo P, Loukola A, et al. Biophys Res Commun. 2004 Sep 3;321(4):954-60 Association between genes of Disrupted in schizophrenia 1 (DISC1) interactors and schizophrenia supports the role Al Sarraj J, Vinson C, Thiel G. Regulation of asparagine of the DISC1 pathway in the etiology of major mental synthetase gene transcription by the basic region leucine illnesses. Biol Psychiatry. 2009 Jun 15;65(12):1055-62 zipper transcription factors ATF5 and CHOP. Biol Chem. 2005 Sep;386(9):873-9 Wei Y, Ge Y, Zhou F, Chen H, Cui C, Liu D, Yang Z, et al. Identification and characterization of the promoter of Angelastro JM, Mason JL, Ignatova TN, Kukekov VG, et al. human ATF5 gene. J Biochem. 2010 Aug;148(2):171-8 Downregulation of activating transcription factor 5 is required for differentiation of neural progenitor cells into This article should be referenced as such: astrocytes. J Neurosci. 2005 Apr 13;25(15):3889-99 Ching AKK, Wong N. ATF5 (activating transcription factor Dong S, Nutt CL, Betensky RA, Stemmer-Rachamimov 5). Atlas Genet Cytogenet Oncol Haematol. 2011; AO, Denko NC, Ligon KL, Rowitch DH, Louis DN. 15(3):252-254. Histology-based expression profiling yields novel

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Gene Section Review

BRE (brain and reproductive organ-expressed (TNFRSF1A modulator)) Yiu-Loon Chui, Kenneth Ka-Ho Lee, John Yeuk-Hon Chan Department of Chemical Pathology, The Chinese University of Hong Kong, Hong Kong (YLC), School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong (KKHL), Key Lab of Regenerative Medicine, Ministry of Education, Jinan University, Guang Zhou, Guang Dong, China (JYHC)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/BREID839ch2p23.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI BREID839ch2p23.txt

undetermined. In mice, alternative splicing occurs Identity only at the 5' region of the gene. The major Other names: BRCC4; BRCC45 transcript is variant 5 (NCBI nomenclature), which HGNC (Hugo): BRE encodes the ubiquitous 383-amino acid protein that is 99% identical to human BRE. The minor Location: 2p23.2 transcript variants, unlike the human counterparts, Local order: According to GeneLoc and NCBI are expressed differentially among tissues. Their Map Viewer, genes flanking BRE are RBKS functions are undetermined (Ching et al., 2003). 2p23.3 (ribokinase) in the minus strand orientation, Transcription and RPL23AP34 2p23.2 (ribosomal protein L23a pseudogene 34) in the positive strand orientation. Exon 1 is non-coding; its flanking sequences are embedded in a CpG island of 1216 bases long. DNA/RNA Transcription start varies over the region between 35 to 112 bases upstream of the last base of exon 1, Description with the most common site at 40 bases upstream. The gene spans 448284 bases, telomere to No TATA or CAAT box is located within 150 centromere orientation. The first exon is non- bases upstream of any of the transcription start coding. In humans, six transcript variants are sites. BRE mRNA is expressed ubiquitously, and produced by alternative splicing predominantly at was initially found to be highly expressed in brain, either end of the gene. All human cells examined and reproductive organs; hence the name "BRE" (Li co-express all of the splice variants, but at different et al., 1995). Subsequent screens using human ratios to one another. The major transcript is αa, multiple-tissue RNA dot blot and Northern blot also known as variant 3 by NCBI nomenclature. revealed highest transcript expression in adrenal This transcript encodes the ubiquitous 383-amino- and heart (Miao et al., 2001). acid protein, designated by NCBI as protein Pseudogene isoform 2 (NP_954661.1) (Ching et al., 2001). No pseudogene found. Functions of all minor transcript variants are

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BRE (brain and reproductive organ-expressed (TNFRSF1A Chui YL, et al. modulator))

Generation of the major transcript variant of human BRE. Human BRE gene (not drawn in scale) is consisted of 15 exons, three of which are alternatively spliced. The light green boxes, X - Z, are alternative exons which are not present in the major transcript. The asterisked ATG is the translation start. This transcript encodes the major 383-amino-acid BRE protein isoform 2, that has been studied.

Protein Function DNA-repair and anti-apoptosis via regulation of Note ubiquitination. BRE was shown able to bind K48- BRE is a 383-amino-acid protein of no identifiable and K63-linked polyubiquitin chains (Wang et al., functional domain by sequence homology. No 2009). BRE and its mouse ortholog are expressed in crystal structure of BRE is available. This protein cytosolic and nuclear compartments (Li et al., has no paralog. The N-terminal region of 333 2004). In the nucleus, BRE is part of the BRCA1-A residues of human BRE, which is conserved among complex involved in DNA repair and maintaining vertebrate orthologs, has been classified as a single G2/M arrest in response to DNA damage. BRCA1- unique domain, pfam06113. It has been recently A complex consists of BRCA1, BARD1, proprosed that BRE contains 2 ubiquitin E2 variant Abraxas/Abra1/CCDC98, RAP80, BRCC36, BRE, (UEV) domains (Wang et al., 2009). and MERIT40/NBA1 (Dong et al., 2003; Sobhian Description et al., 2007; Feng et al., 2009; Shao et al., 2009; BRE is an evolutionarily highly conserved protein Wang et al., 2009). BRE interacts strongly with with no homolog within the same species. The MERIT40 and is responsible for binding the latter major protein isoform is 383 amino-acids long. to the complex of Abraxas, RAP80 and BRCC36 Based on bioinformatic analysis, BRE was (Feng et al., 2009). BRE may also regulate the K63 proposed to have two ubiquitin-binding UEV deubiquitinase activity of BRCC36 (Sobhian et al., (Ubiquitin E2 variant) domains. One was located in 2007). In conjunction with BRCC36, BRE was the N-terminal region between residues 30 and 147. shown to potentiate the E3 activity of BRCA1- The other one, however, could only be located in BARD1 complex (Dong et al., 2003). Furthermore, the isoform encoded by a rare transcript variant 1, depletion of BRE by siRNA sensitized cells to as the C-terminal one quarter of the putative death induced by ionizing irradiation (Dong et al., domain is encoded by the alternative exon Y (Wang 2003; Feng et al., 2009). This protein also forms et al., 2009). Thus, it is not clear whether the multiprotein BRISC (Brcc36 isopeptidase complex) remaining putative UEV domain sequence from in the cytoplasm. BRISC, containing at least 3 residues 275 to 363 of the major BRE isoform is proteins, FAM175B/ABRO1, BRCC36 and functional. MERIT40/NBA1, in addition to BRE, specifically cleaves K63-linked polyubiquitin chains (Cooper et Expression al., 2009). It is not known whether such cytosolic BRE is ubiquitously expressed. All mammalian cell complex is responsible for attenuating apoptotic lines examined express high levels of BRE. These response emanating from the activated death cell lines include Jurkat, KRC/Y, HeLa, HepG2, receptors, TNF-R1 and Fas. BRE also binds to the HL60, MCF7, NIH3T3, NS0, THP-1, and cytoplasmic region of TNF-R1 and Fas, as well as lymphoblastoid CB14022 cells. Among mouse the death-inducing signaling complex (DISC) tissues, the expression levels of BRE detected by during apoptotic induction (Gu et al., 1998; Li et Western blot analysis showed the following pattern: al., 2004). The anti-apoptotic role of BRE has been lungs = spleen = thymus > adrenal > testis = kidney shown by the increased apoptotic response to TNF- > brain > heart = liver. Human hepatocytes express alpha of HeLa cell line depleted of BRE by siRNA, little BRE as detected by immumnohistochemistry and the attenuated response of HeLa and Jurkat to and Western blot analysis (Chan et al., 2008). TNF-alpha and anti-Fas agonist antibody by over- Localisation expression of the protein. As over-expression of BRE also reduced intrinsic apoptotic response BRE is located in cytoplasm and nucleus. induced by stress-related and genotoxic stimuli, it

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BRE (brain and reproductive organ-expressed (TNFRSF1A Chui YL, et al. modulator)) has been proposed that the death receptor- The other one is from position 28338948 to associating BRE inhibits the recruitment of 28341210, located between coding exons 10 and mitochondrial apoptotic machinery, which is 11. Copy number polymorphisms (CNP) involving necessary for amplifying the death-receptor- a large contiguous region of 163295 bases initiated apoptosis of CD95 type II cell types, encompassing the first 3 coding exons and the which include HeLa, Jurkat, and hepatocytes upstream sequence of the neighbouring ribokinase (Scaffidi et al., 1998; Engels et al., 2000). Ectopic gene and smaller downstream regions have also expression of BRE in mouse Lewis lung carcinoma been identified (see diagram above) (The cells was shown to promote tumor growth in International HapMap Consortium, 2003). footpad injection model, but have no effect on cell Somatic proliferation in culture condition (Chan et al., 2005). Over-expression of BRE was found in 74% One R9L mutation was identified in a lung of 123 samples of human hepatocellular carcinoma, carcinoma cell line, NIH-H2126, and a synonymous and the protein expression level correlated with mutation S182S in a clear cell renal cell carcinoma poor prognosis. Immortalized human cell lines also sample, PD2198a. uniformly express high levels of BRE regardless of the tissue origin of these cell lines. Transgenic Implicated in expression of BRE in mouse liver attenuated acute fulminant hepatitis induced by anti-Fas antibody, Hepatocellular carcinoma (HCC) and promoted diethylnitrosamine-induced, but not Note spontaneous, liver tumors (Chan et al., 2008; Chui Immunohistochemical analysis, supplemented by et al., 2010). Thus, it is likely that BRE over- immunoblotting, has revealed overexpression of expression enhances tumor survival through its BRE in the tumoral regions of 72% of the 123 anti-apoptotic activity, rather than initiates tumor human HCC samples examined. Non-tumoral liver formation. regions, cirrhotic or otherwise, expressed little BRE Homology (Chan et al., 2008). Prognosis No homologous protein of BRE found within the The over-expression levels of BRE correlated with same species. poor differentiation of HCC cells and therefore poor Mutations prognosis. Cytogenetics Note Not determined. According to HapMap genotyped SNP data, there is Hybrid/Mutated gene no SNP polymorphism in any of the coding exons Not determined. of BRE. Abnormal protein Germinal No fusion protein reported. According to the current HapMap_rel27 for all the Oncogenesis 4 populations (CEU, CHB, JPT and YRI), the The transgenic mouse model with liver-specific number of nucleotide positions in BRE gene with over-expression of human BRE showed no HapMap genotyped SNP is 453. Given the size of enhanced spontaneous tumor development, BRE gene of 448284 bases long, the number of indicating that BRE over-expression alone is not bases with SNP fits well to the average genome- tumorigenic. These mice, however, showed wide figure of one SNP per 1000 bases (Dutt and significant attenuation of liver apoptosis induced by Beroukhim, 2007). It is, however, noteworthy that injection anti-Fas agonist antibody. These findings no SNP has been found in any of the coding exons. indicate that the over-expression of BRE in HCC is All of the SNPs, except one located in the 5' UTR, related to the anti-apoptotic activity of the protein are present in the introns. Two recombination which promotes growth of the carcinoma (Chan et hotspots are located in the introns, one of which is al., 2008). from position 28271535 to 28276573, located between coding exons 7 and 8.

Copy number polymorphism (CNP) of BRE gene. Regions with CNP are shown in colored boxes. Blue and red indicate copy loss and gain, respectively. Green indicates loss and gain at different segments of the contiguous region. The largest CNP region on the far left spans the first non-coding and the next 3 coding exons (exons 1, 2, 3 and 4) and extends further upstream into the neighboring ribokinase gene. The copy gain variant at the far right spans the alternative exon Z. Data obtained from HapMap.

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BRE (brain and reproductive organ-expressed (TNFRSF1A Chui YL, et al. modulator))

Recent work on inducing liver carcinoma to the signalosome-like subunit and its role in DNA repair. Mol above transgenic mice by neonatal injection of Cell. 2003 Nov;12(5):1087-99 diethylnitrosamine (DEN) confirmed that BRE Li Q, Ching AK, Chan BC, Chow SK, Lim PL, Ho TC, Ip over-expression in the liver could only promote WK, Wong CK, Lam CW, Lee KK, Chan JY, Chui YL. A death receptor-associated anti-apoptotic protein, BRE, growth of the already initiated tumor, rather than on inhibits mitochondrial apoptotic pathway. J Biol Chem. initiating tumor formation. Interestingly, the DEN- 2004 Dec 10;279(50):52106-16 induced liver tumors of the non-transgenic controls Chan BC, Li Q, Chow SK, Ching AK, Liew CT, Lim PL, Lee also showed up-regulation of endogenous BRE, KK, Chan JY, Chui YL. BRE enhances in vivo growth of suggesting that the BRE is important in liver tumor cells. Biochem Biophys Res Commun. 2005 Jan carcinogenesis through its anti-apoptotic activity 14;326(2):268-73 (Chui et al., 2010). Dutt A, Beroukhim R. Single nucleotide polymorphism array analysis of cancer. Curr Opin Oncol. 2007 References Jan;19(1):43-9 Sobhian B, Shao G, Lilli DR, Culhane AC, Moreau LA, Xia Li L, Yoo H, Becker FF, Ali-Osman F, Chan JY. B, Livingston DM, Greenberg RA. RAP80 targets BRCA1 Identification of a brain- and reproductive-organs-specific to specific ubiquitin structures at DNA damage sites. gene responsive to DNA damage and retinoic acid. Science. 2007 May 25;316(5828):1198-202 Biochem Biophys Res Commun. 1995 Jan 17;206(2):764- 74 Chan BC, Ching AK, To KF, Leung JC, Chen S, Li Q, Lai PB, Tang NL, Shaw PC, Chan JY, James AE, Lai KN, Lim Gu C, Castellino A, Chan JY, Chao MV. BRE: a modulator PL, Lee KK, Chui YL. BRE is an antiapoptotic protein in of TNF-alpha action. FASEB J. 1998 Sep;12(12):1101-8 vivo and overexpressed in human hepatocellular Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, carcinoma. Oncogene. 2008 Feb 21;27(9):1208-17 Tomaselli KJ, Debatin KM, Krammer PH, Peter ME. Two Cooper EM, Cutcliffe C, Kristiansen TZ, Pandey A, Pickart CD95 (APO-1/Fas) signaling pathways. EMBO J. 1998 CM, Cohen RE. K63-specific deubiquitination by two Mar 16;17(6):1675-87 JAMM/MPN+ complexes: BRISC-associated Brcc36 and Engels IH, Stepczynska A, Stroh C, Lauber K, Berg C, proteasomal Poh1. EMBO J. 2009 Mar 18;28(6):621-31 Schwenzer R, Wajant H, Jänicke RU, Porter AG, Belka C, Feng L, Huang J, Chen J. MERIT40 facilitates BRCA1 Gregor M, Schulze-Osthoff K, Wesselborg S. Caspase- localization and DNA damage repair. Genes Dev. 2009 8/FLICE functions as an executioner caspase in anticancer Mar 15;23(6):719-28 drug-induced apoptosis. Oncogene. 2000 Sep 21;19(40):4563-73 Shao G, Patterson-Fortin J, Messick TE, Feng D, Shanbhag N, Wang Y, Greenberg RA. MERIT40 controls Ching AK, Li PS, Li Q, Chan BC, Chan JY, Lim PL, Pang BRCA1-Rap80 complex integrity and recruitment to DNA JC, Chui YL. Expression of human BRE in multiple double-strand breaks. Genes Dev. 2009 Mar 15;23(6):740- isoforms. Biochem Biophys Res Commun. 2001 Nov 54 2;288(3):535-45 Wang B, Hurov K, Hofmann K, Elledge SJ. NBA1, a new Miao J, Panesar NS, Chan KT, Lai FM, Xia N, Wang Y, player in the Brca1 A complex, is required for DNA Johnson PJ, Chan JY. Differential expression of a stress- damage resistance and checkpoint control. Genes Dev. modulating gene, BRE, in the adrenal gland, in adrenal 2009 Mar 15;23(6):729-39 neoplasia, and in abnormal adrenal tissues. J Histochem Cytochem. 2001 Apr;49(4):491-500 Chui YL, Ching AK, Chen S, Yip FP, Rowlands DK, James AE, Lee KK, Chan JY. BRE over-expression promotes . The International HapMap Project. Nature. 2003 Dec growth of hepatocellular carcinoma. Biochem Biophys Res 18;426(6968):789-96 Commun. 2010 Jan 15;391(3):1522-5 Ching AK, Li Q, Lim PL, Chan JY, Chui YL. Expression of a conserved mouse stress-modulating gene, Bre: This article should be referenced as such: comparison with the human ortholog. DNA Cell Biol. 2003 Aug;22(8):497-504 Chui YL, Lee KKH, Chan JYH. BRE (brain and reproductive organ-expressed (TNFRSF1A modulator)). Dong Y, Hakimi MA, Chen X, Kumaraswamy E, Cooch NS, Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):255- Godwin AK, Shiekhattar R. Regulation of BRCC, a 258. holoenzyme complex containing BRCA1 and BRCA2, by a

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in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Mini Review

DDX1 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 1) Takahiko Hara, Kiyoko Tanaka Stem cell project group, The Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan (TH, KT)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/DDX1ID40283ch2p24.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI DDX1ID40283ch2p24.txt

implicated in a number of cellular processes Identity involving alteration of RNA secondary structure Other names: DBP-RB; UKVH5d such as translational initiation, nuclear and HGNC (Hugo): DDX1 mitochondrial RNA splicing, and ribosome/spliceosome assembly (Rocak and Location: 2p24.3 Linder, 2004). DNA/RNA

Genomic organization of human DDX1 gene. Boxes and connecting lines indicate exons (omitted in the middle part) and introns, respectively. The ATG transcription initiation Diagram of conserved motifs among DEAD box RNA site is located in exon 2. helicase family proteins. Description Expression DDX1 gene is located in an approximately 40 kb DDX1 mRNA is widely expressed in many tissues, chromosomal DNA region of 2p24.3 containing at but its expression level is highest in testis (Tanaka least 27 exons. There are alternatively used exons et al., 2009). DDX1 level tends to be higher in for exon 2, 16, 19, 21, 23 and 27. tumor-derived cells than in normal tissues. Transcription Localisation Size of the major mRNA is 2.7 kb. DDX1 protein is localized both in the cytoplasm and nucleus of DDX1 gene-amplified Protein neuroblastoma and retinoblastoma cell lines, but mainly located in the nucleus of normal fibroblasts Description (Godbout et al., 1998). DDX1 protein is a putative RNA helicase Function containing the characteristic Asp-Glu-Ala-Asp DDX1 is believed to regulate translational (DEAD) conserved sequence motif (Linder et al., initiation, nuclear hnRNA splicing, 1989). It is composed of 740 amino acid residues ribosome/spliceosome assembly, and mRNA (82432 Da). Proteins of this family (more than 30 synthesis as a putative ATP-dependent RNA from bacteria to humans) have been described to be helicase. DDX1 is associated with a pre-mRNA 3'-

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end cleavage protein CstF-64 (Bleoo et al., 2001) the DDX1 gene amplification for the clinical and heterogeneous nuclear ribonucleoprotein K outcome is not clear. (hnRNP K) (Chen et al., 2002). hnRNP-K is Testicular tumors involved in cell migration and cytoplasmic accumulation of hnRNP-K is crucial for metastasis Note (Inoue et al., 2007). It was reported that DDX1 In testicular tumors including seminoma and interacts with nuclear diffusion inhibitory signal nonseminoma, significantly higher levels of DDX1 (NIS) motif of HIV-1 Rev protein in human mRNA are expressed. In a nonseminoma-derived astrocytes. Accumulation of DDX1 in astrocytes cell line NEC8, siRNA-mediated knockdown of changed sub-cellular distribution of Rev from DDX1 abrogated their anchorage-independent nuclear to cytoplasmic (Fang et al., 2005). More growth in a semisolid medium and in vivo tumor recently, it was reported that DDX1 is recruited to formation in nude mice. the sites of DNA double strand breaks in cells Breast cancer exposed to ionizing radiation and removes single Note stranded RNAs to facilitate the repair reaction of transcriptionally active regions of the genome (Li et Expression of DDX1 mRNA and cytoplasmic al., 2008). DDX1 levels are significantly elevated in relapsed In retinoblastoma and neuroblastoma cell lines, co- breast cancer samples. Thus, DDX1 can be a amplification of DDX1 and the proto-oncogene prognostic biomarker for early recurrence in MYCN has been demonstrated (Godbout and primary breast cancer. Squire, 1993; Godbout et al., 1998). Similar gene amplification of the genomic region containing References DDX1 and MYCN was observed in alveolar Linder P, Lasko PF, Ashburner M, Leroy P, Nielsen PJ, rhabdomyosarcoma samples (Barr et al., 2009) and Nishi K, Schnier J, Slonimski PP. Birth of the D-E-A-D box. Wilms tumor-derived cells (Noguera et al., 2010). Nature. 1989 Jan 12;337(6203):121-2 Elevated expression of DDX1 mRNA was reported Godbout R, Squire J. Amplification of a DEAD box protein to be a prognostic marker for early recurrence in gene in retinoblastoma cell lines. Proc Natl Acad Sci U S primary breast cancer (Germain et al., 2010). A. 1993 Aug 15;90(16):7578-82 Recently, it was demonstrated that DDX1 is Godbout R, Packer M, Bie W. Overexpression of a DEAD essential for the solid tumor formation of a human box protein (DDX1) in neuroblastoma and retinoblastoma testicular tumor cell line NEC8 in nude mice cell lines. J Biol Chem. 1998 Aug 14;273(33):21161-8 (Tanaka et al., 2009). In this case, DDX1 directly Bléoo S, Sun X, Hendzel MJ, Rowe JM, Packer M, bound to the -348 and -329 promoter region of the Godbout R. Association of human DEAD box protein DDX1 with a cleavage stimulation factor involved in 3'-end cyclin-D2 gene and enhanced its transcription. processing of pre-MRNA. Mol Biol Cell. 2001 Furthermore, siRNA-mediated knockdown of Oct;12(10):3046-59 DDX1 resulted in coordinated down-regulation of Chen HC, Lin WC, Tsay YG, Lee SC, Chang CJ. An RNA stem cell-associated genes located in chromosomal helicase, DDX1, interacting with poly(A) RNA and region 12p13. Therefore, DDX1 may function as an heterogeneous nuclear ribonucleoprotein K. J Biol Chem. essential transcriptional activator for the 2002 Oct 25;277(43):40403-9 tumorigenic capacity of testicular germ line tumor- Rocak S, Linder P. DEAD-box proteins: the driving forces derived cells. In agreement, DDX1 promotes the behind RNA metabolism. Nat Rev Mol Cell Biol. 2004 proliferation of JC virus via the transcriptional Mar;5(3):232-41 activation of its viral promoter (Sunden et al., Fang J, Acheampong E, Dave R, Wang F, Mukhtar M, 2007). It was also reported that DDX1 acts as a co- Pomerantz RJ. The RNA helicase DDX1 is involved in activator to enhance NF-kappaB-mediated restricted HIV-1 Rev function in human astrocytes. Virology. 2005 Jun 5;336(2):299-307 transcription (Ishaq et al., 2009). Inoue A, Sawata SY, Taira K, Wadhwa R. Loss-of-function screening by randomized intracellular antibodies: Implicated in identification of hnRNP-K as a potential target for metastasis. Proc Natl Acad Sci U S A. 2007 May Retinoblastoma and neuroblastoma 22;104(21):8983-8 Note Sunden Y, Semba S, Suzuki T, Okada Y, Orba Y, Coamplication of DDX1 and MYCN genes Nagashima K, Umemura T, Sawa H. DDX1 promotes frequently occurs in both retinoblastoma and proliferation of the JC virus through transactivation of its promoter. Microbiol Immunol. 2007;51(3):339-47 neuroblastoma cell lines. This is because DDX1 gene is located to chromosome 2p24, 400-kb Li L, Monckton EA, Godbout R. A role for DEAD box 1 at telomeric to MYCN gene. This type of gene DNA double-strand breaks. Mol Cell Biol. 2008 Oct;28(20):6413-25 amplification has also been reported in alveolar rhabdomyosarcoma samples and Wilms tumor- Barr FG, Duan F, Smith LM, Gustafson D, Pitts M, Hammond S, Gastier-Foster JM. Genomic and clinical derived cells. However, prognostic significance of analyses of 2p24 and 12q13-q14 amplification in alveolar

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rhabdomyosarcoma: a report from the Children's Oncology prognostic marker for early recurrence in breast cancer. Group. Genes Chromosomes Cancer. 2009 Breast Cancer Res Treat. 2010 May 25; Aug;48(8):661-72 Noguera R, Villamón E, Berbegall A, Machado I, Giner F, Ishaq M, Ma L, Wu X, Mu Y, Pan J, Hu J, Hu T, Fu Q, Guo Tadeo I, Navarro S, Llombart-Bosch A. Gain of MYCN D. The DEAD-box RNA helicase DDX1 interacts with RelA region in a Wilms tumor-derived xenotransplanted cell line. and enhances nuclear factor kappaB-mediated Diagn Mol Pathol. 2010 Mar;19(1):33-9 transcription. J Cell Biochem. 2009 Feb 1;106(2):296-305 This article should be referenced as such: Tanaka K, Okamoto S, Ishikawa Y, Tamura H, Hara T. DDX1 is required for testicular tumorigenesis, partially Hara T, Tanaka K. DDX1 (DEAD (Asp-Glu-Ala-Asp) box through the transcriptional activation of 12p stem cell polypeptide 1). Atlas Genet Cytogenet Oncol Haematol. genes. Oncogene. 2009 May 28;28(21):2142-51 2011; 15(3):259-261. Germain DR, Graham K, Glubrecht DD, Hugh JC, Mackey JR, Godbout R. DEAD box 1: a novel and independent

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Gene Section Review

DIO2 (deiodinase, iodothyronine, type II) Ana Luiza Maia, Simone Magagnin Wajner, Leonardo B Leiria Thyroid Section, Endocrine Division, Hospital de Clinicas de Porto Alegre (HCPA), Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil (ALM, SMW, LBL)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/DIO2ID44390ch14q31.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI DIO2ID44390ch14q31.txt

Identity Protein Other names: 5DII; D2; SelY; TXDI2 Description HGNC (Hugo): DIO2 The protein encoded by this gene belongs to the Location: 14q31.1 iodothyronine deiodinase family. This enzyme activates thyroid hormone by converting the DNA/RNA prohormone thyroxine (T4) by outer ring deiodination to bioactive 3,3',5-triiodothyronine Description (T3). It is highly expressed in the thyroid, and may The Dio2 gene is composed of 3 exons comprising contribute significantly to the relative increase in 14656 bp of the genomic DNA. thyroidal T3 production in patients with Graves' disease and thyroid adenomas. This protein Transcription contains selenocysteine (Sec) residues encoded by The length of transcribed mRNA is about 6,8 kb the UGA codon, which often signals the end of and generates three variants of mRNA. Transcript process of translation. The 3'UTR of Sec-containing variant 1 represents the longest transcript and genes have a common stem-loop structure, the sec encodes isoform a. Transcript variant 2 differs in insertion sequence (SECIS), which is necessary for the 5'UTR when compared to variant 1. Both the recognition of UGA as a Sec codon rather than variants 1 and 2 encode isoform a. Transcript a stop signal. Alternative splicing results in multiple variant 3 includes an alternate in-frame exon in the transcript variants encoding different isoforms. coding region, compared to variant 1. Variant 3 Ubiquitination can also regulate proteins by encodes isoform b, which is longer than isoform a. transiently inactivating enzymatic function through Pseudogene conformational change in a dimeric enzyme, which can be reversed upon deubiquitination (post- No pseudogene have been described. translational).

Organization of the Dio2 gene: Yellow bars represent the coding region (exon) and red bars, the untranslated region.

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DIO2 (deiodinase, iodothyronine, type II) Maia AL, et al.

Schematic representation of D2 peptide structure (not on scale). Isoform a (273 aa) and Isoform b (309 aa). In deep green transmembran domain (position 10-34). In yellow active site (position 133). In deep blue alternative sequence isoform b (position 74).

Expression Mutations Ohba et al. (2001) identified 2 alternatively spliced DIO2 transcripts that include intronic sequences Note between the 2 invariant DIO2 exons. These splice No germinal or somatic mutations has been variants showed tissue-specific expression in brain, described. However, the polymorphism Thr92Ala thyroid, liver, thymus, anterior pituitary gland and in Dio2 gene is associated with increased risk of brown adipose tissue. In mesothelioma cell lysates, mental retardation, insulin resistance in type 2 Curcio et al. (2001) determined that endogenous diabetic patients, reduced glucose availability in DIO2 gene had an apparent molecular mass of 31 obese women, symptomatic osteoarthritis, Graves' kD. In normal tissues, D2 activity/mRNA ratio is disease and arterial hypertension. variable, but the enzyme is expressed in rodents in the developing and adult testis, heart, muscle, Implicated in thyroid, BAT, brain, pituitary, thymus, skin, spinal cord, placenta, liver and pancreas. In humans D2 is Various cancers expressed in brain, BAT, heart, thyroid, muscle, Note placenta, skin and vascular smooth muscle cells. Although not completely understood, Dio2 gene Localisation expression and activity is altered in some tumors. It is under-expressed in papillary thyroid carcinomas Immuno location of the protein in cells showed D2 (PTC). In follicular tumors, D2 activity is similar or as an endoplasmic reticulum resident protein. elevated when compared to non tumoral tissues, Function and augmented in follicular adenomas. D2 is also highly expressed in medullar thyroid carcinoma. A Type 2 deiodinase converts intracellular pro- higher expression of the Dio2 gene was also hormone-3,3',5,5'-tetraiodothyronine (T4) into the described in gliosarcoma, oligoastrocytoma, active thyroid hormone 3,3',5-triiodothyronine (T3) glioblastoma, oligodendroglioma and pituitary thereby regulating intracellular levels of active T3 tumors. In contrast, meningioma does not express in target tissues. D2 activity. These differences might be related to Thermogenesis the embrionary tumor origin. Mesothelioma The expression of D2 is increased in response to expresses higher activity of D2, whereas cold stimulation in brown adipocytes isolated from osteosarcoma has diminished D2 activity. mice. Dio2 activation in the brown adipose tissue (BAT) of human newborns and rodents is known to Insulin resistance play a role in adaptive energy expenditure during Note cold exposure. Dio2 polymorphism Thr92Ala interacts with a Development polymorphism in PPAR gamma 2 gene and is D2 activity is present in human placenta through all associated with insulin resistance in diabetic pregnancy, and is highly expressed during the first patients. This Dio2 polymorphism is associated trimester. The level of activity is low in the non- with a ~20% lower rate of glucose disposal in obese pregnant uterus, but in pregnancy the level rises women than in non-obese women. Although the progressively to a maximum at gestation day 17 association between those two genes occurs in when it is increased threefold. patients with insulin resistance, these results are Homology contradictory in non diabetic population. Several homologues of Dio2 have been identified in Hypothyroidism Pan troglodytes and Macaca mulatta (100%). The Note chicken and mouse have similar domain structures Disruption in mouse Dio2 gene is associated with with human Dio2 (97%). Human Dio2 homology alterations in T4/T3 balance with elevated TSH with D3 is expressed in Sus scrofia, Equus caballus, levels, which demonstrates that the Dio2 gene is of Cricetus cricetus, Oryctolagus cuniculus, Pituophis critical importance in the feedback regulation of deppei (92% similarity) and limited domains with TSH secretion. human D3 and D1.

