Number 11 November 2014

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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, and also more traditional review articles (“deep insights”) on the above subjects and on surrounding topics. It also present case reports in hematology and educational items in the various related topics for students in Medicine and in Sciences.

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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.

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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 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 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 Adriana Zamecnikova (Kuwait) Leukaemia Section

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) Atlas of Genetics and Cytogenetics in Oncology and Haematology

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Volume 18, Number 11, November 2014

Table of contents

Gene Section

ACVRL1 (activin A receptor type II-like 1) 789 Federica Ornati, Luca Vecchia, Claudia Scotti, Sara Plumitallo, Carla Olivieri CTDSPL (CTD (Carboxy-Terminal Domain, RNA Polymerase II, Polypeptide A) Small Phosphatase- Like) 797 Shreya Sarkar, Guru Prasad Maiti, Chinmay Kumar Panda DLX2 (distal-less homeobox 2) 805 Yorick Gitton, Giovanni Levi DLX5 (distal-less homeobox 5) 810 Yorick Gitton, Giovanni Levi DLX6 (distal-less homeobox 6) 817 Yorick Gitton, Giovanni Levi EDIL3 (EGF-Like Repeats And Discoidin I-Like Domains 3) 824 Hisataka Kitano, Chiaki Hidai HELLS (Helicase, Lymphoid-Specific) 829 Kathrin Muegge, Theresa Geiman NUAK1 (NUAK family, SNF1-like kinase, 1) 834 Fumika Inazuka, Hiroyasu Esumi PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2) 838 Ana Rodríguez-García, Pere Fontova, Helga Simon, Anna Manzano, Ramon Bartrons, Àurea Navarro-Sabaté WWTR1 (WW domain containing transcription regulator 1) 849 Yulei Zhao, Xiaolong Yang

Leukaemia Section t(5;11)(q35;q12) NSD1/FEN1 853 Nathalie Douet-Guilbert, Etienne De Braekeleer, Corinne Tous, Nadia Guéganic, Audrey Basinko, Marie-Josée Le Bris, Frédéric Morel, Marc De Braekeleer t(7;9)(q11;p12) PAX5/POM121 856 Jean-Loup Huret t(9;12)(q34;p13) ETV6/ABL1 859 Etienne De Braekeleer, Nathalie Douet-Guilbert, Marc De Braekeleer t(9;22)(p13;q13) PAX5/BRD1 862 Jean-Loup Huret

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Deep Insight Section

Class III beta-tubulin, drug resistance and therapeutic approaches in cancers 865 Roshan Karki, Cristiano Ferlini

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Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11)

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

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Gene Section Review

ACVRL1 (activin A receptor type II-like 1) Federica Ornati, Luca Vecchia, Claudia Scotti, Sara Plumitallo, Carla Olivieri Dept of Molecular Medicine, Unit of General Biology and Medical Genetics, University of Pavia, Italy (FO, SP, CO), Dept of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Italy (LV, CS)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/ACVRL1ID569ch12q13.html DOI: 10.4267/2042/54160 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Transcription Gene database underlines the presence of two Activin A receptor, type II-like kinase 1 (ALK1 is a different ACVRL1 transcripts, which both translate serine-threonine kinase) predominantly expressed into the same protein isoform. The second transcript on endothelial cells surface. Mutations in its variant is the shortest one and differs from the first ACVRL1 encoding gene (12q11-14) cause type 2 one in the 5'UTR region, due to the presence of an Hereditary Haemorrhagic Telangiectasia (HHT2), upstream in-frame start codon, poorly conserved in an autosomal dominant multisystem vascular the population. Nevertheless, in 2010 two new dysplasia. Its involvement in cancer transcripts were discovered in HUVEC cells. These neoangiogenesis has lead to the recent development new variants, called mRNA3 and mRNA4, begin of novel anti-cancer drugs, which are now in the transcription +1 nucleotide upstream , clinical trials. respectively at -510 and -470 positions, adding a cryptic non translated exon, that doesn't affect the Identity protein ORF (Garrido-Martin et al., 2010). Other names: ACVRLK1, ALK-1, ALK1, HHT, The promoter region of ACVRL1 (5' proximal HHT2, ORW2, SKR3, TSR-I region: -1035/+210) was characterized by Garrido- Martin et al., 2010. This region lacks TATA/CAAT HGNC (Hugo): ACVRL1 boxes but contains a high number of GC-rich Sp1 Location: 12q13.13 consensus sites. It also shares different putative regulatory elements with other endothelial-specific DNA/RNA genes. These motifs includes: Ets (E26- Note Transformation-Specific), KLF (Krüppel-Like Starts at 52300692 and ends at 52307134 bp from Factor), NFkB (Nuclear Factor kappa-light-chain- pter (according to hg19- Feb_2009). enhancer of activated B cells), E2F (Elongation Factor 2), one Smad binding element (SBE), RXR Description (Retinoid X Receptor) and HIF (Hypoxia Inducible ACVRL1 is a protein coding gene and in human it Factor). Moreover, the authors demonstrated that is constituted by 10 exons. All exons but the first methylation status of CpG islands modulates Sp1 are coding exons. ACVRL1 transcript variants transcription of ACVRL1 in endothelial cells. mRNA3 and mRNA4 include 11 exons, through the In 2013, it has been demonstrated that ubiquitin E3 presence of a cryptic non-translated exon upstream ligase, EDD, can down-regulate ACVRL1 of the canonical exon 1 (Garrido-Martin et al., expression in HeLa and HUVEC cells (Chien et al., 2010). 2013).

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Protein Description Activin A receptor, type II-like kinase 1 (also called ALK1, Uniprot entry P37023, protein family (pfam) 01064 of Activin types I and II receptor domains), is a serine-threonine kinase and it acts as a type I receptor for the Transforming Growth Factor-β / Bone Morphogenetic Protein (TGF- β/BMP) superfamily of ligands. It includes 503 amino acids, with residues 1-21 forming a leader sequence which targets the protein to the membrane. The extracellular domain includes amino acids 22-118 and it is followed by a 23 amino acid long transmembrane domain (residues 119-141). The intracellular domain comprises residues 142-503, with a GS domain (residues 172- 201) and the protein kinase domain (residues 202- 492). The crystal structure of ALK1 ectodomain (Figure 1a) and of the intracellular kinase domain (Figure 1b) have been recently determined (PDB ID: 4FAO and 3MY0, respectively) (Townson et al., 2012). Like all type I and type II receptors, ALK1 shows a general fold resembling a class of neurotoxins known as three-finger toxins and hence called "three-finger toxin fold". This fold is comprised from β-strands stabilised by disulphide bonds formed by conserved Cys residues. Three pairs of anti-parallel β-strands are curved to generate a concave surface. Despite the common architecture and the cluster of conserved Cys residues, very little sequence identity and no functional overlap exist between the two types of receptors. BMPs consist of a Cys knot characterised by three Figure 1: Structures of ALK1 ectodomain (a) and of the pairs of highly conserved disulphide bonds in kinase domain (b). which one traverses through a ring formed by the other 2. This fold can be described as a hand with a Function concave palm side and two parallel β-sheet forming ALK1 activation, triggered by its physiological 4 fingers, with each β-strand being likened to a ligand BMP-9, can be pro-angiogenic or anti- finger. Finger 2 leads to a helix "wrist" region. In angiogenic, depending on the experimental system the dimeric ligand the 4 fingers extend from the considered. Thus, inhibition of primary cells Cys core of the protein like butterfly wings. (HMVEC-D, HUVEC and endothelial cells) Binding of type I receptors occurs near the α-helix proliferation was observed upon activation of the on the concave side at the junction between the two receptor, suggesting that this signaling pathway is subunits (Kirsch et al., 2000), whereas binding to involved in the resolution phase of angiogenesis, type II receptors happens on the convex side of the during which endothelial cell proliferation and hand near the "fingertips" (Greenwald et al., 2003; migration stop. Disruption of the pathway would Thompson et al., 2003). therefore lead to persistent proliferation of endothelial cells with the lack of a correct Expression morphogenesis. ALK1 is predominantly expressed on the On the other hand, MESEC (mouse embryonic- endothelial cells surface of arteries. According to stem-cell-derived endothelial cells) and MEEC EBI gene expression database, ALK1 levels are (mouse embryonic endothelial cells) cells are reduced in non-small cell lung cancer tissue, and stimulated to proliferate by ALK1 activation and increased in monocytes exposed to infections by BMP9 stimulates angiogenesis in a matrigel plug Francisella tularensis novocida and by assay and in a tumour model in vivo. Also, cancer Porphyromonas gengivalis. cells produced tumours whose size and

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vascularization were reduced by 50% in ALK1+/- heterozygous mice compared with tumours Implicated in implanted in wild-type littermates. Solid tumours In addition, a soluble ALK1-Fc fusion protein known as Dalantercept (ACE-041) showed an anti- Note angiogenic effect by reducing vascular density and As a receptor mainly expressed on the surface of perfusion of the tumour burden in model mice of endothelial cells, ALK1 overexpression and endocrine pancreatic tumorigenesis and mice unbalances in its signalling are implicated in many bearing 786-0 and A498 human renal cell solid tumours, despite the origin and specific carcinoma (Wang et al., 2012). features of the latter. Thus, they will be discussed in This contradictory findings may be explained by a single paragraph. the site- and context-dependent balance of the In a study (Hu-Lowe et al., 2011) performed on synergic proangiogenic effects of BMP-9 and the 3000 human tumour specimens representing more lower affinity ALK1 ligand TGF-β, but the than 100 tumour types, ALK1 resulted particularly assumption has to be confirmed. expressed in the vasculature of prostate cancers, Recent studies also report a role for ALK1 in cancer malignant melanomas of the skin, follicular cancers independent from its effects on angiogenesis, of the tyroid, renal clear cell cancers and enhancing the cell migration and invasion potential endometrioid ovarian cancers. in cancers like squamous cell carcinomas of the A reduced expression of ACVRL1 by qRT-PCR head and the neck or haepatocarcinomas (Hu-Lowe and immunohistochemistry was demonstrated in et al., 2011; Chien et al., 2013; Sun et al., 2013). nasopharingeal carcinomas by Zhang et al., 2012. ALK1 signalling through SMAD 1/SMAD An increased ALK1 expression in papillary thyroid 5/SMAD 8 seems to induce chondrocytes carcinomas with bone formation was increased if hypertrophy in cartilages by an effect mediated by compared to that in normal thyroid tissue and the interaction with the canonical Wnt signaling tumors without bone formation, as assessed using (van den Bosch et al., 2014). immunohistochemistry and quantitative real-time Again, in other kind of cancers ALK1 activation polymerase chain reaction (Na et al., 2013). seems to be protective, as assessed for instance in in In Head and Neck Squamous Cell Carcinomas vitro models of pancreatic cancers (Ungefroren et (HNSCC), using immunohistochemistry and qRT- al., 2007). PCR, Chien et al. found a correlation between a high ACVRL1 expression and an advanced T Homology classification, a positive N classification, an ALK1 shares with other type I receptors a high advanced TNM stage, the presence of degree of similarity in the GS domain, in the lymphovascular invasion, an extracapsular spread following serine-threonine kinase subdomains and of lymph node metastasis and a poorer prognosis in the short C-terminal tail (ten Dijke et al., 1994), (Chien et al., 2013). but the extracellular domain shows a peculiar amino As a therapeutic target, anti-ALK1 drugs (both in acidic sequence. the form of an Fc-fusion protein acting as a soluble receptor for BMP9 and of an anti-ALK1 Mutations monoclonal antibody) are under investigation in phase I and phase II clinical trials in a wide range of Germinal solid tumours (Vecchia et al., 2013). Phase II Mutations in the ACVRL1 gene result in Hereditary studies clinical trials encompass particularly Hemorrhagic Telangiectasia Type 2 (HHT2). A squamous cell carcinoma of the head and neck, germinal mosaic with two mutant alleles in endometrial cancer, epithelial ovarian cancer, hereditary hemorrhagic telangiectasia associated fallopian tube cancer and primary peritoneal with pulmonary arterial hypertension was described carcinoma for ACE-041 (also known as (Eyries et al., 2011). Dalantercept, the Fc-receptor fusion protein). A germline heterozygous ACVRL1 polymorphisms Dalantercept displayed promising antitumour (p. A482V) has been reported in a patient with a activity particularly in patients with advanced gonadotroph pituitary tumour by D'Abronzo et al., refractory cancer (Bendell et al., 2014). PF- 1999. 03446962 (the anti-ALK1 monoclonal antibody), is up to now studied in phase II clinical trials Somatic particularly in malignant mesoteliomas of the No somatic mutations of ACVRL1 have been found pleura and transitional cell carcinomas of the in human cancers. bladder.

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A recent study showed that PF-03446962 has no penetrance. HHT is usually not apparent at birth, activity as a single drug in refractory urothelial but evolves with age into a recognizable phenotypic cancer as is thus suggested, for this kind of cancer, pattern. Spontaneous recurrent nosebleeds are the only as a combination therapy with other agents most common and usually earliest clinical against the VEGF receptor axis (Necchi et al., manifestation. HHT telangiectases develop and get 2014). Both Dalantercept and PF-03446962 are worse with age. Complete penetrance was found to currently under investigation in phase II trials be by 40 years of age (Porteous et al., 1992). HHT particularly in advanced and refractory patients show approximately 15-50% of pulmonary hepatocarcinomas. AVMs (PAVMs), 32-78% of liver AVMs As assessed by Hosman et al., 2013, mutations in (HAVMS) and approximately 23% will harbor ACVRL1 gene, as the ones observed in HHT2 AVMs in the brain (CAVMs). Although 80% of patients, seem to reduce the prevalence of some patients with HHT have gastric or small intestinal types of solid tumours and account for the telangiectases, only 25-30% of patients will unexpected good life expectancy of HHT patients develop symptomatic GI bleeding which usually older than 60 years of age. does not present until the fifth or sixth decades of Although it is important to take with care the results life (Faughnan et al., 2011). of the study due to the methodology used for the HHT arises from heterozygous mutations in ENG assessment (for the statistical and logistic (HHT1, OMIM #187300) coding for ENDOGLIN difficulties to perform a longitudinal study in a rare (ENG) (McAllister et al., 1994) and ACVRL1 disease, the authors used a questionnaire, inevitably (HHT2, OMIM #600376) coding for ALK1 biased), HHT patients older than 60 presented an (Johnson et al., 1996), Type III and Type I TGF-β apparent reduction in lung, liver and colorectal receptors, respectively. Certain HHT2 patients cancer compared to controls. develop a Pulmonary Artery Hypertension (PAH)- This could potentially be related to the ALK1 like syndrome, suggesting that ACVRL1 mutations haploinsuffiency present in ALK1 HHT mutations, are also likely to be involved in PAH (Trembath et opposite to the overexpression usually showed in al., 2001; Olivieri et al., 2006). A subset of patients cancers. with juvenile polyposis, carrying mutations in On the other hand, colorectal cancer was instead SMAD4/MADH4 (JPHT, OMIM #175050), can more frequent in younger HHT patients, also develop HHT (Gallione et al., 2004). Recently, particularly in the subgroup with SMAD4 mutations in BMP9 were reported in three unrelated mutations and juvenile polyposis. families affected by a vascular-anomaly syndrome Hereditary hemorrhagic presenting with phenotypic overlap with HHT (Wooderchak-Donahue et al., 2013). Additional as- telangiectasia type 2 (HHT2) yet-unknown HHT genes have been suggested by Note linkage analysis in two affected kindred on Hereditary Hemorrhagic Telangiectasia (HHT), or chromosome 5 and on chromosome 7 (Cole et al., Rendu-Osler-Weber disease, is a vascular dysplasia 2005; Bayrak-Toydemir et al., 2006). Molecular inherited as an autosomal dominant trait (Shovlin, genetic testing of the three known genes detects 2010; McDonald et al., 2011). It affects mutations in approximately 85% of patients. As approximately 1 in 5-8000 individuals (Faughnan et reported above, the mutated genes encode proteins al., 2011) with regional differences due to founder that mediate signaling by TGF-β family. effects (Westermann et al., 2003; Lesca et al., More than 375 ACVRL1 variants are present in the 2008). The clinical diagnosis of HHT is based on international HHT mutation database and more than the presence of at least three of the following 185 are demonstrated to be pathogenic for HHT. "Curaçao criteria" (Shovlin et al., 2000): (1) TGF-β ligands regulate angiogenesis through their spontaneous, recurrent epistaxis; (2) actions either on endothelial cells (EC) and/or mucocutaneous telangiectases at characteristic sites mural cell, demonstrating that they play important as nose, lips, oral cavity, finger tips and roles in both activation (via ALK1) and resolution gastrointestinal (GI) mucosa; (3) visceral (via ALK5) phases of angiogenesis. It has been arteriovenous malformations (AVMs) in lungs, reported that BMP9, rather than BMP10, might be liver, GI, brain and spinal cord; (4) family history the specific ALK1 ligand and activator of the of first-degree relative in whom HHT has been Smad1/5/8 signaling pathway in endothelial cells diagnosed using these criteria. Significant clinical and that they are potent inhibitors of EC migration variability was observed in HHT (Lesca et al., and growth (David et al., 2007). Previous studies 2007; Govani and Shovlin, 2009), with both intra- have suggested the synergy between Notch and and interfamilial variations in age-of-onset, TGF-β, and that Notch signaling modulates the localization of lesions, and severity of balance between TGF-β/ALK1 and TGF-β/ALK5 complications, whereas it usually shows a high signaling pathways (Fu et al., 2009).

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ALK1-1 and pulmonary arterial PAH. This observation was further confirmed by hypertension other studies (Olivieri et al., 2006) and extensively discussed by Machado et al. (Machado et al., 2009). Note The exact prevalence of PAH in the HHT Pulmonary arterial hypertension (PAH) is a severe population has not been systematically evaluated, and rare disease affecting small pulmonary arteries, but most authors agree that it is a rare complication with progressive remodeling leading to elevated found in less than 1% of HHT patients (Cottin et pulmonary vascular resistance and right ventricular al., 2007). In rare cases, ACVRL1 mutations have failure, and is a major cause of progressive right- been reported to cause IPAH or HPAH without sided heart failure and premature death (Trembath HHT (Harrison et al., 2003). et al., 2001). PAH is defined as the sustained Both ALK1 and BMPR2 belong to the family of elevation of mean pulmonary artery pressure (PA) TGF-β receptors, they have different specific above 25 mmHg at rest or 30 mmHg during ligands but share a common intracellular pathway exercise (Rabinovitch, 2012). The histopathology is based on the activation of the SMAD proteins 1/5/8 marked by vascular proliferation/fibrosis, (Faughnan et al., 2009). remodeling, and vessel obstruction (Chan and The formation of an heteromeric complex with Loscalzo, 2008). BMPR2 and ALK1 could at least in part explain In the second World Symposium held in Evian, why any dysregulation of this pathway may France, in 1998, was proposed a clinical promote pulmonary endothelial and/or smooth classification for pulmonary hypertension. The first muscle cell dysfunction and proliferative category was defined PAH and includes two characteristic of PAH, in subjects carrying mutation subgroups, the first incorporates both the idiopathic either in BMPR2 or in ACVRL1 gene. form (IPAH) that the inherited (HPAH) of the Mutations identified in several studies on ALK1 disease. The second subgroup includes a number of associated with PAH are all likely to disrupt conditions associated with various diseases activation of this intracellular pathway and the (APAH), including connective tissue diseases, majority of these comprise missense mutations. human immunodeficiency virus infection, Particularly, mutations in exon 10 of ACVRL1 are congenital heart disease, and portal hypertension relevant because they occur in functional domains (Simonneau et al., 2004; Machado et al., 2009). of the receptor within a conserved carboxyl- Heterozygous mutations in the transforming growth terminal region of ALK1 (the non-activating non- factor-β receptor (TGF-β receptor) super family down regulating box) NANDOR BOX (Faughnan have been genetically linked to PAH and likely play et al., 2009; Machado et al., 2009). a causative role in the development of disease. Of note, the NANDOR BOX, located from codon Particularly, mutations in the bone morphogenetic 479 to 489, is necessary for regulation of TGF-β factor receptor type 2 (BMPR2) gene account for signaling, accordingly any alteration may have approximately 70% of all familial pedigrees of effects on TGF-β-induced receptor signaling PAH (HPAH) and 10-30% of idiopathic PAH cases (Girerd et al., 2010). (IPAH) (Chan and Loscalzo, 2008; Machado et al., Moreover, recent studies in animal models have 2009). shown that Alk1 heterozygous mice spontaneously Much less commonly (5%) two other members of develop signs of pulmonary hypertension in the the TGF-β superfamily are also recognized as early months of life, and with increasing age show uncommon causes of PAH: activine A receptor type more occluded vessels and pulmonary vascular II-like kinase 1 (ALK1) and, at significant lower remodeling, indicating a progression of the disease. frequency, endoglin (ENG) (Harrison et al., 2003). These mice had also higher ROS levels in adult Heterozygous mutations of these genes cause the lungs contributing to PAH development compared autosomal dominant vascular disorder hereditary to control mice. haemorrhagic teleangiectasia (HHT) (Shovlin, Whereas Bmpr2 heterozygous mouse model 2010). In fact, in a small proportion of HHT requires additional factors, such as hypoxia and patients, was observed a form of pulmonary arterial serotonin or inflammation, to elicit a pulmonary hypertension that is associated with a model of hypertensive phenotype (Jerkic et al., 2011). precapillary pulmonary hypertension that is Finally, Girerd B. et al. hypothesized that mutated histopathologically indistinguishable from ACVRL1 status might be associated with distinct idiopathic form of PAH. Since the publication by PAH phenotypes, as compared with patients PAH Trembath et al. in 2001 (Trembath et al., 2001), without ALK1 mutations. who first reported patients with a mutation in the The authors analyzed clinical, functional gene ACVRL1 with clinical features of both PAH characteristic, hemodynamic features and outcomes and HHT, subsequently, have been recognized for patients with PAH carrying ACVRL1 mutation. several other mutations in the ALK1 gene that seem Of notice, these patients were significantly younger to predispose patients with HHT development of at diagnosis (P