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Graves' disease Celi FS, Canettieri G, Yarnall DP, Burns DK, Andreoli M, Shuldiner AR, Centanni M. Genomic characterization of Note the coding region of the human type II 5'-deiodinase gene. It is suggested that the Thr92Ala variant of the Mol Cell Endocrinol. 1998 Jun 25;141(1-2):49-52 Dio2 gene is associated or might be in linkage Araki O, Murakami M, Morimura T, Kamiya Y, Hosoi Y, disequilibrium with a functional DIO2 Kato Y, Mori M. Assignment of type II iodothyronine polymorphism which involves the development of deiodinase gene (DIO2) to human chromosome band 14q24.2-->q24.3 by in situ hybridization. Cytogenet Cell Graves' disease in a Russian population. Genet. 1999;84(1-2):73-4 Mental retardation Bartha T, Kim SW, Salvatore D, Gereben B, Tu HM, Note Harney JW, Rudas P, Larsen PR. Characterization of the 5'-flanking and 5'-untranslated regions of the cyclic A case control study in Chinese patients adenosine 3',5'-monophosphate-responsive human type 2 demonstrated that two allelic intronic SNPs iodothyronine deiodinase gene. Endocrinology. 2000 (rs225010 (T/C) and rs225012 (A/G)) in the DIO2 Jan;141(1):229-37 gene could affect the amount of T3 available and in Campos-Barros A, Amma LL, Faris JS, Shailam R, Kelley an iodine-deficient environment and partially MW, Forrest D. Type 2 iodothyronine deiodinase determine on augmented risk of mental retardation. expression in the cochlea before the onset of hearing. Proc Natl Acad Sci U S A. 2000 Feb 1;97(3):1287-92 They found a positive association with mental retardation and the two intronic Dio2 Murakami M, Araki O, Morimura T, Hosoi Y, Mizuma H, Yamada M, Kurihara H, Ishiuchi S, Tamura M, Sasaki T, polymorphisms but not with Dio2 Thr92Ala alone Mori M. Expression of type II iodothyronine deiodinase in and concluded that the genetic variation in Dio2 brain tumors. J Clin Endocrinol Metab. 2000 determine the risk of development of mental Nov;85(11):4403-6 retardation that could be due to alterations in the Curcio C, Baqui MM, Salvatore D, Rihn BH, Mohr S, local amount of T3 available in the brain. Harney JW, Larsen PR, Bianco AC. The human type 2 iodothyronine deiodinase is a selenoprotein highly Bone metabolism expressed in a mesothelioma cell line. J Biol Chem. 2001 Note Aug 10;276(32):30183-7 Dio2 is expressed in human and mouse osteoblast Mizuma H, Murakami M, Mori M. Thyroid hormone cells. In patients with differentiated thyroid activation in human vascular smooth muscle cells: carcinoma, the Dio2 Thr92Ala polymorphism is expression of type II iodothyronine deiodinase. Circ Res. 2001 Feb 16;88(3):313-8 associated with a decreased femoral neck bone mineral density and higher bone turnover Murakami M, Araki O, Hosoi Y, Kamiya Y, Morimura T, Ogiwara T, Mizuma H, Mori M. Expression and regulation independent of serum thyroid hormone levels. of type II iodothyronine deiodinase in human thyroid gland. Cardiomyopathy and arterial Endocrinology. 2001 Jul;142(7):2961-7 hypertension Murakami M, Kamiya Y, Morimura T, Araki O, Imamura M, Ogiwara T, Mizuma H, Mori M. Thyrotropin receptors in Note brown adipose tissue: thyrotropin stimulates type II Dio2 gene expression is also markedly up-regulated iodothyronine deiodinase and uncoupling protein-1 in in hearts of mice that develops hypothyroidism or brown adipocytes. Endocrinology. 2001 Mar;142(3):1195- 201 eccentric hypertrophy after myocardial infarction. The Dio2 polymorphism Thr92Ala is also Ohba K, Yoshioka T, Muraki T. Identification of two novel splicing variants of human type II iodothyronine deiodinase associated with increased risk for the development mRNA. Mol Cell Endocrinol. 2001 Feb 14;172(1-2):169-75 of hypertension. Schneider MJ, Fiering SN, Pallud SE, Parlow AF, St Germain DL, Galton VA. Targeted disruption of the type 2 References selenodeiodinase gene (DIO2) results in a phenotype of pituitary resistance to T4. Mol Endocrinol. 2001 Campos-Barros A, Hoell T, Musa A, Sampaolo S, Dec;15(12):2137-48 Stoltenburg G, Pinna G, Eravci M, Meinhold H, Baumgartner A. Phenolic and tyrosyl ring iodothyronine Zaninovich AA. [Thyroid hormones, obesity and brown deiodination and thyroid hormone concentrations in the adipose tissue thermogenesis]. Medicina (B Aires). human central nervous system. J Clin Endocrinol Metab. 2001;61(5 Pt 1):597-602 1996 Jun;81(6):2179-85 Mentuccia D, Proietti-Pannunzi L, Tanner K, Bacci V, Croteau W, Davey JC, Galton VA, St Germain DL. Cloning Pollin TI, Poehlman ET, Shuldiner AR, Celi FS. of the mammalian type II iodothyronine deiodinase. A Association between a novel variant of the human type 2 selenoprotein differentially expressed and regulated in deiodinase gene Thr92Ala and insulin resistance: evidence human and rat brain and other tissues. J Clin Invest. 1996 of interaction with the Trp64Arg variant of the beta-3- Jul 15;98(2):405-17 adrenergic receptor. Diabetes. 2002 Mar;51(3):880-3 Salvatore D, Tu H, Harney JW, Larsen PR. Type 2 Curcio-Morelli C, Zavacki AM, Christofollete M, Gereben B, iodothyronine deiodinase is highly expressed in human de Freitas BC, Harney JW, Li Z, Wu G, Bianco AC. thyroid. J Clin Invest. 1996 Aug 15;98(4):962-8 Deubiquitination of type 2 iodothyronine deiodinase by von Hippel-Lindau protein-interacting deubiquitinating enzymes Buettner C, Harney JW, Larsen PR. The 3'-untranslated regulates thyroid hormone activation. J Clin Invest. 2003 region of human type 2 iodothyronine deiodinase mRNA Jul;112(2):189-96 contains a functional selenocysteine insertion sequence element. J Biol Chem. 1998 Dec 11;273(50):33374-8

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Wagner MS, Morimoto R, Dora JM, Benneman A, Pavan Maia AL, Dupuis J, Manning A, Liu C, Meigs JB, Cupples R, Maia AL. Hypothyroidism induces type 2 iodothyronine LA, Larsen PR, Fox CS. The type 2 deiodinase (DIO2) A/G deiodinase expression in mouse heart and testis. J Mol polymorphism is not associated with glycemic traits: the Endocrinol. 2003 Dec;31(3):541-50 Framingham Heart Study. Thyroid. 2007 Mar;17(3):199- 202 Chistiakov DA, Savost'anov KV, Turakulov RI. Screening of SNPs at 18 positional candidate genes, located within Sagar GD, Gereben B, Callebaut I, Mornon JP, Zeöld A, the GD-1 locus on chromosome 14q23-q32, for da Silva WS, Luongo C, Dentice M, Tente SM, Freitas BC, susceptibility to Graves' disease: a TDT study. Mol Genet Harney JW, Zavacki AM, Bianco AC. Ubiquitination- Metab. 2004 Nov;83(3):264-70 induced conformational change within the deiodinase dimer is a switch regulating enzyme activity. Mol Cell Biol. Guo TW, Zhang FC, Yang MS, Gao XC, Bian L, Duan SW, 2007 Jul;27(13):4774-83 Zheng ZJ, Gao JJ, Wang H, Li RL, Feng GY, St Clair D, He L. Positive association of the DIO2 (deiodinase type 2) Wajner SM, dos Santos Wagner M, Melo RC, Parreira GG, gene with mental retardation in the iodine-deficient areas Chiarini-Garcia H, Bianco AC, Fekete C, Sanchez E, of China. J Med Genet. 2004 Aug;41(8):585-90 Lechan RM, Maia AL. Type 2 iodothyronine deiodinase is highly expressed in germ cells of adult rat testis. J Arnaldi LA, Borra RC, Maciel RM, Cerutti JM. Gene Endocrinol. 2007 Jul;194(1):47-54 expression profiles reveal that DCN, DIO1, and DIO2 are underexpressed in benign and malignant thyroid tumors. Gereben B, Zavacki AM, Ribich S, Kim BW, Huang SA, Thyroid. 2005 Mar;15(3):210-21 Simonides WS, Zeöld A, Bianco AC. Cellular and molecular basis of deiodinase-regulated thyroid hormone Bianco AC, Maia AL, da Silva WS, Christoffolete MA. signaling. Endocr Rev. 2008 Dec;29(7):898-938 Adaptive activation of thyroid hormone and energy expenditure. Biosci Rep. 2005 Jun-Aug;25(3-4):191-208 Maia AL, Hwang SJ, Levy D, Larson MG, Larsen PR, Fox CS. Lack of association between the type 2 deiodinase Canani LH, Capp C, Dora JM, Meyer EL, Wagner MS, A/G polymorphism and hypertensive traits: the Harney JW, Larsen PR, Gross JL, Bianco AC, Maia AL. Framingham Heart Study. Hypertension. 2008 The type 2 deiodinase A/G (Thr92Ala) polymorphism is Apr;51(4):e22-3 associated with decreased enzyme velocity and increased insulin resistance in patients with type 2 diabetes mellitus. Meyer EL, Goemann IM, Dora JM, Wagner MS, Maia AL. J Clin Endocrinol Metab. 2005 Jun;90(6):3472-8 Type 2 iodothyronine deiodinase is highly expressed in medullary thyroid carcinoma. Mol Cell Endocrinol. 2008 Jul Galton VA. The roles of the iodothyronine deiodinases in 16;289(1-2):16-22 mammalian development. Thyroid. 2005 Aug;15(8):823-34 Watanabe M, Yamamoto T, Mori C, Okada N, Yamazaki Gouveia CH, Christoffolete MA, Zaitune CR, Dora JM, N, Kajimoto K, Kataoka M, Shinohara Y. Cold-induced Harney JW, Maia AL, Bianco AC. Type 2 iodothyronine changes in gene expression in brown adipose tissue: selenodeiodinase is expressed throughout the mouse implications for the activation of thermogenesis. Biol skeleton and in the MC3T3-E1 mouse osteoblastic cell line Pharm Bull. 2008 May;31(5):775-84 during differentiation. Endocrinology. 2005 Jan;146(1):195- 200 Williams AJ, Robson H, Kester MH, van Leeuwen JP, Shalet SM, Visser TJ, Williams GR. Iodothyronine Maia AL, Kim BW, Huang SA, Harney JW, Larsen PR. deiodinase enzyme activities in bone. Bone. 2008 Type 2 iodothyronine deiodinase is the major source of Jul;43(1):126-34 plasma T3 in euthyroid humans. J Clin Invest. 2005 Sep;115(9):2524-33 Panicker V, Saravanan P, Vaidya B, Evans J, Hattersley AT, Frayling TM, Dayan CM. Common variation in the Morimura T, Tsunekawa K, Kasahara T, Seki K, Ogiwara DIO2 gene predicts baseline psychological well-being and T, Mori M, Murakami M. Expression of type 2 response to combination thyroxine plus triiodothyronine iodothyronine deiodinase in human osteoblast is therapy in hypothyroid patients. J Clin Endocrinol Metab. stimulated by thyrotropin. Endocrinology. 2005 2009 May;94(5):1623-9 Apr;146(4):2077-84 Heemstra KA, Hoftijzer H, van der Deure WM, Peeters RP, Canani LH, Leie MA, Machado WE, Capp C, Maia AL. Hamdy NA, Pereira A, Corssmit EP, Romijn JA, Visser TJ, Type 2 deiodinase Thr92Ala polymorphism is not Smit JW. The type 2 deiodinase Thr92Ala polymorphism is associated with arterial hypertension in type 2 diabetes associated with increased bone turnover and decreased mellitus patients. Hypertension. 2007 Jun;49(6):e47; femoral neck bone mineral density. J Bone Miner Res. author reply e48 2010 Jun;25(6):1385-91 Fiorito M, Torrente I, De Cosmo S, Guida V, Colosimo A, Wang YY, Morimoto S, Du CK, Lu QW, Zhan DY, Prudente S, Flex E, Menghini R, Miccoli R, Penno G, Tsutsumi T, Ide T, Miwa Y, Takahashi-Yanaga F, Sasaguri Pellegrini F, Tassi V, Federici M, Trischitta V, Dallapiccola T. Up-regulation of type 2 iodothyronine deiodinase in B. Interaction of DIO2 T92A and PPARgamma2 P12A dilated cardiomyopathy. Cardiovasc Res. 2010 Sep polymorphisms in the modulation of metabolic syndrome. 1;87(4):636-46 Obesity (Silver Spring). 2007 Dec;15(12):2889-95 Gumieniak O, Perlstein TS, Williams JS, Hopkins PN, This article should be referenced as such: Brown NJ, Raby BA, Williams GH. Ala92 type 2 deiodinase Maia AL, Wajner SM, Leiria LB. DIO2 (deiodinase, allele increases risk for the development of hypertension. iodothyronine, type II). Atlas Genet Cytogenet Oncol Hypertension. 2007 Mar;49(3):461-6 Haematol. 2011; 15(3):262-265.

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Gene Section Review

GFI1B (growth factor independent 1B transcription repressor) Lothar Vassen, Tarik Möröy Research Unit Hematopoiesis and Cancer, Institut de recherches cliniques de Montreal, 110 avenue des Pins Ouest, Montreal (Quebec) H2W 1R7, Canada (TM, LV)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/GFI1BID40707ch9q34.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI GFI1BID40707ch9q34.txt

Identity Pseudogene HGNC (Hugo): GFI1B Unknown. Location: 9q34.13 Protein Local order: The human GFI1B gene is telomeric to TSC1 (tuberous sclerosis 1 protein) and Note centromeric to GTF3C5 (general transcription The longer GFI1B variant 1 is the protein refered to factor 3C polypeptide 5). in most cases. GFI1B variant 2 shows only a restricted expression in normal cells and could be DNA/RNA preferentially associated with leukemic diseases. Functional differences between both proteins are Description not described yet. The GFI1B gene structure is composed of at least 7 Description exons (ranging from 120 to 824 bp), six of which GFI1B (isoform 1) is a protein of 330 aa residues are coding. Spliced ESTs and human mRNAs may and has a predicted molecular mass of 37492.38 define up to eight additional 5'exons which possibly Da. Isoelectric point: 9.3076, charge: 25.0, average point to an alternative promoter close to the residue weight: 113613. promoter of TSC1. GFI1B is composed of a 20-amino-acid N-terminal Transcription SNAG (SNAIL-GFI) transcription repressor Two human mRNA transcripts arise from domain, and intermediary domain of largely alternative splicing. GFI1B mRNA variant one unknown function and six c-terminal C2H2 zinc- encodes the more frequent full length GFI1B (330 finger domains encompassing residues 163-327. aa), while variant two lacks the in frame exon 5 Zinc-fingers 3-5 are involved in sequence specific (according to the NCBI RefSeq for hGFI1B) DNA binding and recognize a taAATCaca/tgca/t leading to a shorter isoform (284 aa) lacking zinc- core motif. The bases flanking the AATC core finger two and parts of zinc-finger one and three. motif seem to be poorly conserved. Predictions of The residual parts of Znf one and three are joined to true GFI1B binding sites in the genome based only form a new zinc-finger (see mRNA and protein on this sequence have to be validated below). independently.

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GFI1B (growth factor independent 1B transcription repressor) Möröy T, Vassen L

Model for the generation of the two major GFI1B isoforms. GFI1B-V2 is translated from a shorter mRNA splice variant where exon 9 is skipped (aa 171-216 missing). There are many potential 5' transcriptional start sites, but the major start site seems to be in exon 5, corresponding to exon 1 (122 bp) in the RefSeq database. SD: SNAG domain necessary for repression of transcription by GFI1B. ID: Intermediary domain with very low homology to the GFI1B homolog GFI1 and unknown function, which is presumably important for specific protein-protein interactions unique to GFI1B. Znf 1-6: C2H2 zinc-finger domains which are highly conserved between all members of the GFI1 protein family. Znf 3-5 bind to the major groove of target DNA. Znf 4-5 of GFI1B (almost identical to GFI1) recognize a AATC DNA core sequence in the GFI1/GFI1B predicted binding site.

Expression produce functional erythrocytes and megakaryocytes and increases the apoptosis rate in GFI1B is mainly expressed in the fetal and adult leukemic cell lines. The GFI1B gene locus can be hematopoietic system, where it is detected in autoregulated by autorepression of its own hematopoietic stem cells, megakaryocyte/erythroid promoter in hematopoietic cells (Vassen et al., precursors (MEP), common myeloid precursors 2005; Anguita et al., 2010), most likely by (CMP), erythroblasts and early erythrocytes, interaction with GATA1 (GATA binding protein 1) megakaryocytes and megakaryocyte precursors, B- (Huang et al., 2005), an activator of GFI1B cell precursors and a small subset of T-cell transcription that is also essential for erythroid and precursors (Vassen et al., 2007). GFI1B is lso megakaryocytic development. GFI1B and its detected in fetal thymus and testes. GFI1B homolog GFI1 show cross-repression, resulting in expression varies throughout the maturation of an enhanced expression of the respective these cells, with the highest expression levels in counterpart, when one of these genes is deleted. MEPs, megakaryocytes and erythrocytes and seems The repressory activity of GFI1B is achieved by to be tightly regulated. The shorter isoform 2 of recruiting histone deacetylases (HDAC1 and GFI1B is lowly expressed in normal cells, but HDAC2), lysine specific demethylase 1 (LSD1 or upregulated in several types of leukemia (e.g. KDM1) and the REST corepressor (CoREST) to chronic myelogenous leukemia, acute myeloid target DNA sequences (Saleque et al., 2007). leukemia, erythroleukemia, megakaryocytic GFI1B alters histone methylation at target gene leukemia). The expression of GFI1B is described to promoters and is associated with sites of gamma- be positively regulated by GATA-1, NF-Y, E2- satellite containing heterochromatin (Vassen et al., alpha/TCF3, and HMGB2 and to be repressed by 2006). GFI1B interacts also with the histone Oct1, GFI1B and GFI1. GFI1B expression is down- methyltransferases G9a and SUV39H1 and a role in regulated by erythropoietin (EPO) in a signal- heterochromatin formation is hypothesized (Vassen transducer-and-activator-of-transcription-5 et al., 2006). GFI1B target genes (e.g. BCL2L1, (STAT5) dependent manner. SOCS1, SOCS3, CDKN1A, GATA3) are Localisation frequently also GATA1 target genes (e.g. GFI1B, Almost exclusively nuclear, frequently GATA2, Myb, Myc) and GFI1B is overrepresented accumulating in foci of pericentric heterochromatin. at sites where GATA1 binds to repress its target genes (Yu et al., 2009). GATA2 needs to be Function repressed by GATA1 in developing erythroid cells Negative regulation of transcription. GFI1B is an pointing to an involvement of a GATA1-GFI1B essential factor in erythroid and megakaryocytic repressory complex in this process. GATA1 and development and differentiation, very likely with GFI1B have been found in a complex with SUZ12, proto-oncogenic potential. GFI1B deficiency leads a member of the polycomb repressory complex 2 to embryonic lethality in mice due to failure to (PRC2, SUZ12 and Eed) on repressed genes in

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GFI1B (growth factor independent 1B transcription repressor) Möröy T, Vassen L

MEL (erythroleukemia) cells (Yu et al., 2009). et al., 2009). Knock down of GFI1B in leukemia Since GFI1B is also expressed in hematopoietic cell lines markedly increased the apoptosis rate of stem cells (HSC), the existence of such a repressory these cells (Elmaagacli et al., 2007), pointing to a complex might point to a role of GFI1B in role of GFI1B in protection against apoptosis. maintaining HSC self renewal, where PCR Overexpression of GFI1B in human CD34+ complexes play a major role. Consistent with this hematopoietic progenitors induced an expansion of hypothesis, GFI1B can functionally replace GFI1 erythroblasts, independent from erythropoietin, during hematopoiesis but not in the development of pointing to a role of GFI1B in regulation of inner ear hair cells, where GFI1 exerts a critical proliferation (Osawa et al., 2002). Since reduced survival function (Fiolka et al., 2006; Wallis et al., apoptosis and enhanced proliferation both are 2003). An implication of GFI1B in sensory involved in leukemogenesis, GFI1B may play an epithelial cells similar to GFI1 remains to be important role in these diseases. Additionally, the elucidated. In megakaryocytes GFI1B is found in a GFI1B locus was found in a retroviral insertion complex with GATA1 and ETO2, another mutagenesis screen for factors, cooperating with corepressor protein that is also implicated in human EGR1 haploinsufficiency to induce myeloid leukemogenesis (Hamlett et al., 2008), but how leukemias in the mouse (Quian et al., 2010). GFI1B regulates megakaryocytic development is Disease not clear yet. GFI1B regulates TGF-beta signaling Chronic myeloid leukemia (CML), essential in bipotent erythroid-megakaryocytic progenitors thrombocythemia (ET), myelodysplastic syndrome (Randrianarison-Huetz, 2010), which is involved in (MDS), myeloproliferative syndrome (MPS), B-cell the control of their differentiation. Finally, GFI1B acute lymphocytic leukemia (B-ALL), acute regulates the expression of GATA3 in T-cell myeloid leukemia (AML), erythroleukemia (EL), lymphomas, a critical factor for survival of megakaryocytic/megakaryoblastic leukemias. lymphomas and T-cell progenitors (Wei and Kee, 2007). Breakpoints Positive regulation of transcription. GFI1B can activate transcription from a promoter containing Note four GFI1 consensus-sites in the erythroid cell line Translocated along with ABL1 in chronic myeloid K562 (Osawa et al., 2002). leukemia with translocation t(9:22). Homology References GFI1B is highly homologous to its closest relative GFI1. Highly conserved GFI1(B) proteins have Rödel B, Wagner T, Zörnig M, Niessing J, Möröy T. The been detected in many species from C. elegans to human homologue (GFI1B) of the chicken GFI gene maps to chromosome 9q34.13-A locus frequently altered in drosophila and human. hematopoietic diseases. Genomics. 1998 Dec 15;54(3):580-2 Mutations Tong B, Grimes HL, Yang TY, Bear SE, Qin Z, Du K, El- Deiry WS, Tsichlis PN. The Gfi-1B proto-oncoprotein Germinal represses p21WAF1 and inhibits myeloid cell A single base mutation in the GFI1B promoter (T- differentiation. Mol Cell Biol. 1998 May;18(5):2462-73 C) was detected affecting a potential Oct-1 binding Jegalian AG, Wu H. Regulation of Socs gene expression site in an acute lymphoblastic leukemia patient. The by the proto-oncoprotein GFI-1B: two routes for STAT5 mutation was shown to affect the promoter activity, target gene induction by erythropoietin. J Biol Chem. 2002 Jan 18;277(3):2345-52 leading to an increased expression of GFI1B. (Hernández et al., 2010). Osawa M, Yamaguchi T, Nakamura Y, Kaneko S, Onodera M, Sawada K, Jegalian A, Wu H, Nakauchi H, Iwama A. Somatic Erythroid expansion mediated by the Gfi-1B zinc finger protein: role in normal hematopoiesis. Blood. 2002 Oct A natural variant (p.R231H) was detected in a 15;100(8):2769-77 colorectal cancer sample. A GFI1B promoter Wallis D, Hamblen M, Zhou Y, Venken KJ, Schumacher A, mutation was detected in an acute myeloid Grimes HL, Zoghbi HY, Orkin SH, Bellen HJ. The zinc leukemia M5a patient, affecting a GATA1 binding finger transcription factor Gfi1, implicated in site which was previously shown to be involved in lymphomagenesis, is required for inner ear hair cell the regulation of GFI1B expression. (Hernández et differentiation and survival. Development. 2003 Jan;130(1):221-32 al., 2010). Huang DY, Kuo YY, Lai JS, Suzuki Y, Sugano S, Chang ZF. GATA-1 and NF-Y cooperate to mediate erythroid- Implicated in specific transcription of Gfi-1B gene. Nucleic Acids Res. Leukemia 2004;32(13):3935-46 Garçon L, Lacout C, Svinartchouk F, Le Couédic JP, Note Villeval JL, Vainchenker W, Duménil D. Gfi-1B plays a GFI1B was shown to be highly overexpressed in critical role in terminal differentiation of normal and various leukemias (Elmaagacli et al., 2007; Vassen

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transformed erythroid progenitor cells. Blood. 2005 Feb Koldehoff M, Zakrzewski JL, Klein-Hitpass L, Beelen DW, 15;105(4):1448-55 Elmaagacli AH. Gene profiling of growth factor independence 1B gene (Gfi-1B) in leukemic cells. Int J Huang DY, Kuo YY, Chang ZF. GATA-1 mediates auto- Hematol. 2008 Jan;87(1):39-47 regulation of Gfi-1B transcription in K562 cells. Nucleic Acids Res. 2005;33(16):5331-42 Tsiftsoglou AS, Vizirianakis IS, Strouboulis J. Erythropoiesis: model systems, molecular regulators, and Rodriguez P, Bonte E, Krijgsveld J, Kolodziej KE, Guyot B, developmental programs. IUBMB Life. 2009 Heck AJ, Vyas P, de Boer E, Grosveld F, Strouboulis J. Aug;61(8):800-30 GATA-1 forms distinct activating and repressive complexes in erythroid cells. EMBO J. 2005 Jul Vassen L, Khandanpour C, Ebeling P, van der Reijden BA, 6;24(13):2354-66 Jansen JH, Mahlmann S, Dührsen U, Möröy T. Growth factor independent 1b (Gfi1b) and a new splice variant of Vassen L, Fiolka K, Mahlmann S, Möröy T. Direct Gfi1b are highly expressed in patients with acute and transcriptional repression of the genes encoding the zinc- chronic leukemia. Int J Hematol. 2009 May;89(4):422-30 finger proteins Gfi1b and Gfi1 by Gfi1b. Nucleic Acids Res. 2005;33(3):987-98 Yu M, Riva L, Xie H, Schindler Y, Moran TB, Cheng Y, Yu D, Hardison R, Weiss MJ, Orkin SH, Bernstein BE, Fiolka K, Hertzano R, Vassen L, Zeng H, Hermesh O, Fraenkel E, Cantor AB. Insights into GATA-1-mediated Avraham KB, Dührsen U, Möröy T. Gfi1 and Gfi1b act gene activation versus repression via genome-wide equivalently in haematopoiesis, but have distinct, non- chromatin occupancy analysis. Mol Cell. 2009 Nov overlapping functions in inner ear development. EMBO 25;36(4):682-95 Rep. 2006 Mar;7(3):326-33 Anguita E, Villegas A, Iborra F, Hernández A. GFI1B Vassen L, Fiolka K, Möröy T. Gfi1b alters histone controls its own expression binding to multiple sites. methylation at target gene promoters and sites of gamma- Haematologica. 2010 Jan;95(1):36-46 satellite containing heterochromatin. EMBO J. 2006 Jun 7;25(11):2409-19 Hernández A, Villegas A, Anguita E. Human promoter mutations unveil Oct-1 and GATA-1 opposite action on Elmaagacli AH, Koldehoff M, Zakrzewski JL, Steckel NK, Gfi1b regulation. Ann Hematol. 2010 Aug;89(8):759-65 Ottinger H, Beelen DW. Growth factor-independent 1B gene (GFI1B) is overexpressed in erythropoietic and Laurent B, Randrianarison-Huetz V, Maréchal V, Mayeux megakaryocytic malignancies and increases their P, Dusanter-Fourt I, Duménil D. High-mobility group proliferation rate. Br J Haematol. 2007 Jan;136(2):212-9 protein HMGB2 regulates human erythroid differentiation through trans-activation of GFI1B transcription. Blood. Kuo YY, Chang ZF. GATA-1 and Gfi-1B interplay to 2010 Jan 21;115(3):687-95 regulate Bcl-xL transcription. Mol Cell Biol. 2007 Jun;27(12):4261-72 Qian Z, Joslin JM, Tennant TR, Reshmi SC, Young DJ, Stoddart A, Larson RA, Le Beau MM. Cytogenetic and Saleque S, Kim J, Rooke HM, Orkin SH. Epigenetic genetic pathways in therapy-related acute myeloid regulation of hematopoietic differentiation by Gfi-1 and Gfi- leukemia. Chem Biol Interact. 2010 Mar 19;184(1-2):50-7 1b is mediated by the cofactors CoREST and LSD1. Mol Cell. 2007 Aug 17;27(4):562-72 Randrianarison-Huetz V, Laurent B, Bardet V, Blobe GC, Huetz F, Duménil D. Gfi-1B controls human erythroid and Vassen L, Okayama T, Möröy T. Gfi1b:green fluorescent megakaryocytic differentiation by regulating TGF-beta protein knock-in mice reveal a dynamic expression pattern signaling at the bipotent erythro-megakaryocytic progenitor of Gfi1b during hematopoiesis that is largely stage. Blood. 2010 Apr 8;115(14):2784-95 complementary to Gfi1. Blood. 2007 Mar 15;109(6):2356- 64 Yue P, Forrest WF, Kaminker JS, Lohr S, Zhang Z, Cavet G. Inferring the functional effects of mutation through Wickrema A, Crispino JD. Erythroid and megakaryocytic clusters of mutations in homologous proteins. Hum Mutat. transformation. Oncogene. 2007 Oct 15;26(47):6803-15 2010 Mar;31(3):264-71 Xu W, Kee BL. Growth factor independent 1B (Gfi1b) is an E2A target gene that modulates Gata3 in T-cell This article should be referenced as such: lymphomas. Blood. 2007 May 15;109(10):4406-14 Möröy T, Vassen L. GFI1B (growth factor independent 1B Hamlett I, Draper J, Strouboulis J, Iborra F, Porcher C, transcription repressor). Atlas Genet Cytogenet Oncol Vyas P. Characterization of megakaryocyte GATA1- Haematol. 2011; 15(3):266-269. interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation. Blood. 2008 Oct 1;112(7):2738-49

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in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

LRP5 (low density lipoprotein receptor-related protein 5) Zhendong Alex Zhong, Bart O Williams Laboratory of Cell Signaling and Carcinogenesis, Van Andel Research Institute, Grand Rapids, Michigan 49503-2518, USA (ZAZ, BOW)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/LRP5ID44282ch11q13.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI LRP5ID44282ch11q13.txt

Identity Protein Other names: BMND1, EVR1, EVR4, HBM, Description LR3, LRP7, OPPG, OPS, OPTA1, VBCH2 LRP5 contains a large extracellular domain (ECD) HGNC (Hugo): LRP5 making up over 85% of the approximately 1600- Location: 11q13.2 amino-acid protein. At the amino terminus of the ECD, four beta-propeller motifs and four epidermal DNA/RNA growth factor (EGF)-like repeats create the binding sites for extracellular ligands. These domains are Description followed by three LDLR type A (LA) domains. The Genomic size: 136636; genomic sequence: (chr11: intracellular domain of LRP5 contains 5 PPPSP 67 836 684-67 973 319). motifs, to which Axin preferentially binds after phosphorylation of the PPPSP motif induced by Transcription Wnt ligands. Tamai et al. showed that Wnt activates 5161 bp mRNA; (NM_002335, 05-oct-2009). LRP5's homologue, LRP6, by inducing LRP6 Pseudogene phosphorylation at the PPP(S/T)P motifs, which serve as inducible docking sites for Axin, thereby Homo sapiens low density lipoprotein receptor- recruiting Axin to the plasma membrane. related protein 5-like (LRP5L), transcript variant 1, Aliases: DKFZp434O0213, Expression NCBI Reference Sequence: NM_182492.2, Widely expressed, with the highest level of Location: 22q11.23, expression in the liver. HGNC ID: HGNC:25323.

Exon count: 23; coding exon count: 23.

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LRP5 (low density lipoprotein receptor-related protein 5) Zhong ZA, Williams BO

Mutations Germinal

The heterozygous LRP5V171 mutation cosegregated with high bone density. This gain-of-function mutation in LRP5 causes an autosomal dominant disorder characterized by high bone density, torus palatinus, and a wide, deep mandible. In 2001, Gong et al. reported that they identified a total of six different homozygous frame-shift or nonsense mutations in affected offspring from consanguineous families affected by osteoporosis pseudoglioma syndrome. They also found homozygous missense mutations in affected patients from two other consanguineous families and heterozygous nonsense, frame-shift, and missense mutations in affected patients from four nonconsanguineous families. Many patients with this syndrome are also born with severe disruption of the ocular structure, phthisis bulbi. Schematic diagram of human LRP5, 1615 aa. (from He et Jiao et al. reported that homozygous mutations al., Development. 2004 Apr;131(8):1663-77). R570Q, R752G, and E1367K in LRP5 cosegregated with familial exudative vitreoretinopathy (FEVR). There are many other papers reporting LRP5 gene Post-translational modification: Phosphorylation of mutations and SNP polymorphisms that are the PPPSP motif creates an inducible docking site associated with bone density variation, familial for Axin. Palmitoylation is required for LRP6 to exudative vitreoretinopathy, obesity, etc. exit the endoplasmic reticulum (ER). Localisation Somatic Westin's group reported that the tumor-associated Membrane; single-pass type I membrane protein. shorter transcript of LRP5 containing an in-frame Function deletion of 142 amino acids (D666-809) was Involved in the Wnt/beta catenin signaling strongly implicated in deregulated activation of the pathway, acting as a co-receptor together with Wnt/beta-catenin signaling pathway in Frizzled for Wnt ligands. hyperparathyroid tumors and mammary gland tumorigenesis.

Schematic representation of LRP5 mutations; those associated with osteoporosis pseudoglioma (OPPG) syndrome, autosomal- dominant familial exudative vitreoretinopathy (FEVR), and various high-bone-density diseases are shown in red, purple, and green, respectively. Arrows indicate mutation locations: *, nonsense mutation; fs, frame-shift mutation. (from He et al., Development. 2004 Apr;131(8):1663-77).

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LRP5 (low density lipoprotein receptor-related protein 5) Zhong ZA, Williams BO

Implicated in Cytogenetics Gong et al. found that OPPG carriers have reduced Hyperparathyroid tumors, breast bone mass when compared with age- and gender- cancer matched controls. They demonstrated LRP5 expression by osteoblasts in situ and showed that Note LRP5 can transduce Wnt signaling in vitro via the According to Bjorklund's reports, the internally canonical pathway. They also showed that a mutant truncated human LRP5 receptor is strongly secreted form of LRP5 can reduce bone thickness in implicated in deregulated activation of the mouse calvarial explant cultures. These data Wnt/beta-catenin signaling pathway in indicate that Wnt-mediated signaling via LRP5 hyperparathyroid tumors and mammary gland affects bone accrual during growth and is important tumorigenesis, and thus presents a potential target for the establishment of peak bone mass. for therapeutic intervention. Ai et al. sequenced the coding exons of LRP5 in 37 probands suspected of having OPPG on the basis of the co-occurrence of severe congenital or childhood-onset visual impairment and bone fragility or osteoporosis recognized by young adulthood. They measured the ability of wild-type The truncation version of LRP5 (LRP5Δ666-809) missed and mutant LRP5 to transduce Wnt and Norrin the last 93 bp of exon 9, all 227 bp of exon 10, and the first signals ex vivo. Each of the seven OPPG mutations 106 bp of exon 11. tested had reduced signal transduction relative to Oncogenesis wild-type controls. These results indicate that early Reverse transcription PCR and Western blot bilateral vitreoretinal eye pathology coupled with analysis showed expression of truncated LRP5 in skeletal fragility is a strong predictor of LRP5 32 out of 37 primary hyperparathyroidism (pHPT) mutation and that mutations in LRP5 cause OPPG tumors (86%) and 20 out of 20 secondary by impairing Wnt and Norrin signal transduction. hyperparathyroidism (SHPT) tumors (100%). In 2008, Yadav et al. identified Tph1, which Truncated LRP5 frequently expressed in breast encodes the rate-limiting enzyme in serotonin tumors of different cancer stages (58-100%), synthesis, as the most highly overexpressed gene in -/- including carcinoma in situ and metastatic LRP5 mice. Tph1 expression was also elevated in -/- carcinoma. Truncated LRP5 was required in MCF7 LRP5 duodenal cells. Decreasing serotonin blood breast cancer cells for the nonphosphorylated active levels normalized bone formation and bone mass in -/- beta-catenin level, transcription activity of beta- LRP5 mice, and gut-specific LRP5 inactivation catenin, cell growth in vitro, and breast tumor decreased bone formation in a beta-catenin- growth in a xenograft SCID mouse model. independent manner. They concluded that LRP5 inhibits bone formation by inhibiting serotonin Other cancers production in the gut. Note Cheung et al. identified a family with osteoporosis LRP5 is required for maintaining the basal lineage pseudoglioma syndrome due to compound of mouse mammary tissue (Badders et al., 2009) heterozygosity of two novel mutations in the LRP5 and for mammary ductal stem cell activity and gene (W478R and W504C). Wnt1-induced tumorigenesis (Lindvall et al., 2006). In 2007, Drenser et al. found familial exudative LRP5 is a novel marker for disease progression in vitreoretinopathy and osteoporosis pseudoglioma high-grade osteosarcoma (Hoang et al., 2004). syndrome caused by a mutation in the LRP5 gene. Dominant negative LRP5 showed inhibition of Xiong et al. found that LRP5 gene polymorphisms osteosarcoma tumorigenicity and metastasis in are associated with bone mass density in both mouse model (Guo et al., 2008). Chinese and whites. The Chinese sample consisted Osteoporosis-pseudoglioma of 733 unrelated subjects and the white sample was made up of 1873 subjects from 405 nuclear syndrome (OPPG) families. Note The most frequently studied polymorphisms in Children with the autosomal recessive disorder LRP5 are two amino acid substitutions, Val667Met osteoporosis pseudoglioma syndrome (OPPG) and Ala1330Val. A common variant of LRP6, (Gong et al., 1996) have very low bone mass and Ile1062Val, contributes to fracture risk in elderly are prone to developing fractures and deformation. men, and is linked to coronary heart disease and In addition to the skeletal phenotype, many low BMD. In 2008, Joyce et al. confirmed that the individuals with OPPG have eye involvement in the two common LRP5 variants are consistently form of severe disruption of the ocular structure, associated with BMD and fracture risk across called phthisis bulbi. different white populations, but the LRP6 variant is not.

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LRP5 (low density lipoprotein receptor-related protein 5) Zhong ZA, Williams BO

High bone mass (HBM) cell surfaces, resulting in fewer LRP5 molecules on the cell surface. So they think that the G171V Note mutation may cause an increase in Wnt activity in Bone mass density (BMD) and fracture rates vary osteoblasts by reducing the number of targets for among women of differing ethnicities. Most reports paracrine DKK1 to antagonize without affecting the had suggested that BMD is highest in African activity of autocrine Wnt. Americans, lowest in Asians, and intermediate in Ai et al. expressed seven different HBM-LRP5 Caucasians, yet Asians have lower fracture rates missense mutations, including G171V, to delineate than Caucasians. Finkelstein et al. (2002) assessed the mechanism by which they alter Wnt signaling. lumbar spine and femoral neck BMD by dual- Each mutant receptor was able to reach the cell energy x-ray absorptiometry in 2277 (lumbar) and surface, albeit in differing amounts, and transduce 2330 (femoral) premenopausal or early exogenously supplied Wnt1 and Wnt3a signals. The perimenopausal women (mean age, 46.2 yr) affinities between the mutant forms of LRP5 and participating in the Study of Women's Health Mesd did not correlate with their abilities to reach Across the Nation. When BMD was assessed in a the cell surface. All HBM mutant proteins had subset of women weighing less than 70 kg and then reduced physical interaction with and reduced adjusted for covariates, lumbar spine BMD was inhibition by DKK1. These data suggest that HBM similar in African American, Chinese, and Japanese mutant proteins can transit to the cell surface in women and was lowest in Caucasian women. sufficient quantity to transduce Wnt signal and that Femoral neck BMD was highest in African the likely mechanism for the HBM mutations' Americans and similar in Chinese, Japanese, and physiologic effects is via reduced affinity to and Caucasians. They also suggested that these findings inhibition by DKK1. may explain why Caucasian women have higher Semenov further showed that LRP5 HBM mutant fracture rates than African Americans and Asians. proteins exhibit reduced binding to a secreted bone- Cytogenetics specific LRP5 antagonist, SOST, and consequently Little et al. also identified the same Gly171Val are more refractory to inhibition by SOST. Further, mutation in the LRP5 gene (G171V; 603506.0013) Bhat used structure-based mutation analysis to that results in an autosomal dominant high bone show the importance of LRP5 beta-propeller 1 in mass trait. modulating Dkk1-mediated inhibition of Wnt Van Wesenbeeck et al. performed mutation analysis signaling. of the LRP5 gene in 10 families or isolated patients with various conditions of an increased bone Familial exudative vitreoretinopathy density, including endosteal hyperostosis. Direct (FEVR) sequencing of the LRP5 gene revealed 19 sequence Note variants. Six novel missense mutations (D111Y, Familial exudative vitreoretinopathy (FEVR) is a G171R, A214T, A214V, A242T, and T253I) are well-defined inherited disorder of retinal vessel located in the amino-terminal part of the gene, development (Benson, 1995). It is reported to have before the first epidermal growth factor-like a penetrance of 100%, but clinical features can be domain, which is the same as for the G171V highly variable even within the same family. mutation that causes the high-bone-mass phenotype Severely affected patients may be legally blind and most likely is disease-causing. during the first decade of life, whereas mildly Boyden et al. found that the expression of LRP5V171 affected individuals may not even be aware of did not activate signaling in the absence of Wnt-1. symptoms and may receive a diagnosis only by use The activation of the signaling pathway in response of fluorescein angiography. to Wnt-1 was the same with normal and mutant Cytogenetics LRP5.They also tested the action of the endogenous As reported by Toomes et al., mutations in LRP5 antagonist of Wnt signaling, Dkk-1. Although Dkk- within the EVR1 locus can cause FEVR, 1 inhibited Wnt signaling in conjunction with wild- accounting for 15% of the patients and indicating type LRP5, Dkk-1 inhibition of Wnt signaling was that other unidentified FEVR genes may be a more virtually abolished in cells expressing LRP5 . V171 significant cause of the disease than previously These findings indicated that the mutation G171V, thought. located in the first YWTD repeat of LRP5, results Jiao et al. studied three consanguineous families of in increased Wnt signaling because of loss of Dkk European descent in which autosomal recessive antagonism to LRP5. FEVR was diagnosed in multiple individuals. However, Zhang et al. found that the third YWTD Sequencing of LRP5 showed, in all three families, repeat (but not the first repeat domain) was required homozygosity for mutation in LRP5: R570Q, for DKK1-mediated antagonism. They found that R752G, and E1367K. Thus, mutations in the LRP5 the G171V mutation disrupted the interaction of gene can cause autosomal recessive as well as LRP5 with Mesd, a chaperone protein for LRP5/6 autosomal dominant FEVR. that is required for transport of the co-receptors to

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LRP5 (low density lipoprotein receptor-related protein 5) Zhong ZA, Williams BO

Qin et al. screened 56 unrelated patients with FEVR (31 familial and 25 simplex cases) for possible References mutations in LRP5 and Frizzled 4 (FZD4). Six Benson WE. Familial exudative vitreoretinopathy. Trans novel mutations in either LRP5 or FZD4 were Am Ophthalmol Soc. 1995;93:473-521 identified in six familial cases. Four novel Gong Y, Vikkula M, Boon L, Liu J, Beighton P, Ramesar R, mutations in LRP5 and one known mutation in Peltonen L, Somer H, Hirose T, Dallapiccola B, De Paepe FZD4 were detected in three simplex cases, and two A, Swoboda W, Zabel B, Superti-Furga A, Steinmann B, Brunner HG, Jans A, Boles RG, Adkins W, van den of these patients carried compound heterozygous Boogaard MJ, Olsen BR, Warman ML. Osteoporosis- mutations in LRP5. They also demonstrated that pseudoglioma syndrome, a disorder affecting skeletal reduced bone density is a common feature in strength and vision, is assigned to chromosome region patients with FEVR who harbor LRP5 mutations. 11q12-13. Am J Hum Genet. 1996 Jul;59(1):146-51 Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick Obesity MA, Wu D, Insogna K, Lifton RP. High bone density due to Note a mutation in LDL-receptor-related protein 5. N Engl J Obesity is a growing health care problem and a risk Med. 2002 May 16;346(20):1513-21 factor for common diseases such as diabetes, heart Hsieh JC, Lee L, Zhang L, Wefer S, Brown K, DeRossi C, disease, and hypertension. Wines ME, Rosenquist T, Holdener BC. Mesd encodes an LRP5/6 chaperone essential for specification of mouse LRP5 is highly expressed in many tissues, embryonic polarity. Cell. 2003 Feb 7;112(3):355-67 including hepatocytes and pancreatic beta cells. Some evidence has shown that LRP5 can bind Van Wesenbeeck L, Cleiren E, Gram J, Beals RK, Bénichou O, Scopelliti D, Key L, Renton T, Bartels C, apolipoprotein E (apoE), which raises the Gong Y, Warman ML, De Vernejoul MC, Bollerslev J, Van possibility that LRP5 plays a role in the hepatic Hul W. Six novel missense mutations in the LDL receptor- clearance of apoE-containing chylomicron related protein 5 (LRP5) gene in different conditions with remnants, a major plasma lipoprotein carrying diet- an increased bone density. Am J Hum Genet. 2003 Mar;72(3):763-71 derived cholesterol. Using LRP5 knock-out mice model, Fujino et al. Ferrari SL, Deutsch S, Choudhury U, Chevalley T, Bonjour showed that LRP5-deficient islets had a marked JP, Dermitzakis ET, Rizzoli R, Antonarakis SE. 2+ Polymorphisms in the low-density lipoprotein receptor- reduction in the levels of intracellular ATP and Ca related protein 5 (LRP5) gene are associated with variation in response to glucose, and thereby glucose-induced in vertebral bone mass, vertebral bone size, and stature in insulin secretion was decreased. The intracellular whites. Am J Hum Genet. 2004 May;74(5):866-75 inositol 1,4,5-trisphosphate (IP3) production in He X, Semenov M, Tamai K, Zeng X. LDL receptor-related response to glucose was also reduced in LRP5-/- proteins 5 and 6 in Wnt/beta-catenin signaling: arrows islets. The authors suggested that Wnt/LRP5 point the way. Development. 2004 Apr;131(8):1663-77 signaling contributes to the glucose-induced insulin Hoang BH, Kubo T, Healey JH, Sowers R, Mazza B, Yang secretion in the islets. R, Huvos AG, Meyers PA, Gorlick R. Expression of LDL receptor-related protein 5 (LRP5) as a novel marker for Cytogenetics disease progression in high-grade osteosarcoma. Int J Guo et al. performed genotyping of 27 single Cancer. 2004 Mar;109(1):106-11 nucleotide polymorphisms (SNPs), spaced 5 kb Jiao X, Ventruto V, Trese MT, Shastry BS, Hejtmancik JF. apart on average and covering the full transcript Autosomal recessive familial exudative vitreoretinopathy is length of the LRP5 gene, using samples of 1873 associated with mutations in LRP5. Am J Hum Genet. Caucasian people from 405 nuclear families. They 2004 Nov;75(5):878-84 found that SNP4 (rs4988300) and SNP6 Tamai K, Zeng X, Liu C, Zhang X, Harada Y, Chang Z, He (rs634008), located in block 2 (intron 1), showed X. A mechanism for Wnt coreceptor activation. Mol Cell. 2004 Jan 16;13(1):149-56 significant associations with obesity and BMI after Bonferroni correction (SNP4: p < 0.001 and p = Toomes C, Bottomley HM, Jackson RM, Towns KV, Scott 0.001, respectively; SNP6: p = 0.002 and 0.003, S, Mackey DA, Craig JE, Jiang L, Yang Z, Trembath R, Woodruff G, Gregory-Evans CY, Gregory-Evans K, Parker respectively). The common allele A for SNP4 and MJ, Black GC, Downey LM, Zhang K, Inglehearn CF. minor allele G for SNP6 were associated with an Mutations in LRP5 or FZD4 underlie the common familial increased risk of obesity. Significant associations exudative vitreoretinopathy locus on chromosome 11q. Am were also observed between the common haplotype J Hum Genet. 2004 Apr;74(4):721-30 A-G-G-G of block 2 and obesity, BMI, fat mass, Zhang Y, Wang Y, Li X, Zhang J, Mao J, Li Z, Zheng J, Li and PFM, with global empirical values of p < L, Harris S, Wu D. The LRP5 high-bone-mass G171V mutation disrupts LRP5 interaction with Mesd. Mol Cell 0.001, p < 0.001, p = 0.003 and p = 0.074, Biol. 2004 Jun;24(11):4677-84 respectively. They concluded that intronic variants of the LRP5 gene are markedly associated with Ai M, Holmen SL, Van Hul W, Williams BO, Warman ML. Reduced affinity to and inhibition by DKK1 form a common obesity, possibly due to the role of LRP5 in the Wnt mechanism by which high bone mass-associated signaling pathway or lipid metabolism. missense mutations in LRP5 affect canonical Wnt signaling. Mol Cell Biol. 2005 Jun;25(12):4946-55

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LRP5 (low density lipoprotein receptor-related protein 5) Zhong ZA, Williams BO

Cheung WM, Jin LY, Smith DK, Cheung PT, Kwan EY, disease and metabolic risk factors. Science. 2007 Mar Low L, Kung AW. A family with osteoporosis pseudoglioma 2;315(5816):1278-82 syndrome due to compound heterozygosity of two novel mutations in the LRP5 gene. Bone. 2006 Sep;39(3):470-6 Xiong DH, Lei SF, Yang F, Wang L, Peng YM, Wang W, Recker RR, Deng HW. Low-density lipoprotein receptor- Guo YF, Xiong DH, Shen H, Zhao LJ, Xiao P, Guo Y, related protein 5 (LRP5) gene polymorphisms are Wang W, Yang TL, Recker RR, Deng HW. Polymorphisms associated with bone mass in both Chinese and whites. J of the low-density lipoprotein receptor-related protein 5 Bone Miner Res. 2007 Mar;22(3):385-93 (LRP5) gene are associated with obesity phenotypes in a large family-based association study. J Med Genet. 2006 Guo Y, Rubin EM, Xie J, Zi X, Hoang BH. Dominant Oct;43(10):798-803 negative LRP5 decreases tumorigenicity and metastasis of osteosarcoma in an animal model. Clin Orthop Relat Res. Lindvall C, Evans NC, Zylstra CR, Li Y, Alexander CM, 2008 Sep;466(9):2039-45 Williams BO. The Wnt signaling receptor Lrp5 is required for mammary ductal stem cell activity and Wnt1-induced Yadav VK, Ryu JH, Suda N, Tanaka KF, Gingrich JA, tumorigenesis. J Biol Chem. 2006 Nov 17;281(46):35081-7 Schütz G, Glorieux FH, Chiang CY, Zajac JD, Insogna KL, Mann JJ, Hen R, Ducy P, Karsenty G. Lrp5 controls bone Semenov MV, He X. LRP5 mutations linked to high bone formation by inhibiting serotonin synthesis in the mass diseases cause reduced LRP5 binding and inhibition duodenum. Cell. 2008 Nov 28;135(5):825-37 by SOST. J Biol Chem. 2006 Dec 15;281(50):38276-84 Badders NM, Goel S, Clark RJ, Klos KS, Kim S, Bafico A, Bhat BM, Allen KM, Liu W, Graham J, Morales A, Lindvall C, Williams BO, Alexander CM. The Wnt receptor, Anisowicz A, Lam HS, McCauley C, Coleburn V, Cain M, Lrp5, is expressed by mouse mammary stem cells and is Fortier E, Bhat RA, Bex FJ, Yaworsky PJ. Structure-based required to maintain the basal lineage. PLoS One. 2009 mutation analysis shows the importance of LRP5 beta- Aug 12;4(8):e6594 propeller 1 in modulating Dkk1-mediated inhibition of Wnt signaling. Gene. 2007 Apr 15;391(1-2):103-12 Björklund P, Svedlund J, Olsson AK, Akerström G, Westin G. The internally truncated LRP5 receptor presents a Björklund P, Akerström G, Westin G. An LRP5 receptor therapeutic target in breast cancer. PLoS One. with internal deletion in hyperparathyroid tumors with 2009;4(1):e4243 implications for deregulated WNT/beta-catenin signaling. PLoS Med. 2007 Nov 27;4(11):e328 Williams BO, Insogna KL. Where Wnts went: the exploding field of Lrp5 and Lrp6 signaling in bone. J Bone Miner Res. Drenser KA, Trese MT. Familial exudative 2009 Feb;24(2):171-8 vitreoretinopathy and osteoporosis-pseudoglioma syndrome caused by a mutation in the LRP5 gene. Arch This article should be referenced as such: Ophthalmol. 2007 Mar;125(3):431-2 Zhong ZA, Williams BO. LRP5 (low density lipoprotein Mani A, Radhakrishnan J, Wang H, Mani A, Mani MA, receptor-related protein 5). Atlas Genet Cytogenet Oncol Nelson-Williams C, Carew KS, Mane S, Najmabadi H, Wu Haematol. 2011; 15(3):270-275. D, Lifton RP. LRP6 mutation in a family with early coronary

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Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

MIF (macrophage migration inhibitory factor (glycosylation-inhibiting factor)) Jan-Philipp Bach, Michael Bacher, Richard Dodel Department of Neurology, Philipps-University Marburg, Rudolf-Bultmann-Strasse 8, 35039 Marburg, Germany (JPB, MB, RD)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/MIFID41365ch22q11.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI MIFID41365ch22q11.txt

Identity Localisation Other names: GIF, GLIF, MMIF Intracellular, cytoplasm, cytosolic, near the plasma membrane, perinuclear. HGNC (Hugo): MIF Function Location: 22q11.23 MIF monomers are able to align in order to form a DNA/RNA homotrimeric molecule that is homologous to the enzyme D-Dopachrome-tautomerase (Sun et al., Description 1996). From this structural analysis, some 0,84 kb; mRNA: 561 bp; 3 Exons. researches suggested that MIF may also display enzymatic activity (Rosengren et al., 1996). To Transcription date, the physiological importance of this The promoter region contains no TATA box. enzymatic activity has not yet been revealed. Pseudogene Interestingly, using ISO-1, a known inhibitor of the enzyme D-Dopachrome-tautomerase led to reduced There are no MIF pseudogenes in the human activity of MIF (Lubetsky et al., 2002). Therefore, genome. In contrast 5 pseudogenes have been it was hypothesized that this enzymatic activity described in the murine genome. may be related to its proper functioning. The protein MIF is involved in inducing angiogenesis, Protein promoting cell cycle progression, inhibiting apoptosis and inhibiting lysing of tumor cells by Description NK cells (Takahashi et al., 1998; Shimizu et al., MIF is comprised of 115 amino acids with a 1999; Mitchell and Bucala, 2000; Morrison et al., molecular weight of 12,5 kDa (Weiser et al., 1989). 2001; Fingerle-Rowson et al., 2003). Recently, the In addition, research on the secondary structure CD74 molecule has been suggested to act as a revealed the existence of two antiparallel alpha- potential receptor for MIF (Leng et al., 2003). helices and six beta-pleated sheets with a high degree of similarity to MHC molecules (Suzuki et Homology al., 1996). MIF acts as a pro-inflammatory protein, MIF shows homology to the enzyme D- exists as a homo-trimer and displays enzymatic Dopachrome tautomerase. action (Rosengren et al., 1996). Expression Mutations Widely. Note Not known.

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MIF (macrophage migration inhibitory factor (glycosylation- Bach JP, et al. inhibiting factor))

Implicated in Prognosis To date, there is no clear association between MIF Colon cancer concentration and prognosis in melanoma cells. Disease Prostate cancer MIF is upregulated in tumors as well as Disease precancerous lesions (Wilson et al., 2005). They MIF was shown to be abundant in prostate cancer examined patients suffering from adenomas. In (Meyer-Siegler et al., 1998). In addition, MIF was addition, an animal model of adenomatous shown to influence cell viability and invasiveness. polyposis coli was used. In both settings, the Specifically in prostate cancer, androgen authors describe an increase in MIF mRNA levels independent cancer cells relied on MIF activated in the diseases group when this was compared to pathways in order to grow and for their healthy controls. Interestingly, in a mouse model of invasiveness. Androgen dependent tumor cells did intestinal tumorigenesis, MIF deletion leads to a not require these signal transduction pathways. significant decrease in tumor size. In addition, Meyer-Siegler et al. were able to show that CD74, a reduced angiogenesis was taking place. The potential MIF surface receptor, was abundant in involvement of MIF in angiogenesis was also androgen-independent tumor cells (Meyer-Siegler shown by Ogawa et al., using a mouse model of et al., 2006). Receptor blockage as well as strategies colon cancer (Ogawa et al., 2000). The application to reduce MIF resulted in decreased cell of MIF antibodies leads to suppression of proliferation, MIF secretion and invasion of tumor angiogenesis in this disease model. Further work by cells. Sun et al. demonstrated an involvement of MIF in Prognosis tumor cell migration (Sun et al., 2005). They used siRNA technique and were able to show inhibition MIF expression clearly correlates with disease of cell migration after addition of siRNA directed progression (Meyer-Siegler et al., 2002). In against MIF. In an animal model, they injected androgen independent prostate cancer, cells require colon cancer cells into mice portal vein after MIF activated signal transduction pathways for pretreatment with siRNA. The number of liver invasion and growth, in contrast to androgen metastases was significantly reduced in the dependent tumor cells. In a study by Meyer-Siegler, pretreated model. In summary, there is sufficient serum MIF concentration was measured in evidence to assume a role of MIF in both dependence of Gleason score in patients with angiogenesis and tumor cell migration in colon prostate cancer (Meyer-Siegler et al., 2002). They cancer (Bach et al., 2009). were able to show that increased MIF concentration is positively associated with a Gleason Score Prognosis greater than 5. In addition, even in patients with MIF expression is correlated with outcome normal prostate-specific antigen, MIF concentration (Legendre et al., 2003). Legendre et al. (2003) was increased. From their data, they conclude that examined MIF distribution in 99 specimens of MIF may be a suitable biomarker for prostate colorectal cancer. Primarily, they applied cancer. immunohistochemistry. They describe that the expression of MIF (and also galectin-3) were Lung adenocarcinoma increased in tumor tissue compared to normal Note tissue. For MIF, they could provide evidence that in Treatment with siRNA leads to a significant Dukes C or D tumors with high concentration of reduction in cell invasiveness and cell migration MIF, this was associated with significantly better (Rendon et al., 2007), paralled by a reduction of a prognosis than in tumors with low MIF Rho GTPase. MIF overexpression led to adverse concentrations. They suggest that MIF could be effects. used to identify patients at risk needing more Prognosis aggressive treatment strategies. Kamimura and colleagues analysed the role of MIF Melanoma with respect to prognosis in lung cancer (Kamimura Disease et al., 2000). They used immunofluorescence MIF inhibits lysis of melanoma cells by NK cells staining in primary lung tissue that had been (Repp et al., 2000). This was shown by Apte et al., obtained surgically. They were able to demonstrate who demonstrated that NK cells are prevented from a diffuse staining pattern within the cytoplasm. cell lysis of melanoma cells (Apte et al., 1998). Furthermore, sometimes, a nuclear staining was This underlines the influence of MIF on the also observed. Interestingly, the authors were able immune system. In addition, it enhances tumor cell to demonstrate a correlation between a lack of proliferation. nuclear staining and a poorer prognosis.