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Pulmonary arterial hypertension is therefore a the activin receptor-like kinase 1 gene in hereditary complex disease that involves the interaction haemorrhagic telangiectasia type 2. Nat Genet. 1996 Jun;13(2):189-95 between genetic predisposition and environmental risk factors. The identification of human mutations D'Abronzo FH, Swearingen B, Klibanski A, Alexander JM. Mutational analysis of activin/transforming growth factor- in components of the TGF-β receptor different from beta type I and type II receptor kinases in human pituitary each other but somehow bound by common tumors. J Clin Endocrinol Metab. 1999 May;84(5):1716-21 intracellular signaling pathways, which may lead to Kirsch T, Sebald W, Dreyer MK. Crystal structure of the the development of pulmonary vascular disease, has BMP-2-BRIA ectodomain complex. Nat Struct Biol. 2000 provided important targets for further investigation. Jun;7(6):492-6 Hematological malignancies Shovlin CL, Guttmacher AE, Buscarini E, Faughnan ME, Hyland RH, Westermann CJ, Kjeldsen AD, Plauchu H. Note Diagnostic criteria for hereditary hemorrhagic Roughly 80% of non-Hodgkin's lymphomas and telangiectasia (Rendu-Osler-Weber syndrome). Am J Med 60% of Hodgin lymphomas express ALK1 in their Genet. 2000 Mar 6;91(1):66-7 vasculature (Hu-Lowe et al., 2011). The expression Trembath RC, Thomson JR, Machado RD, Morgan NV, of ALK1 in haematological cancers was further Atkinson C, Winship I, Simonneau G, Galie N, Loyd JE, confirmed in an exploratory study on patients Humbert M, Nichols WC, Morrell NW, Berg J, Manes A, McGaughran J, Pauciulo M, Wheeler L. Clinical and affected with Acute Myeloid leukemia (AML) molecular genetic features of pulmonary hypertension in (Otten et al., 2011). patients with hereditary hemorrhagic telangiectasia. N Engl Using qRT-PCR, ALK1 was demonstrated to be J Med. 2001 Aug 2;345(5):325-34 expressed by 82% of patients' samples Greenwald J, Groppe J, Gray P, Wiater E, Kwiatkowski W, (pretherapeutic bone marrow or peripheral blood Vale W, Choe S. The BMP7/ActRII extracellular domain from 93 patients with newly diagnosed AML). complex provides new insights into the cooperative nature Furthermore, formalin-fixed, paraffin-embedded of receptor assembly. Mol Cell. 2003 Mar;11(3):605-17 trephine bone marrow specimens from two Harrison RE, Flanagan JA, Sankelo M, Abdalla SA, Rowell arbitrarily selected patients with AML and from J, Machado RD, Elliott CG, Robbins IM, Olschewski H, McLaughlin V, Gruenig E, Kermeen F, Halme M, two patients with non-leukemic reactive changes Räisänen-Sokolowski A, Laitinen T, Morrell NW, Trembath were analyzed for ALK-1 expressions by RC. Molecular and functional analysis identifies ALK-1 as immunohistochemistry. Endothelial cells from two the predominant cause of pulmonary hypertension related AML patients and those with reactive disorders to hereditary haemorrhagic telangiectasia. J Med Genet. were strongly positive and a fraction of AML blasts 2003 Dec;40(12):865-71 stained positively for ALK-1 in AML bone Thompson TB, Woodruff TK, Jardetzky TS. Structures of marrows, whereas normal hematopoietic cells were an ActRIIB:activin A complex reveal a novel binding mode for TGF-beta ligand:receptor interactions. EMBO J. 2003 negative. Apr 1;22(7):1555-66 Anyway, ALK1 alterations, opposite to those in ALK5, seemed not to have a significant impact on Westermann CJ, Rosina AF, De Vries V, de Coteau PA. The prevalence and manifestations of hereditary survival. 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Bendell JC, Gordon MS, Hurwitz HI, Jones SF, Mendelson This article should be referenced as such: DS, Blobe GC, Agarwal N, Condon CH, Wilson D, Pearsall AE, Yang Y, McClure T, Attie KM, Sherman ML, Sharma Ornati F, Vecchia L, Scotti C, Plumitallo S, Olivieri C. S. Safety, pharmacokinetics, pharmacodynamics, and ACVRL1 (activin A receptor type II-like 1). Atlas Genet antitumor activity of dalantercept, an activin receptor-like Cytogenet Oncol Haematol. 2014; 18(11):789-796.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 796

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

CTDSPL (CTD (Carboxy-Terminal Domain, RNA Polymerase II, Polypeptide A) Small Phosphatase-Like) Shreya Sarkar, Guru Prasad Maiti, Chinmay Kumar Panda Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37 S P Mukherjee Road, Kolkata - 700026, West Bengal, India (SS, GPM, CKP)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/CTDSPLID40189ch3p22.html DOI: 10.4267/2042/54161 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Identity Review on CTDSPL, with data on DNA/RNA, on Other names: C3orf8, HYA22, PSR1, RBSP3, the protein encoded and where the gene is SCP3 implicated. HGNC (Hugo): CTDSPL

Location: 3p22.2

Diagram shows the different transcripts of CTDSPL (Brown, Blue, Grey and Maroon boxes). Beginning of boxes represents transcription start sites. Filled areas represent translated regions. The larger form, CTDSPL B is shown as CTDSPL 001, whereas the smaller form, CTDSPL A is shown as CTDSPL 002. Image adapted from Ensembl.org.

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Schematic diagram of full length RBSP3 protein, showing different domains. Adapted from PDB O15194. Data origin/ Colour codes: Data in Green originates from UniProtKB; Data in Yellow originates from Pfam, by interacting with the HMMER3 website; Data in Grey has been calculated using BioJava. Protein disorder predictions are based on JRONN, a Java implementation of RONN. (a. Red- Potentially disordered region. b. Blue- Probably ordered region. Hydropathy has been calculated using a sliding window of 15 residues and summing up scored from standard hydrophobicity tables. a. Red- Hydrophobic. b. Blue- Hydrophilic); Data in blue originates from PDB. Secstruc- Secondary structure projected from representative PDB entries onto the UniProt sequence. (a. Red box - Helix. b. Yellow box - Sheet. c. Grey tube- Coil); Data in red indicates combined ranges of Homology Models from SBKB and the Protein Model Portal.

common. Difference in the size of the 5'UTR may DNA/RNA account for differential splicing between the Description isoforms. Located in the short (p) arm of chromosome 3, the Pseudogene length of the CTDSPL gene is about 122.5 kb, None reported. contains 8 exons and is arranged in a telomere to centromere orientation. Protein Transcription Description The full length transcript of CTDSLP is 4459 bp (Ensembl, Transcript ID ENST00000443503). A The full length CTDSPL protein (CTDSPL B) is total of 8 transcripts can be generated, out of which 276 amino acids in length, with a molecular weight 5 are protein coding, 1 undergoes nonsense of 31 kD. mediated decay, while the rest 2 do not code for a The smaller protein, CTDSPL A is 265 amino acids protein product. However, two splice variants of in length (amino acids 79- 89 missing) and 29.9 kD CTDSPL, the smaller CTDSPL A (lacking exon 3, in weight. Amino acids 102-260 contain the FCP1 therefore short of 33 bp, 11 amino acids) and the homology domain, which contains an essential full length CTDSPL B were identified by Kashuba protein serine phosphatase that dephosphorylates et al., 2004. the C-terminal domain (CTD) of RNA polymerase Interesting observation: The transcription start II. site for CTDSPL A and CTDSPL B are different Localisation (from ensemble.org). While the larger B form has a shorter 5'UTR, the smaller A form has a larger Both nuclear and cytoplasmic (Maiti et al., 2012; 5'UTR, although their translation start sites remain Sarkar et al., 2013).

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CTDSPL Protein expression data from MOPED 1, PaxDb 2 and MAXQB 3. 1. MOPED - Eugene Kolker, Bioinformatics & High- throughput Analysis Lab, Seattle Children's Research Institute. 2. PaxDb - Christian von Mering, Bioinformatics Group, Institute of Molecular Life Sciences, University of Zurich. 3. MAXQB - Matthias Mann, Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Germany. The data was normalized as follows: 1. For each sample, ppm protein values were calculated, if not provided so by data sources. For each sample from MAXQB, iBAQ expression values were divided by sum of values of each sample, and multiplied by 1000000. For all samples, data was gene centrically aggregated by summing expression values of all isoforms for each gene. 2. For better visualization of graphs, expression values are drawn on a root scale, which is an intermediate between log and linear scales as used for our mRNA expression graphs (Safran et al., 2003).

Function phosphatase of Smad1, Smad2/Smad3 and Snail (Wu et al., 2009; Sapkota et al., 2006). - CTDSPL is a serine phosphatase which regulates cell growth and differentiation. It dephosphorylates Homology RB at serine 807/ 811 (hence called RB1 serine Chimpanzee, Rhesus monkey, dog, cow, mouse, phosphatase from human chromosome 3), thereby chicken, zebrafish, S.cerevisiae, K.lactis, increasing RB-E2F interaction and halting the cell E.gossypii, S.pombe, M.oryzae, and N.crassa show cycle at G1/S boundary (Kashuba et al., 2004). conserved RBSP3 gene (Source NCBI - It also inactivates RNA polymerase-II by homologene). preferential dephosphorylation of 'Ser-5' within the RBSP3 and miRNAs tandem 7 residues repeats in the C-terminal domain 1. miRNA 100 (CTD) of the largest RNA polymerase II subunit, - RBSP3 is a bonafide target for miRNA 100. thus controlling the transcription machinery (hence - miRNA 100 binds to the 3`UTR of RBSP3 in called carboxy-terminal domain, RNA polymerase regions conserved in humans, rats and mice. II, polypeptide A small phosphatase-like) (Yeo et - RBSP3 expression is inversely co-related with the al., 2003). expression of miRNA 100 in 76.5% AML cases. - Studies also suggest that CTDSPL/RBSP3 might 2. miRNA 26a (has-miR-26a-1) function as a transcriptional co-repressor, inhibiting - miRNA 26a resides in the intron of RBSP3. transcription of neuronal genes in non-neuronal - It is concomitantly expressed with RBSP3 during cells (Yeo et al., 2005), and may also act as a the cell cycle.

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Different organisms showing homology in RBSP3 protein.

Interacting proteins of RBSP3, using String network.

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Mutations and copy number variations in different organs. Red bar: Loss. Grey bar: Gain. Adapted from COSMIC gene analysis.

variation (p<0.05) between metastatic (64%) and Mutations non-metastatic (32%) cases (Anedchenko et al., Germinal 2007) None reported. - Altogether, copy number change was seen in 51% cases (Anedchenko et al., 2007). Implicated in - Decreased expression was seen in 64% cases, with significant difference between metastatic (83%) and Cervical cancer non-metastatic (52%) cases (Anedchenko et al., Note 2007). - High deletion (48%, 45% cases) and methylation - Increase in expression was also observed in some (26%, 25% cases) was seen in CIN and CACX cases (Anedchenko et al., 2007). respectively (Mitra et al., 2010). - Altogether, change in expression was in 79% of - Reduced mRNA expression was seen in CACX cases (Anedchenko et al., 2007). (Mitra et al., 2010). Prognosis - RBSP3B (larger, active isoform) was under- RBSP3 alterations (deletion, methylation) were expressed in CACX (Mitra et al., 2010). significantly associated with poor patient outcome - In HPV infected cervical cancer, high deletion and posed 4.5-13 times risk of survival (42% cases) was observed, with significant (Anedchenko et al., 2007).

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Oncogenesis et al., 2008). Inactivation of RBSP3 was an early event in - Fold decrease in expression of RBSP3 in AC and cervical carcinogenesis (Mitra et al., 2010). SCC was 78% and 88% respectively, with overall Breast cancer 85% decrease in expression of RBSP3 in NSCLC (Senchenko et al., 2010). Note Oncogenesis - Study population was divided into two groups, Deletion and methylation of promoter of RBSP3 Group A ( ≤40 yrs, early onset) and Group B (>40 are responsible for reduction in expression of the yrs, late onset) (Sinha et al., 2008). protein and play important roles in progression of - High deletion (30%, 24% cases) and methylation NSCLC (Senchenko et al., 2008). (38%, 32% cases) were observed in Groups A and Reduction of expression of RBSP3 is required for B respectively (Sinha et al., 2008). development of lung adenocarcinomas (Senchenko - 28.9 ± 39.1 fold reduction in expression of RBSP3 et al., 2010). was observed in about 33 - 40% of the tumors (Sinha et al., 2008). Ovarian cancer - Homozygous deletion (10-18%) was observed for Note RBSP3 (Senchenko et al., 2004). Deletion/Methylation of RBSP3 were observed in Prognosis 33% cases. Patients belonging lower to age of onset ( ≤40 yrs) Oncogenesis with alterations of RBSP3 had poor disease RBSP3 deletion/methylation can be used as a outcome (Sinha et al., 2008). biomarker for ovarian cancer in combination with Oncogenesis other studied markers. Higher alterations of RBSP3 were observed in Head and neck squamous cell patients belonging to the lower age of onset (Group A) (Sinha et al., 2008). carcinoma (HNSCC) Acute lymphoid leukemia (ALL) Note - Deletion of RBSP3 in dysplasia and HNSCC was Note in 24% and 32% cases respectively (Ghosh et al., Promoter methylation was seen in RBSP3 in 24% 2010). of ALL patients. - Promoter methylation was observed in 39% and Prostate cancer 38% cases of dysplasia and HNSCC samples respectively (Ghosh et al., 2010). Note - Fold reduction of mRNA in the tumors was 33.6 ± GWAS study using Affymetrix 100K SNP 9.4 (Ghosh et al., 2010). GeneChip with GEE model showed that the SNP, - While normal tissues expressed the larger RBSP3 rs9311171 (G/ T), located within RBSP3, had a B form, tumors either showed no expression of notable GEE p value (1.8x 10 -6). RBSP3, or preferentially expressed the smaller, less Oncogenesis active form, RBSP3 A (Ghosh et al., 2010). -6 GEE p value 1.8x 10 indicates that this SNP Expression of RBSP3 decreases from pre-malignant within RBSP3 plays a role in tumor progression. to malignant lesions (Maiti et al., 2012). Non - small cell lung cancer (NSCLC) Expression of RBSP3 was seen to be increased from pre-neoadjuvant chemotherapy tumors to Note post-therapy tumors (Sarkar et al., 2013). - Reduction of expression of RBSP3 was obtained for both adenocarcinoma (AC) and squamous cell Prognosis carcinoma (SCC) (Senchenko et al., 2008). Patients with RBSP3 alterations show poor survival - Downregulation was both genetic and epigenetic (Ghosh et al., 2010). (Senchenko et al., 2008). Oncogenesis - For ACs, decrease in level of expression was in Early alteration of RBSP3 takes place in head and 88% cases and 70% cases of metastatic and non- neck cancers (Ghosh et al., 2010). metastatic tumors respectively, whereas for SCCs, Loss of expression of RBSP3 was seen to be it was in 88% cases for both metastatic and non- required for progression from malignant to invasive metastatic tumors (Senchenko et al., 2008). cancer (Maiti et al., 2012). - Decrease in mRNA in ACs was due to deletion Regain of expression of RBSP3 in post-therapy (25% cases) and promoter methylation (38% cases), tumors may be one of the reasons of shrinkage of whereas for SCCs, it was in 30% and 80% cases for tumors due to neoadjuvant chemotherapy (Sarkar et deletion and methylation respectively (Senchenko al., 2013).

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Lung, renal, breast, cervical and Yeo M, Lee SK, Lee B, Ruiz EC, Pfaff SL, Gill GN. Small CTD phosphatases function in silencing neuronal gene ovarian cancers expression. Science. 2005 Jan 28;307(5709):596-600 Note Sapkota G, Knockaert M, Alarcón C, Montalvo E, Brivanlou High frequencies of somatic mutations in RBSP3 in AH, Massagué J. Dephosphorylation of the linker regions different cancers suggesting it may underlay the of Smad1 and Smad2/3 by small C-terminal domain phosphatases has distinct outcomes for bone mutator phenotype of cancer. morphogenetic protein and transforming growth factor-beta Acute myeloid leukemia (AML) pathways. J Biol Chem. 2006 Dec 29;281(52):40412-9 Note Shu J, Jelinek J, Chang H, Shen L, Qin T, Chung W, Oki Y, Issa JP. Silencing of bidirectional promoters by DNA RBSP3 might have a crucial role in myeloid cell methylation in tumorigenesis. Cancer Res. 2006 May differentiation towards granulocyte/monocyte 15;66(10):5077-84 lineages through pRB-E2F pathway. Anedchenko EA, Kiseleva NP, Dmitriev AA, Kiselev FL, Cell lines Zabarovski ĭ ER, Senchenko VN. [Tumor suppressor gene RBSP3 in cervical carcinoma: copy number and Note transcriptional level]. Mol Biol (Mosk). 2007 Jan- Leukemia cell lines RAJI, BJAB (B cell leukemia) Feb;41(1):86-95 and HL-60 (myeloid leukemia) showed Murabito JM, Rosenberg CL, Finger D, Kreger BE, Levy D, hypermethylation of RBSP3 promoter. Splansky GL, Antman K, Hwang SJ. A genome-wide association study of breast and prostate cancer in the Hepatocellular carcinoma (HCC) in NHLBI's Framingham Heart Study. BMC Med Genet. 2007 mouse model system Sep 19;8 Suppl 1:S6 Anedchenko EA, Dmitriev AA, Krasnov GS, Kondrat'eva Note TT, Kopantsev EP, Vinogradova TV, Zinov'eva MV, RBSP3 shows increase in expression (RNA, Zborovskaia IB, Polotski ĭ BE, Sakharova OV, Kashuba VI, protein) upon treatment with the chemopreventive Zabarovski ĭ ER, Senchenko VN. [Down-regulation of agent Amarogentin. RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1 and HYAL2 genes in non-small cell lung cancer]. Mol Biol Oncogenesis (Mosk). 2008 Nov-Dec;42(6):965-76 Increase in expression of RBSP3 might play a role Sinha S, Singh RK, Alam N, Roy A, Roychoudhury S, in chemoprevention upon treatment with Panda CK. Frequent alterations of hMLH1 and amarogentin. RBSP3/HYA22 at chromosomal 3p22.3 region in early and late-onset breast carcinoma: clinical and prognostic References significance. Cancer Sci. 2008 Oct;99(10):1984-91 Kashuba VI, Pavlova TV, Grigorieva EV, Kutsenko A, Protopopov A, Kashuba V, Zabarovska VI, Muravenko OV, Yenamandra SP, Li J, Wang F, Protopopov AI, Lerman MI, Klein G, Zabarovsky ER. An integrated Zabarovska VI, Senchenko V, Haraldson K, Eshchenko T, physical and gene map of the 3.5-Mb chromosome 3p21.3 Kobliakova J, Vorontsova O, Kuzmin I, Braga E, Blinov (AP20) region implicated in major human epithelial VM, Kisselev LL, Zeng YX, Ernberg I, Lerman MI, Klein G, malignancies. Cancer Res. 2003 Jan 15;63(2):404-12 Zabarovsky ER. High mutability of the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) in cancer. PLoS Safran M, Chalifa-Caspi V, Shmueli O, Olender T, Lapidot One. 2009 May 29;4(5):e5231 M, Rosen N, Shmoish M, Peter Y, Glusman G, Feldmesser E, Adato A, Peter I, Khen M, Atarot T, Groner Y, Lancet D. Ghosh A, Ghosh S, Maiti GP, Sabbir MG, Zabarovsky ER, Human Gene-Centric Databases at the Weizmann Institute Roy A, Roychoudhury S, Panda CK. Frequent alterations of Science: GeneCards, UDB, CroW 21 and HORDE. of the candidate genes hMLH1, ITGA9 and RBSP3 in early Nucleic Acids Res. 2003 Jan 1;31(1):142-6 dysplastic lesions of head and neck: clinical and prognostic significance. Cancer Sci. 2010 Jun;101(6):1511-20 Yeo M, Lin PS, Dahmus ME, Gill GN. A novel RNA polymerase II C-terminal domain phosphatase that Mitra S, Mazumder Indra D, Bhattacharya N, Singh RK, preferentially dephosphorylates serine 5. J Biol Chem. Basu PS, Mondal RK, Roy A, Zabarovsky ER, 2003 Jul 11;278(28):26078-85 Roychoudhury S, Panda CK. RBSP3 is frequently altered in premalignant cervical lesions: clinical and prognostic Kashuba VI, Li J, Wang F, Senchenko VN, Protopopov A, significance. Genes Chromosomes Cancer. 2010 Malyukova A, Kutsenko AS, Kadyrova E, Zabarovska VI, Feb;49(2):155-70 Muravenko OV, Zelenin AV, Kisselev LL, Kuzmin I, Minna JD, Winberg G, Ernberg I, Braga E, Lerman MI, Klein G, Senchenko VN, Anedchenko EA, Kondratieva TT, Krasnov Zabarovsky ER. RBSP3 (HYA22) is a tumor suppressor GS, Dmitriev AA, Zabarovska VI, Pavlova TV, Kashuba VI, gene implicated in major epithelial malignancies. Proc Natl Lerman MI, Zabarovsky ER. Simultaneous down- Acad Sci U S A. 2004 Apr 6;101(14):4906-11 regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G21 and RASSF1A in primary non-small cell lung Senchenko VN, Liu J, Loginov W, Bazov I, Angeloni D, cancer. BMC Cancer. 2010 Mar 1;10:75 Seryogin Y, Ermilova V, Kazubskaya T, Garkavtseva R, Zabarovska VI, Kashuba VI, Kisselev LL, Minna JD, Kashuba V, Dmitriev AA, Krasnov GS, Pavlova T, Ignatjev Lerman MI, Klein G, Braga EA, Zabarovsky ER. Discovery I, Gordiyuk VV, Gerashchenko AV, Braga EA, of frequent homozygous deletions in chromosome 3p21.3 Yenamandra SP, Lerman M, Senchenko VN, Zabarovsky LUCA and AP20 regions in renal, lung and breast E. NotI Microarrays: Novel Epigenetic Markers for Early carcinomas. Oncogene. 2004 Jul 29;23(34):5719-28 Detection and Prognosis of High Grade Serous Ovarian

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Cancer. Int J Mol Sci. 2012 Oct 18;13(10):13352-77 Zhu Y, Lu Y, Zhang Q, Liu JJ, Li TJ, Yang JR, Zeng C, Zhuang SM. MicroRNA-26a/b and their host genes Maiti GP, Ghosh A, Chatterjee R, Roy A, Sharp TV, cooperate to inhibit the G1/S transition by activating the Roychoudhury S, Panda CK. Reduced expression of pRb protein. Nucleic Acids Res. 2012 May;40(10):4615-25 LIMD1 in ulcerative oral epithelium associated with tobacco and areca nut. Asian Pac J Cancer Prev. Sarkar S, Maiti GP, Jha J, Biswas J, Roy A, Roychoudhury 2012;13(9):4341-6 S, Sharp T, Panda CK. Reduction of proliferation and induction of apoptosis are associated with shrinkage of Pal D, Sur S, Mandal S, Das A, Roy A, Das S, Panda CK. head and neck squamous cell carcinoma due to Prevention of liver carcinogenesis by amarogentin through neoadjuvant chemotherapy. Asian Pac J Cancer Prev. modulation of G1/S cell cycle check point and induction of 2013;14(11):6419-25 apoptosis. Carcinogenesis. 2012 Dec;33(12):2424-31 Zheng YS, Zhang H, Zhang XJ, Feng DD, Luo XQ, Zeng This article should be referenced as such: CW, Lin KY, Zhou H, Qu LH, Zhang P, Chen YQ. MiR-100 Sarkar S, Maiti GP, Panda CK. CTDSPL (CTD (Carboxy- regulates cell differentiation and survival by targeting Terminal Domain, RNA Polymerase II, Polypeptide A) RBSP3, a phosphatase-like tumor suppressor in acute Small Phosphatase-Like). Atlas Genet Cytogenet Oncol myeloid leukemia. Oncogene. 2012 Jan 5;31(1):80-92 Haematol. 2014; 18(11):797-804.

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DLX2 (distal-less homeobox 2) Yorick Gitton, Giovanni Levi Evolution des Regulations Endocriniennes, CNRS, UMR7221, Museum National d'Histoire Naturelle, Paris, France (YG, GL)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/DLX2ID52177ch2q31.html DOI: 10.4267/2042/54162 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract integrity (Kraus and Lufkin, 2006). DLX2 belongs to the six-member family of DLX Identity genes characterized by a homeobox related to that Other names: TES-1, TES1 found in the insect Distal-less (Dll) gene. It was the first human homologue to be discovered (Selski et HGNC (Hugo): DLX2 al., 1993; McGuinness et al., 1996). Location: 2q31.1 The six DLX genes are organized as three bigenic Local order pairs with a tail-to-tail orientation (Zerucha et al., Reverse strand of human chromosome 2, from 2000) and located on chromosomes where HOX 172964167 to 172967628 - see Figure 1 below. clusters are also found (DLX5/DLX6; 7q21.3, DLX2 forms a bigenic cluster with DLX1 at 2q31. syntenic to the HOXA cluster), (DLX1/DLX2; 2q32 syntenic to the HOXD cluster; Simeone et al., DNA/RNA 1994; Zerucha et al., 2000) and (DLX3/DLX4; 17q21.33 syntenic to the HOXB cluster). Note During embryonic development DLX genes are DLX2 is in an inverted convergent orientation from involved in the control of appendage and DLX1, with both exons 3 separated by 10.7 kb craniofacial morphogenesis and in the where two enhancers have been identified and differentiation of reproductive organs; in the adult functionally characterized (Zerucha et al., 2000; they play a role in bone homeostasis and in the Sumiyama et al., 2002; Ghanem et al., 2003; Park maintenance of tissue et al., 2004).

Figure 1. Genomic context of the human DLX1/DLX2 bigenic locus.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 805 DLX2 (distal-less homeobox 2) Gitton Y, Levi G

Figure 2. The two known human DLX2 transcripts.

While one regulatory element is enough and sufficient to drive proper and full expression of Protein Dlx1/Dlx2 genes based upon mouse reporter Description assays, other, extragenic elements are involved in the dual regulation of Dlx1 and Dlx2. The major DLX2 isoform is a 328 AA helix-turn- The imprinting status of the DLX1/DLX2 locus has helix homeodomain transcription factor (34.2 kDa received much less attention than that of their and pI 9.5). The ultraconserved homeobox domain DLX5/DLX6 paralogs (see their respective cards). spans exons 1 and 2 at 153-211. A second N- However, an epigenetic mechanism linking Dlx2 terminal DNA binding domain, specific to the function with neural stem cell maintenance has DLX2/3/5 clade within the distal-less family, is been demonstrated in adult mice (Lim et al., 2009). encoded by exon 1 at AA 51-132. Local Chromatin immunoprecipitation assays composition biases include three poly-glycine and differentiating subventricular zone neural stem cells one poly-histidine stretches, and a phosphorylation has shown that the Dlx2 locus is a direct bivalent target serine at 232 (see Figure 3 below). target of the histone methyltransferase Mll1 Expression (mixed-lineage leukemia 1). Whether such DLX2 is a predominantly nuclear transcription methylation process has a clinical relevance factor, from the helix-turn-helix group. It remains to be determined, as altered methylation of transactivates target gene expression in DLX2 has been observed in pathogenic conditions, heterodimeric association with other DLX and including in primary cells from brain astrocytomas, MSX transcription factors. Its consensus binding where hypermethylation of DLX2 CpG island has site is TTA(G/A)TTGA. Chromatin been observed (Wu et al., 2010). immunoprecitpitation assays have shown that Transcription during mouse forebrain development, Dlx2 (along with Dlx1) specifically binds an intergenic Transcription from DLX2 yields two splice variants enhancer within the Dlx5/Dlx6 locus (see their which share the first two exons, the homeodomain respective cards), and transactivates their and the N-terminal DLL domain (see Figure 2). The expression (Zerucha et al., 2000; Zhou et al., 2004). major, mature isoform encodes a 328 AA long Interestingly, regulation of the Dlx5/Dlx6 locus in transcription factor (34.2 kDa and pI 9.25). Sense the developing retina occurs through transactivation isoforms have not been reported for this gene. by Dlx2 but not Dlx1.

Figure 3. Structure of the major DLX2 protein isoform. Note the N- and C-terminal poly-glycine stretches, and the N-terminal DLL-like domain.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 806 DLX2 (distal-less homeobox 2) Gitton Y, Levi G

Figure 4. NCBI/COBALT alignment of DLX homeoproteins. Note the disposition according to the DLX 1/4/6 versus DLX 2/3/5 clades. Indicated by a yellow box is the ultraconserved glutamine featured by most homeoproteins at position 50 of the homeodomain.

Function Homology A particularity of Dlx2 is its ability to cooperate With regards to other members of the DLX family, with a nuclear non-coding RNA (sense and single- DLX2 belongs to the DLX2/3/5 clade based on stranded, 440 b), Evf-2, transcribed from the sequence homology (see Figure 4). It shares an N- Dlx5/Dlx6 locus, to form a stable complex which terminal DLL-like domain specific to this clade. binds and transactivates both Dlx5/6 intergenic The homeodomain sequence remains close to the enhancers (Feng et al., 2006). other DLX proteins. While such an RNA/homeoprotein cooperativity has been demonstrated for other factors (Dubnau Implicated in and Struhl, 1996), it has not been reported so far for other Dlx family members. Breast tumors and their metastases On the other hand, Dlx2 has been shown to to bone and lung tissues autorepress its expression during mouse tooth formation when expressed alone, while Note autoactivating it when expressed in combination Neoplastic processes often result from with PitX2 (Venugopalan et al., 2011). combinatorial activity of developmental genes This observation lends support to the notion that the (Abate-Shen, 2002). Dysregulated expression of transcriptional activity of DLX factors often homeobox-containing genes of the distal-less depends upon cooperative binding with other family, arranged as three bigenic pairs in mammals homeproteins, including from the PTX and MSX (DLX1/2, DLX3/4 and DLX5/6; Kraus and Lufkin, families (Zhang et al., 1997; Vieux-Rochas et al., 2006), has been reported to correlate with distinct 2013). oncogenic mechanisms.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 807 DLX2 (distal-less homeobox 2) Gitton Y, Levi G

DLX2 is expressed and necessary but insufficient to examined for association with autism spectrum initiate metabolic stress-induced necrosis within disorder. Extensive coverage of both coding and several solid human high-grade tumors (Lee et al., intergenic sequences among a large cohort of 2011). DLX2 is strongly expressed in human autistic probands has uncovered only a handful of primary breast tumors, its expression is associated non-synonymous variants - which nevertheless with better prognosis and fewer relapses (Morini et provides a strong set of functional candidates to al., 2010). In contrast, DLX2 expression is lost by assess whether disrupted DLX2 expression might breast tumor-derived metastatic cells found in lung play a role in autism (Hamilton et al., 2005). More or bone tissues - a poor prognosis marker (Morini et recently, a large cohort study has pinpointed al., 2010). The combined downregulation of DLX2 stronger candidate sites of polymorphism correlated and upregulation of DLX5 might thus prove a with increased susceptibility to develop the valuable prognostic marker. neurologic condition (Liu et al., 2009) - however a DLX2 has been observed to be one of several direct functional impact remains to be evidenced. homeogenes whose CpG islands were Interestingly, DLX2 was found to harbour hypermethylated in luminal breast cancer cells, at 1 trinucleotide repeats but as for its DLX6 paralog, kb from the transcription start site (Kamalakaran et family-based association analysis ruled out this al., 2011). This status has been found to correlate polymorphism as a risk variant, in either autism or significantly with higher expression level of DLX2, schizophrenia patients (Laroche et al., 2008). which lends support to the notion that DLX2 may serve as a candidate prognosis marker in breast Dysmorphogenesis cancer (Morini et al., 2010). Note Solid tumors involving other organs Although mouse embryos invalidated for Dlx1 and/or Dlx2 display craniofacial abnormalities, a Note direct involvement of DLX1/DLX2 mutation in Induction of DLX2 expression has been further human malformations remains to be demonstrated. reported in other solid tumors, including promoting For instance, while synpolydactyly has been tightly advanced gastric adenocarcinoma (Tang et al., linked to DLX2 (Sarfarazi et al., 1995), it is the 2013), promoting growth from lung, prostate and lack of induction of its promoter by defective glioma tumors, and correlates with melanoma PITX2 which has been demonstrated to directly malignancy (Yilmaz et al., 2011; Yan et al., 2013). cause Axenfeld-Rieger syndrome (ARS, OMIM It appears that at least one member of each DLX #180500) - an autosomal dominant condition bigenic pair (DLX2, DLX5 and DLX4 : see Hara et featuring a wide range of tooth anomalies, al., 2007) is closely implicated with solid maxillary hypoplasia, and eye malformation tumorigenicty. (Espinoza et al., 2002). Acute lymphoblastic leukemia Note References Conversely, DLX2 expression is lost along with Selski DJ, Thomas NE, Coleman PD, Rogers KE. The DLX3 and DLX4 in samples from patients afflicted human brain homeogene, DLX-2: cDNA sequence and alignment with the murine homologue. Gene. 1993 Oct by acute lymphoblastic leukemia with 15;132(2):301-3 t(4;11)(q21;q23) chromosomal abnormality (Ferrari et al., 2003). In the same paper it is also shown that Simeone A, Acampora D, Pannese M, D'Esposito M, Stornaiuolo A, Gulisano M, Mallamaci A, Kastury K, Druck Dlx genes participate to the regulatory cascade T, Huebner K. Cloning and characterization of two initiated by acute lymphoblastic leukemia (ALL)-1, members of the vertebrate Dlx gene family. Proc Natl Acad a recurring partner of translocations involving Sci U S A. 1994 Mar 15;91(6):2250-4 chromosome band 11q23 in human biphenotypic Sarfarazi M, Akarsu AN, Sayli BS. Localization of the leukemias. syndactyly type II (synpolydactyly) locus to 2q31 region and identification of tight linkage to HOXD8 intragenic Autism spectrum disorder marker. Hum Mol Genet. 1995 Aug;4(8):1453-8 Note Dubnau J, Struhl G. RNA recognition and translational Autism has been recognized as a condition which regulation by a homeodomain protein. Nature. 1996 Feb may result from an imbalance between inhibitory 22;379(6567):694-9 and excitatory processes in the developing and McGuinness T, Porteus MH, Smiga S, Bulfone A, Kingsley mature brain. Dlx1 and Dlx2 control the C, Qiu M, Liu JK, Long JE, Xu D, Rubenstein JL. specification, fate and metabolic function of a Sequence, organization, and transcription of the Dlx-1 and Dlx-2 locus. Genomics. 1996 Aug 1;35(3):473-85 subset of neurons known to exert an inhibitory role in the brain. As part of a cascade of homeobox- Zhang H, Hu G, Wang H, Sciavolino P, Iler N, Shen MM, Abate-Shen C. Heterodimerization of Msx and Dlx containing genes controlling neuronal specification homeoproteins results in functional antagonism. Mol Cell in the brain, the DLX1/2 locus has thus been Biol. 1997 May;17(5):2920-32

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Zerucha T, Stühmer T, Hatch G, Park BK, Long Q, Yu G, Genet. 2008 Dec;18(6):295-301 Gambarotta A, Schultz JR, Rubenstein JL, Ekker M. A highly conserved enhancer in the Dlx5/Dlx6 intergenic Lim DA, Huang YC, Swigut T, Mirick AL, Garcia-Verdugo region is the site of cross-regulatory interactions between JM, Wysocka J, Ernst P, Alvarez-Buylla A. Chromatin Dlx genes in the embryonic forebrain. J Neurosci. 2000 remodelling factor Mll1 is essential for neurogenesis from Jan 15;20(2):709-21 postnatal neural stem cells. Nature. 2009 Mar 26;458(7237):529-33 Abate-Shen C. Deregulated homeobox gene expression in cancer: cause or consequence? Nat Rev Cancer. 2002 Liu X, Novosedlik N, Wang A, Hudson ML, Cohen IL, Oct;2(10):777-85 Chudley AE, Forster-Gibson CJ, Lewis SM, Holden JJ. The DLX1and DLX2 genes and susceptibility to autism Espinoza HM, Cox CJ, Semina EV, Amendt BA. A spectrum disorders. Eur J Hum Genet. 2009 molecular basis for differential developmental anomalies in Feb;17(2):228-35 Axenfeld-Rieger syndrome. Hum Mol Genet. 2002 Apr 1;11(7):743-53 Morini M, Astigiano S, Gitton Y, Emionite L, Mirisola V, Levi G, Barbieri O. Mutually exclusive expression of DLX2 Sumiyama K, Irvine SQ, Stock DW, Weiss KM, Kawasaki and DLX5/6 is associated with the metastatic potential of K, Shimizu N, Shashikant CS, Miller W, Ruddle FH. the human breast cancer cell line MDA-MB-231. BMC Genomic structure and functional control of the Dlx3-7 Cancer. 2010 Nov 25;10:649 bigene cluster. Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):780-5 Wu X, Rauch TA, Zhong X, Bennett WP, Latif F, Krex D, Pfeifer GP. CpG island hypermethylation in human Ferrari N, Palmisano GL, Paleari L, Basso G, Mangioni M, astrocytomas. Cancer Res. 2010 Apr 1;70(7):2718-27 Fidanza V, Albini A, Croce CM, Levi G, Brigati C. DLX genes as targets of ALL-1: DLX 2,3,4 down-regulation in Kamalakaran S, Varadan V, Giercksky Russnes HE, Levy t(4;11) acute lymphoblastic leukemias. J Leukoc Biol. 2003 D, Kendall J, Janevski A, Riggs M, Banerjee N, Aug;74(2):302-5 Synnestvedt M, Schlichting E, Kåresen R, Shama Prasada K, Rotti H, Rao R, Rao L, Eric Tang MH, Satyamoorthy K, Ghanem N, Jarinova O, Amores A, Long Q, Hatch G, Park Lucito R, Wigler M, Dimitrova N, Naume B, Borresen-Dale BK, Rubenstein JL, Ekker M. Regulatory roles of AL, Hicks JB. DNA methylation patterns in luminal breast conserved intergenic domains in vertebrate Dlx bigene cancers differ from non-luminal subtypes and can identify clusters. Genome Res. 2003 Apr;13(4):533-43 relapse risk independent of other clinical variables. Mol Oncol. 2011 Feb;5(1):77-92 Park BK, Sperber SM, Choudhury A, Ghanem N, Hatch GT, Sharpe PT, Thomas BL, Ekker M. Intergenic Lee SY, Jeon HM, Kim CH, Ju MK, Bae HS, Park HG, Lim enhancers with distinct activities regulate Dlx gene SC, Han SI, Kang HS. Homeobox gene Dlx-2 is implicated expression in the mesenchyme of the branchial arches. in metabolic stress-induced necrosis. Mol Cancer. 2011 Dev Biol. 2004 Apr 15;268(2):532-45 Sep 14;10:113 Zhou QP, Le TN, Qiu X, Spencer V, de Melo J, Du G, Venugopalan SR, Li X, Amen MA, Florez S, Gutierrez D, Plews M, Fonseca M, Sun JM, Davie JR, Eisenstat DD. Cao H, Wang J, Amendt BA. Hierarchical interactions of Identification of a direct Dlx homeodomain target in the homeodomain and forkhead transcription factors in developing mouse forebrain and retina by optimization of regulating odontogenic gene expression. J Biol Chem. chromatin immunoprecipitation. Nucleic Acids Res. 2011 Jun 17;286(24):21372-83 2004;32(3):884-92 Yilmaz M, Maass D, Tiwari N, Waldmeier L, Schmidt P, Hamilton SP, Woo JM, Carlson EJ, Ghanem N, Ekker M, Lehembre F, Christofori G. Transcription factor Dlx2 Rubenstein JL. Analysis of four DLX homeobox genes in protects from TGF β-induced cell-cycle arrest and autistic probands. BMC Genet. 2005 Nov 2;6:52 apoptosis. EMBO J. 2011 Sep 6;30(21):4489-99 Feng J, Bi C, Clark BS, Mady R, Shah P, Kohtz JD. The Tang P, Huang H, Chang J, Zhao GF, Lu ML, Wang Y. Evf-2 noncoding RNA is transcribed from the Dlx-5/6 Increased expression of DLX2 correlates with advanced ultraconserved region and functions as a Dlx-2 stage of gastric adenocarcinoma. World J Gastroenterol. transcriptional coactivator. Genes Dev. 2006 Jun 2013 May 7;19(17):2697-703 1;20(11):1470-84 Vieux-Rochas M, Bouhali K, Mantero S, Garaffo G, Kraus P, Lufkin T. Dlx homeobox gene control of Provero P, Astigiano S, Barbieri O, Caratozzolo MF, Tullo mammalian limb and craniofacial development. Am J Med A, Guerrini L, Lallemand Y, Robert B, Levi G, Merlo GR. Genet A. 2006 Jul 1;140(13):1366-74 BMP-mediated functional cooperation between Dlx5;Dlx6 and Msx1;Msx2 during mammalian limb development. Hara F, Samuel S, Liu J, Rosen D, Langley RR, Naora H. PLoS One. 2013;8(1):e51700 A homeobox gene related to Drosophila distal-less promotes ovarian tumorigenicity by inducing expression of Yan ZH, Bao ZS, Yan W, Liu YW, Zhang CB, Wang HJ, vascular endothelial growth factor and fibroblast growth Feng Y, Wang YZ, Zhang W, You G, Zhang QG, Jiang T. factor-2. Am J Pathol. 2007 May;170(5):1594-606 Upregulation of DLX2 confers a poor prognosis in glioblastoma patients by inducing a proliferative Laroche F, Ramoz N, Leroy S, Fortin C, Rousselot-Paillet phenotype. Curr Mol Med. 2013 Mar;13(3):438-45 B, Philippe A, Colleaux L, Bresson JL, Mogenet A, Golse B, Mouren-Simeoni MC, Gorwood P, Galli T, Simonneau This article should be referenced as such: M, Krebs MO, Robel L. Polymorphisms of coding trinucleotide repeats of homeogenes in Gitton Y, Levi G. 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DLX5 (distal-less homeobox 5) Yorick Gitton, Giovanni Levi Evolution des Regulations Endocriniennes, CNRS, UMR7221, Museum National d'Histoire Naturelle, Paris, France (YG, GL)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/DLX5ID44295ch7q21.html DOI: 10.4267/2042/54163 This article is an update of : Xu J, Testa JR. DLX5 (distal-less homeobox 5). Atlas Genet Cytogenet Oncol Haematol 2012;16(8):526-528.