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MIF (macrophage migration inhibitory factor (glycosylation- Bach JP, et al. inhibiting factor))

From this observation, they concluded that MIF Kieser A, Weich HA, Brandner G, Marmé D, Kolch W. might play different roles with respect to Mutant p53 potentiates protein kinase C induction of vascular endothelial growth factor expression. Oncogene. subcellular localization. It needs to be mentioned 1994 Mar;9(3):963-9 however, that only thirty-eight cases were Rosengren E, Bucala R, Aman P, Jacobsson L, Odh G, reviewed. Metz CN, Rorsman H. The immunoregulatory mediator Glioblastoma macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction. Mol Med. 1996 Jan;2(1):143-9 Note Sun HW, Swope M, Cinquina C, Bedarkar S, Bernhagen J, MIF is involved in angiogenesis and cell cycle Bucala R, Lolis E. The subunit structure of human regulation. It was demonstrated that MIF macrophage migration inhibitory factor: evidence for a expression is induced following hypoxia and trimer. Protein Eng. 1996 Aug;9(8):631-5 hypoglycemia (Bacher et al., 2003), which are both Suzuki M, Sugimoto H, Nakagawa A, Tanaka I, Nishihira J, considered activators of angiogenesis. Using Sakai M. Crystal structure of the macrophage migration immunofluorescence techniques, MIF could be inhibitory factor from rat liver. Nat Struct Biol. 1996 demonstrated around necrotic tissue and in blood Mar;3(3):259-66 vessels surrounding tumor cells. Therefore it was Apte RS, Sinha D, Mayhew E, Wistow GJ, Niederkorn JY. concluded that neovascularization is enhanced by Cutting edge: role of macrophage migration inhibitory factor in inhibiting NK cell activity and preserving immune MIF. In line with this observation is the work privilege. J Immunol. 1998 Jun 15;160(12):5693-6 published by Munaut et al. (2002). They demonstrated that there is a correlation between Meyer-Siegler K, Fattor RA, Hudson PB. Expression of macrophage migration inhibitory factor in the human MIF expression and the expression of vascular prostate. Diagn Mol Pathol. 1998 Feb;7(1):44-50 endothelial growth factor (VEGF). They analysed Takahashi N, Nishihira J, Sato Y, Kondo M, Ogawa H, primary glioblastoma tissue using RT-PCR and Ohshima T, Une Y, Todo S. Involvement of macrophage immmunohistochemistry and they were able to migration inhibitory factor (MIF) in the mechanism of tumor show a strong correlation between MIF expression cell growth. Mol Med. 1998 Nov;4(11):707-14 and VEGF mRNA concentration. >From these data, Shimizu T, Abe R, Nakamura H, Ohkawara A, Suzuki M, it was suggested that there may be a common Nishihira J. High expression of macrophage migration triggering factor. Inactivation of p53 may be of inhibitory factor in human melanoma cells and its role in central importance, as it is a common event in tumor cell growth and angiogenesis. Biochem Biophys Res Commun. 1999 Nov 2;264(3):751-8 glioblastoma progression (Bach et al., 2009). In addition, p53 inactivation is also involved in Kamimura A, Kamachi M, Nishihira J, Ogura S, Isobe H, increased VEGF expression (Kieser et al., 1994). Dosaka-Akita H, Ogata A, Shindoh M, Ohbuchi T, Kawakami Y. Intracellular distribution of macrophage Since the angiogenic potential of a tumor is vital for migration inhibitory factor predicts the prognosis of patients formation of metastases, it may be a potential target with adenocarcinoma of the lung. Cancer. 2000 Jul for antitumor therapy. 15;89(2):334-41 Hepatocellular carcinoma Mitchell RA, Bucala R. Tumor growth-promoting properties of macrophage migration inhibitory factor (MIF). Semin Note Cancer Biol. 2000 Oct;10(5):359-66 MIF expression and increased angiogenesis were Ogawa H, Nishihira J, Sato Y, Kondo M, Takahashi N, also demonstrated in hepatocellular carcinoma Oshima T, Todo S. An antibody for macrophage migration following hypoxia (Hira et al., 2005). inhibitory factor suppresses tumour growth and inhibits tumour-associated angiogenesis. Cytokine. 2000 Prognosis Apr;12(4):309-14 MIF overexpression correlates with negative Repp AC, Mayhew ES, Apte S, Niederkorn JY. Human prognosis (Hira et al., 2005). 56 samples of uveal melanoma cells produce macrophage migration- hepatocellular carcinoma were analysed using inhibitory factor to prevent lysis by NK cells. J Immunol. Western blot and correlated these values with 2000 Jul 15;165(2):710-5 clinical parameters. In addition, they used Morrison H, Sherman LS, Legg J, Banine F, Isacke C, immunohistochemistry. They showed that MIF Haipek CA, Gutmann DH, Ponta H, Herrlich P. The NF2 expression was correlated with high alpha tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44. Genes fetoprotein level and the recurrence of hepatic Dev. 2001 Apr 15;15(8):968-80 tumor. In addition, tumor free survival was reduced when MIF expression was increased. In addition, Lubetsky JB, Dios A, Han J, Aljabari B, Ruzsicska B, Mitchell R, Lolis E, Al-Abed Y. The tautomerase active site increased mRNA concentrations can be seen (Ren of macrophage migration inhibitory factor is a potential et al., 2003). target for discovery of novel anti-inflammatory agents. J Biol Chem. 2002 Jul 12;277(28):24976-82 References Meyer-Siegler KL, Bellino MA, Tannenbaum M. Macrophage migration inhibitory factor evaluation Weiser WY, Temple PA, Witek-Giannotti JS, Remold HG, compared with prostate specific antigen as a biomarker in Clark SC, David JR. Molecular cloning of a cDNA encoding patients with prostate carcinoma. Cancer. 2002 Mar a human macrophage migration inhibitory factor. Proc Natl 1;94(5):1449-56 Acad Sci U S A. 1989 Oct;86(19):7522-6

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MIF (macrophage migration inhibitory factor (glycosylation- Bach JP, et al. inhibiting factor))

Munaut C, Boniver J, Foidart JM, Deprez M. Macrophage hepatocellular carcinoma. Cancer. 2005 Feb 1;103(3):588- migration inhibitory factor (MIF) expression in human 98 glioblastomas correlates with vascular endothelial growth factor (VEGF) expression. Neuropathol Appl Neurobiol. Sun B, Nishihira J, Yoshiki T, Kondo M, Sato Y, Sasaki F, 2002 Dec;28(6):452-60 Todo S. Macrophage migration inhibitory factor promotes tumor invasion and metastasis via the Rho-dependent Bacher M, Schrader J, Thompson N, Kuschela K, Gemsa pathway. Clin Cancer Res. 2005 Feb 1;11(3):1050-8 D, Waeber G, Schlegel J. Up-regulation of macrophage migration inhibitory factor gene and protein expression in Wilson JM, Coletta PL, Cuthbert RJ, Scott N, MacLennan glial tumor cells during hypoxic and hypoglycemic stress K, Hawcroft G, Leng L, Lubetsky JB, Jin KK, Lolis E, indicates a critical role for angiogenesis in glioblastoma Medina F, Brieva JA, Poulsom R, Markham AF, Bucala R, multiforme. Am J Pathol. 2003 Jan;162(1):11-7 Hull MA. Macrophage migration inhibitory factor promotes intestinal tumorigenesis. Gastroenterology. 2005 Fingerle-Rowson G, Petrenko O, Metz CN, Forsthuber TG, Nov;129(5):1485-503 Mitchell R, Huss R, Moll U, Müller W, Bucala R. The p53- dependent effects of macrophage migration inhibitory Meyer-Siegler KL, Iczkowski KA, Leng L, Bucala R, Vera factor revealed by gene targeting. Proc Natl Acad Sci U S PL. Inhibition of macrophage migration inhibitory factor or A. 2003 Aug 5;100(16):9354-9 its receptor (CD74) attenuates growth and invasion of DU- 145 prostate cancer cells. J Immunol. 2006 Dec Legendre H, Decaestecker C, Nagy N, Hendlisz A, 15;177(12):8730-9 Schüring MP, Salmon I, Gabius HJ, Pector JC, Kiss R. Prognostic values of galectin-3 and the macrophage Rendon BE, Roger T, Teneng I, Zhao M, Al-Abed Y, migration inhibitory factor (MIF) in human colorectal Calandra T, Mitchell RA. Regulation of human lung cancers. Mod Pathol. 2003 May;16(5):491-504 adenocarcinoma cell migration and invasion by macrophage migration inhibitory factor. J Biol Chem. 2007 Leng L, Metz CN, Fang Y, Xu J, Donnelly S, Baugh J, Oct 12;282(41):29910-8 Delohery T, Chen Y, Mitchell RA, Bucala R. MIF signal transduction initiated by binding to CD74. J Exp Med. 2003 Bach JP, Deuster O, Balzer-Geldsetzer M, Meyer B, Dodel Jun 2;197(11):1467-76 R, Bacher M. The role of macrophage inhibitory factor in tumorigenesis and central nervous system tumors. Cancer. Ren Y, Tsui HT, Poon RT, Ng IO, Li Z, Chen Y, Jiang G, 2009 May 15;115(10):2031-40 Lau C, Yu WC, Bacher M, Fan ST. Macrophage migration inhibitory factor: roles in regulating tumor cell migration This article should be referenced as such: and expression of angiogenic factors in hepatocellular carcinoma. Int J Cancer. 2003 Oct 20;107(1):22-9 Bach JP, Bacher M, Dodel R. MIF (macrophage migration inhibitory factor (glycosylation-inhibiting factor)). Atlas Hira E, Ono T, Dhar DK, El-Assal ON, Hishikawa Y, Genet Cytogenet Oncol Haematol. 2011; 15(3):276-279. Yamanoi A, Nagasue N. Overexpression of macrophage migration inhibitory factor induces angiogenesis and deteriorates prognosis after radical resection for

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Gene Section Mini Review

NEU3 (sialidase 3 (membrane sialidase)) Kazunori Yamaguchi, Taeko Miyagi Division of Biochemistry, Miyagi Cancer Center Research Institute, Natori 981-1293, Japan (KY), Cancer Glycosylation Research, Tohoku Pharmaceutical University, Sendai 981-8558, Japan (TM)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/NEU3ID44505ch11q13.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI NEU3ID44505ch11q13.txt

Identity Protein Other names: FLJ12388, SIAL3 Description HGNC (Hugo): NEU3 Deduced amino acid sequence of human NEU3 Location: 11q13.4 comprises of 428 amino acids and contains RIP box at N-terminal region and three Asp boxes in the DNA/RNA center of the amino acid sequence. These motifs are commonly found in sialidases of microorganisms Description and vertebrates. The RIP motif is a part of active The NEU3 gene spans 22 kb, consists of 4 exons site and mutation of this motif led to decreased and 3 introns. It is a member of sialidase family enzymatic activity. The Asp box is thought to consisting of NEU1, NEU2, NEU3, and NEU4. participate in proper 3D structure formation of NEU3. Transcription Northern blot analysis reveals 2.5 kb and 7 kb Expression transcripts of NEU3 gene, possessing a common The gene is ubiquitously expressed with relatively open reading frame of 1284 bp. NEU3 gene higher levels in skeletal muscle, heart, and testis. expression is diversely regulated by Sp1/Sp3 The expression is upregulated during transcription factors which are recently considered tumorigenesis, neuronal differentiation, T cell to play critical roles in regulating the transcription activation, and monocyte differentiation. Abnormal of genes involved in cell growth and tumorigenesis. upregulation of NEU3 is observed in various

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NEU3 (sialidase 3 (membrane sialidase)) Yamaguchi K, Miyagi T

human neoplasms including colon, renal, ovarian keratinocytes or fibroblasts does not cause growth and prostate cancers, except for the down- arrest nor apoptosis. Transgenic mice ectopically regulation in acute lymphoblastic leukemia. expressing human NEU3 shows upregulation of It is interesting to note that the transgenic mice EGFR signaling, lower induction of apoptosis, and ectopically expressing human NEU3 develop high incidence of ACF (aberrant crypt foci) impaired insulin signaling and insulin-resistant formation in colon mucosa cells upon diabetes mellitus by 18-22 weeks. administration of azoxymethane (AOM), a Localisation carcinogen for colonic tumorigenesis. Biochemical fractionation shows NEU3 to be Renal cell carcinoma localized in membrane fractions, especially in raft Note or caveolae membrane microdomains. In immuno- Renal cell carcinoma shows upregulation of NEU3 fluorescence studies, the bovine and mouse Neu3 along with high expression of IL6. IL6 appears to sialidases are mostly detected on the cell surface, increase NEU3 gene expression in ACHN cells, and but the human NEU3 may exist also in other the increase brings about enhanced activation of cellular membrane components and can mobilize to PI3K and Akt upon IL6 administration, resulting in membrane ruffles together with Rac-1 in response suppression of apoptosis. to growth stimuli such as EGF, enhancing cell Type II diabetes mellitus movement. Note Function NEU3 transgenic mice develop impaired insulin NEU3 sialidase removes sialic acid moiety of signaling and insulin-resistant diabetes mellitus by gangliosides. It hardly acts on glycoproteins, 18-22 weeks, associated with hyper-insulinemia, oligosaccharides, and an artificial substrate 4MU- islet hyperplasia and increase in the beta-cell mass. NeuAc. NEU3 alters gangliosides composition of As compared to the wild type, insulin-stimulated tissues and cells, and leads to modulation of signal phosphorylation of the insulin receptor and insulin transduction such as EGFR (epidermal growth receptor substrate I is significantly reduced, and factor receptor) and IR (insulin receptor) signaling. activities of phosphatidylinositol 3-kinase and Besides, NEU3 has been shown to associate with glycogen synthase are also decreased. In muscle signaling molecules including EGFR, Grb2, extracts, association of tyrosine-phosphorylated caveolin-1, and Rac. In addition to the catalytic NEU3 with Grb2 occurs in response to insulin, reaction as a sialidase, the interaction with the together with accumulation of ganglioside GM1 protein molecules may also give an influence on the and GM2. NEU3 function. Disturbance of signaling by Involvement of NEU3 in cancer progression and abnormal upregulation of NEU3 is likely a possible development of diabetes suggests that these cause of tumorigenesis or diabetes mellitus. diseases might be closely related to each other in Homology pathogenesis, given the recent epidemiological reports of higher cancer risk in diabetic patients The NEU3 amino acid sequence shows 28% than in controls. identity to NEU1, 42% to NEU2, and 45% to NEU4. NEU3 orthologs are identified in bovine, mouse, and rat. References Wada T, Yoshikawa Y, Tokuyama S, Kuwabara M, Akita Implicated in H, Miyagi T. Cloning, expression, and chromosomal mapping of a human ganglioside sialidase. Biochem Colon cancer Biophys Res Commun. 1999 Jul 22;261(1):21-7 Monti E, Bassi MT, Papini N, Riboni M, Manzoni M, Note Venerando B, Croci G, Preti A, Ballabio A, Tettamanti G, NEU3 shows higher enzymatic activity and mRNA Borsani G. Identification and expression of NEU3, a novel level in colon tumors as compared to adjacent human sialidase associated to the plasma membrane. normal mucosa. In tumor cells, lactosylceramide, Biochem J. 2000 Jul 1;349(Pt 1):343-51 one of the products of NEU3 enzymatic reaction, Kakugawa Y, Wada T, Yamaguchi K, Yamanami H, Ouchi accumulates in tumor cells. Over expression of K, Sato I, Miyagi T. Up-regulation of plasma membrane- NEU3 or exogenous addition of lactosylceramide to associated ganglioside sialidase (Neu3) in human colon cancer and its involvement in apoptosis suppression. Proc the cell culture confer resistance to sodium Natl Acad Sci U S A. 2002 Aug 6;99(16):10718-23 butyrate-induced apoptosis. On the other hand, Monti E, Preti A, Venerando B, Borsani G. Recent silencing of NEU3 by siRNA causes induction of development in mammalian sialidase molecular biology. apoptosis in cancer cells accompanied with Neurochem Res. 2002 Aug;27(7-8):649-63 suppression of EGFR signaling, suggesting that Wang Y, Yamaguchi K, Wada T, Hata K, Zhao X, Fujimoto survival of tumor cells is addictive to NEU3 T, Miyagi T. A close association of the ganglioside-specific expression. Interestingly, NEU3-knock down in sialidase Neu3 with caveolin in membrane microdomains. normal cells including primary culture of J Biol Chem. 2002 Jul 19;277(29):26252-9

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Ueno S, Saito S, Wada T, Yamaguchi K, Satoh M, Arai Y, treated transgenic mice. Cancer Sci. 2009 Apr;100(4):588- Miyagi T. Plasma membrane-associated sialidase is up- 94 regulated in renal cell carcinoma and promotes interleukin- 6-induced apoptosis suppression and cell motility. J Biol Tringali C, Lupo B, Cirillo F, Papini N, Anastasia L, Chem. 2006 Mar 24;281(12):7756-64 Lamorte G, Colombi P, Bresciani R, Monti E, Tettamanti G, Venerando B. Silencing of membrane-associated sialidase Wada T, Hata K, Yamaguchi K, Shiozaki K, Koseki K, Neu3 diminishes apoptosis resistance and triggers Moriya S, Miyagi T. A crucial role of plasma membrane- megakaryocytic differentiation of chronic myeloid leukemic associated sialidase in the survival of human cancer cells. cells K562 through the increase of ganglioside GM3. Cell Oncogene. 2007 Apr 12;26(17):2483-90 Death Differ. 2009 Jan;16(1):164-74 Miyagi T. Aberrant expression of sialidase and cancer Mandal C, Tringali C, Mondal S, Anastasia L, Chandra S, progression. Proc Jpn Acad Ser B Phys Biol Sci. Venerando B, Mandal C. Down regulation of membrane- 2008;84(10):407-18 bound Neu3 constitutes a new potential marker for childhood acute lymphoblastic leukemia and induces Miyagi T, Wada T, Yamaguchi K. Roles of plasma apoptosis suppression of neoplastic cells. Int J Cancer. membrane-associated sialidase NEU3 in human cancers. 2010 Jan 15;126(2):337-49 Biochim Biophys Acta. 2008 Mar;1780(3):532-7 Yamaguchi K, Koseki K, Shiozaki M, Shimada Y, Wada T, Miyagi T, Wada T, Yamaguchi K, Hata K, Shiozaki K. Miyagi T. Regulation of plasma-membrane-associated Plasma membrane-associated sialidase as a crucial sialidase NEU3 gene by Sp1/Sp3 transcription factors. regulator of transmembrane signalling. J Biochem. 2008 Biochem J. 2010 Aug 15;430(1):107-17 Sep;144(3):279-85 Miyagi T, Wada T, Yamaguchi K, Shiozaki K, Sato I, This article should be referenced as such: Kakugawa Y, Yamanami H, Fujiya T. Human sialidase as Yamaguchi K, Miyagi T. NEU3 (sialidase 3 (membrane a cancer marker. Proteomics. 2008 Aug;8(16):3303-11 sialidase)). Atlas Genet Cytogenet Oncol Haematol. 2011; Shiozaki K, Yamaguchi K, Sato I, Miyagi T. Plasma 15(3):280-282. membrane-associated sialidase (NEU3) promotes formation of colonic aberrant crypt foci in azoxymethane-

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Gene Section Review

NPY1R (neuropeptide Y receptor Y1) Massimiliano Ruscica, Elena Dozio, Luca Passafaro, Paolo Magni Dipartimento di Endocrinologia, Fisiopatologia e Biologia Applicata, Universita degli Studi di Milano, Italy (MR, LP, PM), Dipartimento di Morfologia Umana e Scienze Biomediche "Citta Studi", Universita degli Studi di Milano, Italy (ED)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/NPY1RID44260ch4q32.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI NPY1RID44260ch4q32.txt

164253748 bp. The overall sequence of the gene Identity consists of approximately 10 kb. The genomic Other names: NPYR structure presents a 6-kb intron situated HGNC (Hugo): NPY1R approximately 150 bp upstream of the start codon within the 5'-untranslated region (5'UTR), as well Location: 4q32.2 as a small intron within the coding region. The human NPY1R gene is divided into three DNA/RNA exons: exon 1 (115 bp), exon 2 (850 bp), and exon Note 3 (1749 bp). In particular, the NPY1R gene History: The human NPY1R cDNA was cloned contains three alternative exon 1 sequences (80, from a human brain cDNA library. The NPY1R 110, and 106 bp) located 6.4, 18.4, and 23.9 kb was the first to be characterized, when the upstream of exon 2. Exon 1A is located 6.4 kb expression pattern of an orphan receptor was upstream of exon 2; exon 1B was found a further 12 recognized to overlap with the distribution of NPY kb upstream exon 1A, and exon 1C another 5.5 kb in brain. NPY receptors belong to the large upstream of exon 1B. These alternative 5' exons superfamily of G-protein-coupled receptors. Many allow the regulation of tissue-specific expression of of these receptor genes lack introns, supporting the the receptor. The first 57 nucleotides of the 5'UTR proposition that they were created via RNA- of the human NPY1R mRNA are separated by a 6- mediated transpositional events. Differently from kb intron from the second exon. The second intron the other NPY receptor isoforms NPY1R is the only 97 bp, containing an in-frame stop codon, is located one containing a single 97-base pairs (bp) intron in at nucleotide 908 in the protein coding region after the coding region following the fifth the fifth transmembrane domain between exon 2 transmembrane domain. and 3. Moreover, as shown by Nakamura, mouse NPY1R gene contains an alternate exon 4 located Description over 15 kb downstream of exon 3. A 14-kilobase pair (kb) region of genomic DNA Transcription encoding the human neuropeptide Y Y1-receptor gene including 3'- and 5'- flanking sequences is The human cDNA encodes a protein of 384 amino- localized to chromosome 4. It encompasses 8632 bp acids (aa) in lenght that is preceded by of DNA (4q32.2) between 164245117 and approximately 200 bp of 5'UTR sequence.

Exon/intron structure and splice sites in the 5'UTR of the human NPY1R gene.

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NPY1R (neuropeptide Y receptor Y1) Ruscica M, et al.

Figure A. Y1R affinity for various PP-family hormones and their C-terminal sequences. Figure B. Homo sapiens Neuropeptide Y receptor type 1 (384 aa).

central nervous system (i.e., cerebral cortex, Protein thalamus and amigdala) and periphery (i.e., heart, Note kidneys, gastrointestinal tract, as well as blood The NPY Y1 receptor subtype (Y1R) was the first vessels). to be cloned in the rat, and subsequently in human Nervous system and mouse. This receptor is as conserved as its The NPY Y1R is widely distributed in the central ligand (NPY) throughout evolution and mammalian nervous system. A study conducted on four normal and non-mammalian species. The complete NPY1-36 human brains revealed that high levels of Y1R molecule is necessary for NPY to bind to Y1R. Any receptor mRNA were expressed in cortical areas proteolytic process leading to alterations in the and in the claustrum, while moderate levels were NH2-terminal domain essentially abolishes the present in the nucleus accumbens, caudate nucleus, ability of NPY to bind to Y1R. Therefore, NH2- putamen, amygdaloid nuclei and arcuate and terminally truncated NPY fragments such as NPY2- paraventricular nuclei of the hypothalamus. 36, or NPY3-36 have little or no affinity for the Y1R. Moreover, a study conducted on prefrontal cortex Modification of COOH-terminal residues does not of subjects affected by bipolar disorder, major affect agonist binding. Thus, it has been established depression, or schizophrenia revealed a progressive that the NH2 terminus is essential for NPY to age-related decline in the expression of Y1R activate Y1R. The pharmacological profile of the mRNA associated with a lack of coexpression with Y1R is characterized by high affinity for NPY, NPY neurons. Interestingly, there was no PYY and the corresponding analogs containing significant effect of suicide as a cause of death on Pro34 and low affinity for the N-terminally Y1R mRNA expression levels. In fact, subjects truncated analogs and for PP. with suicide as a cause of death tended to have higher Y1R mRNA expression levels, but these Description individuals were among the youngest ones (45 Y1R has seven putative transmembrane domains years old) in the population studied. associated with G-protein (GPCR). In the N- Periphery terminal portion Y1R presents potential sites of Peripherally, Y1Rs are expressed mainly in arteries glycosylation and in the second extracellular loop, and veins, where they are associated with four extracellular cysteines in position 33, 113, 198 vasoconstriction and potentiation of other and 296 which may form two disulfide bridges (Cys vasoconstrictors of neurogenic origin. Although 33 and 296; Cys 133 and 198). Phosphorylation limited, there is evidence of prejunctional Y1R sites are present in the intracellular domain inhibition of neurotransmitter release. Nonetheless, (cysteine in the C-terminal portion at position 338). NPY Y1R is primarily located postjunctionally on These cysteines may also explain the capability of vascular smooth muscle cells. palmitate residues to bind to the receptor. As 1) Colon observed for many GPCR, Y1R is internalized In vitro receptor autoradiography ([125I]PYY) together with its ligand into endosomes and performed on normal human colonic tissue obtained recycled to the cell surface within 60 minutes upon from nine patients showed that Y1R is distributed agonist stimulation. Moreover, Y1R is able to form only in vessels. No measurable levels of subtype homodimers. Y1 was identified in smooth muscle, mucosa, Expression muscularis mucosae, as well as in lymphoid follicles, myoenteric and submucosal plexus. The expression of the human NPY Y1 receptor has 2) Heart been studied extensively by using A study conducted on 20-week old fetal human immunohistochemical methods, in situ hearts showed that Y1R is present on right hybridization experiments and reverse-transcription ventricular endocardial endothelial cells. In polymerase chain reaction (RT-PCR; mRNA particular, it is highly expressed at the level of the detection). The human NPY1R is expressed in both nucleus specifically at the perinucleoplasm and

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2+ nuclear membrane levels, while lower levels were ([Ca ]i), and modulation of the MAPK pathway via detected in the cytoplasm and the plasma several signaling molecules, including the protein membrane. kinase C (PKC). 3) Dental pulp Y1R has been involved in several NPY-induced NPY Y1R proteins were present in solubilized responses, such as activation of neuroendocrine membrane preparations of both healthy and axes, vasoconstriction, anxiolysis, as well as the inflamed human gingival tissue by Western stimulation of food intake. Moreover, Y1R blotting. Major immunoreactive bands were mediates emotional behavior, stress response, and detected at approximately 55 kDa due to a ethanol consumption. glycosylated form of the native receptor protein. By The prototype of NPY Y1R-mediated responses is using the SwissProt glycosylation prediction vasoconstriction. Specifically, the physiological packages NetNGlyc and NetOGly, authors role of the Y1R subtype was demonstrated in mice confirmed that the human Y1R has potential N- and lacking Y1R expression, which show no blood O-glycosylation sites. The expression of Y1R pressure response to NPY, but a normal response to protein in both healthy and inflamed gingival tissue norepinephrine. Y1R knockout mice have normal suggests that NPY could act via the Y1R to exert its blood pressure, suggesting that the Y1R does not tonic effects. Moreover, Y1R was expressed in play a crucial role in maintaining blood pressure human dental pulp with evidence of increased homeostasis in unstimulated conditions. However, expression in carious compared with noncarious Y1R has been also involved in other NPY-induced teeth. Y1R were localized to nerve fibres and responses, such as stimulation of food intake and inflammatory cells in the dental pulp of carious activation of neuroendocrine axes. In particular, teeth. Y1R and Y5R, both expressed in hypothalamic 4) Achilles tendons regions involved in the control of feeding, represent Y1R is expressed in the tenocytes in the Achilles the most likely candidates for mediating the tendon. Specifically, Y1R is present within the appetite stimulatory capacity of NPY. Mice lacking smooth muscle of the blood vessel walls, but not in Y1R showed an increased body weight due to a the endothelial layer of calcaneal tendons. low-energy expenditure rather than high-energy 5) Skin intake. In fact, these mice had a decreased In human tissues, RT-PCR and metabolic rate secondary to decreased locomotor immunocytochemistry studies suggested that Y1R activity and movement associated thermogenesis. is the primary receptor in human cutaneous Homology circulation, supporting the findings that local non- noradrenergic mechanisms are entirely Y1R-based. The human Y1R subtype shares closest aa identity Skin blood flow in humans is controlled through with the Y4R subtype (42%) and the non-active, two branches of the sympathetic nervous system: a human form of the y6 subtype (51%). vasoconstrictor system and an active vasodilator system of uncertain neurotransmitter. In this Mutations context, NPY showed a vasoconstrictor effect in Note human subcutaneous arteries that had been In 2004 Ramanathan described a case of autism in dissected out of the abdominal regions from which a 19 megabase on chromosome 4q, spanning patients who underwent nonvascular disease 4q32 to 4q34, was detected. Being involved in the surgeries (e.g., hernia). NPY decreased cutaneous deletion, those genes which are abundantly blood flow via Y1R, with evidence for the expressed in the brain, Y1R and Y5R resulted additional involvement of postjunctional Y2R. This implicated. In this context, being the ability of NPY and Y1R to affect skin vascular neuroproliferative effect of NPY in the conductance varies in accordance with relative hippocampus mediated through the neuropeptide Y innervations at specific sites. Y1R, the authors postulate that the effect of NPY Localisation on learning and memory may be mediated through NPY Y1R is a seven transmembrane receptor NPY neurogenesis. which has all the characteristics of the GPCR Okahisa et al. described that genetic variants of family including potential glycosylation sites in the rs7687423 of the NPY1R gene may alter the N-terminal portion and in the second extra-cellular subjective effects of methamphetamine and result in loop. susceptibility to dependence. Because NPY1R mRNA changes were observed in peripheral tissues Function and the brain in schizophrenia patients, these NPY has been demonstrated to be involved in findings may also indicate that the NPY1R gene is mitogenic pathways and stimulate cell proliferation involved in vulnerability to methamphetamine- via the Y1R. The activation of Y1R is generally induced psychosis because almost all of the associated with reduction of cAMP accumulation, analyzed subjects with methamphetamine increase of intracellular free calcium concentration

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dependence had comorbid methamphetamine Moreover, a functional interplay between estrogen psychosis. and Y1R has been shown in a human breast cancer cell line responsive to this steroid, where estrogen Implicated in was found to increase Y1R expression, which in turn negatively regulated estrogen-stimulated cell Various tumors proliferation. Note Pheochromocytoma and NPY receptors are mainly expressed in specific paraganglioma endocrine tumors and epithelial malignancies as well as in embryonal tumors. In endocrine tumors, Note NPY receptors are present in steroid hormone The frequency of NPY receptors (NPYRs) producing tumors, namely adrenal cortical expression in pheochromocytomas and adenomas, carcinomas, ovarian granulosa cell paragangliomas was found to be 35% and 61%, tumors, Sertoli-Leydig cell tumors, and in respectively. Both Y1R and Y2R are expressed, catecholamine producing tumors, i.e. with a higher density of Y2R in paragangliomas pheochromocytomas and paragangliomas. Based on than in pheochromocytomas, whereas the density of pharmacological displacement experiments, in Y1R is comparably low in both tumor categories. addition to tumor cells, intra- and peritumoral blood NPYRs, mainly Y1R and Y5R, are also expressed vessels express Y1Rs. The Y1R-expressing blood in the Ewing's sarcoma family of tumors, other vessels are mainly small and medium-sized arteries. related neural crest-derived tumors, where activation of these receptors has been reported to Prostate cancer regulate cell proliferation, as shown in the SK-N- Note MC cell line, an Ewing's sarcoma family of tumors Prostate cancer represents one of the most common expressing Y1R, where NPY has been shown to malignant diseases among men in the Western inhibit cell growth. world. It is initially androgen dependent and it may later progress to the androgen-independent stage, References which is associated with a lack of efficacy of the Soares MB, Schon E, Henderson A, Karathanasis SK, available hormonal therapy. This tumoral Cate R, Zeitlin S, Chirgwin J, Efstratiadis A. RNA-mediated progression appears to be promoted at least in part gene duplication: the rat preproinsulin I gene is a functional by several growth factors and neurohormones. retroposon. Mol Cell Biol. 1985 Aug;5(8):2090-103 Within this context, we showed that Y1R protein is Wahlestedt C, Yanaihara N, Håkanson R. Evidence for expressed in three human prostate cancer cell lines different pre-and post-junctional receptors for neuropeptide (LNCaP -androgen dependent-, DU145 and PC3 - Y and related peptides. Regul Pept. 1986 Feb;13(3-4):307- androgen independent-) and that NPY treatment 18 reduced the proliferation of LNCaP and DU145 Eva C, Oberto A, Sprengel R, Genazzani E. The murine cells and increased that of PC3 cells. Interestingly, NPY-1 receptor gene. Structure and delineation of tissue- the Y1R antagonist BIBP3226 abolished such specific expression. FEBS Lett. 1992 Dec 21;314(3):285-8 effects, suggesting a mandatory role of Y1-R in this Grundemar L, Jonas SE, Mörner N, Högestätt ED, process. Moreover, these effects are associated with Wahlestedt C, Håkanson R. Characterization of vascular neuropeptide Y receptors. Br J Pharmacol. 1992 a clone-specific pattern of intracellular signaling Jan;105(1):45-50 activation, including a peculiar time-course of MAPK/ERK1/ERK2 phosphorylation (long-lasting Larhammar D, Blomqvist AG, Yee F, Jazin E, Yoo H, Wahlested C. Cloning and functional expression of a in DU145 and transient in PC3 cells). human neuropeptide Y/peptide YY receptor of the Y1 type. Breast cancer J Biol Chem. 1992 Jun 5;267(16):10935-8 Herzog H, Hort YJ, Shine J, Selbie LA. Molecular cloning, Note characterization, and localization of the human homolog to Breast cancer accounts for almost 1/3 of all incident the reported bovine NPY Y3 receptor: lack of NPY binding cases of cancer in women. Interestingly, the and activation. DNA Cell Biol. 1993 Jul-Aug;12(6):465-71 expression of NPY-Rs has been found in 85% of Ball HJ, Shine J, Herzog H. Multiple promoters regulate primary breast cancer in a series of 95 cases, and in tissue-specific expression of the human NPY-Y1 receptor 100% of lymph node metastases of receptor- gene. J Biol Chem. 1995 Nov 10;270(45):27272-6 positive primaries, where Y1R expression Nakamura M, Sakanaka C, Aoki Y, Ogasawara H, Tsuji T, predominated and was often present in high density Kodama H, Matsumoto T, Shimizu T, Noma M. and great homogeneity. In normal breast tissue, Identification of two isoforms of mouse neuropeptide Y-Y1 receptor generated by alternative splicing. Isolation, however, Y1R was only found in a minority of the genomic structure, and functional expression of the cases and concomitantly with Y2R, which seemed receptors. J Biol Chem. 1995 Dec 15;270(50):30102-10 to be predominant in non-neoplastic breast. The Ammar DA, Eadie DM, Wong DJ, Ma YY, Kolakowski LF neoplastic condition of breast tissue may thus Jr, Yang-Feng TL, Thompson DA. Characterization of the induce a switch of expression from Y2R to Y1R. human type 2 neuropeptide Y receptor gene (NPY2R) and localization to the chromosome 4q region containing the

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NPY1R (neuropeptide Y receptor Y1) Ruscica M, et al.