Abstract DNA/RNA DLX5 belongs to the six-member family of DLX Description genes characterized by a homeobox related to that found in the insect Distal-less (Dll) gene. The six The DLX5 gene is composed of 3 exons spanning a DLX genes are organized as three bigenic pairs genomic region of 4442 bp. with a tail-to-tail orientation (Zerucha et al., 2000) Genomic features and located on chromosomes where HOX clusters Mutations: Breakpoint analyses of genomic are also found (DLX5/DLX6; 7q21.3, syntenic to deletions and chromosomal rearrangements in the the HOXA cluster), (DLX1/DLX2; 2q32, syntenic congenital split-hand/split-foot malformation to the HOXD cluster; Simeone et al., 1994) and (SHFM type 1D, OMIM #220600), have shown (DLX3/DLX4; 17q21.33, syntenic to the HOXB that positional effect and disrupted regulatory cluster). During embryonic development DLX elements controlling DLX5/DLX6 activity are genes are involved in the control of appendage and involved in the pathogenesis of this developmental craniofacial morphogenesis and in the disorder (see further "dysmorphologies"). differentiation of reproductive organs; in the adult In-depth sequencing of the candidate regions has they play a role in bone homeostasis and in the shown that the expression of DLX6 depends upon maintenance of tissue integrity. the activity of conserved regulatory elements shared with DLX5, and located both within the Identity DLX5/DLX6 intergenic territory and outside of the Other names: SHFM1D locus (Lango Allen et al., 2014). Furthermore these enhancers have been identified HGNC (Hugo): DLX5 in all examined species - including in mouse where Location: 7q21.3 transgenic analyses have allowed the functional Local order: -DLX6-DLX5 -ACN9- characterization of their tissue-specificity.

Local order of DLX5 and flanking genes DLX6 and ACN9 is shown, with centromere at left and telomere (qter) at right. Arrows indicate transcriptional orientation of individual genes. DLX6 gene range: 96635290 - 96640352; DLX5 gene range: 96649702 - 96654143; ACN9 gene range: 96745905 - 96811075 (Hillier et al., 2003).

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Exon 1: 1 - 563; exon 2: 2463 - 2647; exon 3: 3767 - 4442.

Moreover, recent analyses of genomic integrity in homeodomain. The shorter transcript (DLX5-003) SHFM1D probands have unravelled two new encodes a predicted 191 AA-long, 20.9 kDa intragenic, non-synonymous mutations within the isoform with both N-terminal DLL domain and a open reading frame of DLX5. homeodomain. Imprinting: The status of parental imprinting of It should be observed that in vitro 35S-primed the DLX5/DLX6 locus has recently gained strong expression from full-length murine Dlx5 yields interest as these genes have been considered to be only one isoform at 32 kDa (Zhang et al., 1997). putative methylation targets of the methyl-CpG The latter study provided evidence for an binding protein-2 (MECP2), and thus might be incompatibility between Dlx5 DNA binding to its indirectly involved in the aetiology of the Rett target homeodomain-responsive element syndrome, a severe X-linked neurodevelopmental (TAATTA) and heterodimerization with its partner disorder afflicting girls with MECP2 mutation (see Msx factor. further "Rett syndrome"). It further showed that both events were mutually Transcription antagonistic, suggesting a regulatory role during Dlx/Msx-controlled morphogenetic processes such The DLX5 coding sequence consists of 870 bp as branchial arch and limb formation. During bone from the start of the first codon to the stop codon formation, Dlx5 (pI 9.3) transactivation activity is (Simeone et al., 1994). enhanced through serine phosphorylation in the Pseudogene nucleus by p38 MAP-kinase upon BMP2 signaling (Ulsamer et al., 2008). None known. Dlx5 has further been shown to be subjected to threonine phosphorylation by PKA during BMP2- Protein induced osteoblast differentiation, which increases Description Dlx5 nuclear levels by improving its stability (Han et al., 2011). The DLX5 protein consists of 289 amino acids with a calculated molecular weight of 31,5 kDa. Homology The protein contains two motifs, one dubbed None reported to date. homeobox protein distal-less-like N terminal and a second known as a homeodomain. Mutations Localisation Germinal Nucleus. A novel DLX5 mutation (c.A533C: p.Q178P) was Function identified in a family with autosomal recessive split Transcription factor important in the control of hand and foot malformation (Shamseldin et al., bone formation in embryonic development (Hassan 2012). et al., 2004). Transcription from DLX5 yields three Recently, a second rare familial case of SHFM1 has splice variants, which range from 1062 b to 1688 b been demonstrated to result, with highest (major isoform) due to alternative splicing sites probability, from intragenic missense mutations of throughout the precursor transcript. The shortest a critical glutamine residue in the third helix of the (Dlx5-002) is not processed. The other two share DLX5 homeodomain - Q186H (Wang et al., 2014). exon 1 which encodes an N-terminal DLL domain, The encoded mutant DLX5 has been demonstrated and exon 2 which encodes a part of the to fail at transactivating a bona fide MYC target.

DLL_N: homeobox protein distal-less-like N terminal; Homeodomain: homeobox DNA binding domain.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 811 DLX5 (distal-less homeobox 5) Gitton Y, Levi G

NCBI/COBALT alignment of DLX homeoproteins. Note the disposition according to the DLX 1/4/6 versus DLX 2/3/5 clades. Indicated by a yellow box is the ultraconserved glutamine featured by most homeoproteins at position 50 of the homeodomain.

Such an observation is not unexpected as this Furthermore, immunohistochemical studies mutation affects Q50, the most conserved residue of revealed that positive immunostaining for DLX5 all homeoproteins (see diagram above), which correlated with tumor size and poorer prognosis. numerous biochemical studies have demonstrated A DLX5 transcript isoform has been shown to be to be responsible for the specificity of the DNA strongly overexpressed in a large panel of primary recognition at the TAATT homeo-element (for lung cancer samples, providing a reliable prognosis review, Galliot et al., 1999). marker (Kato et al., 2008). In this study, the Somatic detected isoform (1.8 kb on Northern blot using a probe spanning exon 3 and 3'UTR) was claimed to A DLX5 mutation (c.C119G: p.S40C) was be found only in the placenta, among 23 normal observed in an ovarian carcinoma (Cancer Genome adult tissues. It should be observed that other Atlas Research Network, 2011). studies have reported numerous expression sites in Overexpression of DLX5 has been reported in adult human, including bone (osteoblasts and several types of human malignancy including lung marrow), ear, tooth, fat and brain. With regards to cancer (Kato et al., 2008; Xu and Testa, 2009), T- function, down-regulation of DLX5 through RNA cell lymphoma (Tan et al., 2008), and ovarian interference compromised the growth or survival of cancer (Tan et al., 2010), etc. two lung cancer cell lines, suggesting that controling DLX5 expression levels might be Implicated in clinically relevant (Kato et al., 2008). Lung cancer Lymphoma Note Note The DLX5 gene was reported to be overexpressed DLX5 was found to be highly expressed in 3 of 7 in the great majority of human non-small cell lung (42%) patient-derived T-cell lymphomas compared cancers examined by Kato et al., 2008. with that observed in nonmalignant lymph node

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samples (Tan et al., 2008). In addition, these has been demonstrated to result, with highest investigators found repeated upregulation of Dlx5 probability, from an intragenic missense mutation in T-cell lymphomas from transgenic mice in which of the DLX5 homeodomain (Q178P; Shamseldin et the Lck promoter was used to drive expression of a al., 2012). In the latter case, a causal link between constitutively active form of Akt2 in the thymus. defective DLX5/DLX6 expression and the Dlx5 was overexpressed due to a novel pathogenic mechanism impairing limb development chromosome inversion that placed the T-cell remains to be elucidated (Lango Allen et al., 2014). receptor beta (Tcrb) enhancer region near the Dlx5 On a further note, SHFM cases have often been locus. reported to include hearing loss, a trait consistent Breast cancer with a developmental role demonstrated for Dlx5/Dlx6 during ear formation in mouse Note embryogenesis (Acampora et al., 1999; Merlo et al., Both DLX5 and DLX6 were found to be 2002; Robledo and Lufkin, 2006; Chatterjee et al., upregulated during metastasis formation after 2010; Frenz et al., 2010). Moreover, both genes are intravenous injection of MDA-MB-231 breast major targets of two regulator genes whose cancer cells. The in vitro treatment of MDA-MB- deficiencies are responsible for a related pathogenic 231 cells with endothelin 1, a peptide associated condition, the auriculo-condylar syndrome (ACS, with breast cancer invasive phenotype, resulted in a Rieder et al., 2012; Brown et al., 2010). switch from DLX2 to DLX5 expression. Mutually Anorectal malformation associated with SHFM has exclusive expression of DLX2 and DLX5 was been reported in a family with a missense mutation found in both MDA-MB-231 cells and human in the P63 gene, a known direct upstream regulator breast cancer specimens. This evidence suggested of DLX5/DLX6 during morphogenesis (Su et al., that DLX genes are involved in human breast 2013). Whether DLX5/DLX6 expression is cancer progression, and that expression of DLX2 dysregulated in this condition, and whether this trait and DLX5 genes might serve as prognostic markers can be functionaly associated with the phenotype, (Morini et al., 2010). remains to be elucidated. Astrocytoma Rett syndrome Note Note Transcriptional profiling in search for prognosis DLX5 and DLX6 (OMIM 600028) have been markers has identified DLX5 as an upregulated controversial candidates for neurodevelopmental candidate for high-grade astrocytomas (Phillips et defects progressively afflicting young girls al., 2006). suffering of Rett syndrome (OMIM 312750). This Various cancers late onset disorder features fatal motor abnormalities, seizures, autism and mental Note retardation. While the genomic sequence of the DLX5 mRNA is abundantly expressed in many DLX5/DXL6 locus remains unaffected in all cancer cell lines derived from malignant tissues of reported cases, it is a direct target of the breast, brain, lung, skin, and ovarian cancer transcriptional regulator methyl-CpG-binding patients, whereas expression of DLX5 was low or protein 2 (MeCP2), which has been strongly undetectable in tumor cells from patients with associated to this syndrome by linkage analysis leukemia or with colorectal, prostate, and kidney (Horike et al., 2005). While still debated (Horike et cancers (Tan et al., 2010). al., 2005; Schüle et al., 2007; LaSalle, 2007; Dysmorphologies Miyano et al., 2008), initial MeCP2 deficiency is considered as causing defective neurogenesis Note through dysregulated expression of DLX5/DLX6, Split hand-foot malformation (SHFM) type 1 with due to altered chromatin state at this target locus sensory-neural hearing loss (SHFM1D; (Horike et al., 2005; Lilja et al., 2013). Mouse MIM:220600). This malformative syndrome affects mutagenesis has substantiated this hypothesis by hands and feet alike, resulting in moderate to severe pinpointing GABA ( γ-aminobutyric acid)-releasing median ray deficiency with syndactily. Among the neurons as a major cellular target expressing Dlx5 described six non-syndromic SHFM loci, one spans and Dlx6, whose deficiency impairs neurogenesis the DLX5/DLX6 bigenic cluster (Scherer et al., in MeCP2 null mutant (Chao et al., 2010). 1994; Crackower et al., 1996). However, numerous reported mutations spare DLX5 or DLX6 open Osteoporosis reading frames, suggesting it may rather be their Note common regulatory elements which is impacted Mouse mutational studies have demonstrated a role (Robledo et al., 2002; Lo Iacono et al., 2008). for Dlx5 and Dlx6 as a major determinant of However recently, one rare familial case of SHFM1 chondrogenesis and chondrocyte hypertrophy in the

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endochondral skeleton, throughout embryogenesis Simeone A, Acampora D, Pannese M, D'Esposito M, and adulthood (Samee et al., 2007; Samee et al., Stornaiuolo A, Gulisano M, Mallamaci A, Kastury K, Druck T, Huebner K. Cloning and characterization of two 2008; Samee et al., 2009). These observations pave members of the vertebrate Dlx gene family. Proc Natl Acad the way for a better understanding of human Sci U S A. 1994 Mar 15;91(6):2250-4 osteoporosis, in particular in patients with Coberly S, Lammer E, Alashari M. Retinoic acid dysfunctional regulation of bone-remodeling embryopathy: case report and review of literature. Pediatr hormonal levels (Prall et al., 2013). Pathol Lab Med. 1996 Sep-Oct;16(5):823-36 Reproductive tract Crackower MA, Scherer SW, Rommens JM, Hui CC, Poorkaj P, Soder S, Cobben JM, Hudgins L, Evans JP, Note Tsui LC. Characterization of the split hand/split foot Dlx5 and Dlx6 are involved in the development and malformation locus SHFM1 at 7q21.3-q22.1 and analysis function of the reproductive tract. The dual mouse of a candidate gene for its expression during limb development. Hum Mol Genet. 1996 May;5(5):571-9 mutant for Dlx5 and Dlx6 displays abnormal urethra formation (Suzuki et al., 2007), reduced Zhang H, Hu G, Wang H, Sciavolino P, Iler N, Shen MM, testicular steroidogenesis with feminization Abate-Shen C. Heterodimerization of Msx and Dlx homeoproteins results in functional antagonism. Mol Cell (Nishida et al., 2008), and early ovarian follicular Biol. 1997 May;17(5):2920-32 depletion (Bouhali et al., 2011). A human mutation Galliot B, de Vargas C, Miller D. Evolution of homeobox in a genomic region including DLX5 and DLX6 has genes: Q50 Paired-like genes founded the Paired class. been associated to a case of familial premature Dev Genes Evol. 1999 Mar;209(3):186-97 ovarian failure (Caburet et al., 2012). Acampora D, Merlo GR, Paleari L, Zerega B, Postiglione Teratology MP, Mantero S, Bober E, Barbieri O, Simeone A, Levi G. Craniofacial, vestibular and bone defects in mice lacking Note the Distal-less-related gene Dlx5. Development. 1999 With regards to pharmacologically-induced Sep;126(17):3795-809 teratogenesis, dysregulation of DLX5/DLX6 gene Zerucha T, Stühmer T, Hatch G, Park BK, Long Q, Yu G, expression has been demonstrated to be a major Gambarotta A, Schultz JR, Rubenstein JL, Ekker M. A step during craniofacial embryopathy induced by highly conserved enhancer in the Dlx5/Dlx6 intergenic region is the site of cross-regulatory interactions between two compounds: Dlx genes in the embryonic forebrain. J Neurosci. 2000 i) retinoic acid, a vitamin A derivative found in the Jan 15;20(2):709-21 RoAccutane ® drug, which indirectly prevents the Abate-Shen C. Deregulated homeobox gene expression in induction of DLX5/DLX6 in human (Lammer et cancer: cause or consequence? Nat Rev Cancer. 2002 al., 1985; Coberly et al., 1996) and in all animal Oct;2(10):777-85 models investigated (Vieux-Rochas et al., 2007), Merlo GR, Paleari L, Mantero S, Zerega B, Adamska M, which share a wide range of jaw and ear Rinkwitz S, Bober E, Levi G. The Dlx5 homeobox gene is malformations; essential for vestibular morphogenesis in the mouse ii) the food contaminant ochratoxin A, a fungal embryo through a BMP4-mediated pathway. Dev Biol. toxin demonstrated to prevent Dlx5 activation in 2002 Aug 1;248(1):157-69 exposed mouse embryos, which later develop Robledo RF, Rajan L, Li X, Lufkin T. The Dlx5 and Dlx6 craniofacial malformations (Wei and Sulik, 1993; homeobox genes are essential for craniofacial, axial, and appendicular skeletal development. Genes Dev. 2002 May Napoletano et al., 2010). Although a causal link 1;16(9):1089-101 between Dlx5, Dlx6 and the toxin remains to be functionally demonstrated, this observation may Hillier LW, Fulton RS, Fulton LA, Graves TA, Pepin KH, Wagner-McPherson C, Layman D, Maas J, Jaeger S, account for teratogenesis observed in human Walker R, Wylie K, Sekhon M, Becker MC, O'Laughlin MD, embryos maternally exposed to the toxin (Hope and Schaller ME, Fewell GA, Delehaunty KD, Miner TL, Nash Hope, 2012; Thrasher et al., 2012). 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DNA sequence of human chromosome 7. Nature. 2003 Jul models of split-hand/split-foot malformation type 1 and 10;424(6945):157-64 type 4. Eur J Hum Genet. 2008 Jan;16(1):36-44 Hassan MQ, Javed A, Morasso MI, Karlin J, Montecino M, Tan Y, Timakhov RA, Rao M, Altomare DA, Xu J, Liu Z, van Wijnen AJ, Stein GS, Stein JL, Lian JB. Dlx3 Gao Q, Jhanwar SC, Di Cristofano A, Wiest DL, Knepper transcriptional regulation of osteoblast differentiation: JE, Testa JR. A novel recurrent chromosomal inversion temporal recruitment of Msx2, Dlx3, and Dlx5 implicates the homeobox gene Dlx5 in T-cell lymphomas homeodomain proteins to chromatin of the osteocalcin from Lck-Akt2 transgenic mice. Cancer Res. 2008 Mar gene. Mol Cell Biol. 2004 Oct;24(20):9248-61 1;68(5):1296-302 Horike S, Cai S, Miyano M, Cheng JF, Kohwi-Shigematsu Ulsamer A, Ortuño MJ, Ruiz S, Susperregui AR, Osses N, T. Loss of silent-chromatin looping and impaired imprinting Rosa JL, Ventura F. 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Activation of placenta-specific transcription factor distal-less homeobox Frenz DA, Liu W, Cvekl A, Xie Q, Wassef L, Quadro L, 5 predicts clinical outcome in primary lung cancer patients. Niederreither K, Maconochie M, Shanske A. Retinoid Clin Cancer Res. 2008 Apr 15;14(8):2363-70 signaling in inner ear development: A "Goldilocks" phenomenon. Am J Med Genet A. 2010 Lo Iacono N, Mantero S, Chiarelli A, Garcia E, Mills AA, Dec;152A(12):2947-61 Morasso MI, Costanzo A, Levi G, Guerrini L, Merlo GR. Regulation of Dlx5 and Dlx6 gene expression by p63 is Napoletano M, Pennino D, Izzo G, de Maria S, Ottaviano involved in EEC and SHFM congenital limb defects. R, Ricciardi M, Mancini R, Schiattarella A, Farina E, Development. 2008 Apr;135(7):1377-88 Metafora S, Cartenì M, Ritieni A, Minucci S, Morelli F. Ochratoxin A induces craniofacial malformation in mice Miyano M, Horike S, Cai S, Oshimura M, Kohwi- acting on Dlx5 gene expression. Front Biosci (Elite Ed). Shigematsu T. 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Thrasher JD, Gray MR, Kilburn KH, Dennis DP, Yu A. A This article should be referenced as such: water-damaged home and health of occupants: a case study. J Environ Public Health. 2012;2012:312836 Gitton Y, Levi G. DLX5 (distal-less homeobox 5). Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11):810-816. Lilja T, Wallenborg K, Björkman K, Albåge M, Eriksson M,

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 816

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

DLX6 (distal-less homeobox 6) Yorick Gitton, Giovanni Levi Evolution des Regulations Endocriniennes, CNRS, UMR7221, Museum National d'Histoire Naturelle, Paris, France (YG, GL)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/DLX6ID52195ch7q21.html DOI: 10.4267/2042/54164 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract DNA/RNA DLX6 belongs to the six-member family of DLX Description genes characterized by a homeobox related to that In contrast to DLX5, no intragenic mutations have found in the insect Distal-less (Dll) gene. The six been found for DLX6. It is considered that DLX genes are organized as three bigenic pairs disruption of distant regulatory elements is most with a tail-to-tail orientation (Zerucha et al., 2000), usually responsible for DLX5/DLX6-related and located on chromosomes where HOX clusters disorders in human. Breakpoint analyses of are also found (DLX5/DLX6; 7q21.3, syntenic to genomic deletions and chromosomal the HOXA cluster), (DLX1/DLX2; 2q32 syntenic rearrangements in the congenital split-hand/split- to the HOXD cluster) and (DLX3/DLX4; 17q21.33 foot malformation (SHFM type 1D, OMIM syntenic to the HOXB cluster). During embryonic #220600), have shown that positional effect and development DLX genes are involved in the control disrupted regulatory elements controlling of appendage and craniofacial morphogenesis and DLX5/DLX6 activity are involved in the in the differentiation of reproductive organs; in the pathogenesis of this developmental disorder (see adult they play a role in bone homeostasis and in further "dysmorphologies"). In-depth sequencing of the maintenance of tissue integrity. the candidate regions has shown that the expression of DLX6 depends upon the activity of conserved Identity regulatory elements shared with DLX5, and located HGNC (Hugo): DLX6 both within the DLX5/DLX6 intergenic territory and outside of the locus (see Figure 1; Lango Allen Location: 7q21.3 et al., 2014). Furthermore these enhancers have Local order been identified in all examined species - including Forward strand of human chromosome 7, from in mouse where transgenic analyses have allowed 96634860 to 96640352 - see Figure 1 below. DLX6 the functional characterization of their tissue- forms a bigenic cluster with DLX5 at 7q21.31. specificity.

Figure 1. Genomic context of the human DLX5/DLX6 bigenic locus.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 817 DLX6 (distal-less homeobox 6) Gitton Y, Levi G

Figure 2. The four known human DLX6 transcripts.

The status of parental imprinting of the proline stretch, 11-20 CAG/CCG repeat long, DLX5/DLX6 locus has recently gained strong which has been found to be conserved in mouse interest as these genes have been considered to be (Pfeffer et al., 2001; see further "trinucleotide putative methylation targets of the methyl-CpG repeats"). The functional consequences of these binding protein-2 (MECP2), and thus might be expansions upon DLX6 activity remain to be indirectly involved in the aetiology of the Rett determined. syndrome, a severe X-linked neurodevelopmental disorder afflicting girls with MECP2 mutation (see Protein further "Rett syndrome"). Description Transcription DLX6 is a 175 AA helix-turn-helix homeodomain Transcription from DLX6 yields four splice transcription factor (19.7 kDa and pI 9.9). The variants, one transcript being untranslated (see homeodomain spans AA 49-108 across exons 2 and Figure 2). The three coding ones range from 666 b 3 (see Figure 3). to 2304 b (major isoform) due to alternative splicing sites throughout the precursor transcript. Function Furthermore, two antisense non-coding transcripts During mouse craniofacial morphogenesis, Dlx6 have been characterized - one of which, Evf2 acts as transactivator of the helix-loop-helix dHand (Dlx6as/Dlx6os1; HNGC#37151), has been gene through a regulatory element, [ATTA/TAAT], demonstrated to regulate transactivation from an which does not bind other Dlx factors. Noticeably, intergenic enhancer of Dlx5/Dlx6 (Feng et al., this binding is a specifically endothelin-1 signaling- 2006; Berghoff et al., 2013). dependent mechanism. Thus, despite sharing DLX6 sequence analysis of one sporadic SHFM regulatory elements and subsequent expression patient (Ferro et al., 2001) has led to the discovery patterns with Dlx5, the Dlx6 factor appears to be of a longer transcript endowing the N-terminus of competent to exert selective roles depending upon DLX6 with an unusual dual poly-glutamine/poly- specific cellular signalling contexts.

Figure 3. Structure of the three DLX6 protein isoforms. Sequence below belongs to the longest isoform. Note the N-terminal series of poly-residue stretches.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 818 DLX6 (distal-less homeobox 6) Gitton Y, Levi G

Figure 4. NCBI/COBALT alignment of DLX homeoproteins. Note the disposition according to the DLX 1/4/6 versus DLX 2/3/5 clades. Indicated by a yellow box is the ultraconserved Glutamine featured by most homeoproteins at position 50 of the homeodomain.

As other DLX factors, DLX6 modulates target combinatorial activity of developmental genes genes expression through a domain which is (Abate-Shen, 2002). distinct from the DNA-binding homeodomain, and Dysregulated expression of homeobox-containing in association within transactivating complexes genes of the distal-less family, arranged as three which include MSX. bigenic pairs in mammals (DLX1/2, DLX3/4 and Composition biases in DLX6 include one poly-Gly DLX5/6; Kraus and Lufkin, 2006), has been and one-His stretches (see Figure 3). reported to correlate with distinct oncogenic Of note, DLX6 encodes for one long isoform mechanisms. endowed with a contiguous series of residue DLX6 along with DLX5 is a direct MYC oncogene stretches including glutamine, proline, alanine and inducer, responsible for neoplastic initiation in histidine (see Figure 3). many cancers, including lymphoma and lung Homology cancers (Xu and Testa, 2009). With regards to other members of the DLX family, Breast cancers and their bone DLX6 belongs to the DLX1/4/6 clade based on metastases sequence homology (see Figure 4). It shares a lack Note of N-terminal DLL-like domain specific to the other DLX6, together with DLX5, is upregulated in lung clade constituted by DLX2/3/5. The homeodomain and bone metastatic cells derived from primary remains close to all other DLX proteins. breast tumors in human - a pattern associated with tumour agressivity and thus, poor prognosis and Implicated in increased relapses (Morini et al., 2010). Lung cancers Transcriptional profiling in search for prognosis markers has identified DLX6 as an upregulated Note candidate for high-grade astrocytomas (Phillips et Neoplastic processes often result from al., 2006).

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Dysmorphologies Anorectal malformation associated with SHFM has been reported in a family with a missense mutation Note in the P63 gene, a known direct upstream regulator DLX6 is often regarded as a functional substitute of of DLX5/DLX6 during morphogenesis (Su et al., DLX5 and autonomous regulation as been seldom 2013). Whether DLX5/DLX6 expression is observed; one rare such situation being the dysregulated in this condition, and whether this trait Endothelin-1→Endothelin-Ra →Dlx6 →Hand2 can be functionaly associated with the phenotype, signalling cascade which specifies lower jaw remains to be elucidated. identity in the mouse embryo (Charité et al., 2001). As such, malformations described for DLX5 are Trinucleotide repeats commonly regarded as involving DLX6. In most Note mouse mutant models, severe phenotypes result The first DLX6 exon harbours a trinucleotide repeat from dual invalidation of Dlx5 and Dlx6. region of 11 to 20 CAG triplets in normal, Malformative processes implying DLX6 will thus heterozygous subjects. This CAG repeat is highly be simultaneously described on the DLX5 gene polymorphic (Pfeffer et al., 2001). While no card. obvious phenotype was associated with this newly Split hand-foot malformation (SHFM) type 1 with discovered polymorphism in the investigated sensory-neural hearing loss (SHFM1D; cohort, such repeat length variations are critical MIM:220600). This malformative syndrome affects determinants of colon carcinogenesis and hands and feet alike, resulting in moderate to severe neurodegenerative disorder when occurring in the median ray deficiency with syndactily. Among the androgen receptor and huntingtin genes, described six non-syndromic SHFM loci, one spans respectively. the DLX5/DLX6 bigenic cluster (Scherer et al., 1994; Crackower et al., 1996). Numerous reported Rett syndrome mutations spare DLX5 or DLX6 open reading Note frames, suggesting it may rather be their common DLX6 and DLX5 (OMIM #600028) have been regulatory elements which is impacted (Robledo et controversial candidates for neurodevelopmental al., 2002 ; Lo Iacono et al., 2008). However defects progressively afflicting young girls recently, two rare familial cases of SHFM1 have suffering of Rett syndrome (OMIM #312750). This been demonstrated to result, with highest late onset disorder features fatal motor probability, from intragenic missense mutations of abnormalities, seizures, autism and mental two critical glutamine residues in the third helix of retardation. While the genomic sequence of the the DLX5 homeodomain (Q178P reported in DLX5/DXL6 locus remains unaffected in all Shamseldin et al., 2012; and Q186H characterized reported cases, it is a direct target of the in Wang et al., 2014). In the first case, a causal link transcriptional regulator methyl-CpG-binding between defective DLX5/DLX6 expression and the protein 2 (MeCP2), which has been strongly pathogenic mechanism impairing limb development associated to this syndrome by linkage analysis remains to be elucidated. In the second case, the (Horike et al., 2005). While still debated (Horike et mutated DLX5 has been demonstrated to fail at al., 2005; Schüle et al., 2007; LaSalle, 2007; transactivating its bona fide MYC target. Such an Miyano et al., 2008), initial MeCP2 deficiency is observation is not unexpected as the mutation considered as causing defective neurogenesis affects Q50, the most conserved residue of all through dysregulated expression of DLX5/DLX6, homeoproteins (see diagram), which numerous due to altered chromatin state at this target locus biochemical studies have demonstrated to be (Horike et al., 2005; Lilja et al., 2013). Mouse responsible for the specificity of the DNA mutagenesis has substantiated this hypothesis by recognition at the TAATT homeo-element (for pinpointing GABA ( γ-aminobutyric acid)-releasing review, Galliot et al., 1999). neurons as a major cellular target expressing Dlx5 Other pathogenetic processes: on a further note, and Dlx6, whose deficiency impairs neurogenesis SHFM cases have often been reported to include in MeCP2 null mutant (Chao et al., 2010). hearing loss, a trait consistent with a developmental role demonstrated for Dlx5/Dlx6 during ear Osteoporosis formation in mouse embryogenesis (Acampora et Note al., 1999; Merlo et al., 2002; Robledo and Lufkin, Mouse mutational studies have demonstrated a role 2006; Chatterjee et al., 2010; Frenz et al., 2010). for Dlx5 and Dlx6 as a major determinant of Moreover, both genes are major targets of two chondrogenesis and chondrocyte hypertrophy in the regulator genes whose deficiencies are responsible endochondral skeleton, throughout embryogenesis for a related pathogenic condition, the auriculo- and adulthood (Samee et al., 2007; Samee et al., condylar syndrome (ACS, Rieder et al., 2012). 2008; Samee et al., 2009).

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 820 DLX6 (distal-less homeobox 6) Gitton Y, Levi G

These observations pave the way for a better Scherer SW, Poorkaj P, Massa H, Soder S, Allen T, Nunes understanding of human osteoporosis, in particular M, Geshuri D, Wong E, Belloni E, Little S. Physical mapping of the split hand/split foot locus on chromosome 7 in patients with dysfunctional regulation of bone- and implication in syndromic ectrodactyly. Hum Mol Genet. remodeling hormonal levels (Prall et al., 2013). 1994 Aug;3(8):1345-54 Reproductive tract Crackower MA, Scherer SW, Rommens JM, Hui CC, Poorkaj P, Soder S, Cobben JM, Hudgins L, Evans JP, Note Tsui LC. Characterization of the split hand/split foot Dlx5 and Dlx6 are involved in the development and malformation locus SHFM1 at 7q21.3-q22.1 and analysis function of the reproductive tract. of a candidate gene for its expression during limb The dual mouse mutant for Dlx5 and Dlx6 displays development. Hum Mol Genet. 1996 May;5(5):571-9 abnormal urethra formation (Suzuki et al., 2008), Acampora D, Merlo GR, Paleari L, Zerega B, Postiglione reduced testicular steroidogenesis with feminization MP, Mantero S, Bober E, Barbieri O, Simeone A, Levi G. Craniofacial, vestibular and bone defects in mice lacking (Nishida et al., 2008), and early ovarian follicular the Distal-less-related gene Dlx5. Development. 1999 depletion (Bouhali et al., 2011). Sep;126(17):3795-809 A human mutation in a genomic region including Galliot B, de Vargas C, Miller D. Evolution of homeobox DLX5 and DLX6 has been associated to a case of genes: Q50 Paired-like genes founded the Paired class. familial premature ovarian failure (Caburet et al., Dev Genes Evol. 1999 Mar;209(3):186-97 2012). Zerucha T, Stühmer T, Hatch G, Park BK, Long Q, Yu G, Teratology Gambarotta A, Schultz JR, Rubenstein JL, Ekker M. A highly conserved enhancer in the Dlx5/Dlx6 intergenic Note region is the site of cross-regulatory interactions between With regards to pharmacologically-induced Dlx genes in the embryonic forebrain. J Neurosci. 2000 teratogenesis, dysregulation of Dlx5/Dlx6 gene Jan 15;20(2):709-21 expression has been demonstrated to be a major Charité J, McFadden DG, Merlo G, Levi G, Clouthier DE, step during craniofacial embryopathy induced by Yanagisawa M, Richardson JA, Olson EN. Role of Dlx6 in regulation of an endothelin-1-dependent, dHAND branchial two compounds : arch enhancer. Genes Dev. 2001 Nov 15;15(22):3039-49 i) retinoic acid, a vitamin A derivative found in the Ferro P, dell'Eva R, Pfeffer U. Are there CAG repeat RoAccutane ® drug, which prevents the induction expansion-related disorders outside the central nervous of Dlx5/Dlx6 in all animal models investigated system? Brain Res Bull. 2001 Oct-Nov 1;56(3-4):259-64 (Vieux-Rochas et al., 2007; Vieux-Rochas et al., Pfeffer U, Ferro P, Pavia V, Trombino S, Dell'Eva R, Merlo 2010); this discovery has given strong insight into G, Levi G. The coding region of the human DLX6 gene the aetiology of teratologic impact of fetal exposure contains a polymorphic CAG/CCG repeat. Int J Oncol. to RoAccutane medication in man. 2001 Jun;18(6):1293-7 Analyses of retinoic acid-induced embryopathy in Abate-Shen C. Deregulated homeobox gene expression in mouse neurulas have demonstrated that retinoic cancer: cause or consequence? Nat Rev Cancer. 2002 acid exposure prevents proper induction of both Oct;2(10):777-85 Dlx5 and Dlx6 by endothelin-1 signalling. Merlo GR, Paleari L, Mantero S, Zerega B, Adamska M, This disruption has been found to be finely tuned Rinkwitz S, Bober E, Levi G. The Dlx5 homeobox gene is during a surprisingly short timeframe spanning a essential for vestibular morphogenesis in the mouse embryo through a BMP4-mediated pathway. Dev Biol. critical period of neurulation. 2002 Aug 1;248(1):157-69 This exposure creates a functional invalidation of Dlx5/Dlx6-controlled cranio-facial morphogenesis Robledo RF, Rajan L, Li X, Lufkin T. The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and (reviewed in Gitton et al., 2010); appendicular skeletal development. Genes Dev. 2002 May ii) the food contaminant ochratoxin A, a fungal 1;16(9):1089-101 toxin demonstrated to prevent Dlx5 activation in Horike S, Cai S, Miyano M, Cheng JF, Kohwi-Shigematsu exposed mouse embryos, which later develop T. Loss of silent-chromatin looping and impaired imprinting craniofacial malformations (Wei and Sulik, 1993; of DLX5 in Rett syndrome. Nat Genet. 2005 Jan;37(1):31- Napoletano et al., 2010). 40 Although a causal link between Dlx5, Dlx6 and the Feng J, Bi C, Clark BS, Mady R, Shah P, Kohtz JD. The toxin remains to be functionally demonstrated, this Evf-2 noncoding RNA is transcribed from the Dlx-5/6 observation may account for teratogenesis observed ultraconserved region and functions as a Dlx-2 transcriptional coactivator. Genes Dev. 2006 Jun in human embryos maternally exposed to the toxin 1;20(11):1470-84 (Hope and Hope, 2012; Thrasher et al., 2012). Kraus P, Lufkin T. Dlx homeobox gene control of mammalian limb and craniofacial development. Am J Med References Genet A. 2006 Jul 1;140(13):1366-74 Wei X, Sulik KK. Pathogenesis of craniofacial and body Phillips HS, Kharbanda S, Chen R, Forrest WF, Soriano wall malformations induced by ochratoxin A in mice. Am J RH, Wu TD, Misra A, Nigro JM, Colman H, Soroceanu L, Med Genet. 1993 Nov 1;47(6):862-71 Williams PM, Modrusan Z, Feuerstein BG, Aldape K.

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Molecular subclasses of high-grade glioma predict Frenz DA, Liu W, Cvekl A, Xie Q, Wassef L, Quadro L, prognosis, delineate a pattern of disease progression, and Niederreither K, Maconochie M, Shanske A. Retinoid resemble stages in neurogenesis. Cancer Cell. 2006 signaling in inner ear development: A "Goldilocks" Mar;9(3):157-73 phenomenon. Am J Med Genet A. 2010 Dec;152A(12):2947-61 Robledo RF, Lufkin T. Dlx5 and Dlx6 homeobox genes are required for specification of the mammalian vestibular Gitton Y, Heude E, Vieux-Rochas M, Benouaiche L, apparatus. Genesis. 2006 Sep;44(9):425-37 Fontaine A, Sato T, Kurihara Y, Kurihara H, Couly G, Levi G. Evolving maps in craniofacial development. Semin Cell LaSalle JM. The Odyssey of MeCP2 and parental Dev Biol. 2010 May;21(3):301-8 imprinting. Epigenetics. 2007 Jan-Mar;2(1):5-10 Morini M, Astigiano S, Gitton Y, Emionite L, Mirisola V, Samee N, de Vernejoul MC, Levi G. Role of DLX Levi G, Barbieri O. Mutually exclusive expression of DLX2 regulatory proteins in osteogenesis and chondrogenesis. and DLX5/6 is associated with the metastatic potential of Crit Rev Eukaryot Gene Expr. 2007;17(3):173-86 the human breast cancer cell line MDA-MB-231. BMC Schüle B, Li HH, Fisch-Kohl C, Purmann C, Francke U. Cancer. 2010 Nov 25;10:649 DLX5 and DLX6 expression is biallelic and not modulated Napoletano M, Pennino D, Izzo G, de Maria S, Ottaviano by MeCP2 deficiency. Am J Hum Genet. 2007 R, Ricciardi M, Mancini R, Schiattarella A, Farina E, Sep;81(3):492-506 Metafora S, Cartenì M, Ritieni A, Minucci S, Morelli F. Vieux-Rochas M, Coen L, Sato T, Kurihara Y, Gitton Y, Ochratoxin A induces craniofacial malformation in mice Barbieri O, Le Blay K, Merlo G, Ekker M, Kurihara H, acting on Dlx5 gene expression. Front Biosci (Elite Ed). Janvier P, Levi G. Molecular dynamics of retinoic acid- 2010 Jan 1;2:133-42 induced craniofacial malformations: implications for the Vieux-Rochas M, Bouhali K, Baudry S, Fontaine A, Coen origin of gnathostome jaws. PLoS One. 2007 Jun L, Levi G. Irreversible effects of retinoic acid pulse on 6;2(6):e510 Xenopus jaw morphogenesis: new insight into cranial Lo Iacono N, Mantero S, Chiarelli A, Garcia E, Mills AA, neural crest specification. Birth Defects Res B Dev Reprod Morasso MI, Costanzo A, Levi G, Guerrini L, Merlo GR. Toxicol. 2010 Dec;89(6):493-503 Regulation of Dlx5 and Dlx6 gene expression by p63 is Bouhali K, Dipietromaria A, Fontaine A, Caburet S, involved in EEC and SHFM congenital limb defects. Barbieri O, Bellessort B, Fellous M, Veitia RA, Levi G. Development. 2008 Apr;135(7):1377-88 Allelic reduction of Dlx5 and Dlx6 results in early follicular Miyano M, Horike S, Cai S, Oshimura M, Kohwi- depletion: a new mouse model of primary ovarian Shigematsu T. DLX5 expression is monoallelic and Dlx5 is insufficiency. Hum Mol Genet. 2011 Jul 1;20(13):2642-50 up-regulated in the Mecp2-null frontal cortex. J Cell Mol Caburet S, Zavadakova P, Ben-Neriah Z, Bouhali K, Med. 2008 Aug;12(4):1188-91 Dipietromaria A, Charon C, Besse C, Laissue P, Chalifa- Nishida H, Miyagawa S, Vieux-Rochas M, Morini M, Ogino Caspi V, Christin-Maitre S, Vaiman D, Levi G, Veitia RA, Y, Suzuki K, Nakagata N, Choi HS, Levi G, Yamada G. Fellous M. Genome-wide linkage in a highly Positive regulation of steroidogenic acute regulatory consanguineous pedigree reveals two novel loci on protein gene expression through the interaction between chromosome 7 for non-syndromic familial Premature Dlx and GATA-4 for testicular steroidogenesis. Ovarian Failure. PLoS One. 2012;7(3):e33412 Endocrinology. 2008 May;149(5):2090-7 Hope JH, Hope BE. A review of the diagnosis and Samee N, Geoffroy V, Marty C, Schiltz C, Vieux-Rochas treatment of Ochratoxin A inhalational exposure M, Levi G, de Vernejoul MC. Dlx5, a positive regulator of associated with human illness and kidney disease osteoblastogenesis, is essential for osteoblast-osteoclast including focal segmental glomerulosclerosis. J Environ coupling. Am J Pathol. 2008 Sep;173(3):773-80 Public Health. 2012;2012:835059 Suzuki K, Haraguchi R, Ogata T, Barbieri O, Alegria O, Rieder MJ, Green GE, Park SS, Stamper BD, Gordon CT, Vieux-Rochas M, Nakagata N, Ito M, Mills AA, Kurita T, Johnson JM, Cunniff CM, Smith JD, Emery SB, Lyonnet S, Levi G, Yamada G. Abnormal urethra formation in mouse Amiel J, Holder M, Heggie AA, Bamshad MJ, Nickerson models of split-hand/split-foot malformation type 1 and DA, Cox TC, Hing AV, Horst JA, Cunningham ML. A type 4. Eur J Hum Genet. 2008 Jan;16(1):36-44 human homeotic transformation resulting from mutations in PLCB4 and GNAI3 causes auriculocondylar syndrome. Am Samee N, Geoffroy V, Marty C, Schiltz C, Vieux-Rochas J Hum Genet. 2012 May 4;90(5):907-14 M, Clément-Lacroix P, Belleville C, Levi G, de Vernejoul MC. Increased bone resorption and osteopenia in Dlx5 Shamseldin HE, Faden MA, Alashram W, Alkuraya FS. heterozygous mice. J Cell Biochem. 2009 Aug Identification of a novel DLX5 mutation in a family with 1;107(5):865-72 autosomal recessive split hand and foot malformation. J Med Genet. 2012 Jan;49(1):16-20 Xu J, Testa JR. DLX5 (distal-less homeobox 5) promotes tumor cell proliferation by transcriptionally regulating MYC. Thrasher JD, Gray MR, Kilburn KH, Dennis DP, Yu A. A J Biol Chem. 2009 Jul 31;284(31):20593-601 water-damaged home and health of occupants: a case study. 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in lymphocytes of Rett syndrome patients. Epigenetics. Turnpenny PD, Turner CL, Weedon MN, Ellard S. Next 2013 Mar;8(3):246-51 generation sequencing of chromosomal rearrangements in patients with split-hand/split-foot malformation provides Prall WC, Haasters F, Heggebö J, Polzer H, Schwarz C, evidence for DYNC1I1 exonic enhancers of DLX5/6 Gassner C, Grote S, Anz D, Jäger M, Mutschler W, expression in humans. J Med Genet. 2014 Apr;51(4):264-7 Schieker M. Mesenchymal stem cells from osteoporotic patients feature impaired signal transduction but sustained Wang X, Xin Q, Li L, Li J, Zhang C, Qiu R, Qian C, Zhao osteoinduction in response to BMP-2 stimulation. Biochem H, Liu Y, Shan S, Dang J, Bian X, Shao C, Gong Y, Liu Q. Biophys Res Commun. 2013 Nov 1;440(4):617-22 Exome sequencing reveals a heterozygous DLX5 mutation in a Chinese family with autosomal-dominant split- Su P, Yuan Y, Huang Y, Wang W, Zhang Z. Anorectal hand/foot malformation. Eur J Hum Genet. 2014 Feb 5; malformation associated with a mutation in the P63 gene in a family with split hand-foot malformation. Int J This article should be referenced as such: Colorectal Dis. 2013 Dec;28(12):1621-7 Gitton Y, Levi G. DLX6 (distal-less homeobox 6). Atlas Lango Allen H, Caswell R, Xie W, Xu X, Wragg C, Genet Cytogenet Oncol Haematol. 2014; 18(11):817-823.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 823