type 1 neuropeptide Y receptor gene. Genomics. 1996 Dec units (AMPA 2, GLRA3, GLRB) and neuropeptide 15;38(3):392-8 receptors NPY1R, NPY5R. BMC Med Genet. 2004 Apr 16;5:10 Jacques D, Tong Y, Dumont Y, Shen SH, Quirion R. Expression of the neuropeptide Y Y1 receptor mRNA in Jemal A, Ward E, Thun MJ. Contemporary lung cancer the human brain: an in situ hybridization study. trends among U.S. women. Cancer Epidemiol Biomarkers Neuroreport. 1996 Apr 10;7(5):1053-6 Prev. 2005 Mar;14(3):582-5 Blomqvist AG, Herzog H. Y-receptor subtypes--how many Kitlinska J, Abe K, Kuo L, Pons J, Yu M, Li L, Tilan J, more? Trends Neurosci. 1997 Jul;20(7):294-8 Everhart L, Lee EW, Zukowska Z, Toretsky JA. Differential effects of neuropeptide Y on the growth and Cabrele C, Beck-Sickinger AG. Molecular characterization vascularization of neural crest-derived tumors. Cancer of the ligand-receptor interaction of the neuropeptide Y Res. 2005 Mar 1;65(5):1719-28 family. J Pept Sci. 2000 Mar;6(3):97-122 Amlal H, Faroqui S, Balasubramaniam A, Sheriff S. Caberlotto L, Hurd YL. Neuropeptide Y Y(1) and Y(2) Estrogen up-regulates neuropeptide Y Y1 receptor receptor mRNA expression in the prefrontal cortex of expression in a human breast cancer cell line. Cancer Res. psychiatric subjects. Relationship of Y(2) subtype to 2006 Apr 1;66(7):3706-14 suicidal behavior. Neuropsychopharmacology. 2001 Jul;25(1):91-7 Ruscica M, Dozio E, Boghossian S, Bovo G, Martos Riaño V, Motta M, Magni P. Activation of the Y1 receptor by Rettenbacher M, Reubi JC. Localization and neuropeptide Y regulates the growth of prostate cancer characterization of neuropeptide receptors in human colon. cells. Endocrinology. 2006 Mar;147(3):1466-73 Naunyn Schmiedebergs Arch Pharmacol. 2001 Oct;364(4):291-304 Ruscica M, Dozio E, Motta M, Magni P. Relevance of the neuropeptide Y system in the biology of cancer Reubi JC, Gugger M, Waser B, Schaer JC. Y(1)-mediated progression. Curr Top Med Chem. 2007;7(17):1682-91 effect of neuropeptide Y in cancer: breast carcinomas as targets. Cancer Res. 2001 Jun 1;61(11):4636-41 El Karim IA, Lamey PJ, Linden GJ, Lundy FT. Neuropeptide Y Y1 receptor in human dental pulp cells of Balasubramaniam A. Neuropeptide Y (NPY) family of noncarious and carious teeth. Int Endod J. 2008 hormones: progress in the development of receptor Oct;41(10):850-5 selective agonists and antagonists. Curr Pharm Des. 2003;9(15):1165-75 Bjur D, Alfredson H, Forsgren S. Presence of the neuropeptide Y1 receptor in tenocytes and blood vessel Dinger MC, Bader JE, Kobor AD, Kretzschmar AK, Beck- walls in the human Achilles tendon. Br J Sports Med. 2009 Sickinger AG. Homodimerization of neuropeptide y Dec;43(14):1136-42 receptors investigated by fluorescence resonance energy transfer in living cells. J Biol Chem. 2003 Mar Hodges GJ, Jackson DN, Mattar L, Johnson JM, 21;278(12):10562-71 Shoemaker JK. Neuropeptide Y and neurovascular control in skeletal muscle and skin. Am J Physiol Regul Integr Pheng LH, Dumont Y, Fournier A, Chabot JG, Beaudet A, Comp Physiol. 2009 Sep;297(3):R546-55 Quirion R. Agonist- and antagonist-induced sequestration/internalization of neuropeptide Y Y1 Lundy FT, El Karim IA, Linden GJ. Neuropeptide Y (NPY) receptors in HEK293 cells. Br J Pharmacol. 2003 and NPY Y1 receptor in periodontal health and disease. Jun;139(4):695-704 Arch Oral Biol. 2009 Mar;54(3):258-62 Körner M, Waser B, Reubi JC. High expression of Okahisa Y, Ujike H, Kotaka T, Morita Y, Kodama M, Inada neuropeptide y receptors in tumors of the human adrenal T, Yamada M, Iwata N, Iyo M, Sora I, Ozaki N, Kuroda S. gland and extra-adrenal paraganglia. Clin Cancer Res. Association between neuropeptide Y gene and its receptor 2004 Dec 15;10(24):8426-33 Y1 gene and methamphetamine dependence. Psychiatry Clin Neurosci. 2009 Jun;63(3):417-22 Pedrazzini T. Importance of NPY Y1 receptor-mediated pathways: assessment using NPY Y1 receptor knockouts. This article should be referenced as such: Neuropeptides. 2004 Aug;38(4):267-75 Ruscica M, Dozio E, Passafaro L, Magni P. NPY1R Ramanathan S, Woodroffe A, Flodman PL, Mays LZ, (neuropeptide Y receptor Y1). Atlas Genet Cytogenet Hanouni M, Modahl CB, Steinberg-Epstein R, Bocian ME, Oncol Haematol. 2011; 15(3):283-287. Spence MA, Smith M. A case of autism with an interstitial deletion on 4q leading to hemizygosity for genes encoding for glutamine and glycine neurotransmitter receptor sub-

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Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

REPS2 (RALBP1 associated Eps domain containing 2) Salvatore Corallino, Luisa Castagnoli Department of Biology, University of Rome Tor Vergata, via ricerca scientifica, 00133 Rome, Italy (SC, LC)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/REPS2ID44120chXp22.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI REPS2ID44120chXp22.txt

strong expression in rat cerebrum, cerebellum, lung, Identity and testis, with weak expression in kidney and no Other names: POB1 expression in heart, thymus, liver, spleen, or HGNC (Hugo): REPS2 adrenal gland. Relatively highly expressed in androgen-dependent as compared to androgen- Location: Xp22.2 independent prostate cancer cell lines. Local order: Forward strand: before 16804550- REPS2/POB1 is down-regulated during progression 16862642 CXorf15 (ENSG00000086712) and after of prostate cancer. 17300683-17301216. Known pseudogene RP11- 674N8.1 (ENSG00000214321). Protein Note: This gene is a member of the human CCDS set: CCDS14180, CCDS43919. Description REPS2/POB1, Swiss-Prot Q8NFH8, is expressed as DNA/RNA two isoforms, the short isoform is 521 residues long, while the 660 residues one differs by having a Description 139 amino acid extension at the amino-terminus. 18 exons in REPS2/POB1 gene. The most prominent structural/functional features, which are common to both isoforms, include an Transcription amino-terminal EH (Eps15 homology) domain, a The transcript length of REPS2/POB1 is 7945 bp. central region containing two adjacent proline-rich REPS2 is not differentially expressed in regions and a carboxy-terminal portion mediating monozygotic twins. Northern blot analysis reveal the binding to RalBP1.

REPS2/POB1: synopsis of protein structure, interactors, functions.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3) 288

REPS2 (RALBP1 associated Eps domain containing 2) Corallino S, Castagnoli L

In the figure (not in scale), are described the main epsin and their binding regulates receptor-mediated motifs and domains of the long isoform of endocytosis (Morinaka et al., 1999). REPS2/POB1, the interacting proteins and the Augmented expression of full-length POB1 in cellular functions, that are described in the text. A431 cells does not affect either binding or Expression internalization of EGF, on the contrary, over- expression of either the EH domain or the C- The POB1 mRNA is expressed in cerebrum, terminal region of REPS2/POB1 affects the ligand cerebellum, lung, weakly in kidney, and testis dependent internalization pathway of EGF and (Ikeda et al., 1998). It is relatively highly expressed insulin without interfering with the constitutive in androgen-dependent as compared to androgen- transferrin pathway (Nakashima et al., 1999). independent prostate cancer cell lines and Santonico et al. have demonstrated that the EH xenografts and it is down-regulated during domain of REPS2/POB1 binds Eps15 through an progression of prostate cancer (Oosterhoff et al., unconventional recognition specificity, since it 2003). binds to both NPF and DPF (Asp-Prp-Phe) motifs. Localisation The region of Eps15 responsible for the interaction with the EH domain of REPS2/POB1 maps within a REPS2/POB1 localizes in the cytosol, in different 18 amino acid peptide (residues 623-640) that sub cellular compartments: it colocalizes with includes three DPF repeats. Accordingly, the clathrin CHC in coated pits, with CD63 in late authors identify a cluster of solvent exposed Lys endosomes, with GM130 in golgi and with LAMP2 residues, which are only found in the EH domain of in lysosomes. Localization is not EGF dependent REPS2/POB1, and influence binding to both NPF and POB1 doesn't localize with EEA1 in early and DPF motifs (Santonico et al., 2007). endosomes (Tomassi et al., 2008). RalBP1, REPS2/POB1, epsin, and Eps15 form a Function complex with alpha-adaptin of AP-2 in Chinese REPS2/POB1 is part of a protein complex that hamster ovary cells and this complex is reduced in regulates the endocytosis and down regulation of mitotic phase, when REPS2/POB1 and epsin are growth factor receptors. Its expression can found phosphorylated. They are both negatively affect growth factor signaling. Multiple phosphoryated by p34 cdc2 kinase, in vitro. POB1 transcript variants encoding different isoforms have is found phosphorylated in Ser551 and Ser493, in been found for this gene and posttranslational vivo. Phosphorylation of epsin in Ser 357 inhibits modifications have been described, such as binding to POB1, causing disassembly of the phosphorylation of Ser493 and Ser549 (Oppermann complex, thus inhibiting receptor mediated et al., 2009). The REPS2/POB1 has two amino- endocytosis (Kariya et al., 2000). This data explains terminal EH domains. The structure of the second the contribution of the EH domain of POB1 to the EH domain that extends from 265 to 367 has been formation of a protein complex that favours solved by NMR and consists of two EF-hand receptor internalization and that it is dismantled in structures, and the second one binds a calcium ion mitosis. It is suggested that EGF stimulation (Koshiba et al., 1999). The EH domain binds epsin, induces also tyrosine-phosphorylation of POB1 and Eps15 and NF-kappaB p65, and it is associated to subsequent formation of a EGFR-POB1 complex in endocytosis and apoptosis. The central proline rich COS cells (Ikeda et al., 1998). region of POB1/REPS2 binds to 14-3-3, REPS2/POB1 shorter isoform2 is downregulated amphiphysin II and Grb2 and it is associated to during human prostate cancer progression from receptor downregulation and signaling. The androgen-dependent to androgen-independent carbossi-terminal proline rich binds PAG2 and (Oosterhoff et al., 2003). It was observed that a influences paxillin localization in focal adhesion. high level of REPS2 correlates with reduced EGF- POB1 C-terminus (514-660) directly interacts with internalisation and signaling since the induced a GTPase activating protein that functions expression of REPS2 exerts an inhibiting effect on downstream of the small G protein Ral, RalBP1. several EGF-responsive genes (EGF-receptor, Their interaction induces apoptosis. EGR-1, Fos and Jun) (Oosterhoff et al., 2005). REPS2 downregulates receptor signaling and Accordingly, increased expression of POB1 endocytosis: REPS2/POB1 interacts with Eps15, isoform 2 correlates with a decrease of EGF- epsin EPN1, 14-3-3 isoforms, Grb2, amphiphysin induced phosphorylation of Erk1-Erk2 and Shc The presence of EH domains in REPS2/POB1 is (Tomassi et al., 2008). symptomatic of a role of this gene in receptor From these experiments, it can be concluded that endocytosis. In fact, REPS2/POB1 EH domain increased REPS2/POB1 expression negatively binds to Eps15 and to epsin that are both proteins affects EGF receptor internalisation and subsequent present in clathrin-coated pits, involved in receptor signaling. Therefore, the decreased REPS2 endocytosis and receptor down regulation. The EH expression observed during prostate cancer domain interacts specifically with the three Asn- progression, results in enhanced EGF receptor Pro-Phe (NPF) motifs in the C-terminal region of expression and signaling, which could add to the

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REPS2 (RALBP1 associated Eps domain containing 2) Corallino S, Castagnoli L

androgen-independent state of advanced prostate on the POB1-binding domain, of PAG2. On the cancer. other end, POB1, but not POB1(PA), can suppress The central region of REPS2/POB1 plays a the inhibitory action of PAG2 on paxillin regulatory role in epidermal growth factor receptor localization to focal adhesion (Oshiro et al., 2002). endocytosis and signaling. Overexpression of the These results suggest that POB1, by binding to central region of POB1 (447-504), inhibits EGF PAG2, suppresses the inhibitory action of PAG2 on endocytosis, titrating essential proteins away, thus the paxillin recruitment to focal contacts. This depauperating the receptor down-regulation suggests that POB1 may function as a scaffold machinery. In fact, this region of POB1/REPS2 protein that interacts with proteins involved in plays its regulatory role in EGFR endocytosis by endocytosis and migration, thus regulating binding: (i) to 14-3-3 proteins in a phosphorylation signaling and motility. PAG2/ASAP1 gene was dependent way (i.e., phospho-Ser493 of POB1), (ii) found associated with prostate cancer metastasis to the C-SH3 domain of Grb2 and (iii) to the SH3 since it is up-regulated in a human metastatic of amphiphysin II. The target of the SH3 domain of prostate subline and immunohistochemistry of amphiphysin and of the carboxy-terminal SH3 of xenograft sections show a significantly strong Grb2 is a short peptide flanking Arg483 in POB1. cytoplasmic ASAP1 protein staining in tumor- These interactions are not EGF dependent and are nonmetastatic PCa2 tissue, compared to a non- probably exclusive, since the binding motifs are staining in benign tissue, and an even stronger only nine amino acids apart. These findings suggest staining in PCa1-metastatic tissue. Moreover, that 14-3-3 could work by bridging the EGF additional ASAP1 gene copies are detected in 58% receptor and the scaffold protein POB1/REPS2. of the primary prostate cancer clinical specimens. A The 14-3-3 binding motif HSRASSLD, flanking the small interfering RNA reducing ASAP1 protein Ser493 of POB1, is conserved in the mouse expression, can suppress in vitro PC-3 cell orthologs and in the 14-3-3 binding motif that migration and matrigel invasion. Therefore flanks the Ser510 of human REPS1 protein, found PAG2/ASAP1 represents a therapeutic target and a phosphorylated in vivo in A431 cells (Stover DR et biomarker for metastatic disease (Lin et al., 2008) al. Phosphosite). The POB1 Ser493 is predicted to while REPS2/POB1 can suppress PAG2 oncogenic- be phosphorylated by Akt. In agreement, when cells metastatic activity. are treated with PI3K/Akt inhibitor wortmannin, 14-3-3 binding to REPS2/POB1 is abolished Mutations (Tomassi et al., 2008). The 14-3-3 zeta has already been reported as associating with the EGFR, Somatic epidermal growth factor receptor, cytoplasmic tail - S324F, cds mutation 971C>T heterozygous in and co-localizing along the plasma membrane with glioblastoma multiforme (Parsons et al., 2008). EGFR upon EGF stimulation (Jin et al., 2004). - V67M, cds mutation 199G>A homozygous in Thus a 14-3-3 dimer could bridge REPS2/POB1 to malignant melanoma. the EGFR upon EGF induction. - No high level gene amplification (>7), 1 Cell migration and paxillin localization: homozygous deletion in breast cancer, 559 LOH REPS2/POB1 antagonises PAG2/ASAP1 (Loss of Heterozygosity). POB1 forms a complex with PAG2/ASAP1 in intact cells. PAG2 is a paxillin-associated protein Implicated in with ADP-ribosylation factor GTPase-activating protein activity, also called ASAP1 (ArfGAP with Non-small cell lung cancer (NSLC) SH3 domain, ankyrin repeat and PH domain and prostate cancer UniProtKB: Q9ULH1). The SH3 of PAG2 binds - Apoptosis in non-small cell lung cancer (NSLC) the proline motif (562PSKPIR567) at the carboxyl- and prostate cancer: REPS2 binds and inhibits terminal region of POB1. This motif is essential for RalBP1. PAG2-POB1 interaction since substitution of the Ikeda et al. (1998) cloned POB1 (partner of two proline residues with alanines in mutant Ralbp1) as the first known binding partner of POB1(PA), impaired its binding to PAG2. POB1 Ralbp1 by the yeast two-hybrid method using may therefore form a complex with paxillin through Ralbp1/RLIP76 as bait and clearly demonstrated PAG2. Paxillin is a focal adhesion-associated specific binding and complex formation between scaffolding protein that recruits signaling molecules Ralbp1 and REPS2/POB1. The binding to RalBP1 to the focal adhesions and forms protein complexes did not affect the GTPase activating activity of that coordinate signaling, cell spreading and RalBP1. The interaction of POB1 with RalBP1 motility. PAG2 overexpression causes loss of induces cell death in human prostate cancer cell endogenous paxillin recruitment to focal contacts ines LNCaP-FGC and LNCaP-LNO. Oosterhoff et and also impaires cell migratory activities. The al. show that REPS2/POB1-induces apoptosis in ability to suppress fibronectin-dependent migration 45% of transfected cells, within 48 hours. When depends on the ArfGAP domain of PAG2, but not prostate cancer cell lines are transfected with a

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REPS2 (RALBP1 associated Eps domain containing 2) Corallino S, Castagnoli L

deletion mutant of REPS2/POB1, lacking the apoptosis (Penninkhof et al., 2004). Hence, the RalBP1 binding domain, only 30-40% of the authors suggest that a decreased expression of transfected cells became apoptotic after 72-96 hours REPS2 might be a key factor in causing prostate (Oosterhoff et al., 2003). cancer cells to avoid apoptosis. REPS2/POB1 (514-660) binds RalBP1 C-terminal Breast cancer amino acids, 499-655, while an almost overlapping region of RalBP1 (440-655) binds the heat shock Note factor 1 Hsf-1 (Hu and Mivechi, 2003). Shingal et Doolan et al. (2009) suggest that REPS2 mRNAs al. show a ternary complex formation between may be useful as favourable prognostic and RalBP1, Hsf-1, and REPS2/POB1. RalBP1, Hsf1, predictive markers for breast cancer. Univariate and HSP90 and tubulin make a complex in cell (Singhal multivariate analyses were used to identify et al., 2008). Hsf-1 and REPS2/POB1 induce drug associations between expression of these transcripts sensitivity and apoptosis by inhibiting RalBP1. and patients' clinicopathological and survival data. Binding of REPS2/POB1 to RALBP1 inhibits the transport activity of the Ral-binding nucleotidase, References which functions as an energy-dependent transporter Ikeda M, Ishida O, Hinoi T, Kishida S, Kikuchi A. for GSH-conjugates as well as unrelated Identification and characterization of a novel protein xenobiotics. RALBP1 (RLIP76) is the major interacting with Ral-binding protein 1, a putative effector transporter of the anthracycline antibiotic, protein of Ral. J Biol Chem. 1998 Jan 9;273(2):814-21 doxorubicin, which is one of the most widely used Koshiba S, Kigawa T, Iwahara J, Kikuchi A, Yokoyama S. anticancer drugs (Singhal et al., 2007). Therefore, Solution structure of the Eps15 homology domain of a REPS2/POB1 is a specific and saturable inhibitor human POB1 (partner of RalBP1). FEBS Lett. 1999 Jan 15;442(2-3):138-42 of the glutathione-electrophile conjugates and of the doxorubicin transport activity of RalBP1. Yadav et Morinaka K, Koyama S, Nakashima S, Hinoi T, Okawa K, Iwamatsu A, Kikuchi A. Epsin binds to the EH domain of al. show that REPS2/POB1 can regulate the POB1 and regulates receptor-mediated endocytosis. transport function of RalBP1/RLIP76 and, in Oncogene. 1999 Oct 21;18(43):5915-22 agreement with previous studies, show that Nakashima S, Morinaka K, Koyama S, Ikeda M, Kishida M, inhibition of RalBP1 induces apoptosis in cancer Okawa K, Iwamatsu A, Kishida S, Kikuchi A. Small G cells through the accumulation of endogenously protein Ral and its downstream molecules regulate formed GSH-conjugates (Yadav et al., 2005). endocytosis of EGF and insulin receptors. EMBO J. 1999 Hence, REPS2/POB1 over-expression inhibits Jul 1;18(13):3629-42 RalBP1-mediated transport of glutathione- Kariya K, Koyama S, Nakashima S, Oshiro T, Morinaka K, conjugates thus promoting apoptosis and can Kikuchi A. Regulation of complex formation of POB1/epsin/adaptor protein complex 2 by mitotic influence drug-efflux mechanisms that cause phosphorylation. J Biol Chem. 2000 Jun resistance to cancer treatment. Hsf-1 also causes 16;275(24):18399-406 specific and saturable inhibition of the transport Oshiro T, Koyama S, Sugiyama S, Kondo A, Onodera Y, activity of RalBP1. The combined augmentation of Asahara T, Sabe H, Kikuchi A. Interaction of POB1, a Hsf-1 and REPS2/POB1 causes nearly complete downstream molecule of small G protein Ral, with PAG2, a inhibition of RalBP1 and a dramatic apoptosis in paxillin-binding protein, is involved in cell migration. J Biol NSLC (non-small cell lung cancer) cell line H358 Chem. 2002 Oct 11;277(41):38618-26 through Ralbp1 binding (Singhal et al., 2008). The Hu Y, Mivechi NF. HSF-1 interacts with Ral-binding protein marked apoptotic effect caused by the increase of 1 in a stress-responsive, multiprotein complex with HSP90 Hsf-1 and REPS2/POB1 in lung cancer cells, in vivo. J Biol Chem. 2003 May 9;278(19):17299-306 suggests a novel targeted therapy in which Oosterhoff JK, Penninkhof F, Brinkmann AO, Anton liposomally encapsulated Hsf-1 and POB1 could be Grootegoed J, Blok LJ. REPS2/POB1 is downregulated during human prostate cancer progression and inhibits used clinically as a therapeutic agent. growth factor signalling in prostate cancer cells. - Apoptosis in prostate cancer cells: Oncogene. 2003 May 15;22(19):2920-5 REPS2/POB1 counteracts the apoptosis Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni inhibitor NF-kappaB p65 S, Metalnikov P, O'Donnell P, Taylor P, Taylor L, Zougman The NF-kappaB subunit p65 is identified as a A, Woodgett JR, Langeberg LK, Scott JD, Pawson T. human REPS2/POB1 protein partner, since the Proteomic, functional, and domain-based analysis of in NPF-motif in p65 acts as binding site for the EH vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization. Curr Biol. 2004 Aug domain in REPS2. However, in cultured prostate 24;14(16):1436-50 cancer cells, the REPS2-p65 interaction is triggered upon stimulation with the phorbol ester, phorbol- Penninkhof F, Grootegoed JA, Blok LJ. Identification of REPS2 as a putative modulator of NF-kappaB activity in 12-myristate-13-acetate (PMA). During prostate prostate cancer cells. Oncogene. 2004 Jul 22;23(33):5607- cancer progression from androgen-dependent to 15 androgen-independent growth, the observed Oosterhoff JK, Kühne LC, Grootegoed JA, Blok LJ. EGF downregulation of REPS2 is accompanied by signalling in prostate cancer cell lines is inhibited by a high upregulation of NF-kappaB activity, that inhibits expression level of the endocytosis protein REPS2. Int J Cancer. 2005 Feb 10;113(4):561-7

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Yadav S, Zajac E, Singhal SS, Singhal J, Drake K, Awasthi An integrated genomic analysis of human glioblastoma YC, Awasthi S. POB1 over-expression inhibits RLIP76- multiforme. Science. 2008 Sep 26;321(5897):1807-12 mediated transport of glutathione-conjugates, drugs and promotes apoptosis. Biochem Biophys Res Commun. Singhal SS, Yadav S, Drake K, Singhal J, Awasthi S. Hsf-1 2005 Mar 25;328(4):1003-9 and POB1 induce drug sensitivity and apoptosis by inhibiting Ralbp1. J Biol Chem. 2008 Jul Santonico E, Panni S, Falconi M, Castagnoli L, Cesareni 11;283(28):19714-29 G. Binding to DPF-motif by the POB1 EH domain is responsible for POB1-Eps15 interaction. BMC Biochem. Tomassi L, Costantini A, Corallino S, Santonico E, 2007 Dec 21;8:29 Carducci M, Cesareni G, Castagnoli L. The central proline rich region of POB1/REPS2 plays a regulatory role in Singhal SS, Singhal J, Nair MP, Lacko AG, Awasthi YC, epidermal growth factor receptor endocytosis by binding to Awasthi S. Doxorubicin transport by RALBP1 and ABCG2 14-3-3 and SH3 domain-containing proteins. BMC in lung and breast cancer. Int J Oncol. 2007 Biochem. 2008 Jul 22;9:21 Mar;30(3):717-25 Doolan P, Clynes M, Kennedy S, Mehta JP, Germano S, Lin D, Watahiki A, Bayani J, Zhang F, Liu L, Ling V, Sadar Ehrhardt C, Crown J, O'Driscoll L. TMEM25, REPS2 and MD, English J, Fazli L, So A, Gout PW, Gleave M, Squire Meis 1: favourable prognostic and predictive biomarkers JA, Wang YZ. ASAP1, a gene at 8q24, is associated with for breast cancer. Tumour Biol. 2009;30(4):200-9 prostate cancer metastasis. Cancer Res. 2008 Jun 1;68(11):4352-9 Oppermann FS, Gnad F, Olsen JV, Hornberger R, Greff Z, Kéri G, Mann M, Daub H. Large-scale proteomics analysis Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, of the human kinome. Mol Cell Proteomics. 2009 Angenendt P, Mankoo P, Carter H, Siu IM, Gallia GL, Olivi Jul;8(7):1751-64 A, McLendon R, Rasheed BA, Keir S, Nikolskaya T, Nikolsky Y, Busam DA, Tekleab H, Diaz LA Jr, Hartigan J, This article should be referenced as such: Smith DR, Strausberg RL, Marie SK, Shinjo SM, Yan H, Riggins GJ, Bigner DD, Karchin R, Papadopoulos N, Corallino S, Castagnoli L. REPS2 (RALBP1 associated Parmigiani G, Vogelstein B, Velculescu VE, Kinzler KW. Eps domain containing 2). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):288-292.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3) 292

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Mini Review

XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6) Sabina Pucci, Maria Josè Zonetti Lab of Molecular Pathology, Dept of Biopathology, University of Rome "Tor Vergata", Via Montpellier, 00133 Rome, Italy (SP, MJZ)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/XRCC6ID246ch22q13.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI XRCC6ID246ch22q13.txt

Identity Localisation Other names: CTC75, CTCBF, G22P1, KU70, Ku was originally reported to be a nuclear protein, ML8, TLAA consistent with its functions as a subunit of DNA- PK involved in DNA double strand breaks repair. HGNC (Hugo): XRCC6 However, several studies have revealed the Location: 22q13.2 cytoplasmic or cell surface localization of Ku proteins in various cell types (Prabhakar et al., DNA/RNA 1990). Recently, it has been demonstrated that the shift from the nucleus to the cytoplasm of the Description Ku70/Ku80 proteins in tumor cells could represents The KU70 gene is composed of 13 exons. a mechanism to inhibit cell death through the Ku70- Transcription Bax-sCLU interactions, giving rise to a new chemoresistant clone with a more aggressive 2156 bp mRNA. phenotype. Protein Function Ku is a heterodimeric protein composed of two Description subunits with molecular weight of 70 and 86 kDa. The Ku70 protein is 609 amino acid long and its Ku forms a complex with the DNA-dependent molecular weight is 69.8 kDa. It is composed of 3 protein kinase catalytic subunit (DNA-PKcs) to domains: an amino (N) amino-terminal alpha/beta form the full DNA-dependent protein kinase, DNA- domain, a central beta-barrel domain and a helical PK, consisting of 470 kDa and required for the non- C-terminal arm (Rivera-Calzada et al., 2007). The homologous end joining (NHEJ) pathway of DNA C-terminal region consists of a 5 kDa SAP domain repair. The Ku heterodimer binds the ends of (Ku70-SAP) which involved in DNA binding various types of DNA discontinuity, and is involved during NHEJ reaction. in the repair of DNA breaks caused by an incorrect DNA replication, V(D)J recombination, Expression physiological oxidations, ionizing irradiation, and Ku70 is ubiquitously expressed. Changes in Ku70 some chemotherapeutic drug effects (Featherstone expression correlated to a pathological state. and Jackson, 1999).

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XRCC6 (X-ray repair complementing defective repair in Pucci S, Zonetti MJ Chinese hamster cells 6)

Ku70/80-CLU-Bax interactions. (A) Bax is localized inactive in the cytoplasm in normal, undamaged cell interacting with the Ku70 protein C-terminus. sCLU stabilizes the Ku70-Bax interaction in the cytoplasm acting as cytoprotectant. (B) After DNA damage inducing DNA double-strand breaks repair (UV treatment, ionizing radiation, etc.) Ku70 allows the translocation of Bax to the mitochondria inducing apoptosis (Mazzarelli et al., 2009).

The principal role of Ku proteins is to take care of colon cancer. DNA repair is inhibited in high the homeostasis of the genome being involved in infiltrative colon carcinoma by Ku80 loss and Ku70 telomere maintenance, specific gene transcription, cell compartment shift (from the nucleus to the DNA replication, cell-cycle regulation and cytoplasm). regulation of apoptosis induction. Ku70 has been Moreover in colorectal carcinoma was shown to bind to the pro-apoptotic protein BAX in demonstrated a very important role of Ku70 the cytoplasm in normal, undamaged cell. After expression, localization, and physical interaction DNA damage inducing DNA double-strand breaks with CLU and Bax. In fact the Ku70-CLU-Bax repair (UV treatment, ionizing radiation, etc.) Ku70 colocalization in the cytoplasm and an increase in allows the translocation of Bax to the mitochondria Ku70-CLU-Bax binding were observed in highly leading to the release of death-promoting factors, aggressive human colon cancer (Pucci et al., 2004; such as cytochrome c, in the cytoplasmic Pucci et al., 2009c), confirming that these compartment. interactions regulate the Bax-dependent cell death. In normal cells, after an irreversible cell damage, Breast cancer nCLU cooperates with Ku70 to induce apoptotic death, activating the translocation of Bax to Note mitochondria whereas the sCLU protein stabilizes Experimental data further reported an inactivation the Ku70-Bax interaction in the cytoplasm acting as of Ku DNA-binding activity, essential for genomic cytoprotectant. The Ku70-Bax-sCLU interaction in stability in breast and in bladder carcinomas. A the cytoplasm seems to play an important role in dysfunction of this protective activity let the cell survival pathways and in cell death escape, that aberrant cell clone growing. In highly infiltrative in pathological condition could lead to the survival and metastatic tumors of the breast and bladder, the of the aberrant cell clone. Overall, the dynamic impaired DNA-repair activity is due to the loss of interactions among CLU, Ku70, and Bax seems to Ku86 (Pucci et al., 2001) and to the Ku70 shifting have an important role in both tumor insurgence from the nucleus to the cytoplasm. The shift from and its progression (Pucci et al., 2009a; Pucci et al., the nucleus to the cytoplasm of the Ku70/80 2009b). proteins in tumor cells could represents a mechanism to inhibit cell death through the Implicated in cooperative interaction with sCLU, giving rise to a new chemoresistant clone with a more aggressive Colon cancer phenotype. Note The colon cancer expression and the localization of Ku70 and Ku80 are related to tumor progression in

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XRCC6 (X-ray repair complementing defective repair in Pucci S, Zonetti MJ Chinese hamster cells 6)

Tumor-specific modulation of Ku70/80 in human colon cancer. Ku70 staining was strongly positive in the nuclei of normal mucosa. In node-negative carcinomas (pT3N0) Ku70 expression slightly decreased and it localized mainly in the nucleus. In node-positive carcinomas (pT3N1) Ku70 staining was distributed mainly in cytoplasm. The expression of Ku86 was positive in the nuclei of control tissues (normal mucosa). Nuclear Ku86 expression was strongly decreased in node-negative tumors (pT3N0). No staining for Ku86 was found in the nucleus or in the cytoplasm of node-positive carcinomas (pT3N1).

Ku70-Bax-CLU pathological interaction. Apoptosis escaping. The shift of clusterin forms production, the loss of Ku80, and the cytoplasmic relocalization of Ku70 are related to cell death inhibition and cancer progression.