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

EDIL3 (EGF-Like Repeats And Discoidin I-Like Domains 3) Hisataka Kitano, Chiaki Hidai Division of Oral surgery and Department of Phisiology, Nihon university school of medicine, Tokyo 173-8610, Japan (HK, CH)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/EDIL3ID46078ch5q14.html DOI: 10.4267/2042/54165 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

appears to be a ligand for alpha-v beta-3 integrin Abstract receptor. Review on EDIL3, with data on DNA/RNA, on the Similarly, EDIL3 shares consensus sequences with protein encoded and where the gene is implicated. the fate determining notch proteins and coagulation factor IX, coagulation factor V, and coagulation Identity factor VIII. Other names: DEL1 Description HGNC (Hugo): EDIL3 442.48 kb, mRNA: 2974 bp, 11 Exons. Location: 5q14.3 Transcription Note The major transcript is represented by the most EDIL3 is developmentally regulated and expressed common cDNA clones, and it encodes a 480 amino during embryogenesis. It is expressed in acid protein in human. The major transcript encodes extraembryonic mesoderm, specifically the yolk sac a protein that consists of a signal peptide blood islands. EDIL3 is detected in angioblasts and comprising three epidermal growth factor- (EGF)- early endothelial cells in four developing organs: like repeats and two discoidin I-like domains. A heart, lung, kidney, and eye. It is also expressed in less frequently represented minor transcript hypertrophic chondrocytes. Additionally, EDIL3 designated the minor transcript encodes a signal has a function that affects endocytosis, apoptosis, peptide comprising three EGF repeats and a portion cell migration, or some combination thereof. of the amino-terminal discoidin I-like domain. There is additional complexity among other minor DNA/RNA transcripts; specifically, the variation is in the inclusion or exclusion of sequences that encode 10 Note amino acids in the spacer region between EGF1 and EDIL3 is an extracellular matrix protein that EGF2. contains three EGF-like repeats and two discoidin Pseudogene I-like domains. The second EGF-like repeat contains an RGD integrin binding motif that Not known.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 824 EDIL3 (EGF-Like Repeats And Discoidin I-Like Domains 3) Kitano H, Hidai C

Protein Expression EDIL3 is differentially expressed depending on the Note tissue and cell culture conditions. EDIL3 contains three Ca-binding EGF-like repeats Organism parts: (see below). and two discoidin I-like domains. There is an Kidney, myometrium, bone, spinal cord, stomach integrin-binding site in the second EGF-like repeat; fundus, heart, colon cecum, urethra, islet of specifically, this EGF-like repeat has an RGD Langerhans, synovial membrane, lung, peptide that binds to integrin alpha v beta 3. atherosclerotic aortic wall, internal mammary artery, liver tumor tissue, accumbens, amygdale, Description brain, caudate nucleus, central nervous system, EDIL3 has 480 amino acids, and its molecular cerebral cortex, colon mucosa, corpus callosum, weight is 52 kDa protein. dorsal root ganglion, frontal cortex, primary motor EDIL3 comprises 480 amino acids, and its cortex, Brodmann's Area 4, frontal lobe, molecular weight is 52 kDa protein. hippocampus, hypothalamus, medulla, midbrain, - The domain of 1-23 is an initio prediction. The nodose nucleus, homogenized, occipital lobe, subsequent domain is 24-470 which EGF-like parietal lobe, putamen, saphenous vein, skin, repeat and discoidin I-like domain-containing substantia nigra, subthalamic nucleus, temporal protein 3 isoform 2. lobe, testis, thalamus, trigeminal ganglion, The region name is EGF1 which Calcium-binding umbilical cord, ventral tegmental area, vestibular EGF-like domain, present in a large number of nuclei superior, cervix, ovary, stomach, liver, membrane-bound and extracellular proteins. smooth muscle. - Location: 27-59; many of these proteins require The above data derive from the EDIL3 entry in the calcium for their biological function and calcium- Expression Atlas. binding sites. Diseases: (see below) The region name is EGF2 which Calcium-binding Breast carcinoma, sarcoma, Combined EGF-like domain, present in a large number of Hepatocellular Cholangiocarcinoma (CHC), membrane-bound and extracellular proteins. Intrahepatic Cholangiocarcinoma (ICC), - Location: 69-107; Many of these proteins require leiomyosarcoma, pituitary cancer, extranodal calcium for their biological function and calcium- NK/T-cell lymphoma, osteosarcoma, binding sites. undifferentiated sarcoma, invasive ductal The region name is EGF3 which Calcium-binding carcinoma, mucosa-associated lymphoid tissue EGF-like domain, present in a large number of lymphoma, papillary thyroid carcinoma, peripheral membrane-bound and extracellular proteins. T-cell lymphoma, pancreatic cancer, cervical - Location: 109-145; many of these proteins require cancer, clear cell renal carcinoma, engineered calcium for their biological function and calcium- invasive esophageal squamous cell carcinoma, binding sites. prostate carcinoma. - Location: 109112126; Ca2+ binding site. The above data derive from the EDIL3 entry in the The region (147-304) name is FA58C which note - Expression Atlas. Coagulation factor 5/8 C-terminal domain, Cell lines: (see below) discoidin domain. DU145, ACHN, CaOv3, Caki2, H460, HeLa, KG1, The region name is FA58C which note - MDAMB231, GM10833, GM13883, HCC70, U87 Coagulation factor 5/8 C-terminal domain, CuFi, U251, NCIH1623, SCC-25, 786-O, ITM, discoidin domain; Cell surface-attached HCC-1428, HuNS1, AG10750, LNCAP, SW780, carbohydrate-binding domain, present in eukaryotes AG11498, KTCL26, NCIH1436, Capan2, and assumed to have horizontally transferred to GM10842, HMESO, SKRC54, UMRC3, 639V, eubacterial genomes (150-303), sugar binding site A172, A7, Acute Lymphoblastic Leukemia cell line (196224231). SEM-K2, BE2C, BHT101, BM1604, C32TG, C4II, The region (308-466) name is FA58C which note - CCFSTTG1, CESS, CGTHW1, CHP212, Coagulation factor 5/8 C-terminal domain, COLO320DM, COLO320HSR, COLO704, discoidin domain. CORL279, CORL88, Calu1, D283Med, The region name is FA58C which note - DBTRG05MG, DKMG, DMS114, DMS273, Coagulation factor 5/8 C-terminal domain, Detroit562, GDM1, GLI60 glioblastoma cell line, discoidin domain; Cell surface-attached H1975, H4, H460a, HCC1143, HCC1395, carbohydrate-binding domain, present in eukaryotes HDMYZ, HOS, HT1080, HT3, HuPT4, IM9, and assumed to have horizontally transferred to KHOS240S, KU812, L591, MCF- eubacterial genomes (311-465), sugar binding site 7/LTED,,MCF10-T1k (MII), MDA-MB-231, MT4, (358386393). NCIH1048, NCIH1355, NCIH1395, NCIH1563,

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NCIH1651, NCIH1694, NCIH1770, CIH1792, NC_000015.9), coagulation factors V (Accession: NCIH1975,NCIH1993, NCIH2009, NCIH2030 NC_000001.10) and VIII (Accession: ,NCIH2052, NCIH2228, NCIH226, NCIH2347, NP_000123.1), the extracellular domain of a group NCIH358, NCIH446, NCIH524, NCIH69, of tumor-associated orphan receptor tyrosine NCIH716, NCIH748, NCIH810, NCIH82, OE21, kinases (Accession: Q01973, Q01974), and PC-3, PC-3/Mc, RCC4, RDES, SCaBER, SHP77, discoidin domain receptor tylosin kinase SJRH30, SJSA1, SKLMS1, SKMES1, SKNEP1, (Accession: AB_202100, BC_052998). SNB19, SNU18, 2SNU387, SNU423, SNU449, SNU475, SW1088, SW1353, SW1783, SW1990, Mutations SW756, SW900, SW954, SW982, T98G glioma, TaY-E10, UT-15, WI38, WM115, Y79, YPAC, Note A498, BT474, PC3, SKOV3, HL-60, A549. Not known in human. The above data derive from the EDIL3 entry in the Expression Atlas Expression Atlas. Implicated in Function Hepatocellular carcinoma (HCC) EDIL3 promotes adhesion of endothelial cells to Note extracellular matrix through interaction with the When immunocytochemistry and tumor tissue alpha-v/beta-3 integrin receptor. It inhibits the samples from 101 patients with HCC were used to formation of vascular-like structures in vitro. examine expression level of EDIL3 protein in Exogenous EDIL3 causes abnormal vasculature in HCCs, EDIL3 was detected in the cytoplasm of chick embryos. Overexpression of EDIL3 decreases HCC cells. Overall, 95 (94.06%) of the 101 patients vasculature in mesenteric vessels. It may be exhibited EDIL3-positive expression in the involved in the regulation of vascular respective HCC samples, and 6 (5.94%) exhibited morphogenesis and remodeling during embryonic EDIL3-negative expression in the HCC samples. development. Among these 101 patients with HCC, 49 (48.5%) Reportedly, high expression of EDIL3 by cancer exhibited high levels of EDIL3 expression in cells is an indicator of poor prognosis, possibly normal somatic tissue, and 52 (51.5%) exhibited because EDIL3 can enhance vascular formation in low levels of EDIL3 expression. The high hepatocellular carcinoma, colon cancer, and expression level of EDIL3 protein in HCC samples experimental models with an osteosarcoma cell line was a significant prognostic factor for poor overall or with Lewis lung carcinoma. However, an EDIL3 survival among patients with HCC. The 5-year fragment containing the third EGF-like repeat survival rate among patients with HCC and a high induces apoptosis. In a mouse model of cancer or a low EDIL3 protein expression level was 32.4% involving an explanted tumor, gene therapy with and 53.2%, respectively. (Sun et al., 2010). DNA encoding a recombinant protein comprising the third EGF-like repeat and the first discoidin 1 Colon cancer like domain efficiently induces apoptosis, reduces Note tumor growth, and improves prognosis. Tumor tissues from 10 patients with colon cancer Homology were immunostained with anti-Del1 antibody, and Del1 was found to be expressed in each colon The EGF repeats of EDIL3 are homologous to cancer tissue sample (100%) at various levels. molecules such as Notch (Accession: Using a mouse model of colon cancer involving NM_017617.3) and its ligands Crumbs (Accession: explanted cells from a culture line, both Del1- CH471090.1) and Delta (Accession: shRNA and VEGF-shRNA gene therapies showed a NT_033777.2). There is also considerable synergic effect in suppressing growth of explanted homology in this region to the following four other tumors by anti-angiogenesis and anti-proliferation. proteins: a developmental sea urchin protein (Zou et al., 2009). fibropellin (Accession: NW_003578619.1); a factor shown to function in lineage commitment of Other tumors adipocytes (Accession: NC_000003.11); an Note endothelial cell-specific receptor tyrosine kinase In cases of basal cell carcinoma or astrocytoma, (Accession: NC_000070.6) known to be essential Del1 expression was detected via for embryonic blood vessel development; and immunohistochemistry, and Del1 expression was coagulation factor IX (Accession: NM_000133), primarily evident in the matrix and basement which has a role in the coagulation cascade. In its membrane that surrounded tumor cells. Low-level discoidin I-like domains, EDIL3 is homologous to Del1 staining was also evident over some of the cell five proteins: the mammary epithelial cell marker bodies. Del1 signal was not detected in tumor- milk fat globule membrane protein (Accession: associated endothelial cells.

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A large percentage of cells in primary breast the alphavbeta3 integrin receptor. Genes Dev. 1998 Jan carcinomas, melanomas, and colon cancers in 1;12(1):21-33 humans reportedly express Del1. Additionally, Aoka Y, Johnson FL, Penta K, Hirata Ki K, Hidai C, endothelial cells also reportedly express Del1 in Schatzman R, Varner JA, Quertermous T. The embryonic angiogenic factor Del1 accelerates tumor growth by these tumors. enhancing vascular formation. Microvasc Res. 2002 Aoka et al. have reported that Del1 accelerates Jul;64(1):148-61 tumor growth by enhancing vascular formation in a Chen C, Huang YL, Yang ST. A fibrous-bed bioreactor for mouse explanted tumor model involving an continuous production of developmental endothelial locus- osteosarcoma cell line (Aoka et al., 2002). 1 by osteosarcoma cells. J Biotechnol. 2002 Jul 17;97(1):23-39 References Rezaee M, Penta K, Quertermous T. Del1 mediates VSMC adhesion, migration, and proliferation through interaction Toole JJ, Knopf JL, Wozney JM, Sultzman LA, Buecker JL, with integrin alpha(v)beta(3). Am J Physiol Heart Circ Pittman DD, Kaufman RJ, Brown E, Shoemaker C, Orr EC. Physiol. 2002 May;282(5):H1924-32 Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature. 1984 Nov 22- Hanayama R, Tanaka M, Miwa K, Nagata S. Expression of 28;312(5992):342-7 developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells. J Immunol. Hursh DA, Andrews ME, Raff RA. A sea urchin gene 2004 Mar 15;172(6):3876-82 encodes a polypeptide homologous to epidermal growth factor. Science. 1987 Sep 18;237(4821):1487-90 Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Jenny RJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Hewick RM, Kaufman RJ, Mann KG. Complete cDNA and Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, derived amino acid sequence of human factor V. Proc Natl Nakamura Y, Nagahari K, Murakami K, Yasuda T, Acad Sci U S A. 1987 Jul;84(14):4846-50 Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri Tepass U, Theres C, Knust E. crumbs encodes an EGF- T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi like protein expressed on apical membranes of Drosophila M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe epithelial cells and required for organization of epithelia. K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki Cell. 1990 Jun 1;61(5):787-99 M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Stubbs JD, Lekutis C, Singer KL, Bui A, Yuzuki D, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Srinivasan U, Parry G. cDNA cloning of a mouse Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii mammary epithelial cell surface protein reveals the A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, existence of epidermal growth factor-like domains linked to Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki factor VIII-like sequences. Proc Natl Acad Sci U S A. 1990 H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Nov;87(21):8417-21 Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Rebay I, Fleming RJ, Fehon RG, Cherbas L, Cherbas P, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Artavanis-Tsakonas S. Specific EGF repeats of Notch Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, mediate interactions with Delta and Serrate: implications Takemoto M, Kawakami B, Yamazaki M, Watanabe K, for Notch as a multifunctional receptor. Cell. 1991 Nov Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama 15;67(4):687-99 M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Partanen J, Armstrong E, Mäkelä TP, Korhonen J, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Sandberg M, Renkonen R, Knuutila S, Huebner K, Alitalo Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, K. A novel endothelial cell surface receptor tyrosine kinase Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, with extracellular epidermal growth factor homology Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki domains. Mol Cell Biol. 1992 Apr;12(4):1698-707 M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, del Amo FF, Gendron-Maguire M, Swiatek PJ, Jenkins NA, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi Copeland NG, Gridley T. Cloning, analysis, and H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura chromosomal localization of Notch-1, a mouse homolog of Y, Ohara O, Isogai T, Sugano S. Complete sequencing Drosophila Notch. Genomics. 1993 Feb;15(2):259-64 and characterization of 21,243 full-length human cDNAs. Nat Genet. 2004 Jan;36(1):40-5 Smas CM, Sul HS. Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation. Cell. 1993 May Aoki M, Kanamori M, Ohmori K, Takaishi M, Huh NH, 21;73(4):725-34 Nogami S, Kimura T. Expression of developmentally regulated endothelial cell locus 1 was induced by tumor- Alves J, Selent U, Wolfes H. Accuracy of the EcoRV derived factors including VEGF. Biochem Biophys Res restriction endonuclease: binding and cleavage studies Commun. 2005 Aug 5;333(3):990-5 with oligodeoxynucleotide substrates containing degenerate recognition sequences. Biochemistry. 1995 Hidai C, Kawana M, Kitano H, Kokubun S. Discoidin Sep 5;34(35):11191-7 domain of Del1 protein contributes to its deposition in the extracellular matrix. Cell Tissue Res. 2007 Oct;330(1):83- Artavanis-Tsakonas S, Matsuno K, Fortini ME. Notch 95 signaling. Science. 1995 Apr 14;268(5208):225-32 Niu JX, Zhang WJ, Ye LY, Wu LQ, Zhu GJ, Yang ZH, Grau Hidai C, Zupancic T, Penta K, Mikhail A, Kawana M, GE, Lou JN. The role of adhesion molecules, alpha v beta Quertermous EE, Aoka Y, Fukagawa M, Matsui Y, Platika 3, alpha v beta 5 and their ligands in the tumor cell and D, Auerbach R, Hogan BL, Snodgrass R, Quertermous T. endothelial cell adhesion. 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Choi EY, Chavakis E, Czabanka MA, Langer HF, Thiagarajan P. Developmental endothelial locus-1 (Del-1) Fraemohs L, Economopoulou M, Kundu RK, Orlandi A, mediates clearance of platelet microparticles by the Zheng YY, Prieto DA, Ballantyne CM, Constant SL, Aird endothelium. Circulation. 2012 Apr 3;125(13):1664-72 WC, Papayannopoulou T, Gahmberg CG, Udey MC, Vajkoczy P, Quertermous T, Dimmeler S, Weber C, Kitano H, Mamiya A, Kokubun S, Hidai C. Efficient nonviral Chavakis T. Del-1, an endogenous leukocyte-endothelial gene therapy with FasL and Del1 fragments in mice. J adhesion inhibitor, limits inflammatory cell recruitment. Gene Med. 2012 Nov;14(11):642-50 Science. 2008 Nov 14;322(5904):1101-4 Schürpf T, Chen Q, Liu JH, Wang R, Springer TA, Wang Kitano H, Hidai C, Kawana M, Kokubun S. An epidermal JH. The RGD finger of Del-1 is a unique structural feature growth factor-like repeat of Del1 protein increases the critical for integrin binding. FASEB J. 2012 efficiency of gene transfer in vitro. Mol Biotechnol. 2008 Aug;26(8):3412-20 Jul;39(3):179-85 Kanczkowski W, Chatzigeorgiou A, Grossklaus S, Sprott Zou X, Qiao H, Jiang X, Dong X, Jiang H, Sun X. D, Bornstein SR, Chavakis T. Role of the endothelial- Downregulation of developmentally regulated endothelial derived endogenous anti-inflammatory factor Del-1 in cell locus-1 inhibits the growth of colon cancer. J Biomed inflammation-mediated adrenal gland dysfunction. Sci. 2009;16:33 Endocrinology. 2013 Mar;154(3):1181-9 Kitano H, Kokubun S, Hidai C. The extracellular matrix Mamiya A, Kitano H, Takao K, Kokubun S, Komiya M, protein Del1 induces apoptosis via its epidermal growth Hidai C. An epidermal growth factor motif from Del1 factor motif. Biochem Biophys Res Commun. 2010 Mar protein increases the efficiency of in vivo gene transfer 19;393(4):757-61 with a non-viral vector. Mol Biotechnol. 2013 Jun;54(2):445-50 Sun JC, Liang XT, Pan K, Wang H, Zhao JJ, Li JJ, Ma HQ, Chen YB, Xia JC. High expression level of EDIL3 in HCC This article should be referenced as such: predicts poor prognosis of HCC patients. World J Gastroenterol. 2010 Sep 28;16(36):4611-5 Kitano H, Hidai C. EDIL3 (EGF-Like Repeats And Discoidin I-Like Domains 3). Atlas Genet Cytogenet Oncol Dasgupta SK, Le A, Chavakis T, Rumbaut RE, Haematol. 2014; 18(11):824-828.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 828

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

HELLS (Helicase, Lymphoid-Specific) Kathrin Muegge, Theresa Geiman Laboratory of Cancer Prevention, SAIC-Frederick, National Cancer Institute, Frederick, Maryland 21701, USA (KM, TG)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/HELLSID40811ch10q23.html DOI: 10.4267/2042/54166 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

methylation and histone modifications alter cellular Abstract processes such as transcription, mitosis, meiosis, Review on HELLS, with data on DNA/RNA, on the and DNA repair. protein encoded and where the gene is implicated. HELLS has been shown to be important for stem cell gene control, Hox gene control, DNA Identity methylation, DNA repair, meiosis, and chromatin Other names: LSH, PASG, SMARCA6 packaging of repetitive DNA (Dennis et al., 2001; HGNC (Hugo): HELLS Yan et al., 2003b; De La Fuente et al., 2006; Myant et al., 2008; Burrage et al., 2012). Location: 10q23.33 HELLS belongs to the SNF2 family of chromatin Note remodelling proteins. HELLS (helicase, lymphoid specific) is a member This group of proteins are involved in altering of the SNF2 family of chromatin remodelling chromatin structure by hydrolyzing ATP and proteins that utilize ATP to alter the structure and moving nucleosomes. HELLS has orthologues in packaging of chromatin (Jarvis et al., 1996). These mouse, Xenopus laevis, Danio rerio, Arabidopsis changes in chromatin together with changes in thaliana, and Saccharomyces cerevisiae among other epigenetic mechanisms such as DNA others.

Figure 1. The HELLS gene is located on the long arm of human chromosome 10 at position 24.2. HELLS is found from base pair 94545767 to 94602871 base pair (GRCh38 assembly). Neighboring genes are shown.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 829 HELLS (Helicase, Lymphoid-Specific) Muegge K, Geiman T

Figure 2. The HELLS protein contains the helicase ATP binding and helicase C-terminal domains as identified by Prosite. It additionally contains a nuclear localization signal.

The best studied is the mouse gene called Lsh Function (lymphoid specific helicase) that resides on a syntenic region of mouse chromosome 19 (Geiman HELLS functions as a chromatin remodeling family et al., 1998). member linking multiple epigenetic mechanisms including histone modifications and DNA DNA/RNA methylation (Dennis et al., 2001; Yan et al., 2003a; Yan et al., 2003b; Huang et al., 2004; Fan et al., Description 2005; Zhu et al., 2006; Xi et al., 2007; Myant et al., The HELLS gene consists of 22 exons spanning 2008). Knockout mice die perinatally with defects 57104 base pairs. It is highly expressed in in lymphocyte proliferation, embryonic growth, and embryonic stem cells, proliferating lymphoid kidney development (Geiman et al., 2001; Geiman cells/tissue, and germ cells. In stem cells, and Muegge, 2000). expression decreases upon differentiation. HELLS Loss leads to global DNA methylation changes is additionally expressed at a lower level in many including global hypomethylation at repetive tissues of the developing embryo. In normal adult sequences, as well as both hypo and somatic tissue, expression is low with the exception hypermethylation of single copy genes (Myant et of proliferating lymphocytes. al., 2011; Tao et al., 2011b; Dunican et al., 2013; Yu et al., 2014). Transcription HELLS has been identified as a marker of The HELLS gene produces a transcript of 3163 bp. mammalian stem cells with expression decreasing Ten alternatively spliced isoforms have been upon differentiation. detected. The functional relevance of most of these Reduction in expression of HELLS leads to splice variants is currently unknown. One, variant 1 prolonged expresssion of stem cell genes containing a 44 ntd insertion between exons 3 and implicating this protein as having a role in stem cell 4, creates an additional exon and has been gene silencing (Xi et al., 2009). associated with non-small cell lung cancer HELLS has been also implicated in DNA repair (NSCLC) (Yano et al., 2004). An additional (Burrage et al., 2012). variant, variant 9, contains a 75 ntd deletion in exon HELLS is additionally involved in meiosis with its 18 and has been associated with acute myelogenous loss leading to reduced proliferation and leukemia an acute lymphoblastic leukemia (Lee et differentiation of germ cells with defects in al., 2000). synapsis (de la Fuente et al., 2006; Zeng et al., 2011). Pseudogene Homology None. HELLS exhibits homology with other SNF2 family Protein members like SNF2H, SNF2L, CHD1, CHD2, CHD3, CHD4, CHD5, CHD6, CHD7, CHD8, Description CHD9, BRG1, and BRAHMA. While other SNF2 family members contain additional domains such as HELLS protein consists of 838 amino acids with a chromodomains, bromodomains, BRK, PHD, and predicted size of 97 kD. Since Lsh is a member of RING fingers, HELLS protein does not. Because of the SNF2 chromatin remodeling protein family, it this lack of additional domains, Lsh does not fit into contains the ATP binding and C-terminal helicase any of the other subfamilies of SNF2 members but domains of this subfamily of helicases. appears to represent a distinct subfamily of SNF2 Localisation factors. By homology, it is most closely related to Nuclear. the CHD and SNF2H/L (ISWI) subfamily.

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Mutations Implicated in

Note Acute myelogenous leukemia, acute Hells tm1Kmu lymphoblastic leukemia There are two mutant mouse models of HELLS. Note The original knockout mouse generated by the tm1Kmu When the HELLS gene was first characterized, it Muegge lab (Hells ) deleting the helicase was found to be located in a break point region of domains of I, Ia, and part of two that are included in the human genome frequently associated with exons 6 and 7. leukemia at 10q23-10q24 (Geiman et al., 1998). These mutant mice die perinatally with embryonic Subsequently, an in-frame 75 ntd deletion in exon growth retardation (Geiman et al., 2001). These null 18 (variant 9) of the HELLS gene was found in embryos have kidney defects such as necrosis and 57% of accute myelogenous leukemia and 37% of globule formation in tubules (Geiman et al., 2001). acute lymphoblastic leukemia patient samples Murine embryonic fibroblasts display early tested but not in normal lymphoid tissue examined senescence in culture and mitotic defects (Fan et al., (Lee et al., 2000). This deletion leads to the loss of 2003). 25 amino acids and includes part of one of the Since HELLS is highly expressed in developing conserved SNF2 family helicase domain. This same and activated lymphoid tissue, lymphoid variant was also detected in both normal and non- development and function was studied using a small cell lung cancer patient samples raising the radiation chimera. possibility that variant 9 is also expressed in some Null embryos display lymphoid defects in normal tissue (Yano et al., 2004). thymocyte development with a partial blockage in transitioning from CD4/CD8 double negative to Erythroleukemia double positive T lymphocytes. Note This partial arrest in lymphocyte development leads The mouse HELLS gene (Lsh) has been implicated to a reduction in mature T and B lymphocytes in the development of erythroleukemia based on (Geiman et al., 2000). animal studies involving the original knockout In addition, activation of lymphocytes leads to mouse model (Hells tm1Kmu ) (Fan et al., 2008). Since apoptosis intead of cell proliferation (Geiman et al., these mice die at birth, hematopoiesis was studied 2000). using a reconstitution model (Geiman et al., 2001). Molecular studies on this mouse led to identifying Hells -/- mice had defective hematopoiesis (Fan et HELLS as a gene necessary for DNA methylation al., 2008). In a subset of mice reconstituted with and important for correct chromatin packaging and Hells-/- cells, erythroleukemia developed which is histone methylation (Dennis et al., 2001; Yan et al., normally a rare spontaneous event in mice. Hells 2003b ; Huang et al., 2004; Myant et al., 2011; Tao loss leads to hypomethylation of repetitive elements et al., 2011b; Yu et al., 2014). throughout the genome (Huang et al., 2004; tm1Rarc Hells Dunican et al., 2013). This hypomethylation was Another mutant mouse model of Hells was found to occur in these Hells null mouse tm1Rarc developed in the Arceci lab designated Hells . hemaotopoietic progenitor cells at retroviral This mouse model was generated from a elements within the PU.1 oncogene leading to hypomorphic allele of HELLS by deleting exons overexpression of the gene (Fan et al., 2008). 10, 11, and 12 (Sun et al., 2004). The deleted Increased PU.1 gene expression is linked to section includes several helicase domains of erythroleukemia. HELLS (III, IV, and part of II) but not the ATPase domain. This mutant mouse also shows growth Non-small cell lung cancer (NSCLC) retardation but approximately 40% of mice survive Note to several weeks of age unlike the Muegge mouse A 44 ntd insertion between exons 3 and 4 (variant model which die at birth. Additionally, these mice 1) was detected in 26% of non-small cell lung exhibit signs of premature aging with defects such cancer samples but in none of the normal tissue as graying hair and balding, low fat deposition, and samples (Yano et al., 2004). This insertion through unstable gate among others (Sun et al., 2004). The exon creation of a sequence of intronic origin is premature aging defects seen in this mouse model predicted to lead to premature termination of are likely the result of replicative senescence caused translation leading to a 97 amino acid protein. The by increased expression of the p16 tumor truncated protein may act as a dominant negative suppressor gene in these HELLS mutant mice. protein. This truncated HELLS may also lose its Molecular analysis demonstrated profound DNA normal nuclear localization since at least part of the methylation loss and aberrant expression of repeat nuclear localization signal (NLS) would be sequences (Sun et al., 2004; Dunican et al., 2013). affected.

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Head and neck cancer Melanoma Note Note HELLS is a downstream target of the FOXM1 HELLS was identified as a gene having elevated transcription factor (Waseem et al., 2010). Both expression in aggressive metastatic tumor cells HELLS and FOXM1 have been found to be lines compared to cell lines from less aggressive overexpressed in head and neck cancer such as primary tumors (Ryu et al., 2007). oraopharyngeal squamous cell carcinoma (Janus et HELLS mRNA was also detected in the blood of al., 2011). High expression of both genes correlates patients with different stages of melanoma. The with tumor stage 3 or higher. Additionally, HELLS level of HELLS mRNA in patient blood was expression was found to increase in progression significantly higher in patients with metastatic from normal to dysplasia and then squamous cell melanoma than those with localized primary tumors carcinoma and metastasis in head and neck cancer (Kim et al., 2010). (Waseem et al., 2010). Because of this, HELLS has This work also found that HELLS expression in been proposed as a biomarker for early detection blood appears to be a better biomarker for and progression of head and neck cancer. metastatic melanoma than the currently used Additionally, HELLS is known to be involved in standard (Kim et al., 2010). control of p16 tumor suppressor gene expression These results suggest that HELLS may be a useful through repression with reduction in HELLS biomarker of melanoma presence and progression leading to increased p16 expression. FOXM1 also and a possible target for intervention. suppresses p16, likely through HELLS induction (Teh et al., 2012). This also leads to a DNA Prostate cancer methylation profile in primary oral keratinocytes Note that is similar to human head and neck cancer (Teh The HELLS protein specifically interacts with et al., 2012). E2F3, a transcription factor uniquely amplified in Breast cancer some human tumors. Note The expression of E2F3 is inversely correlated with patient survival. HELLS and E2F3 are co- The HELLS gene has been found to be mutated in overexpressed in prostate carcinomas at the most 3% of invasive breast cancer samples. Most are an aggressive stages. amplification or upregulation of the HELLS gene The association of HELLS and E2F3 occurs at but there are also some that have deletion or several E2F target genes that control cell cycle downregulation (Colak et al., 2013). HELLS was entry. proposed to be a possible marker for progression Depletion of HELLS in a prostate cancer cell line from pre-invasive ductal carcinoma in situ (DCIS) reduced the induction of E2F target genes and to invasive ductal carcinoma (IDC). Additionally, impaired growth suggesting that HELLS may the HELLS gene is located in a copy number contribute to the malignant progression of tumors alteration chromosomal region (Colak et al., 2013). (von Eyss et al., 2012). Furthermore, depletion of HELLS in a breast cancer cell line reduced DNA methylation at several tumor suppressor genes. This resulted in de-repression of References genes involved in proliferation, and altered several Geiman TM, Durum SK, Muegge K. Characterization of growth characteristics of breast cancer cells in vitro gene expression, genomic structure, and chromosomal including anchorage independent growth in soft localization of Hells (Lsh). Genomics. 1998 Dec agar and the ability to migrate in a wound healing 15;54(3):477-83 assay (Tao et al., 2011a). Jarvis CD, Geiman T, Vila-Storm MP, Osipovich O, Akella Skin squamous cell carcinoma U, Candeias S, Nathan I, Durum SK, Muegge K. A novel putative helicase produced in early murine lymphocytes. Note Gene. 1996 Mar 9;169(2):203-7 The role of HELLS in skin tumorigenesis was Geiman TM, Muegge K. Lsh, an SNF2/helicase family explored by overexpressing a deltaNp63alpha member, is required for proliferation of mature T isoform of the p63 gene (Keyes et al., 2011). lymphocytes. Proc Natl Acad Sci U S A. 2000 Apr HELLS is a direct transcriptional target of 25;97(9):4772-7 deltaNp63alpha. HELLS had been implicated in Lee DW, Zhang K, Ning ZQ, Raabe EH, Tintner S, senescence before by targeting the p16 tumor Wieland R, Wilkins BJ, Kim JM, Blough RI, Arceci RJ. Proliferation-associated SNF2-like gene (PASG): a SNF2 supressor gene. In this study, p16 was found not to family member altered in leukemia. Cancer Res. 2000 Jul be involved. Instead, increased deltaNp63alpha led 1;60(13):3612-22 to upregulation of HELLS expression. This caused Dennis K, Fan T, Geiman T, Yan Q, Muegge K. Lsh, a senescence bypass, increased stem like cells and member of the SNF2 family, is required for genome-wide tumorigenesis in human primary keratinocytes. methylation. Genes Dev. 2001 Nov 15;15(22):2940-4

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Geiman TM, Tessarollo L, Anver MR, Kopp JB, Ward JM, Waseem A, Ali M, Odell EW, Fortune F, Teh MT. Muegge K. Lsh, a SNF2 family member, is required for Downstream targets of FOXM1: CEP55 and HELLS are normal murine development. Biochim Biophys Acta. 2001 cancer progression markers of head and neck squamous May 3;1526(2):211-20 cell carcinoma. Oral Oncol. 2010 Jul;46(7):536-42 Fan T, Yan Q, Huang J, Austin S, Cho E, Ferris D, Janus JR, Laborde RR, Greenberg AJ et al.. Linking Muegge K. Lsh-deficient murine embryonal fibroblasts expression of FOXM1, CEP55 and HELLS to show reduced proliferation with signs of abnormal mitosis. tumorigenesis in oropharyngeal squamous cell carcinoma. Cancer Res. 2003 Aug 1;63(15):4677-83 Laryngoscope. 2011 Dec;121(12):2598-603 Yan Q, Cho E, Lockett S, Muegge K. Association of Lsh, a Keyes WM, Pecoraro M, Aranda V et al.. ∆Np63 α is an regulator of DNA methylation, with pericentromeric oncogene that targets chromatin remodeler Lsh to drive heterochromatin is dependent on intact heterochromatin. skin stem cell proliferation and tumorigenesis. Cell Stem Mol Cell Biol. 2003a Dec;23(23):8416-28 Cell. 2011 Feb 4;8(2):164-76 Yan Q, Huang J, Fan T, Zhu H, Muegge K. Lsh, a Myant K, Termanis A, Sundaram AY, Boe T et al.. LSH modulator of CpG methylation, is crucial for normal histone and G9a/GLP complex are required for developmentally methylation. EMBO J. 2003b Oct 1;22(19):5154-62 programmed DNA methylation. Genome Res. 2011 Jan;21(1):83-94 Huang J, Fan T, Yan Q, Zhu H, Fox S, Issaq HJ, Best L, Gangi L, Munroe D, Muegge K. Lsh, an epigenetic Tao Y, Liu S, Briones V, Geiman TM, Muegge K. guardian of repetitive elements. Nucleic Acids Res. Treatment of breast cancer cells with DNA demethylating 2004;32(17):5019-28 agents leads to a release of Pol II stalling at genes with DNA-hypermethylated regions upstream of TSS. Nucleic Sun LQ, Lee DW, Zhang Q, Xiao W, Raabe EH, Meeker A, Acids Res. 2011a Dec;39(22):9508-20 Miao D, Huso DL, Arceci RJ. Growth retardation and premature aging phenotypes in mice with disruption of the Tao Y, Xi S, Shan J, Maunakea A, Che A, Briones V, Lee SNF2-like gene, PASG. Genes Dev. 2004 May EY, Geiman T, Huang J, Stephens R, Leighty RM, Zhao K, 1;18(9):1035-46 Muegge K. Lsh, chromatin remodeling family member, modulates genome-wide cytosine methylation patterns at Yano M, Ouchida M, Shigematsu H et al.. Tumor-specific nonrepeat sequences. Proc Natl Acad Sci U S A. 2011b exon creation of the HELLS/SMARCA6 gene in non-small Apr 5;108(14):5626-31 cell lung cancer. Int J Cancer. 2004 Oct 20;112(1):8-13 Zeng W, Baumann C, Schmidtmann A et al.. Lymphoid- Fan T, Hagan JP, Kozlov SV, Stewart CL, Muegge K. Lsh specific helicase (HELLS) is essential for meiotic controls silencing of the imprinted Cdkn1c gene. progression in mouse spermatocytes. Biol Reprod. 2011 Development. 2005 Feb;132(4):635-44 Jun;84(6):1235-41 De La Fuente R, Baumann C, Fan T, Schmidtmann A, Burrage J, Termanis A, Geissner A, Myant K, Gordon K, Dobrinski I, Muegge K. Lsh is required for meiotic Stancheva I. The SNF2 family ATPase LSH promotes chromosome synapsis and retrotransposon silencing in phosphorylation of H2AX and efficient repair of DNA female germ cells. Nat Cell Biol. 2006 Dec;8(12):1448-54 double-strand breaks in mammalian cells. J Cell Sci. 2012 Zhu H, Geiman TM, Xi S, Jiang Q, Schmidtmann A, Chen Nov 15;125(Pt 22):5524-34 T, Li E, Muegge K. Lsh is involved in de novo methylation Teh MT, Gemenetzidis E, Patel D, Tariq R, Nadir A, Bahta of DNA. EMBO J. 2006 Jan 25;25(2):335-45 AW, Waseem A, Hutchison IL. FOXM1 induces a global Ryu B, Kim DS, Deluca AM, Alani RM. Comprehensive methylation signature that mimics the cancer epigenome in expression profiling of tumor cell lines identifies molecular head and neck squamous cell carcinoma. PLoS One. signatures of melanoma progression. PLoS One. 2007 Jul 2012;7(3):e34329 4;2(7):e594 von Eyss B, Maaskola J, Memczak S et al.. The SNF2-like Xi S, Zhu H, Xu H, Schmidtmann A, Geiman TM, Muegge helicase HELLS mediates E2F3-dependent transcription K. Lsh controls Hox gene silencing during development. and cellular transformation. EMBO J. 2012 Feb Proc Natl Acad Sci U S A. 2007 Sep 4;104(36):14366-71 15;31(4):972-85 Fan T, Schmidtmann A, Xi S, Briones V et al.. DNA Colak D, Nofal A, Albakheet A, Nirmal M et al.. Age- hypomethylation caused by Lsh deletion promotes specific gene expression signatures for breast tumors and erythroleukemia development. Epigenetics. 2008 May- cross-species conserved potential cancer progression Jun;3(3):134-42 markers in young women. PLoS One. 2013;8(5):e63204 Myant K, Stancheva I. LSH cooperates with DNA Dunican DS, Cruickshanks HA, Suzuki M, Semple CA, methyltransferases to repress transcription. Mol Cell Biol. Davey T, Arceci RJ, Greally J, Adams IR, Meehan RR. Lsh 2008 Jan;28(1):215-26 regulates LTR retrotransposon repression independently of Xi S, Geiman TM, Briones V, Guang Tao Y, Xu H, Muegge Dnmt3b function. Genome Biol. 2013 Dec 24;14(12):R146 K. Lsh participates in DNA methylation and silencing of Yu W, Briones V, Lister R, McIntosh C et al.. CG stem cell genes. Stem Cells. 2009 Nov;27(11):2691-702 hypomethylation in Lsh-/- mouse embryonic fibroblasts is Kim HE, Symanowski JT, Samlowski EE, Gonzales J, Ryu associated with de novo H3K4me1 formation and altered B. Quantitative measurement of circulating lymphoid- cellular plasticity. Proc Natl Acad Sci U S A. 2014 Apr specific helicase (HELLS) gene transcript: a potential 22;111(16):5890-5 serum biomarker for melanoma metastasis. Pigment Cell Melanoma Res. 2010 Dec;23(6):845-8 This article should be referenced as such: Tao Y, Xi S, Briones V, Muegge K. Lsh mediated RNA Muegge K, Geiman T. HELLS (Helicase, Lymphoid- polymerase II stalling at HoxC6 and HoxC8 involves DNA Specific). Atlas Genet Cytogenet Oncol Haematol. 2014; methylation. PLoS One. 2010 Feb 11;5(2):e9163 18(11):829-833.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 833

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

NUAK1 (NUAK family, SNF1-like kinase, 1) Fumika Inazuka, Hiroyasu Esumi Research Institute for Biomedical Sciences, Tokyo University of Science, Japan (FI, HE)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/NUAK1ID46275ch12q23.html DOI: 10.4267/2042/54167 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract 84440597. Review on NUAK1, with data on DNA/RNA, on DNA/RNA the protein encoded and where the gene is implicated. Description The human NUAK1 gene is composed of seven Identity exons and spans approximately 76.7 kbp of genomic DNA. Other names: ARK5 Transcription HGNC (Hugo): NUAK1 The human NUAK1 gene encodes a 6828-bp Location: 12q23.3 mRNA. The coding region contains 1986 bp. Three Note: Details concerning the local order of the short splice variants (547-569 bp) have been human NUAK1 locus can be found at ensembl.org. reported. Human NUAK1 is found on chromosome 12 at position 106457118-106533811. Mouse NUAK1 is Pseudogene located on chromosome 10 at position 84370905- No pseudogenes are known.