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XRCC6 (X-ray repair complementing defective repair in Pucci S, Zonetti MJ Chinese hamster cells 6)

Mazzarelli P, Pucci S, Spagnoli LG. CLU and colon References cancer. The dual face of CLU: from normal to malignant phenotype. Adv Cancer Res. 2009;105:45-61 Prabhakar BS, Allaway GP, Srinivasappa J, Notkins AL. Cell surface expression of the 70-kD component of Ku, a Pucci S, Bonanno E, Sesti F, Mazzarelli P, Mauriello A, DNA-binding nuclear autoantigen. J Clin Invest. 1990 Ricci F, Zoccai GB, Rulli F, Galatà G, Spagnoli LG. Oct;86(4):1301-5 Clusterin in stool: a new biomarker for colon cancer screening? Am J Gastroenterol. 2009 Nov;104(11):2807- Featherstone C, Jackson SP. Ku, a DNA repair protein 15 with multiple cellular functions? Mutat Res. 1999 May 14;434(1):3-15 Pucci S, Mazzarelli P, Nucci C, Ricci F, Spagnoli LG. CLU "in and out": looking for a link. Adv Cancer Res. Pucci S, Mazzarelli P, Rabitti C, Giai M, Gallucci M, 2009;105:93-113 Flammia G, Alcini A, Altomare V, Fazio VM. Tumor specific modulation of KU70/80 DNA binding activity in breast and Pucci S, Mazzarelli P, Sesti F, Boothman DA, Spagnoli bladder human tumor biopsies. Oncogene. 2001 Feb LG. Interleukin-6 affects cell death escaping mechanisms 8;20(6):739-47 acting on Bax-Ku70-Clusterin interactions in human colon cancer progression. Cell Cycle. 2009 Feb 1;8(3):473-81 Pucci S, Bonanno E, Pichiorri F, Angeloni C, Spagnoli LG. Modulation of different clusterin isoforms in human colon This article should be referenced as such: tumorigenesis. Oncogene. 2004 Mar 25;23(13):2298-304 Pucci S, Zonetti MJ. XRCC6 (X-ray repair complementing Rivera-Calzada A, Spagnolo L, Pearl LH, Llorca O. defective repair in Chinese hamster cells 6). Atlas Genet Structural model of full-length human Ku70-Ku80 Cytogenet Oncol Haematol. 2011; 15(3):293-296. heterodimer and its recognition of DNA and DNA-PKcs. EMBO Rep. 2007 Jan;8(1):56-62

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Leukaemia Section Short Communication t(6;22)(p21;q11) Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0622p21q11ID1563.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t0622p21q11ID1563.txt

Identity

t(6;22)(p21;q11) G-banding and FISH - Courtesy Claudia Haferlach.

Clinics and pathology Note So far, the t(6;22)(p21;q11) is heterogeneous, and Disease its significance remains problematic. Hematological malignancies.

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Phenotype/cell stem origin References Four cases availble: one case was a myeloid type Gödde-Salz E, Schmitz N, Bruhn HD. Philadelphia blast crisis of chronic myeloid leukemia (CML) in a chromosome (Ph) positive chronic myelocytic leukemia 36-year-old male patient with a t(9;22)(q34;q11); (CML): frequency of additional findings. Cancer Genet another case was a CML aberrant translocation Cytogenet. 1985 Jan 15;14(3-4):313-22 t(6;22)(p21;q11) without apparent involvement of Fears S, Vignon C, Bohlander SK, Smith S, Rowley JD, chromosome 9 in a 44-year-old male patient; a third Nucifora G. Correlation between the ETV6/CBFA2 case was that of a B-cell precursor (CD10+) L1- (TEL/AML1) fusion gene and karyotypic abnormalities in acute lymphoblastic leukemia in a 2-year-old girl children with B-cell precursor acute lymphoblastic leukemia. Genes Chromosomes Cancer. 1996 who was still in complete remission 72 months after Oct;17(2):127-35 diagnosis. A cryptic 5' ETV6 - 3' RUNX1 was Tanaka H, Tanaka K, Oguma N, Ito K, Ito T, Kyo T, Dohy found; there were accompanying anomalies, of H, Kimura A. Effect of interferon-alpha on chromosome which a +10 and a +21; the last case was a chronic abnormalities in treated chronic myelogenous leukemia lymphocytic leukemia (CLL) stage C with also a patients. Cancer Genet Cytogenet. 2004 Sep;153(2):133- del(11q), and a del(13q). 43 Mayr C, Speicher MR, Kofler DM, Buhmann R, Strehl J, Genes involved and Busch R, Hallek M, Wendtner CM. Chromosomal translocations are associated with poor prognosis in proteins chronic lymphocytic leukemia. Blood. 2006 Jan 15;107(2):742-51 Note Genes involved, if any, are unknown. This article should be referenced as such: Huret JL. t(6;22)(p21;q11). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):297-298.

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Solid Tumour Section Short Communication t(11;22)(q24;q12) in giant cell tumour of bone Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Tumors/t1122q24q12GiantCellID6283.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t1122q24q12GiantCellID6283.txt

Clinics and pathology Protein From N-term to C-term: a transactivation domain Disease (TAD) containing multiple degenerate hexapeptide Giant cell tumour of bone is a locally destructive repeats, 3 arginine/glycine rich domains (RGG tumor, usually seen in patients over 20 years of age, regions), a RNA recognition motif, and a RanBP2 a borderline lesion between benign and malignant type Zinc finger. Role in transcriptional regulation tumours, with a good prognosis, despite of for specific genes and in mRNA splicing. recurrences and, more rarely, pulmonary metastases. The most frequent genetic finding is Result of the chromosomal telomeric associations. anomaly Genetics Hybrid Gene Note Description In a study by Scotlandi et al., 2000, was found that 5' EWSR1 - 3' FLI1. EWSR1 exon 7 is fused in a minor population of cells from giant cell tumour frame to FLI1 exon 6 and/or 5 (type 1 and type 2 of bone samples had an EWSR1/FLI1 transcript, fusions respectively), indicating, when both but this was found in a high percentage of samples transcripts were produced in a given sample, (13/15). genetic heterogeneity within the tumour. Genes involved and Fusion Protein Description proteins Fusion of the N terminal transactivation domain of EWSR1 to the ETS type DNA binding domain of FLI1 FLI1. Location 11q24 References Protein Scotlandi K, Chano T, Benini S, Serra M, Manara MC, From N-term to C-term: a 5' ETS domain, a Fli-1- Cerisano V, Picci P, Baldini N. Identification of EWS/FLI-1 specific transcriptional activation domain, and a 3' transcripts in giant-cell tumor of bone. Int J Cancer. 2000 Aug 1;87(3):328-35 ETS transcriptional activation domain. Member of ETS transcription factor gene family. FLI1 binds to This article should be referenced as such: DNA in a sequence-specific manner. Huret JL. t(11;22)(q24;q12) in giant cell tumour of bone. EWSR1 Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):299. Location 22q12

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Solid Tumour Section Short Communication t(11;22)(q24;q12) in rhabdomyosarcomas (RMS) Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Tumors/t1122q24q12RhabdoID6285.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t1122q24q12RhabdoID6285.txt

Clinics and pathology Genes involved and Disease proteins Rhabdomyosarcomas, the most common pediatric FLI1 soft tissue sarcomas, are tumours related to the skeletal muscle lineage. The 2 major subtypes are Location alveolar rhabdomyosarcoma (ARMS) and 11q24 embryonal rhabdomyosarcoma (ERMS). Other Protein subtypes are botryoid, spindle cell, anaplastic, From N-term to C-term: a 5' ETS domain, a Fli-1- pleomorphic, and undifferentiated RMS. specific transcriptional activation domain, and a 3' Note ETS transcriptional activation domain. Member of Most ARMS cases are characterised by either a ETS transcription factor gene family. FLI1 binds to t(2;13)(q35;q14), resulting in a PAX3/FOXO1 DNA in a sequence-specific manner. hybrid gene, or a t(1;13)(p36;q14) resulting in a EWSR1 PAX7/FOXO1 hybrid gene. Most ERMS are Location characterized by chromosome gains and a loss of 22q12 heterozygocity in 11p15. Protein Epidemiology From N-term to C-term: a transactivation domain Three cases of RMS with t(11;22)(q24;q12) have (TAD) containing multiple degenerate hexapeptide been described to date, including a two-years-old repeats, 3 arginine/glycine rich domains (RGG girl with a mixed embryonal and alveolar RMS, regions), a RNA recognition motif, and a RanBP2 who died 14 months after diagnosis, a 4.5-year-old type Zinc finger. Role in transcriptional regulation girl, also with a mixed embryonal and alveolar for specific genes and in mRNA splicing. RMS, who was alive and well 9 months after diagnosis (Sorensen et al., 1993; Thorner et al., Result of the chromosomal 1996). anomaly Genetics Hybrid Gene Note Description A t(2;13) hybrid transcript was excluded in the two 5' EWSR1 - 3' FLI1. EWSR1 exon 7 is fused in cases described by Thorner et al., 1996. In the 4.5- frame to FLI1 exon 6. year-old girl case, a highly abnormal karyotype was found, with 85 to 200 chromosomes per mitosis, Fusion Protein and MDM2 was amplified more than a hundred Description times.

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Fusion of the N terminal transactivation domain of Thorner P, Squire J, Chilton-MacNeil S, Marrano P, Bayani EWSR1 to the ETS type DNA binding domain of J, Malkin D, Greenberg M, Lorenzana A, Zielenska M. Is the EWS/FLI-1 fusion transcript specific for Ewing sarcoma FLI1. and peripheral primitive neuroectodermal tumor? A report of four cases showing this transcript in a wider range of References tumor types. Am J Pathol. 1996 Apr;148(4):1125-38 Sorensen PH, Liu XF, Delattre O, Rowland JM, Biggs CA, This article should be referenced as such: Thomas G, Triche TJ. Reverse transcriptase PCR amplification of EWS/FLI-1 fusion transcripts as a Huret JL. t(11;22)(q24;q12) in rhabdomyosarcomas diagnostic test for peripheral primitive neuroectodermal (RMS). Atlas Genet Cytogenet Oncol Haematol. 2011; tumors of childhood. Diagn Mol Pathol. 1993 Sep;2(3):147- 15(3):300-301. 57

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Solid Tumour Section Short Communication t(11;22)(q24;q12) in solid pseudopapillary tumour of the pancreas Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Tumors/t1122q24q12PseudopapID6284.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t1122q24q12PseudopapID6284.txt

Clinics and pathology EWSR1 Location Disease 22q12 Solid pseudopapillary tumour of the pancreas is a Protein rare epithelial tumour of low malignancy, typically From N-term to C-term: a transactivation domain occuring in young female patients (mean age 27 (TAD) containing multiple degenerate hexapeptide years, sex ratio is 1 male to 8 or 9 female patients). repeats, 3 arginine/glycine rich domains (RGG It accounts for 1% of all pancreatic tumours. It is a regions), a RNA recognition motif, and a RanBP2 encapsulated lesion with well-defined borders, with type Zinc finger. Role in transcriptional regulation about 15% of cases demonstrating malignant for specific genes and in mRNA splicing. behaviour with recurrence and metastasis (Yu et al., 2010). Result of the chromosomal Cytogenetics anomaly Cytogenetics Morphological Hybrid Gene A t(11;22)(q24;q12), accompanied with +8, was Description found in one case (Maitra et al., 2000). 5' EWSR1 - 3' FLI1. EWSR1 exon 7 is fused in frame to FLI1 exon 6 (type 1 fusion). Genes involved and Fusion Protein proteins Description Fusion of the N terminal transactivation domain of FLI1 EWSR1 to the ETS type DNA binding domain of Location FLI1. 11q24 Protein References From N-term to C-term: a 5' ETS domain, a Fli-1- Maitra A, Weinberg AG, Schneider N, Patterson K. specific transcriptional activation domain, and a 3' Detection of t(11;22)(q24;q12) translocation and EWS-FLI- ETS transcriptional activation domain. Member of 1 fusion transcript in a case of solid pseudopapillary tumor of the pancreas. Pediatr Dev Pathol. 2000 Nov- ETS transcription factor gene family. FLI1 binds to Dec;3(6):603-5 DNA in a sequence-specific manner.

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Yu PF, Hu ZH, Wang XB, Guo JM, Cheng XD, Zhang YL, Xu Q. Solid pseudopapillary tumor of the pancreas: a review of 553 cases in Chinese literature. World J Gastroenterol. 2010 Mar 14;16(10):1209-14

This article should be referenced as such: Huret JL. t(11;22)(q24;q12) in solid pseudopapillary tumour of the pancreas. Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):302-303.

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Deep Insight Section

MTA1 of the MTA (metastasis-associated) gene family and its encoded proteins: molecular and regulatory functions and role in human cancer progression Yasushi Toh, Garth L Nicolson Department of Gastroenterological Surgery, National Kyushu Cancer Center, Fukuoka, 811-1395, Japan (YT), Department of Molecular Pathology, The Institute for Molecular Medicine, Huntington Beach, CA 92647, USA (GLN)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Deep/MTAinCancerID20088.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI MTAinCancerID20088.txt

Abstract MTA1(a metastasis-associated gene) is a newly discovered human gene (residing on chromsome 14q32.3) that belongs to a family of cancer progression-related genes (MTA). The mRNA product of MTA1along with its protein product MTA1 have been reported to be over-expressed in a wide variety of animal and human tumors. For example, the expression of MTA1 and its encoded protein MTA1 correlates with the malignant properties of many human cancers, including cancers of the breast, colon, stomach, liver, prostate and others. The MTA proteins have been shown to be ubiquitinated transcriptional co-repressors that function in histone deacetylation and are part of the NuRD complex, a nucleosome remodeling and histone deacetylating complex whose stability appears to be regulated by ubiquitinated MTA1 binding to E3 ubiquitin ligase constitutive photomorphogenesis protein-1 (COP1). The MTA1 protein plays an essential role in c-MYC-mediated cell transformation, and its expression correlates with mammary gland tumor formation. In the latter, MTA1 helps convert mammary cells to more aggressive phenotypes by repression of the estrogen receptor (ER) via trans-activation through deacetylation of chromatin in the ER-responsive element of ER-responsive genes. Another member of the MTA family, MTA3, is induced by estrogen and represses the expression of the transcriptional repressor Snail, a master regulator of epithelial to mesenchymal transformation, resulting in the expression of the cell adhesion molecule E-cadherinand maintenance of a differentiated, normal epithelial phenotype in mammary cells. An important activity mediated by both MTA1 and MTA2 is deacylation and inactivation of tumor suppressor p53protein, in part by controlling its stability by inhibiting ubiquitination, leading to inhibition of growth arrest and apoptosis. Another factor deacetylated and stabilized by MTA1 NuRD complex is hypoxia-inducible factor- 1α (HIF-1α), which is involved in angiogenesis. Therefore, the MTA proteins represent a possible set of master co-regulatory molecules involved in the carcinogenesis and progression of various malignant tumors. As such, they could be important new tools for cancer diagnosis and treatment.

Although additional cancer-related molecules will 1. Introduction - The MTA gene be identified in the future, these molecules must family fulfill two major requirements in order to be An important advance in cancer research has been clinically useful as molecular targets useful for the the discovery of a wide variety of new molecules diagnosis and treatment of human cancers (Toh and involved in carcinogenesis and cancer progression. Nicolson, 2009).

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MTA1 of the MTA (metastasis-associated) gene family and its Toh Y, Nicolson GL encoded proteins: molecular and regulatory functions and role in human cancer progression

The first is that abnormalities must occur in the examine the significance of the expression of MTA expression or structure of the molecules of interest, family members in human cancers and the and their clinical relevance must be definitely important molecular mechanisms that are currently demonstrated in independent studies on human known by which MTA proteins exert their cellular cancers. The second is that the underlying actions as well as discuss the potential clinical molecular mechanisms necessary for the molecules applications of this protein family for the diagnosis to exert their functions in carcinogenesis or cancer and treatment of human cancers. progression must be determined and confirmed in animal tumor models and clinical specimens. 2. The MTA family of proteins, There have been a number of cancer-related genes their structures and cell location and molecules that have been discovered in the last The MTA proteins represent a family of gene few years. In our laboratory we identified a products encoded by three distinct genes (MTA1, candidate metastasis-associated gene by the use of a MTA2 and MTA3), and six reported isoforms differential cDNA screening method. Using this (MTA1, MTA1s, MTA1-ZG29p, MTA2, MTA3, approach we identified a gene that was and MTA3L). The molecular masses of the gene differentially over-expressed in highly metastatic products of MTA1, MTA2 and MTA3 are rat mammary adenocarcinoma cell lines compared approximately 80 kDa, 70 kDa and 65 kDa, to poorly metastatic lines (Toh et al., 1994; Toh et respectively (Manavathi and Kumar, 2007; Toh and al., 1995). When this gene was sequenced, it was Nicolson, 2009). The nucleotide and protein shown to be a completely new, novel gene without alignment homologies and the phylogenetic any homologous or related genes in the database at comparative analyses have been discussed the time. This gene was named mta1 (metastasis- previously (Bowen et al., 2004; Manavathi and associated gene-1). A homologous gene was also Kumar, 2007; Toh and Nicolson, 2009). expressed in human cancer cell lines, and its human The MTA gene family sequences, with the cDNA counterpart, MTA1, was subsequently cloned exception of ZG-29p, contain several common (Nawa et al., 2000) and found to reside on domain structures (Singh and Kumar, 2007; Toh chromosome 14q32.3 (Cui et al., 2001). and Nicolson, 2009). One of these, the BAH Using surgically removed human tissues we (bromo-adjacent homology) domain is involved in showed that high levels of MTA1mRNA expression protein-protein interactions. Another, the SANT were correlated to the invasive and growth (SWI, ADA2, N-CoR, TFIIIB-B) domain shares a properties of gastrointestinal cancers, including high degree of homology with the DNA-binding esophageal, gastric and colorectal cancers (Toh et domain of the Myb-related proteins, suggesting that al., 1997; Toh et al., 1999). After these studies, this domain may be involved in DNA-binding. The several reports from other groups found similar ELM (egl-27 and MTA1 homology) domain has an correlations between MTA1 expression and the unknown function but could be involved in malignant potentials of human cancers (reviewed in embryonic patterning (Solari et al., 1999). Toh and Nicolson, 2009). The MTA family members contain a highly In addition to MTA1, other genes related to MTA1 conserved GATA-type zinc finger motif, suggesting have now been identified. This gene family, which direct interactions with DNA (Nawa et al., 2000). we termed the MTAfamily, now has several The MTA1 protein has two src-homology (SH)- members plus some splice variants (Toh and binding motifs at its C-terminal end-such binding Nicolson, 2003; Manavathi and Kumar, 2007; Toh domains are known to be important in signal and Nicolson, 2009). Furthermore, studies on the transduction involving kinase, adaptor and molecular biological and biochemical properties of scaffolding proteins (Toh et al., 1994; Toh et al., the MTA family have shown that the gene products 1995; Singh and Kumar, 2007). Similarly, SH2- of the main members of the family (proteins and SH3-binding domains are also found in MTA2 MTA1, MTA2 and MTA3) are tightly associated in and MTA3 protein sequences (Toh et al., 1994; Toh a protein complex called NuRD (nucleosome et al., 1995; Singh and Kumar, 2007). These remodeling and histone deacetylation), which has common domain structures demonstrate that the transcriptional regulatory functions via histone MTA family is involved in protein-protein and deacetylation and chromatin remodeling (Toh et al., protein-DNA interactions, indicating the anticipated 2000; Bowen et al., 2004). Interestingly, histone roles of the MTA family of proteins in signal deacetylase activities correlate with squamous cell transduction and transcriptional regulation (Toh and carcinoma invasion (Toh et al., 2003). At the Nicolson, 2003; Singh and Kumar, 2007; Toh and moment, the MTA family has attracted widespread Nicolson, 2009). attention as one of several key molecules that play In addition to protein-protein and protein-DNA indispensable roles in the pathogenesis and binding, MTA proteins contain basic nuclear progression of a wide variety of cancers (Toh and localization signals (Toh et al., 1994; Toh et al., Nicolson, 2003; Kumar et al., 2003; Manavathi et 1995; Singh and Kumar, 2007). They also localize al., 2007b; Toh and Nicolson, 2009). We will in the nucleus in many human cancer cells (Toh et

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MTA1 of the MTA (metastasis-associated) gene family and its Toh Y, Nicolson GL encoded proteins: molecular and regulatory functions and role in human cancer progression al.,1997; Toh et al., 1999); however, MTA1 MTA1-transgenic mice revealed inappropriate localizes to both the cytoplasm and nucleus in some development of their mammary glands. The MTA1- tumors (Moon et al., 2004; Balasenthil et al., 2006; transgenic mice eventually developed hyperplastic Bagheri-Yarmand et al., 2007). MTA3 also nodules and mammary tumors, including mammary localizes to the nucleus, but without any apparent adenocarcinomas and lymphomas. nuclear localization signal (Fujita et al., 2003). A The involvement of MTA1 in the carcinogenesis short splice-variant of MTA1, called MTA1s, is and progression of breast cancers was also shown predominantly localized in the cytoplasm (Kumar et by Martin et al. (2001; 2006). First, they mapped al., 2002). the chromosomal locus in 14q that might be responsible for axillary lymph node metastases in 3. MTA protein expression in human breast cancers by comparing the rate of loss various cancers and its possible of heterozygosity between node-positive and node- clinical relevance negative breast cancers. The MTA1 gene was found Since the report by Toh et al. (1997) that the over- to be contained in the same gene locus, suggesting expression of MTA1was significantly correlated to that MTA1is a candidate for a breast cancer the malignant properties of human gastric and metastasis-promoting gene. Next, using colorectal cancers, there have been several reports immunohistochemistry they examined MTA1 on the expression levels of MTA family members, protein expression in primary human breast cancer especially of MTA1, in various human cancers samples and demonstrated that node-negative breast (reviewed in Toh and Nicolson, 2009). These cancers with over-expression of MTA1 protein had studies revealed that the expression levels of MTA a higher risk of disease relapse similar to node- family members correlate with pathogenic positive tumors. Therefore, the over-expression of significance and prognosis (Toh and Nicolson, MTA1 was proposed as a potential predictor of 2009). The biological relevance of MTA proteins to early disease relapse independent of node status carcinogenesis and cancer progression has been (Martin et al,. 2006). investigated in a few cancer models, such as breast Using surgically resected human breast cancer and gastrointestinal cancers, and these will be specimens Jang et al. (2006) showed that MTA1 discussed in more detail below. over-expression was closely associated with higher tumor grade and high intratumoral microvessel 3.1 MTA1 protein and breast cancer density. This suggested that MTA1 could be a MTA1 protein was originally identified as a useful predictor of an aggressive phenotype, and the candidate cancer progression molecule that was MTA1 molecule could be considered as a possible associated with breast cancer metastasis (Toh et al., angiogenesis-promoting molecule in breast cancers 1994; Toh et al., 1995). Subsequently, using (Jang et al., 2006). antisense RNA of MTA1 a role for MTA1 in the 3.2 MTA1 protein and gastrointestinal cancers growth properties of metastatic breast cancer cells was investigated. Using MTA1antisense RNA we MTA1 over-expression has been shown to be found that the growth rates of highly metastatic pathogenically significant in human gastrointestinal breast cancer cell lines were inhibited significantly cancers. Using a reverse-transcription polymerase in a dose-dependent manner (Nawa et al., 2000). chain reaction (RT-PCR) method on surgically More direct evidence to demonstrate an association resected human gastric and colorectal cancer of MTA1 expression levels with breast cancer specimens were compared to paired normal malignant properties was obtained by Mazumdar et counterpart tissues, and we found that the higher al. (2001). They demonstrated that forced expression of MTA1 mRNA was significantly expression of the MTA1 protein in the human correlated to the depth of cancer invasion and breast cancer cell line MCF-7 was accompanied by extent of lymph node metastasis (Toh et al., 1997). an enhancement in the ability of MCF-7 cells to This study was the first to demonstrate the clinical invade an artificial matrix and ability to grow in an relevance of MTA1 expression to the malignant anchorage-independent manner. They also showed potentials of human cancers. Over-expression of that the enhancement was associated with the MTA1 mRNA was also shown in colorectal cancers interaction between MTA1 protein and histone compared to the normal counterpart tissues deacetylase, resulting in a repression of estrogen (Giannini et al., 2005). receptor (ER) mediated transcription (Mazumdar et Esophageal cancers have also been investigated for al., 2001). MTA1/MTA1 over-expression. Using a RT-PCR Using an animal model the Mazumdar et al. (2001) method we found that human esophageal squamous study was extended by further experiments cell cancers over-express MTA1mRNA (Toh et al., demonstrating a direct in vivo effect of MTA1 on 1999). The over-expressing cancer cells showed the carcinogenesis of breast cancer cells (Bagheri- significantly higher frequencies of adventitial Yarmand et al., 2004; Singh and Kumar, 2007). invasion and lymph node metastasis and tended to This group established a transgenic mice system have higher rates of lymphatic involvement (Toh et that over-expressed the MTA1 protein, and these al., 1999). Using immunohistochemistry we further

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MTA1 of the MTA (metastasis-associated) gene family and its Toh Y, Nicolson GL encoded proteins: molecular and regulatory functions and role in human cancer progression examined the expression levels of MTA1 protein in resected human HCC specimens and found that human esophageal squamous cell cancers and over-expression of MTA1 was associated with reconfirmed the results obtained by RT-PCR (Toh HCC growth and vascular invasion and that nuclear et al., 2004). In the same study we also localization of ERa inversely correlated with MTA1 demonstrated that MTA1 protein was a predictor of expression. This suggested that MTA1 was poor prognosis after surgery (Toh et al., 2004). involved in negative regulatory mechanisms. The roles of MTA1/MTA1 in small intestinal Recently, Yoo et al. (2008) demonstrated that cancers have also been evaluated. Kidd et al. hepatitis B virus (HBV) X (HBx) protein strongly (2006a; 2007) and Modlin et al. (2006b) showed induced the expression of MTA1 and histone that it was useful to examine the expression of deacetylase 1 (HDAC1). This suggests that positive MTA1mRNA and MTA1 protein in order to crosstalk between HBx and MTA1/HDAC1 determine the malignant potential and the complex may occur, and this could be important in propensity to metastasize of enterochromaffin cell stabilizing hypoxia-inducible factor-1α (HIF1-1α), cancers (small intestinal carcinoid tumors). When which appears to play a critical role in angiogenesis compared to nonmetastatic primary tumors, the and metastasis of HBV-associated HCC (Yoo et al., expression of MTA1 was increased in malignant, 2008). Interestingly, it was reported that MTA1 was invasive small intestinal carcinoid tumors and in closely associated with microvascular invasion, metastases to liver and lymph nodes. In these cells frequent postoperative recurrence, and poor loss of TGFβ expression modified expression, prognosis in patients with HCC, especially in those including increased MTA1 expression, of the genes with HBV-associated HCC (Ryu et al., 2008). involved in malignant behavior (Kidd et al., 2007). 3.3 MTA1 protein and other cancers It was further reported that MTA1was a good The reports on MTA1/MTA1 over-expression in candidate genetic molecular marker to discriminate human cancers have been reinforced by the between gastric carcinoids and other gastric experimental over-expression or under-expression neoplasms (Kidd et al., 2006b) as well as malignant of MTA1 in human cells. For example, Mahoney et appendiceal carcinoids from benign tissue (Modlin al. (2002) transfected MTA1 cDNA into et al., 2006a). In these studies, MTA1 and MTA1 immortalized human keratinocytes and expression were thought to be good markers of the demonstrated that forced over-expression of malignant potential of carcinoid tumors. MTA1contributed to some metastatic cell Other gastrointestinal-linked cancers, such as a properties, such as increased cell migration, pancreatic cancers and hepatocellular carcinomas, invasion and survival in an anchorage independent have also been examined for the involvement of medium. Nawa et al. (2000) used antisense MTA1 MTA1/MTA1 over-expression in carcinogenesis to suppress MTA1 levels and inhibit the growth of and cancer progression. Iguchi et al. (2000) breast cancer cells in vitro. These authors examined MTA1 mRNA expression in pancreatic subsequently showed that in vitro invasion of cancer cell lines and resected pancreatic cancer human MBA-MB-231 cells could be inhibited by tissues and found that increased levels of MTA1 antisense MTA1 (Nicolson et al., 2003). Similarly, mRNA expression in the more progressed using a human esophageal squamous cell carcinoma pancreatic cancers. Direct evidence on the role of cell line, Qian et al. (2005) inhibited MTA1 MTA1/MTA1 in the progression of pancreatic expression by RNA interference and found cancer was provided by Hofer et al. (2004). Using a significant inhibition of the cells' in vitro invasion pancreatic cell line (PANC-1) they transfected and migration properties. MTA1cDNA into the cells and found that enhanced Possible relationships between MTA1/MTA1 expression of MTA1 promoted the acquisition of an expression and malignant cell properties, such as invasive and metastatic phenotype and enhanced invasion and metastasis, have been investigated in the malignant potentials of the transformed cells other carcinoma and sarcoma systems. Using (pancreatic adenocarcinomas) by modulation of the human non-small cell lung cancer cells high cytoskeleton via IQGAP1. In addition, Miyake et expression of MTA1 mRNA was correlated with al. (2008) showed the expression level of the lymph node metastasis (Sasaki et al., 2002). This MTA1 protein correlated with poorer prognosis of has also been found to be the case in human ovarian pancreatic cancer patients. cancers (Yi et al., 2003). Additionally, in thymomas An association between MTA1/MTA1 expression advanced stage and invasiveness was related to and the malignant properties of hepatocellular MTA1 expression (Sasaki et al., 2001). carcinomas (HCC) was first reported by Hamatsu et Using various techniques the relationship between al. (2003). In this study, MTA1 mRNA level was MTA1 expression and malignancy has been assessed by RT-PCR in resected human HCC investigated in various cancers. For example, a tissues, and its high expression predicted a lower potential role for MTA1 protein over-expression in disease-free survival rate after curative HCC the progression of human endometrial hepatectomy. Using immunohistochemistry Moon carcinomashas been found by Balasenthil et al. et al. (2004) examined MTA1 protein expression in (2006). Whereas in prostate cancers Hofer et al.

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(2004) showed that metastatic prostate tumors had Only then can MTA proteins be utilized for significantly higher levels of MTA1 protein diagnosis or treatment of human cancers. There are expression and higher percentages of tissue cores several important cellular functions of MTA staining positive for MTA1 protein over-expression proteins that have been recently clarified, such as than in clinically localized prostate cancers or those that are related to carcinogenesis and cancer benign prostate lesions. Most interestingly, using progression. transgenic mice Kumar's group showed that MTA1 4.1 MTA proteins and the nucleosome over-expression was accompanied by a high remodeling-histone deacetylation (NuRD) incidence of spontaneous B cell lymphomas, complex and transcriptional regulation including diffuse large B cell lymphomas (Bagheri- The molecular and biochemical functions of the Yarmand et al., 2007; Balasenthil et al., 2007). The MTA1 protein were first investigated by four high expression of MTA1 in human diffuse B-cell independent groups. In these studies, two different lymphomashas been reported (Hofer et al., 2006). chromatin-modifying activities, ATP-dependent In the transgene model, mammary adenocarcinomas nucleosome remodeling activity and histone also developed (Bagheri-Yarmand et al., 2004). deacetylation, were functionally and physically Microarrays have also been used to follow linked in the same protein complex. This complex MTA1and other genes' expression. Using DNA has been named the NuRD (Nucleosome microarray analysis Roepman et al. (2006) Remodeling and Histone Deacetylation). The investigated gene expression patterns in lymph NuRD complex contains HDAC1, HDAC2, the node metastases of head and neck squamous cell histone binding proteins RbAp46/48 and the carcinomas. They found that the MTA1 gene was dermatomyositis-specific autoantigen Mi-2, which the only single gene that showed consistent over- has been shown to have transcription repressing expression between large numbers of matched activity (Tong et al., 1998; Xue et al., 1998; Wade paired samples of primary tumor and lymph node et al., 1999; Zhang et al., 1999; Bowen et al., 2004). metastases. The MTA1 protein was found in the NuRD 4. Biological significance of the complex by Xue et al. (1998), and this complex MTA proteins also possessed strong transcription repressing activity. Subsequently, Zhang et al. (1999) reported It has been demonstrated by different laboratories that a protein similar to MTA1 (named the MTA2 (see Section 3) that MTA1/MTA1 over-expression protein) was also a component of the NuRD is closely correlated with cancer progression (and in complex, and they found that MTA2 protein was some cases with carcinogenesis) in a wide range of highly expressed in rapidly dividing cells. Later, different cancers. This strongly indicates that the MTA3 protein was identified as an estrogen- MTA1 protein may be an important functional inducible gene product that is present in a distinct molecule in malignancy. Thus, it is necessary to NuRD complex (Fujita et al., 2003). We also clarify the molecular mechanisms by which the reported the physical interaction between MTA1 MTA protein family members exert their functions. protein and HDAC1 (Toh et al., 2000).

Figure 1. MTA proteins in a chromatin remodeling and histone deacetylation complex (NuRD). This complex has transcription repression properties. The NuRD complex also contains histone deacetylases (HDAC1 and 2), major DNA binding protein 3 (MDB3), histone binding proteins RbAp46/48 and the dermatomyositis-specific autoantigen Mi-2 (from Toh and Nicolson, 2009 with permission).

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The basic functions of the MTA protein family HDAC2 on the chromatin site of an ER-responsive members appear to be exerted through NuRD element (ERE) in the promoter regions of estrogen complexes as chromatin remodeling and histone responsive genes, such as pS2 and c-myc. This deacetylating activities (Figure 1). Although there could explain the activation of the HRG/HER2 are also non-histone deacetylating proteins in pathway in ER-positive breast cancers and the NuRD complexes, MTA proteins appear to be suppression of ERα functions, which could result in among the principal components (Figure 1). In the more invasive and aggressive phenotypes addition, the MTA-NuRD complexes show observed in ER-negative breast cancers (Cui et al., transcriptional repression activities (Feron, 2003; 2006). Kumar et al., 2003; Manavathi et al., 2007b; Singh The repressive function of MTA1 protein on ERα is and Kumar, 2007). Although all MTA protein mediated via histone deacetylation by HDAC1 and family members are found in NuRD complexes, HDAC2, suggesting that MTA1 protien has a each MTA protein may form a distinct NuRD potent co-repressor function during the trans- complex that targets different sets of promoters activation of ERα through histone deacetylation (Bowen et al., 2004). (Figures 2 and 3). MTA2 protein has also been 4.2 MTA protein repression of the trans- shown to physically interact with ERα and to activating activity of estrogen receptor-alpha repress its trans-activating function. This could The involvement of MTA proteins in NuRD explain the over-expression of MTA2 protein in complexes suggested that such complexes might cells that were unresponsive to estrogen as well as function in chromatin remodeling and histone suppression of estrogen-induced colony formation deacetylation, but a direct target of a MTA protein in breast cancer cells (Cui et al., 2006). had to first be identified (Mazumdar et al., 2001). MTA proteins also have other activities. For MTA1 protein was identified as a molecule induced example, Khaleque et al. (2008) showed that MTA1 by heregulin-beta1 (HRG), a growth factor that is a protien binds to a heat shock factor 1 (HSF1), the natural ligand of the human epidermal growth transcriptional activator of the heat shock genes, in factor receptors HER3 and HER4. It can also trans- vitro and in human breast carcinoma samples. They activate HER2 (c-erbB-2) in human breast cancer demonstrated that HSF1-MTA1 complex formation cell lines. Mazumdar et al. (2001) showed that was strongly induced by HRG and that the complex MTA1 protein directly interacted with the ligand- was incorporated into a NuRD complex that binding domain of the estrogen receptor ERα and participated in the repression of estrogen-dependent that HRG stimulated the association of MTA1 and transcription in breast cancer cells treated with HRG (Khaleque et al., 2008).

Figure 2. A possible role for MTA proteins in carcinogenesis and cancer progression. In this scheme the main functions of the MTA family of proteins are presented. (A) MTA1 protein is included in a NuRD complex that represses the transactivation function of estrogen receptor (ER), rendering breast cancer cells more phenotypically aggressive. MTA1 proteins in NuRD complexes are proposed to be one of the first downstream targets of c-MYC function, and it is essential for the transformation potential of c-MYC. MTA1s is a splice-variant of MTA1 that localizes in the cytoplasm where it sequesters ERα, preventing the ligand-induced nuclear translocation of Erα, thus stimulating the development of the malignant phenotype of breast cancer cells. (B) MTA3 protein induced by estrogen represses the expression of the transcriptional repressor Snail, a master regulator of epithelial to mesenchymal transitions, resulting in the expression of the cell adhesion molecule E-cadherin and maintenance of a differentiated, normal epithelial status in breast cells (from Toh and Nicolson, 2009 with permission).

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There are apparently several molecules, such as The absence of MTA3 protein as well as the ménage-à-trois 1 (MAT1), MTA1-interacting co- absence of ER results in an aberrantly increased activator (MICoA) and nuclear receptor interacting expression of the transcriptional repressor Snail, a factor 3 (NRIF3), that can interact with MTA1 master regulator of epithelial-to-mesenchymal protein and repress the trans-activation function of transition (EMT). This increased expression of ERα (Manavathi et al., 2007b). These three MTA1- Snail results in reduction in expression of the cell binding proteins themselves have co-activator adhesion molecule E-cadherin, which subsequently properties upon ERα trans-activation. MAT1, an modifies epithelial cell architecture and enhances assembly and targeting ring finger factor for cyclin- invasive growth. MTA3 protein is a transcriptional dependent kinase-activating kinase (CAK), has target of ERα, and in the presence of estrogen ERα been identified by Talukder et al. (2003) as a directly binds to the MTA3 promoter at the SP1 site MTA1-binding protein. The interactions between in close proximity to the ERE half-site, resulting in CAK and MTA1 protein apparently regulate the stimulation of MTA3 transcription (Fujita et al., trans-activation activity of ERα in a CAK- 2004; Mishra et al., 2004). Thus, MTA3 protein dependent manner in breast cancer cells. In may function to maintain a well-differentiated, contrast, MICoA-mediated ERα trans-activation normal epithelial phenotype in breast cells. This is functions are opposed by MTA1 protein through in stark contrast to MTA1 or MTA1s protein, where the recruitment of HDACs (Mishra et al., 2003). In up-regulation of MTA1 or MTA1s protein in breast addition, the interactions between MTA1 protein cancer cells may repress MTA3 expression through and NRIF3 (an estrogen-inducible gene) may be repression of the ERα function, resulting in up- important in modulating the sensitivity of breast regulation of Snail, down-regulation of E-cadherin, cancer cells to estrogen (Talukder et al., 2004). promotion of an EMT phenotype and potentially an Another MTA1-binding protein partner, Lim-only increase in metastatic potential. protein 4 (LMO4), has been identified by Singh et Forced expression of MTA3 protein inhibits ductal al. (2005). LMO4 was found to be a component of branching in virgin and pregnant mammary glands the MTA1 co-repressor complex, and its over- in MTA3-transgenic mice (Zhang et al., 2006). This expression repressed ERα trans-activation in a property is in marked contrast to MTA1-transgenic HDAC-dependent manner. This has been proposed mice, where there is inappropriate development of to result in the acquisition of an ERα-negative mammary glands, resulting in the development of phenotype with its known increased aggressiveness hyperplastic nodules and mammary tumors, in breast cancer cells (Singh et al., 2005). including adenocarcinomas and lymphomas Variants of MTA1 protein have also been found. (Bagheri-Yarmand et al., 2004; Manavathi and For example, a truncated form of MTA1 protein has Kumar, 2007). MTA3 protein also represses Wnt4 been identified and named MTA1s (Balasenthil et transcription and secretion by inhibiting Wnt-target al., 2006). MTA1s is a splice-variant of MTA1, and genes in mammary epithelial cells. This repression it contains an ER-binding motif (nuclear binding of Wnt4 transcription was found to be mediated motif) without any nuclear localization signals at its through a MTA3-NuRD complex, which interacts C-terminus. This truncated MTA protein localizes with the Wnt4-containing chromatin in an HDAC- in the cytoplasm where it sequesters ERα, resulting dependent process (Zhang et al., 2006). in the blockage of ERα ligand-induced nuclear The fundamental actions of the MTA proteins are translocation and stimulation of acquisition of the exerted via transcriptional repression by histone malignant phenotype of breast cancer cells. This deacetylation; however, a transcriptional activating suggests that the regulation of the cellular function has also been described for MTA localization of ERα by MTA1s protein may complexes. Gururaj et al. (2006a, 2006b) showed represent a mechanism for redirecting nuclear that breast cancer amplified sequence 3 (BCAS3), a receptor signaling by nuclear exclusion. MTA1s gene amplified and over-expressed in breast protein has also been shown to associate with cancers, was a chromatin target of MTA1 protein, casein kinase I-gamma2, which is an estrogen- and the transcription of BCAS3 was stimulated by responsive kinase (Mishra et al., 2004). MTA1 protein. This suggested that MTA1 protein MTA3 protein is the newest addition to the MTA has a transcriptional co-activator function in family. It was identified as an estrogen-dependent addition to its co-repressor function. A similar component of the Mi-2/NuRD transcriptional co- property has been also been suggested for mouse repressor complex in breast epithelial cells (Fujita Mta2 protein (Matsusue et al., 2001). et al., 2003).

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Figure 3. Deacetylation of non-histone proteins by MTA protein family complexes. (A) Tumor suppressor p53 protein is deacetylated and inactivated by both MTA1 and MTA2 proteins in NuRD complexes, resulting in inhibition of growth arrest and apoptosis. (B) Hypoxiainducible factor-1α (HIF-1α) is also deacetylated and stabilized by MTA1 protein, leading to angiogenesis (from Toh and Nicolson, 2009 with permission).

4.3 MTA-NuRD protein complexes and by HDAC1/MTA1 complexes (Figure 3). HIF-1α is deacetylation of non-histone proteins a key regulator of angiogenic factors (Yoo et al., Chromatin histones and non-histone proteins are the 2006). The expression of MTA1 was strongly protein targets for deacetylation by HDAC via induced under hypoxic conditions in breast cancer NuRD complexes containing MTA proteins. The cell lines, and MTA1 protein over-expression tumor suppressor gene p53 protein was the first enhanced the transcriptional activity and stability of non-histone protein that was reported to be HIF-1α protein. MTA1 protein physically bound to deacetylated by MTA protein-containing NuRD HIF-1α and deacetylated it by increasing the complexes. Luo et al. (2000) reported that the expression of HDAC1, leading to the stabilization deacetylation of p53 was mediated by an HDAC1 of HIF-1α (Yoo et al., 2006). These results complex containing MTA2 protein. A MTA2- indicated possible positive cross-talk between associated NuRD complex was involved, and this MTA1 and HIF-1α, mediated by HDAC1 HDAC1/MTA2 complex interacted with p53 in recruitment. vitro and in vivo and reduced significantly the Moon et al. (2006) found a close connection steady-state levels of acetylated p53. Deacetylation between MTA1-associated metastasis and HIF-1α- of p53 causes an increase in its own degradation induced tumor angiogenesis. They showed that through MDM2 and a reduction in p53-dependent MTA1 protein increased the transcriptional activity transcriptional activation. Eventually this results the of HIF-1α and a target molecule of HIF-1α, repression of the normal p53 function that mediates vascular endothelial growth factor (VEGF). cell growth arrest and apoptosis (Figure 3). The Conditioned medium collected from MTA1- same phenomenon was observed between p53 and transfectants increased angiogenesis in vitro and in MTA1 complexes. HDAC1/MTA1 complexes vivo (Moon et al., 2006). Functional links between possessed deacetylation activity against p53 protein HIF-1α and MTA1 protein have been demonstrated in human non-small cell carcinoma and human in clinical samples of pancreatic carcinoma. Using hepatoma cells, and the complexes were found to immunohistochemistry and surgically resected inhibit p53-induced apoptosis by attenuating the pancreatic carcinomas Miyake et al. (2008) trans-activation function of p53 (Moon et al., 2007). examined the expression of HIF-1α, HDAC1 and More recently the stability of p53 was determined MTA1 proteins and suggested that HIF-1α to be affected by MTA1 inhibiting p53 expression, which is associated with a poor ubiquitination by E3 ubiquitin ligases double prognosis in patients with pancreatic cancers, might minute 2 (Mdm2) and constitutive be regulated by HDAC1/MTA1 complexes. The photomorphogenic protein 1 (COP1). MTA1 contribution of MTA1 protein to tumor competes with COP1 to bind to p53 and/or angiogenesis was also demonstrated in human destabilize COP1 and Mdm2 (Li et al., 2009b). breast cancers. Using immunohistochemistry Jang MTA1 stability and degradation itself is controlled et al. (2006) examined MTA1 protein expression by ubiquitination, and degradation of MTA1 is and intra-tumoral microvessel density (MVD) in promoted by COP1-mediated hydrolysis (Li et al., clinical samples of breast cancer and showed that 2009b). MTA1 protein expression was significantly HIF-1α (hypoxia-inducible factor-1α) is another correlated with higher tumor grade and higher important non-histone protein that is deacetylated tumor MVD.

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The relationship between MTA1 protein expression lymphomas were induced in the MTA1-transgenic and MVD was also observed in hepatitis B- mice (Bagheri-Yarmand et al., 2007). associated HCC (Ryu et al., 2008). In this tumor Recently, Molli et al. (2008) reported that system hepatitis B virus X protein (Hbx) induces MTA1/NuRD complexes negatively regulated the expression of MTA1 protein and its HDAC1 BRCA1 transcription by physically associating with complex, which enhances hypoxia signaling in ERE of the BRCA1 promoter in an ERα-dependent HCC (Yoo et al., 2006). This suggests that the manner. This repressive effect of MTA1 on BRCA1 HDAC1 complex containing MTA1 protein may be expression resulted in the acquisition of abnormal important in stabilizing HIF-1α, and thus play a role centrosomes and chromosomal instability (Molli et in angiogenesis and metastasis. al., 2008). The relationship between the protein members of The expression of MTA1 and HDAC1 proteins can NuRD complexes, including MTA1 and MTA2 also be increased by the interaction of hepatitis B proteins, and the ataxia teleangiectasia mutated virus X (HBx) protein at the transcriptional level (ATM)- and Rad3-related protein (ATR) has been (Yoo et al., 2008). Since MTA1 and HDAC1/2 shown by co-immunoprocipitation of these proteins proteins are physically associated with HIF-1α in (Schmidt and Schreiber, 1999). ATR is a vivo in the presence of HBx protein, HBx-induced phosphatidylinositol-kinase-related kinase that has deacetylation stabilizes HIF-1α by inhibiting been implicated in the response of human cells to proteosomal degradation. These results indicated multiple forms of DNA damage and may play a the existence of positive cross-talk between HBx role in the DNA replication checkpoint. This and the MTA1/HDAC complex, and it further suggests that MTA proteins may contribute to the suggests that such cross-talk may play a role in regulation of DNA checkpoints (Toh and Nicolson, angiogenesis and metastasis of HBV-associated 2009). hepatocellular carcinomas. 4.4 MTA proteins: other possible functions in Direct interactions between MTA1 protein and cancer cells endophilin 3 have also been reported by Aramaki et Other reports have been forthcoming suggesting al. (2005). This suggests that MTA1 protein might some possible roles of MTA proteins in be involved in the regulation of endocytosis carcinogenesis and cancer progression. The most mediated by endophilin 3. important of these may be the relationship of An important treatment modality in cancer is the MTA1 protein with c-MYC oncoprotein (Figure 2). use of ionizing radiation. MTA1 protein has been Using expression profiling, Zhang et al. (2005) implicated in ionizing radiation-induced DNA identified the MTA1 protein as a target of the c- damage response by regulating p53-dependent MYC protein in primary human cancer cells. They DNA repair (Li et al., 2009a). showed that c-MYC binds to the genomic MTA1 5. MTA/MTA genes and proteins locus and recruits transcriptional co-activators. as new clinical targets They also found that the MTA1 proteins in NuRD complexes were one of the first downstream targets This review and others (Nicolson et al., 2003; of c-MYC function, and this was essential for the Manavathi and Kumar, 2007; Toh and Nicolson, transformation potential of c-MYC. Indeed, 2009) have discussed the available data on the reduction of MTA1 expression by a short hairpin likelyhood that MTA proteins have important and RNA blocked the ability of c-MYC to transform critical roles in the genesis and progression of a mammalian cells (Zhang et al., 2005). wide variety of cancers. MTA1 protein can be Another milestone was the establishment of a thought of as a master co-regulatory molecule transgenic mice model that over-expressed MTA1 (Manavathi and Kumar, 2007; Toh and Nicolson, protein. Kumar and his collaborators found that the 2009). This clearly suggests the possibility that MTA1-transgenic mice showed inappropriate MTA1 protein (or the MTA1 gene or its RNA development of mammary glands. These mice also product) could be an excellent molecular target for developed hyperplastic nodules and mammary cancer therapy as well as its use in cancer tumors (Bagheri-Yarmand et al., 2004; Singh and diagnosis/prognosis. Kumar, 2007). In this study, the underlying The first studies that suggested the possibility of molecular mechanisms of MTA1 protein action and targeting MTA1 RNA were reported by Nawa et al. its regulation were also examined, and the results (2000) and Nicolson et al. (2003). Using antisense suggested that MTA1 protein dysregulation in phosphorothioate oligonucleotides against MTA1 mammary epithelium and cancer cells triggered mRNA, these authors found growth inhibitory down-regulation of the progesterone receptor-B effects and inhibition of invasion of human isoform and up-regulation of the progesterone metastatic breast cancer cell lines. receptor-A isoform, resulting in the up-regulation Different techniques have been used to regulate of the progesterone receptor-A target genes Bcl-XL MTA1/MTA1 expression in order to determine the and cyclin D1 in mammary glands of virgin mice. effects of MTA1 protein on cellular functions. These authors also found that spontaneous B-cell Using RNA interference (RNAi) Qian et al. (2007)

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MTA1 of the MTA (metastasis-associated) gene family and its Toh Y, Nicolson GL encoded proteins: molecular and regulatory functions and role in human cancer progression inhibited MTA1 expression in a human esophageal made up of transcriptional co-repressors that squamous cell carcinoma cell line and demonstrated function via NuRD complexes containing significant inhibition of in vitro invasion and chromatin remodeling and histone deacetylating migration properties of the cancer cells (Qian et al., molecules. These actions clearly have a role in 2005). In a metastasis model based on murine tumor formation and progression. For example, the melanoma Qian et al. (2007) examined the repression of ERα trans-activation function by therapeutic use of lowering MTA1 protein levels in MTA1 protein through deacetylation of ERE the melanoma cells and demonstrated that down- chromatin of the ER-responsive genes has been the regulation of MTA1 protein by RNAi successfully most extensively investigated, and the data clearly suppressed growth in vitro and experimental demonstrated that MTA1 expression results in metastasis in vivo. Using microRNAs against tumor formation in mammary glands and renders MTA1 Reddy et al. (2009) were able to inhibit the breast cancer cells phenotypically more aggressive expression of MTA1 protein in human breast (reviewed in Manavathi and Kumar, 2007). cancer cells, resulting in decreased cell mobility, In addition to chromatin histones, MTA proteins invasiveness, anchorage-dependent growth and also deacetylate non-histone proteins. For example, tumorigenicity. Results such as these suggest a the tumor suppressor p53 protein is deacetylated potential role of the MTA1 gene as a target for and inactivated by both MTA1 and MTA2 proteins, cancer gene therapy. resulting in inhibition of growth arrest and Other MTA/MTA genes and proteins may also be apoptosis. HIF-1α is also deacetylated and useful targets. For example, MTA1s may be a stabilized by MTA1, leading to angiogenesis. Thus, useful target in the treatment of breast cancer. it has been proposed that MTA proteins, especially MTA1s functions as a repressor of ERα MTA1 protein, represent master co-regulatory transcriptional activity by binding and sequestering molecules involved in the carcinogenesis and the ERα in the cytoplasm (Kumar et al., 2002). progression of various malignant tumors MTA1s has a unique C-terminal 33-amino acid (Manavathi and Kumar, 2007; Toh and Nicolson, region containing a nuclear receptor-box motif that 2009). Since, it is assumed that these properties are mediates the interaction of MTA1s protein with important to the survival and progression of cancer ERα. Singh et al. (2006) showed that the MTA1s cells, ultimately this could lead to novel clinical peptide containing this motif could effectively applications of MTA genes or MTA proteins as new repress the ERα transactivation function, measured molecular targets for cancer therapy. by estrogen-induced proliferation and anchorage- There are other examples of the potential use of independent growth of the human breast cancer cell MTA proteins as therapeutic targets. Inhibition of line MCF-7. Using an animal model they also MTA1 protein expression or function may enhance showed the effect of MTA1s peptide in blocking the chemosensitivity of cancer cells by restoring tumor progression of MCF-7 breast cancer cells tumor suppressor function of p53, or it may inhibit that over-expressed ERα (Singh et al., 2006). tumor angiogenesis by destabilizing the The use of MTA1 protein as a target of angiogenesis promoting function of HIF-1α. immunotherapy has also been considered. MTA1 Moreover, MTA proteins may cooperate with protein is a promising antigen for tumor rejection, HDAC inhibitors, which are now expected to be the because it is over-expressed in many different target of a new class of anticancer agents (Toh and tumors and is only expressed at lower levels in Nicolson, 2009). normal tissues (Toh and Nicolson, 2009). In MTA1 will also be clinically useful for the reviewing a model for immunotherapy Assudani et prognosis or prediction of the malignant potentials al. (2006) proposed using a vector that contained of various human cancers, such as esophageal, disabled infectious single cycle-herpes simplex gastric and colorectal cancers (Toh and Nicolson, virus (DISC-HSV). Their initial studies 2009). Thus, evaluating the expression levels of demonstrated the presence of immunogenic MHC MTA proteins in individual cases of various class I-restricted peptides of MTA1 protein. Next, cancers may provide us with important clues. MTA1 protein was identified as a SEREX antigen, Finally, the MTA proteins are clearly present in and hence it is likely to be capable of inducing a T- completely normal cells to provide them with cell response in cancer patients (Chen and Han, certain necessary functions. Thus, it will be 2001). important to understand their physiological functions and underlying mechanisms in normal 6. MTA/MTA genes and proteins: cells. For example, C. elegans has MTA1 future directions homologues, egl-27 and egr-1, that function in This and previous reviews (Toh and Nicolson, embryonic patterning and development (Solari et 2003; Manavathi and Kumar, 2007; Toh and al., 1999; Chen and Han, 2001), suggesting that Nicolson, 2009) have focused on the clinical and MTA1 protein may have an embryonic biological significance of the newly emerging gene developmental function. MTA1 protein is also family named MTA. The family of MTA proteins is thought to play a crucial role in postnatal testis

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MTA1 of the MTA (metastasis-associated) gene family and its Toh Y, Nicolson GL encoded proteins: molecular and regulatory functions and role in human cancer progression development and spermatogenesis (Li et al., 2007a; activity in pancreatic cancer. Int J Oncol. 2000 Li et al., 2007b), and MTA1 protein is a direct Jun;16(6):1211-4 stimulator of rhodopsin expression (Manavathi et Luo J, Su F, Chen D, Shiloh A, Gu W. Deacetylation of p53 al., 2007a). These are only a few of the known modulates its effect on cell growth and apoptosis. Nature. physiological functions of MTA1 protein (Toh and 2000 Nov 16;408(6810):377-81 Nicolson, 2009), and it is expected that other MTA Nawa A, Nishimori K, Lin P, Maki Y, Moue K, Sawada H, proteins have important roles in normal physiology Toh Y, Fumitaka K, Nicolson GL. Tumor metastasis- associated human MTA1 gene: its deduced protein and development. Thus, determining the normal sequence, localization, and association with breast cancer physiological functions of MTA proteins will be cell proliferation using antisense phosphorothioate absolutely necessary in understanding the oligonucleotides. J Cell Biochem. 2000 Aug 2;79(2):202-12 pathological functions of MTA proteins in human Toh Y, Kuninaka S, Endo K, Oshiro T, Ikeda Y, Nakashima cancers. H, Baba H, Kohnoe S, Okamura T, Nicolson GL, Sugimachi K. Molecular analysis of a candidate metastasis-associated gene, MTA1: possible interaction References with histone deacetylase 1. J Exp Clin Cancer Res. 2000 Toh Y, Pencil SD, Nicolson GL. A novel candidate Mar;19(1):105-11 metastasis-associated gene, mta1, differentially expressed Chen Z, Han M. Role of C. elegans lin-40 MTA in vulval in highly metastatic mammary adenocarcinoma cell lines. fate specification and morphogenesis. Development. 2001 cDNA cloning, expression, and protein analyses. 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Development. 2004 effector of the c-MYC oncoprotein. Proc Natl Acad Sci U S Jul;131(14):3469-79 A. 2005 Sep 27;102(39):13968-73 Bowen NJ, Fujita N, Kajita M, Wade PA. Mi-2/NuRD: Assudani DP, Ahmad M, Li G, Rees RC, Ali SA. multiple complexes for many purposes. Biochim Biophys Immunotherapeutic potential of DISC-HSV and OX40L in Acta. 2004 Mar 15;1677(1-3):52-7 cancer. Cancer Immunol Immunother. 2006 Jan;55(1):104- 11 Fujita N, Kajita M, Taysavang P, Wade PA. Hormonal regulation of metastasis-associated protein 3 transcription Balasenthil S, Broaddus RR, Kumar R. Expression of in breast cancer cells. Mol Endocrinol. 2004 metastasis-associated protein 1 (MTA1) in benign Dec;18(12):2937-49 endometrium and endometrial adenocarcinomas. Hum Pathol. 2006 Jun;37(6):656-61 Hofer MD, Kuefer R, Varambally S, Li H, Ma J, Shapiro GI, Gschwend JE, Hautmann RE, Sanda MG, Giehl K, Menke Cui Y, Niu A, Pestell R, Kumar R, Curran EM, Liu Y, Fuqua A, Chinnaiyan AM, Rubin MA. The role of metastasis- SA. Metastasis-associated protein 2 is a repressor of associated protein 1 in prostate cancer progression. estrogen receptor alpha whose overexpression leads to Cancer Res. 2004 Feb 1;64(3):825-9 estrogen-independent growth of human breast cancer cells. Mol Endocrinol. 2006 Sep;20(9):2020-35 Hofer MD, Menke A, Genze F, Gierschik P, Giehl K. Expression of MTA1 promotes motility and invasiveness of Gururaj AE, Holm C, Landberg G, Kumar R. Breast PANC-1 pancreatic carcinoma cells. Br J Cancer. 2004 cancer-amplified sequence 3, a target of metastasis- Jan 26;90(2):455-62 associated protein 1, contributes to tamoxifen resistance in premenopausal patients with breast cancer. Cell Cycle. Li G, Miles A, Line A, Rees RC. Identification of tumour 2006 Jul;5(13):1407-10 antigens by serological analysis of cDNA expression cloning. Cancer Immunol Immunother. 2004 Gururaj AE, Singh RR, Rayala SK, Holm C, den Hollander Mar;53(3):139-43 P, Zhang H, Balasenthil S, Talukder AH, Landberg G, Kumar R. MTA1, a transcriptional activator of breast Mishra SK, Talukder AH, Gururaj AE, Yang Z, Singh RR, cancer amplified sequence 3. Proc Natl Acad Sci U S A. Mahoney MG, Francí C, Vadlamudi RK, Kumar R. 2006 Apr 25;103(17):6670-5 Upstream determinants of estrogen receptor-alpha regulation of metastatic tumor antigen 3 pathway. J Biol Hofer MD, Tapia C, Browne TJ, Mirlacher M, Sauter G, Chem. 2004 Jul 30;279(31):32709-15 Rubin MA. Comprehensive analysis of the expression of the metastasis-associated gene 1 in human neoplastic Mishra SK, Yang Z, Mazumdar A, Talukder AH, Larose L, tissue. Arch Pathol Lab Med. 2006 Jul;130(7):989-96 Kumar R. Metastatic tumor antigen 1 short form (MTA1s) associates with casein kinase I-gamma2, an estrogen- Jang KS, Paik SS, Chung H, Oh YH, Kong G. MTA1 responsive kinase. Oncogene. 2004 May 27;23(25):4422-9 overexpression correlates significantly with tumor grade and angiogenesis in human breast cancers. Cancer Sci. Moon WS, Chang K, Tarnawski AS. 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Kidd M, Modlin IM, Mane SM, Camp RL, Eick GN, Latich I, Manavathi B, Peng S, Rayala SK, Talukder AH, Wang MH, Zikusoka MN. Utility of molecular genetic signatures in the Wang RA, Balasenthil S, Agarwal N, Frishman LJ, Kumar delineation of gastric neoplasia. Cancer. 2006 Apr R. Repression of Six3 by a corepressor regulates 1;106(7):1480-8 rhodopsin expression. Proc Natl Acad Sci U S A. 2007 Aug 7;104(32):13128-33 Martin MD, Hilsenbeck SG, Mohsin SK, Hopp TA, Clark GM, Osborne CK, Allred DC, O'Connell P. Breast tumors Manavathi B, Singh K, Kumar R. MTA family of that overexpress nuclear metastasis-associated 1 (MTA1) coregulators in nuclear receptor biology and pathology. protein have high recurrence risks but enhanced Nucl Recept Signal. 2007 Nov 30;5:e010 responses to systemic therapies. Breast Cancer Res Treat. 2006 Jan;95(1):7-12 Moon HE, Cheon H, Lee MS. Metastasis-associated protein 1 inhibits p53-induced apoptosis. 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Pancreas. 2008 Apr;36(3):e1-9 motif-containing region of MTA1s. J Biol Chem. 2006 Sep 1;281(35):25612-21 Molli PR, Singh RR, Lee SW, Kumar R. MTA1-mediated transcriptional repression of BRCA1 tumor suppressor Yoo YG, Kong G, Lee MO. Metastasis-associated protein gene. Oncogene. 2008 Mar 27;27(14):1971-80 1 enhances stability of hypoxia-inducible factor-1alpha protein by recruiting histone deacetylase 1. EMBO J. 2006 Ryu SH, Chung YH, Lee H, Kim JA, Shin HD, Min HJ, Seo Mar 22;25(6):1231-41 DD, Jang MK, Yu E, Kim KW. Metastatic tumor antigen 1 is closely associated with frequent postoperative Zhang H, Singh RR, Talukder AH, Kumar R. Metastatic recurrence and poor survival in patients with hepatocellular tumor antigen 3 is a direct corepressor of the Wnt4 carcinoma. Hepatology. 2008 Mar;47(3):929-36 pathway. Genes Dev. 2006 Nov 1;20(21):2943-8 Yoo YG, Na TY, Seo HW, Seong JK, Park CK, Shin YK, Bagheri-Yarmand R, Balasenthil S, Gururaj AE, Talukder Lee MO. 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E3 ubiquitin ligase COP1 Kidd M, Modlin IM, Pfragner R, Eick GN, Champaneria regulates the stability and functions of MTA1. Proc Natl MC, Chan AK, Camp RL, Mane SM. Small bowel carcinoid Acad Sci U S A. 2009 Oct 13;106(41):17493-8 (enterochromaffin cell) neoplasia exhibits transforming growth factor-beta1-mediated regulatory abnormalities Reddy SD, Pakala SB, Ohshiro K, Rayala SK, Kumar R. including up-regulation of C-Myc and MTA1. Cancer. 2007 MicroRNA-661, a c/EBPalpha target, inhibits metastatic Jun 15;109(12):2420-31 tumor antigen 1 and regulates its functions. Cancer Res. 2009 Jul 15;69(14):5639-42 Li W, Liu XP, Xu RJ, Zhang YQ. Immunolocalization assessment of metastasis-associated protein 1 in human Toh Y, Nicolson GL. The role of the MTA family and their and mouse mature testes and its association with encoded proteins in human cancers: molecular functions spermatogenesis. Asian J Androl. 2007 May;9(3):345-52 and clinical implications. Clin Exp Metastasis. 2009;26(3):215-27 Li W, Zhang J, Liu X, Xu R, Zhang Y. Correlation of appearance of metastasis-associated protein1 (Mta1) with This article should be referenced as such: spermatogenesis in developing mouse testis. Cell Tissue Res. 2007 Aug;329(2):351-62 Toh Y, Nicolson GL. MTA1 of the MTA (metastasis- associated) gene family and its encoded proteins: Manavathi B, Kumar R. Metastasis tumor antigens, an molecular and regulatory functions and role in human emerging family of multifaceted master coregulators. J Biol cancer progression. Atlas Genet Cytogenet Oncol Chem. 2007 Jan 19;282(3):1529-33 Haematol. 2011; 15(3):304-316.

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Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Deep Insight Section

Role of p38α in apoptosis: implication in cancer development and therapy Almudena Porras, Carmen Guerrero Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, UCM, Ciudad Universitaria, 28040 Madrid, Spain (AP); Centro de Investigación del Cáncer, IBMCC, Universidad de Salamanca-CSIC, 37007 Salamanca, Spain (CG)

Published in Atlas Database: June 2010 Online updated version : http://AtlasGeneticsOncology.org/Deep/MAPK14-p38ainCancerID20089.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI MAPK14-p38ainCancerID20089.txt

1- Introduction MKK3 and MKK6 are in turn activated by The family of p38 mitogen-activated protein phosphorylation by a MAPK kinase kinase kinases (MAPKs) belongs to the MAPK (MKKK) such as MLKs, ASK1, TAK1 or MEKKs, superfamily. They are strongly activated by which are activated by small and heterotrimeric G different stress signals and inflammatory cytokines, proteins (Cuenda and Rousseau, 2007; Wagner and although non-stressful stimuli also activate p38 Nebreda, 2009). MAPKs leading to the regulation of cellular p38α can be also activated through MKK- functions such as proliferation, differentiation and independent pathways such as that involving p38α survival. Four different p38 MAPK family autophospohorylation upon interaction with TAB1 members have been identified so far: p38α, β, γ and (Cuenda and Rousseau, 2007). δ, also known as stress-activated kinase 2a Once p38 MAPKs are activated, they phoshorylate (SAPK2a), SAPK2b, SAPK3 and SAPK4, different transcription factors such as p53, ATF2, respectively, which may have both overlapping and MEF2 or C/EBPb and protein kinases, including specific functions (reviewed by Cuenda and MAPKAP-K2 and MAPKAP-K3 (also known as Rousseau, 2007; Kyriakis and Avruch, 2001; MK-2 and MK-3), MSK-1 (mitogen- and stress- Nebreda and Porras, 2000; Ono and Han, 2000). activated protein kinase 1) and MNK-1 and MNK-2 p38α was initially identified as a 38 KDa protein, (MAP kinase-interacting serine/threonine kinase 1 which became phosphorylated in Tyr in response to and 2) (Cuenda and Rousseau, 2007; Wagner and the bacterial endotoxin LPS in macrophages (Han Nebreda, 2009). et al., 1994). In parallel, other two groups identified The analysis of the function of p38α has been p38α as a kinase activated by stress and IL1, able to initially based on the effect of chemical inhibitors phosphorylate and activate MAPKAP-K2 (MAPK- such as SB203580 and SB202190, which inhibit activated protein kinase 2) (Freshney et al., 1994; p38α but can also inhibit p38β at a higher dose Rouse et al., 1994). p38α (encoded by (reviewed by Nebreda and Porras, 2000). Therefore, MAPK14gene) is broadly expressed and is also the the main effects observed upon treatment with most abundant p38 isoform present in most cell those inhibitors are a consequence of p38α types. Targeted inactivation of the mouse p38α inhibition, sometimes are also due to p38β gene results in embryonic death due to a placental inhibition. The generation of mice with specific defect (Adams et al., 2000; Mudgett et al., 2000; genetic inactivation of the different p38 isoforms or Tamura et al., 2000). the use of interference RNA technology during the p38 MAPKs are mainly activated by MKK3 and last few years has allowed a more precise study of MKK6 through dual phoshorylation in Tyr and Thr, the role played by each isoform and they have been although MKK4 can sometimes activate p38α essential to establish the role of p38α in different (Cuenda and Rousseau, 2007). cellular processes.

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Role of p38α in apoptosis: implication in cancer development Porras A, Guerrero C and therapy

Figure 1. p38 MAPK signaling pathway. A variety of stimuli, mainly stress stimuli, activate p38 MAPK through complex kinase cascades including a MAP3K that phosphorylates a MAPK2 that, in turn, phosphorylates p38MAPKs. Cellular intermediates, like GTPases, receptor adapter proteins or cell cycle checkpoint proteins, among others, transmit the stimulus to the kinase cascades. Once activated, the different p38MAPKs either phosphorylate cytoplasmic targets or translocate into the nucleus leading to the regulation of transcription factors involved in cellular responses. MKK6 is the major activator of all p38 MAPK isoforms, while MKK3 and MKK4 are more specific and can only activate some isoforms.

2003) or oxidative stress (Zhuang et al., 2000) 2- Dual role of p38 MAPK in cell involves one of these p38MAPKs. death: function of p38α in p38α is a mediator of apoptosis in response to a apoptosis number of cellular stresses through transcriptional p38 MAPK can play a dual role as a regulator of and posttranscriptional mechanisms, which can cell death, thus it can either mediate cell survival or involve the regulation of apoptotic and/or survival cell death through different mechanisms, including pathways (reviewed by Wagner and Nebreda, apoptosis. Therefore, the specific function of p38 2009). MAPKs in apoptosis appears to depend on the cell For example, p38α sensitizes cardiomyocytes and type, the stimuli and/or the isoform (reviewed by MEFs-derived cell lines to apoptosis induced by Nebreda and Porras, 2000; Wagner and Nebreda, different stimuli through both, up-regulation of the 2009). pro-apoptotic proteins Fas and Baxand down- As indicated above, some studies have been based regulation of the activity of ERKs and Akt survival on the using of chemical inhibitors such as pathways (Porras et al., 2004; Zuluaga et al., SB203580 which is able to inhibit p38α and p38β, 2007a). According to this, overexpression of p38α thus the participation of the p38β isoform in enhances apoptosis induced by the constitutive apoptosis can not be excluded. In fact, there is a active mutant MKK3bE in cardiomyocytes, while good evidence for a role of p38α and/or p38β overexpression of p38β promotes cell survival MAPKs as mediators of apoptosis in several cell (Nebreda and Porras, 2000). types such as neurons (Ciesielski-Treska et al., p38α is also a negative regulator of survival in 2001; De Zutter and Davis, 2001; Ghatan et al., embryonic stem (ES) (Guo and Yang, 2006). In 2000; Le-Niculescu et al., 1999) or cardiac cells addition, p38α is able to suppress tumor initiation (Mackay and Mochly-Rosen, 1999; Saurin et al., induced by H-rasoncogene through induction of 2000; Wang et al., 1998). Moreover, the induction apoptosis (Dolado et al., 2007). Upon expression of of apoptosis by many types of stimuli such TNF- oncogenic H-Ras, ROS are generated leading to α(Valladares et al., 2000), TGF-β (Edlund et al., p38α activation and apoptotic cell death.

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Moreover, p38α MAPK plays an important role in leukemia) and MCF-7 (breast carcinoma) cell lines the apoptosis induced by some chemotherapeutical (Alsayed et al., 2001). drugs. For example, it is essential for cisplatin- Recent novel findings support the participation of induced apoptosis in the colon carcinoma derived another member of the Ras family, the GTPase cell line, HCT116 (Bragado et al., 2007). Rap1, in the regulation of p38α function in On the other hand, anti-apoptotic roles of p38 apoptosis (Gutiérrez-Uzquiza et al., 2010; Maia et MAPKs have been described in DNA-damaged al., 2009). Rap1 can either activate or inhibit p38α fibroblasts (Héron-Milhavet and LeRoith, 2002), depending on the cell context. In CML cells, the differentiating neurons (Okamoto et al., 2000) and C3G-Rap1 pathway downregulates the pro- activated macrophages (Park et al., 2002). In some apoptotic activity of p38αMAPK as a mediator of cases, p38α has been identified as the isoform STI effects (Maia et al., 2009). In contrast, in MEFs responsible for this survival effect. For example, in C3G is a negative regulator of p38α MAPK activity pulmonary arterial endothelial cells exposed to and Rap1 a positive one, leading to pro- or anti- anoxia-reoxygenation during ischemia-reperfusion, apoptotic effects depending on the stress stimulus carbon monoxide (CO) protects from apoptosis (Gutiérrez-Uzquiza et al., 2010). through p38α activation (Zhang et al., 2003; Zhang There are also evidences of a cross-talk between et al., 2005). In response to H2O2, p38α also PKCs and p38 MAPK in order to regulate mediates cell survival in MEFs, while p38α apoptosis. For example, in LNCaP prostate cancer mediates apoptosis triggered by serum deprivation cells, PMA induces apoptosis through a (Gutiérrez-Uzquiza et al., 2010). In both situations, mechanism, which involves PKCα and d-mediated C3G acts as a negative regulator of p38α. This p38α/β activation (Tanaka et al., 2003). An crosstalk between the C3G and the p38α pathways autocrine pro-apoptotic loop is also generated in has been also observed in CML cell line K562, these cells by PKCδ, which is mediated by death where p38α mediates STI-571-induced apoptosis receptor ligands through a mechanism dependent on (Maia et al., 2009). caspase-8, FADD, p38α/β MAPK and JNK In contrast to the apoptotic role of p38 in CML cells (Gonzalez-Guerrico and Kazanietz, 2005). In treated with STI-571, p38 MAPK pathway exerts addition, in vascular smooth muscle cells activation an antiapoptotic role in CML cells, as well as acute of PKCδ leads to apoptotic cell death involving p53 promyelocytic leukemia cells (APL), treated with induction through a mechanism partially dependent arsenic trioxide (AT) (Verma et al., 2002b). on p38α/β MAPK (Ryer et al., 2005). In some tumor cells p38α can also induce a pro- survival effect known as tumor dormancy, which 4- Targets of p38α in the maintains cells in a quiescent state related to drug regulation of apoptosis resistance (reviewed by Aguirre-Ghiso, 2007). In The mechanisms by which p38α can mediate addition, p38α can also induce cell survival of apoptosis or cell survival, include those involving colorectal cancer cells through inhibition of the regulation of the expression and/or activity of autophagy (Comes et al., 2007). different members of the Bcl-2 family. 3- Pathways regulating p38α Bax is a pro-apoptotic member of the Bcl-2 family, which can be regulated by p38α through different apoptotic function mechanisms. In cardiomyocytes derived cell lines, There are different signaling pathways that are p38α mediates an up-regulation of Bax mRNA and involved in the regulation of p38 apoptotic protein, which sensitizes cells to apoptosis induced function. Some of them can be considered classical by different stimuli (Porras et al., 2004). In and/or canonical, while others such as C3G-Rap1 or addition, in primary neonatal cardiomyocytes p38 PKC are novel or less known. (α/β) mediates Bax translocation to mitochondria Classically, two small GTPases from the Rho upon simulated ischemia (Capano and Crompton, family, Rac1 and Cdc42, have been described to be 2006). This p38 (α/β)-induced Bax translocation activators of p38α MAPK (Nobes and Hall, 1995). was also observed in a human hepatoma cell line The existence of a functional crosstalk between Rac (HepG2) treated with different pro-apoptotic and p38 MAPK has been largely reported in stimuli (Kim et al., 2006). In contrast, in the relation with different cellular functions. p38 can anoikis-induced apoptosis in mammary epithelial act either as an effector or as an activator of Rac. In cells, p38α is not required for Bax mitochondrial particular, in cardiomyocytes p38α acts as a translocation, but it acts as a part of a mitochondrial positive or a negative regulator of Rac1 depending complex allowing Bax activation and cytochrome-c on the presence of growth factors (Zuluaga et al., release (Owens et al., 2009). 2007b). On the other hand, Rac-GTP induces p38 Bimlevels (Cai and Xia, 2008) and activity (Cai et activation in many systems (Nobes and Hall, 1995). al., 2006) are also positively regulated by p38α, For example, Rac-1 is an upstream regulator of p38 which contributes to induce apoptosis. Thus, in in retinoic acid-induced differentiation and PC12 cells treated with sodium arsenite, p38α apoptosis of malignant NB-4 (acute pro-myelocytic induces FOXO3anuclear translocation, which

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Role of p38α in apoptosis: implication in cancer development Porras A, Guerrero C and therapy stimulates Bim transcription, leading to an increase blebbing and nuclear condensation (Deschesnes et in BimEL protein levels (Cai and Xia, 2008). In al., 2001). addition, p38α contributes to BimEL The tumor suppressor protein p53, which is a phosphorylation in Ser65 (Cai et al., 2006). relevant mediator of apoptosis in response to a great Bcl-2 has been shown to be phosphorylated by variety of stimuli, is also an important p38 target. In p38α upon NGF withdrawal in memory B fact, there are a number of evidences supporting a lymphocytes, which leads to inhibition of its anti- role for p38α in the regulation of p53. For example, apoptotic activity and to induction of cytochrome-c in response to different chemotherapeutic drugs release (Torcia et al., 2001). Similarly, H2O2 such as cisplatin p38α phoshorylates and/or induces p38α-mediated Bcl-2 phoshorylation in activates p53 leading to onset of apoptosis adult rat cardiac myocytes contributing to apoptosis (Bragado et al., 2007; Sánchez-Prieto et al., 2000). (Markou et al., 2009). p38α can also induce apoptosis through p53 in p38 can also mediate survival through regulation of response to other stimuli,. Thus, p53 the expression and/or activity of proteins from the phosphorylation by p38α is essential in the Bcl-2 family. For example, carbon monoxide apoptosis induced by the HIV-1 envelope protects from ischemia-reperfusion lung injury (Perfettini et al., 2005). In addition to this role of through a p38α-dependent up-regulation of Bcl-2 p38α as a direct regulator of p53, p38α also acts and Bcl-xLprotein levels (Zhang et al., 2003). through phosphorylation and stabilization of the According to this, UVA-induced p38 (α/β) p53 coactivator, p18Hamlet (Cuadrado et al., 2007). activation in a human keratinocyte cell line (HaCaT In response to DNA damage p18Hamlet is cells) results in an increase in Bcl-xL protein levels accumulated, which contributes to induce apoptosis through post-transcriptional mechanisms mediated through the stimulation of some p53-regulated by the 3'-unstranslated region (UTR) (Bachelor and genes, such as Noxa. Bowden, 2004). In addition, in primary human Other alternative mechanisms are involved in p38α- trophoblasts p38 appears to mediate EGF-mediated mediated apoptosis. For example, phosphorylation Bad phosphorylation in Ser 112, which protects of H2AX by p38α has been shown to be required from hypoxia-induced apoptosis (Humphrey et al., for serum starvation-induced apoptosis (Lu et al., 2008). 2008). p38α is also involved in the stimulation of Fas and In addition to the above referred effects of p38α FasL expression, which can contribute to apoptosis through direct regulation of pro- and/or in a number of cellular systems. p38α was shown to antiapoptotic proteins, p38α is also a negative participate in the anti-CD3-induced upregulation of regulator of PI3K/Akt and ERKs survival Fas and FasL expression in T cells, although p38α pathways. Thus, in p38α-deficient derived alone was unable to increase Fas expression (Hsu et cardiomyocytes or MEFs cell lines ERKs and Akt al., 1999). Similarly, p38 MAPK is a mediator of activities are increased, which contributes to the hepatitis B virus X protein-induced Fas and FasL enhanced survival of these cells (Porras et al., 2004; expression (Wang et al., 2004). p38α activation also Zuluaga et al., 2007a). In particular, in mediates Fas expression through phosphorylation cardiomyocytes p38α can negatively modulate Akt of STAT1 in Ser-727 in cardiac myocytes exposed activity, independently of PI3K, favoring the to ischemia/reperfusion (Stephanou et al., 2001). interaction between caveolin-1 and PP2A and the According to this, Fas expression is down-regulated activation of PP2A through a mechanism dependent in p38α-deficient embryonic cardiomyocytes under on cell attachment (Zuluaga et al., 2007a). basal conditions which contributes to sensitize cells There are additional mechanisms that can be to apoptosis (Porras et al., 2004). In contrast, p38 involved in the p38α-induced survival, such as the downregulates Fas expression through inhibition of activation of the ATF6α-Rheb-mTOR survival NF-kB in human melanoma cells (Ivanov and pathway (Schewe and Aguirre-Ghiso, 2008). Other Ronai, 2000). mechanisms include the p38α mediated induction of Apart from the role of p38α in the regulation of antioxidant enzymes expression in response to Fas/FasL expression, p38α mediates caspase 8 oxidative stress (unpublished data from our group). activation in Fas-activated Jurkat cells by inhibiting In addition, the increase in BNIP-3 levels and the the phoshorylation and presence of c-FLIPs in the low Bim/Bcl-xL ratio induced by p38α would DISC (Tourian et al., 2004). p38 also mediates contribute to this survival effect upon H2O2 TGF-β-induced activation of caspase 8 (Schrantz et treatment (Gutiérrez-Uzquiza et al., 2010). al., 2001) as well as the regulation of membrane

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Role of p38α in apoptosis: implication in cancer development Porras A, Guerrero C and therapy

Figure 2. Mechanisms involved in p38α-mediated apoptosis. p38Î± induces the expression of pro-apoptotic proteins such as the death receptor, Fas and its ligand, FasL, as well as Bax, Bim or Noxa from the Bcl-2 family. This expression is induced by the phosphorylation of transcription factors, such as p53 and/or coactivators such as p18Hamlet. This would lead to the activation of the Fas death receptor pathway and the mitochondrial pathway. p38α can also activate some of these pro-apoptotic proteins, such as Bim, through phosphorylation or through alternative mechanisms as it happens for Bax. Alternatively, p38α can inhibit c- FLIP phosphorylation impairing its function as an inhibitor of caspase 8 activation. p38α can also inactivate the anti-apoptotic protein Bcl-2 through phosphorylation. In addition, p38α negatively regulates ERKs and Akt survival pathways, being PP2A the mediator of Akt inactivation.

in those deficient in this protein. As a consequence, 5- Disregulation of p38α function p38α-deficient MEFs transformed by Ras in apoptosis: involvement in accumulate high levels of ROS which contributes to human diseases tumour progression. This type of response is Different evidences from the literature indicate that produced in the transformation induced by different the pro-apoptotic function of p38α is deregulated in oncogenes able to generate ROS. In fact, many some human diseases such as cancer, although human cancer cell lines have developed recent findings have also linked p38 signaling with mechanisms to uncouple ROS generation to p38α neurodegenerative diseases such as Alzheimer's activation and therefore induction of apoptosis. disease (AD), Parkinson disease (PD) and However, as referred above, p38α can also induce a amyolotrophic lateral sclerosis (ALS) (reviewed by survival state known as tumor dormancy in some Kim and Choi, 2010). cancer cells such as those from squamous carcinoma, which maintains cells in a quiescent 5.1- Role of p38α in cancer: implication in state related to drug resistance through mechanisms cancer development and therapy 5.1.1- p38α as a tumor suppressor and/or mediator including activation of the p38α-ATF6α-Rheb- Several data indicate that p38α can act as a tumour mTOR survival pathway (reviewed by Aguirre- suppressor (Review by Wagner and Nebreda, Ghiso, 2007; Schewe and Aguirre-Ghiso, 2008). 2009). In particular, p38α negatively regulates 5.1.2- Role of p38 in chronic myeloid leukemia and malignant transformation induced by Ras through other hematopoietic malignancies different mechanisms including apoptosis p38 MAPK, mainly the p38α isoform, is a key induction. For example, in MEFs expressing H- player in the maintenance of hematopoiesis RasV12, sustained activation of p38α MAPK homeostasis, as it balances both proliferative and inhibits transformation through a mechanism growth inhibitory signals triggered by the growth involving ROS-induced apoptosis (Dolado et al., factors and cytokines that regulate normal 2007). H-RasV12-induced ROS is only able to hematopoiesis (Feng et al., 2009; Uddin et al., trigger apoptosis in MEFs expressing p38α, but not 2004). Alterations in this p38 MAPK-controlled

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Role of p38α in apoptosis: implication in cancer development Porras A, Guerrero C and therapy balance may result in either overproduction or Alzheimer's disease is an incurable, depletion of myelosuppressive cytokines leading to neurodegenerative disease characterized by a the development of certain bone marrow failure progressive deterioration of the congnitive, memory syndromes. For example, p38α is responsible for and learning ability including lost of motor the enhanced stem cell apoptosis characteristic of coordination at advanced stages. AD is the result of low grade myeolodysplastic syndromes (MDSs) the accumulation of plaques containing (Navas et al., 2006; Zhou et al., 2007). On the other amyloidogenic Aβ proteins and tangles containing hand, imbalance toward the proliferative side may the microtubule-associated protein tau in a conduct to the development of myeloproliferative hiperphosphorylated state (Giacobini and Becker, syndromes (MPSs), such as leukemia, lymphomas 2007). The ASK1-MKK6-p38 signaling pathway and myelomas. participates in amyeloid precursor protein (APP) The role played by different members of the p38 and tau phosphorylation in response to oxidative kinase family in the regulation of acute leukemia stress and contributes to the expression of the β- blasts growth (myeloid and lymphoid), is not well secretase gene (Galvan et al., 2007) and the defined, although they seem to participate in the induction of neuronal apoptosis triggered by ROS generarion of resistance to chemotherapeutic agents (D'Amico et al., 2000; Puig et al., 2004; Tamagno (Feng et al., 2009; Platanias, 2003) In contrast, the et al., 2003). participation of the p38 MAPK pathways in the Parkinson disease is a degenerative disorder of the pathogenesis and pathophysiology of chronic central nervous system characterized by muscle leukemias have been extensively studied, although rigidity, tremor and loss of physical movement its role is contradictory. On one side, p38 is caused by a progressive loss of dopaminergic activated by the Rho-GEF domain of Bcr, neurons. A number of specific genetic mutations contributing to transformation through the causing Parkinson's disease have been discovered, regulation of NF-κB activation (Korus et al., 2002). being the mutations in α-Synuclein (a major On the other side, p38 MAPK is selectively constituent of Lewy bodies) among the best activated by IFNα and mediates the growth characterized (Gandhi and Wood, 2005). α- suppressive effects of IFNα in CML cells (Mayer et Synuclein activates p38 MAPK in human microglia al., 2001) as well as in normal hematopoiesis promoting a potent inflammatory stimulation of (Verma et al., 2002a). microglial cells (Klegeris et al., 2008). The p38 p38 MAPK also seems to play a role in chronic MAPK is also activated in PD cellular and animal lymphocytic leukemia(CLL). p38 and its effector models and plays a role in dopaminergic neural MK-2 (MAP kinase-activated protein kinase-2) are apoptosis through the phosphorylation of p53 and activated by rituximab in cultures of CLL cells and expression of the pro-apoptotic protein Bax contributes to the generation of the antileukemic (Karunakaran et al., 2008; Mathiasen et al., 2004; effects of rituximab in CLL (Pedersen et al., 2002). Silva et al., 2005). Finally, there are evidences supporting the Amyotrophic lateral sclerosis is a progressive, participation of p38 in cell proliferation and lethal, degenerative disorder of motor neurons in adhesion of lymphomas and multiple myelomas in the brain and spinal cord, leading to paralysis of response to growth factors (Feng et al., 2009; voluntary muscles. Aberrant chemistry and Platanias, 2003). oxidative stress have been described to be involved Another important function of p38α MAPK in in ALS development, being SOD1 (superoxide hematopoiesis is to promote cell differentiation. dismutase 1) the most frequently mutated gene in Curiously, while GTP-induced erythroid the inherited cases of ALS. Numerous evidences differentiation of K562 cells is mediated through point to a role of p38 MAPK in the development p38-dependent caspase activation (Moosavi et al., and progression of ALS induced by mutations in 2007), caspases do not participate in STI-571- SOD1 gene (Bendotti et al., 2004; Bendotti et al., induced erythroid differentiation in CML cells, 2005; Dewil et al., 2007; Holasek et al., 2005; where STI-571-mediated apoptosis and Tortarolo et al., 2003). Mutant SOD1 provokes differentiation are independent events (Jacquel et aberrant oxyradical reactions that increase the al., 2007). The differentiation-inducing therapy may activation of p38 MAPK in motor neurons and glial be a good therapeutical alternative for CML cells. This increase in active p38 MAPK may patients that develop resistance to STI-571. phosphorylate cytoskeletal proteins and activate 5.2- Role of p38α in neurodegenerative diseases cytokines and nitric oxide, thus contributing to Recent findings support the involvement of p38 neurodegeneration through different mechanisms MAPK signaling pathway in neurodegenerative including apoptosis (Bendotti et al., 2005; Tortarolo diseases. Thus, persistent activation of the p38 et al., 2003). MAPK signaling has been suggested to contribute 5.3- p38α in cancer therapy to neuronal apoptosis in Alzheimer's disease (AD), p38α can act as a tumor suppressor in the initial Parkinson disease(PD) and Amyotrophic lateral phases of malignant transformation, which involves sclerosis (ALS). apoptosis induction.

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Similarly, p38α is also a mediator of apoptosis upon cancer cell lines (Galán-Moya et al., 2008) and by treatment with many chemotherapeutical drugs such overexpression of Bcr-Abl, Ara-C (1-β-D- as cisplatin, arsenic trioxide or others. Therefore, arabinofuranosylcytosine) is unable to activate p38 p38α can be considered as a target in the treatment MAPK in cells with constitutive expression of Bcr- of cancer, although sometimes is also responsible Abl such as the CML derived cell line K562 for the resistance to some of these treatments. (Sánchez-Arévalo Lobo et al., 2005). In those cells, It is well established that p38 MAPK mediates the Bcr-Abl induces a sustained activation of the p38 responses to several DNA-damaging agents, such MAPK pathway (not related to increased as UV radiation, cisplatin (CDDP) and Ara-C apoptosis), rendering them insensitive to further through activation of p53-dependent and activation in response to Ara-C. Therefore, Bcr-Abl independent responses (Bulavin et al., 1999; Huang mediated p38 MAPK activation is a key mechanism et al., 1997; Sánchez-Prieto et al., 2000). Cisplatin to explain resistance to Ara-C. In fact, lack of p38 is an anti-cancer drug that induces apoptosis in a MAPK activation is a general mechanism for Ara-C number of cancer cell lines through mechanisms resistance, as it had been previously proposed that in many cases are p38 dependent. In the colon (Stadheim et al., 2000). This could provide a clue carcinoma cell line HCT116, the activation of p38α for new therapeutic approaches based on the was shown to be necessary for CDDP-induced combined use of specific Abl and p38 MAPK apoptosis, upon activation by p53-mediated ROS inhibitors. production (Bragado et al., 2007). Once p38α In contrast, p38 MAPK signaling cascade mediates MAPK is activated, it contributes to further the antiproliferative effects of STI-571 (or imatinib, activation of p53, which leads to a positive the current drug used in CML treatment) and feedback loop. So, a p53/ROS/p38α MAPK cascade cisplatin on Bcr-Abl expressing cells (Galán-Moya is essential for cisplatin-induced cell death and the et al., 2008; Parmar et al., 2004). Moreover, we subsequent p38α/p53 positive feedback loop have recently found a functional relationship strongly enhances the initial p53 activation in between C3G and p38 in CML, so that the pro- HCT116 cells. In other cancer cell lines, c-Abl is apoptotic activity of p38α MAPK, as a mediator of required for CDDP-induced p38 activation and for STI-571 effects, is downregulated by the C3G- its antitumoral effect. Thus, CDDP-induced cell Rap1 pathway and the antitumoral effects of STI- death appears to be dependent on c-Abl expression, 571 could be improved by C3G gene silencing as it rather than on its tyrosine kinase activity, and on enhances p38α activation and pro-apoptotic activity stabilization of MKK6 protein levels (Galán-Moya (Maia et al., 2009). et al., 2008). This pro-apoptotic effect of CDDP is In contrast, p38 appears to be a negative regulator not selective for tumoral cells. Hence, in NIH3T3 of the antitumoral effects of all-trans-retinoic acid cells, which are non-transformed cells, an (RA), which induces differentiation and growth involvement of p38(α/β) MAPK was described as arrest in several malignant cell types, including mediator of CDDP-induced apoptosis through a acute promyelocytic leukemia (APL) and breast mechanism dependent on the phosphorylation of carcinoma. The Rac-p38 MAPK-MK2 pathway is p53 in Ser33 and the subsequent p53 transcriptional activated by RA and negatively regulates RA- activation (Sánchez-Prieto et al., 2000). effects in NB-4 APL and MCF-7 breast cancer cell Similarly to CDDP, other anticancer drugs also lead lines, as p38α/β inhibitor SB203580 enhances RA- to the generation of ROS as an important dependent cell differentiation and apoptosis. mechanism to induce apoptosis in cancer cells, Therefore, pharmacological inhibition of p38α/β being p38α a relevant mediator. Thus, diamine, a may prevent resistance to RA that is developed in thiol-oxidizing compound, induces apoptosis in a nearly all cases (Alsayed et al., 2001). gastric human adenocarcinomacell line (AGS) Arsenic trioxide (AT), which also induces through a mechanism dependent on Trx1/p38α/p53 differentiation and apoptosis of leukemia cells in pathway, while CaCo2 cells are resistant to this vitro and in vivo, activates the Rac-p38 MAPK- compound due to the absence of a functional p53 MK2 pathway, which can act as a negative (Piccirillo et al., 2009). regulator of AT functions, suggesting a possible As previously mentioned, activation of p38 MAPK role of p38 MAPK pathways in AT-induced plays a key role in some hematopoietic resistance (Verma et al., 2002b). However, AT can malignancies by different mechanisms including sensitize human promonocytic cells U937 to TNF- increasing resistance to chemotherapeutic agents α-induced apoptosis, which is dependent on p38α/β (Feng et al., 2009). Ara C is a classic anti-neoplasic activation (Amran et al., 2007). AT increases the agent widely used in the treatment of CML blast levels of TNF-α receptor leading to an enhancement crisis, which provides a typical example of of TNF-α-induced apoptosis. genotoxic stress mediated through c-Abl-p38 The role of p38 MAPK in resistance to MAPK pathway (Huang et al., 1997; Pandey et al., chemotherapeutic agents is also observed in an 1996; Stadheim et al., 2000). Although p38 MAPK experimental model of head and neck cancer, where is activated by c-Abl in response to CDDP in some lower activation or lack of activation of p38 MAPK

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Role of p38α in apoptosis: implication in cancer development Porras A, Guerrero C and therapy correlates with a more resistant phenotype (Losa et Pandey P, Raingeaud J, Kaneki M, Weichselbaum R, al., 2003). Davis RJ, Kufe D, Kharbanda S. Activation of p38 mitogen- activated protein kinase by c-Abl-dependent and - Therefore, based on all these data described here independent mechanisms. J Biol Chem. 1996 Sep and other published data, p38α would play a dual 27;271(39):23775-9 role in cancer treatment. Depending on the type of Huang Y, Yuan ZM, Ishiko T, Nakada S, Utsugisawa T, tumour and the chemotherapeutic agent, p38α can Kato T, Kharbanda S, Kufe DW. Pro-apoptotic effect of the either induce apoptosis or survival, leading to the c-Abl tyrosine kinase in the cellular response to 1-beta-D- disappearance of the tumour or making it resistant arabinofuranosylcytosine. Oncogene. 1997 Oct to chemotherapy. 16;15(16):1947-52 Wang Y, Huang S, Sah VP, Ross J Jr, Brown JH, Han J, Concluding remarks Chien KR. Cardiac muscle cell hypertrophy and apoptosis p38α MAPK (encoded by MAPK14 gene) is induced by distinct members of the p38 mitogen-activated protein kinase family. J Biol Chem. 1998 Jan broadly expressed and is also the most abundant 23;273(4):2161-8 p38 isoform present in most cell types. It can be activated by several extracellular signals, including Bulavin DV, Saito S, Hollander MC, Sakaguchi K, Anderson CW, Appella E, Fornace AJ Jr. Phosphorylation stress stimuli, leading to the regulation of different of human p53 by p38 kinase coordinates N-terminal cellular processes, which includes cell death. phosphorylation and apoptosis in response to UV Although in many cases p38α is a mediator of radiation. 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