The human NUAK1 gene has four splice variants. NUAK1-001 is composed of seven exons, which spans 6828 bp and encodes a 661-aa protein. NUAK1-003 is composed of two exons including one non-coding exon, which spans 547 bp and encodes a 118-aa protein. NUAK1-004 is composed of five exons, which spans 560 bp and encodes a 152-aa protein. NUAK1-005 is composed of four exons including two non-coding exons, which spans 569 bp and encodes a 74-aa protein (see Ensembl ENSG00000074590). Open boxes indicate untranslated regions and shaded boxes indicate coding regions of the gene.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 834 NUAK1 (NUAK family, SNF1-like kinase, 1) Inazuka F, Esumi H

The catalytic domain of NUAK1 protein is located in the N-terminal at residues 55 to 306. K84 is an ATP binding site. T211 is phosphorylated by LKB1, which activates NUAK1 (Lizcano et al., 2004). W305 is required for binding of USP9X (Al-Hakim et al., 2008). The GILK motif is a direct binding site for protein phosphatase PPP1CB (Zagorska et al., 2010).

al., 2010). In WI-38 fetal lung fibroblasts and Protein MCF10A immortalized mammary epithelial cells Description NUAK1 phosphorylates the cyclin-dependent protein kinase regulator LATS1, which leads to NUAK1 protein consists of 661 amino acids and destabilization of LATS1 and induces aneuploidy, has a molecular weight of 74 kDa (Nagase et al., resulting in cellular senescence in a cellular 1998). NUAK1 contains a serine/threonine-protein context-dependent manner (Humbert et al., 2010). kinase domain at its N-terminus that is conserved In A549 lung adenocarcinoma cells NUAK1 acts as among AMPKalphas and AMPK-related kinases a transcriptional coactivator in a complex with (AMPK-RKs) (Manning et al., 2002). The same as LKB1 and a tumor suppressor p53, which induces the AMPKalphas and most other AMPK-RKs, cell cycle G1 arrest (Hou et al., 2011). NUAK1 can be phosphorylated by tumor In mice, NUAK1 is essential for closure of the suppressor LKB1 at a conserved threonine residue ventral body wall in developing embryos (Hirano et (corresponding to Thr-211 in NUAK1) in the T- al., 2006). NUAK1 and NUAK2 function in a loop of the catalytic domain, which activates the complementary manner in the apical constriction kinase activity of NUAK1 (Lizcano et al., 2004). and apico-basal elongation during early The phosphorylation at Thr-211 is prevented by embryogenesis (Ohmura et al., 2012). The LKB1- atypical Lys29/ Lys33-linked polyubiquitin chains, NUAK1 pathway regulates cortical axon branching which can be removed by the de-ubiquitinating by immobilizing mitochondria at nascent enzyme USP9X (Al-Hakim et al., 2008). presynaptic sites during postnatal neuronal Expression development (Courchet et al., 2013). NUAK1 has a NUAK1 is preferentially expressed in highly role in the negative feedback regulation of insulin oxidative tissues such as cerebrum, heart, and signaling, which is involved in glucose intolerance soleus muscle in human and mouse (Nagase et al., under high-fat diet conditions (Inazuka et al., 2012). 1998; Inazuka et al., 2012). In mouse In Caenorhabditis elegans, UNC-82, an ortholog of embryogenesis, NUAK1 is prominently expressed NUAK1 and NUAK2, maintains the organization in the neuroectoderm during neurulation, and in the of cytoskeletal structure during cell-shape change cerebral cortex after the late embryonic stage presumably through phosphorylation of myosin and (Ohmura et al., 2012; Courchet et al., 2013). In paramyosin (Hoppe et al., 2010). C2C12 mouse myoblasts, NUAK1 is increasingly Homology expressed as the cells differentiate into myotubes NUAK1 has the highest homology to NUAK2, (Niesler et al., 2007). The elevated expression of whose similarity in the protein as a whole is 58%, NUAK1 has been observed in clinical samples and 82% in the kinase domain in human. Homo obtained from colorectal cancers, pancreatic sapiens NUAK1 is 91% similar to Mus musculus cancers, hepatocellular carcinomas, gliomas, and NUAK1, 75% similar to Xenopus tropicalis angioimmunoblastic T-cell lymphomas (Kusakai et NUAK1, and 65% similar to Caenorhabditis al., 2004a; Morito et al., 2006; Liu et al., 2012; Cui elegans UNC-82. et al., 2013). Localisation Mutations NUAK1 is found in both the cytoplasm and Somatic nucleus. A C-to-T transition at residues 661 in the NUAK1 Function gene that results in a Pro-to-Ser substitution at In HEK293 cells NUAK1 phosphorylates myosin codon 221 (P221S) has been found in tissue phosphatase regulator MYPT1, which inhibits the samples from human colorectal carcinomas and activity of a MYPT1-PPP1CB phosphatase melanomas (Cancer Genome Atlas Network, 2012; complex, enhancing cell detachment (Zagorska et Krauthammer et al., 2012).

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 835 NUAK1 (NUAK family, SNF1-like kinase, 1) Inazuka F, Esumi H

Roh et al., 2010). The expression of NUAK1 is Implicated in closely associated with that of MMP-2 and MMP-9. Glioma Enhanced expression of NUAK1 is more frequent in tumors with RAF-mediated alterations (P = Disease 0.001) or crossover pathways carrying both Malignant gliomas are the most common type of APC/Wnt-activated and mismatch repair/RAF- primary brain tumor in adults. Glioma cells are mediated alterations (P = 0.003) than those without highly proliferative, thereby readily invading them (Roh et al., 2010). surrounding brain structures. Thus, complete surgical resection is practically impossible (Stewart, Oncogenesis 2002). Glioma infiltration occurs through the Higher NUAK1 expression is concordant with activation of matrix metalloproteinases (MMPs). higher invasion activity in human colorectal cancer MMPs also exhibit a function in angiogenesis cell lines (Kusakai et al., 2004a; Kusakai et al., during tumor neovascularization (Forsyth et al., 2004b). Knockdown of NUAK1 suppresses growth 1999; Das et al., 2011). of CRC xenografts in mice (Liu et al., 2012). Prognosis Hepatocellular carcinoma (HCC) Up-regulation of NUAK1 correlates with the World Disease Health Organization grades of glioma (P < 0.001). HCC is the fifth most common cancer and the third High NUAK1 expression was markedly associated most frequent cause of death of cancer worldwide. with reduced overall survival in grade II glioma (P Liver resection is considered to be the mainly < 0.01). The median survival time of patients with curative therapy for HCC, with about 50-70% 5- high NUAK1 expression (18.37 months, 95% year overall survival after curative hepatectomy. confidence interval (CI): 15.95-20.04) was However, the postoperative recurrence rate remains significantly shorter than that of patients with low as high as 70-83.7% (Bruix and Sherman, 2005; NUAK1 expression level (43.80 months, 95% CI: Llovet, 2005). 23.47-48.36). The same conclusions were obtained in grades III and IV gliomas, indicating that Prognosis NUAK1 is a valuable prognostic marker for glioma In HCC, the high expression of NUAK1 is related patients at all disease stages (Lu et al., 2013). to tumor size (P=0.005), histological differentiation (P=0.047), tumor stage (P=0.005), and a Oncogenesis significantly poor prognosis. Multivariate analysis In several kinds of glioma cell lines, NUAK1 identified the level of NUAK1 expression as an promotes IGF-1-induced cell invasion, in which independent predictor of the overall survival rate of NUAK1 mediates cytoskeleton rearrangement patients with HCC (Cui et al., 2013). through activation of LIMK1 and cofilin and activates MMP-2 and MMP-9 through production Oncogenesis of MT1-MMP. Knockdown of NUAK1 reduces Several cancer cell lines including HCC which are brain invasion in mice with glioma xenografts (Lu expressed oncogenic levels of MYC establish a et al., 2013). dependence on NUAK1 for maintaining metabolic homeostasis and for cell survival, in which NUAK1 Colorectal cancer (CRC) restrains mTOR signaling via suppression of Disease proteasomal degradation of AMPKbeta and also CRC is the third leading cancer and the fourth maintains expression of mitochondrial respiratory leading cause of cancer deaths worldwide (Karsa et chain complexes. Knockdown of NUAK1 prevents al., 2010). Various genetic changes, including APC tumorigenesis in MYC-driven mouse models of mutation, mismatch repair defects, Wnt-activated HCC. Depletion of NUAK1 after tumorigenesis alterations, RAF-mediated alterations, and p53 also suppresses tumor growth and provides a alterations are concurrently observed in CRC. survival advantage to the mice (P < 0.01) (Liu et These mutations are interconnected to generate al., 2012). diverse pathways of colorectal tumorigenesis (Conlin et al., 2005; Roh et al., 2010). In addition, References MMPs are overexpressed in colorectal tumors and Nagase T, Ishikawa K, Miyajima N, Tanaka A, Kotani H, involved in degrading components of the basement Nomura N, Ohara O. Prediction of the coding sequences membrane during the progression of CRC (Collins of unidentified human genes. IX. The complete sequences et al., 2001; Dragutinovic et al., 2011). of 100 new cDNA clones from brain which can code for large proteins in vitro. DNA Res. 1998 Feb 28;5(1):31-9 Prognosis Higher expression of NUAK1 is observed in Forsyth PA, Wong H, Laing TD, Rewcastle NB, Morris DG, Muzik H, Leco KJ, Johnston RN, Brasher PM, Sutherland advanced cases, and much higher expression is G, Edwards DR. Gelatinase-A (MMP-2), gelatinase-B observed in liver metastases (Kusakai et al., 2004b; (MMP-9) and membrane type matrix metalloproteinase-1

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(MT1-MMP) are involved in different aspects of the 20;29(2):376-86 pathophysiology of malignant gliomas. Br J Cancer. 1999 Apr;79(11-12):1828-35 Karsa LV, Lignini TA, Patnick J, Lambert R, Sauvaget C. The dimensions of the CRC problem. Best Pract Res Clin Collins HM, Morris TM, Watson SA. Spectrum of matrix Gastroenterol. 2010 Aug;24(4):381-96 metalloproteinase expression in primary and metastatic colon cancer: relationship to the tissue inhibitors of Roh SA, Choi EY, Cho DH, Jang SJ, Kim SY, Kim YS, Kim metalloproteinases and membrane type-1-matrix JC. Growth and invasion of sporadic colorectal metalloproteinase. Br J Cancer. 2001 Jun 15;84(12):1664- adenocarcinomas in terms of genetic change. J Korean 70 Med Sci. 2010 Mar;25(3):353-60 Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam Zagórska A, Deak M, Campbell DG, Banerjee S, Hirano M, S. The protein kinase complement of the human genome. Aizawa S, Prescott AR, Alessi DR. 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ARK5 expression in colorectal preoperative serum as independent prognostic markers in cancer and its implications for tumor progression. Am J patients with colorectal cancer. Mol Cell Biochem. 2011 Pathol. 2004b Mar;164(3):987-95 Sep;355(1-2):173-8 Lizcano JM, Göransson O, Toth R, Deak M, Morrice NA, Hou X, Liu JE, Liu W, Liu CY, Liu ZY, Sun ZY. A new role Boudeau J, Hawley SA, Udd L, Mäkelä TP, Hardie DG, of NUAK1: directly phosphorylating p53 and regulating cell Alessi DR. LKB1 is a master kinase that activates 13 proliferation. Oncogene. 2011 Jun 30;30(26):2933-42 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J. 2004 Feb 25;23(4):833-43 . Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012 Jul Bruix J, Sherman M. Management of hepatocellular 18;487(7407):330-7 carcinoma. Hepatology. 2005 Nov;42(5):1208-36 Inazuka F, Sugiyama N, Tomita M, Abe T, Shioi G, Esumi Conlin A, Smith G, Carey FA, Wolf CR, Steele RJ. The H. Muscle-specific knock-out of NUAK family SNF1-like prognostic significance of K-ras, p53, and APC mutations kinase 1 (NUAK1) prevents high fat diet-induced glucose in colorectal carcinoma. Gut. 2005 Sep;54(9):1283-6 intolerance. J Biol Chem. 2012 May 11;287(20):16379-89 Llovet JM. Updated treatment approach to hepatocellular Krauthammer M, Kong Y, Ha BH, Evans P et al.. Exome carcinoma. J Gastroenterol. 2005 Mar;40(3):225-35 sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat Genet. 2012 Sep;44(9):1006-14 Hirano M, Kiyonari H, Inoue A, Furushima K, Murata T, Suda Y, Aizawa S. A new serine/threonine protein kinase, Liu L, Ulbrich J, Müller J, Wüstefeld T, Aeberhard L, Kress Omphk1, essential to ventral body wall formation. Dev TR, Muthalagu N, Rycak L, Rudalska R, Moll R, Kempa S, Dyn. 2006 Aug;235(8):2229-37 Zender L, Eilers M, Murphy DJ. Deregulated MYC expression induces dependence upon AMPK-related Morito N, Yoh K, Fujioka Y, Nakano T, Shimohata H, kinase 5. Nature. 2012 Mar 28;483(7391):608-12 Hashimoto Y, Yamada A, Maeda A, Matsuno F, Hata H, Suzuki A, Imagawa S, Mitsuya H, Esumi H, Koyama A, Ohmura T, Shioi G, Hirano M, Aizawa S. Neural tube Yamamoto M, Mori N, Takahashi S. Overexpression of c- defects by NUAK1 and NUAK2 double mutation. Dev Dyn. Maf contributes to T-cell lymphoma in both mice and 2012 Aug;241(8):1350-64 human. Cancer Res. 2006 Jan 15;66(2):812-9 Courchet J, Lewis TL Jr, Lee S, Courchet V, Liou DY, Niesler CU, Myburgh KH, Moore F. The changing AMPK Aizawa S, Polleux F. Terminal axon branching is regulated expression profile in differentiating mouse skeletal muscle by the LKB1-NUAK1 kinase pathway via presynaptic myoblast cells helps confer increasing resistance to mitochondrial capture. Cell. 2013 Jun 20;153(7):1510-25 apoptosis. Exp Physiol. 2007 Jan;92(1):207-17 Cui J, Yu Y, Lu GF, Liu C, Liu X, Xu YX, Zheng PY. Al-Hakim AK, Zagorska A, Chapman L, Deak M, Peggie M, Overexpression of ARK5 is associated with poor prognosis Alessi DR. Control of AMPK-related kinases by USP9X in hepatocellular carcinoma. Tumour Biol. 2013 and atypical Lys(29)/Lys(33)-linked polyubiquitin chains. Jun;34(3):1913-8 Biochem J. 2008 Apr 15;411(2):249-60 Lu S, Niu N, Guo H, Tang J, Guo W, Liu Z, Shi L, Sun T, Hoppe PE, Chau J, Flanagan KA, Reedy AR, Schriefer LA. Zhou F, Li H, Zhang J, Zhang B. ARK5 promotes glioma Caenorhabditis elegans unc-82 encodes a cell invasion, and its elevated expression is correlated with serine/threonine kinase important for myosin filament poor clinical outcome. Eur J Cancer. 2013 Feb;49(3):752- organization in muscle during growth. Genetics. 2010 63 Jan;184(1):79-90 This article should be referenced as such: Humbert N, Navaratnam N, Augert A, Da Costa M, Martien S, Wang J, Martinez D, Abbadie C, Carling D, de Launoit Inazuka F, Esumi H. NUAK1 (NUAK family, SNF1-like Y, Gil J, Bernard D. 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Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 837

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

PFKFB2 (6-phosphofructo-2-kinase/fructose- 2,6-biphosphatase 2) Ana Rodríguez-García, Pere Fontova, Helga Simon, Anna Manzano, Ramon Bartrons, Àurea Navarro-Sabaté Departament de Ciencies Fisiologiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, 08907, L'Hospitalet de Llobregat, Barcelona, Spain (ARG, PF, HS, AM, RB, ÀNS)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/PFKFB2ID52100ch1q32.html DOI: 10.4267/2042/54168 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

spanning 22617 bp (GenBank: AJ005577.1). This Abstract gene has 9 transcripts; two of them have been Review on PFKFB2, with data on DNA/RNA, on reported to codify a protein and three contain an the protein encoded and where the gene is open reading frame, but no protein has been implicated. identified. The transcripts are derived from different promoters and vary only in non-coding sequences Identity at the 5' end. Therefore, the resulting proteins differ in their C-terminal amino acid sequence (Heine- Other names: PFK-2/FBPase-2 Suñer et al., 1998). HGNC (Hugo): PFKFB2 The main products of the gene correspond to Location: 1q32.2 mRNAs of 7073 bp and 3529 bp for the variant 1 (isoform a; NM_006212.2) and variant 2 (isoform Local order b; NM_001018053.1), respectively (Fig. 2). The The human PFKFB2 gene is located on the isoform b differs in the 3' UTR and the coding chromosome 1 at position 1q31-q32.2 (GeneCards) region compared to isoform a. The resulting (Fig. 1). isoform b is shorter and has a distinct C-terminus compared to isoform a. However, it is not known DNA/RNA how these different 5' ends are related to the three mRNAs (H1, H2 and H4) that encode the isoform a Description or the H3 mRNA that encodes the isoform b. None The human PFKFB2 is composed of 15 exons of these mRNAs are strictly heart-specific.

Figure 1. Localization of human PFKFB2 gene.

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Figure 2. Schematic representation of the location of PFKFB2 gene in chromosome 1 and its structural organization. Description of the exon/intron splice junctions. Exon sequences are shown in vertical bars numbered 1-15. The sequences of 060825 and 060825-2 correspond to variant 1 and variant 2, respectively (NCBI).

The overall gene structure of the human PFKFB2 PFKFB2 is an enzyme of PFKFB family, as it gene has exons clustered into three groups. The first shares different structure and function with the group contains exons 1 and 2 that are different from others isoenzymes. those in other PFKFB2 genes and contain the ATG PFKFB2 has two distinct catalytic sites in each initiation codon in exon 2. The second group subunit: one for the 6-phosphofruto-2-kinase (PFK- contains exons 3-8 coding for the PFK-2 domain 2) activity and the other for the fructose-2,6- and the third group contains exons 8-15 coding for bisphosphatase (FBPase-2) activity (El-Maghrabi et the FBPase-2 domain and a carboxy-terminal al., 1982; Pilkis et al., 1995; Okar et al., 2001). regulatory domain. Gene structure, exon-intron The sequence of the catalytic core is highly organization, as well as intron sizes, are similar to conserved, whereas the N-terminal and C-terminal those of the rat and bovine homologous genes. regions show more divergence (Rider et al., 2004). Transcription PFK-2/FBPase-2 activities control fructose-2,6- bisphosphate (Fru-2,6-P2) synthesis and The human PFKFB2 coding sequence consists of degradation, regulating the rate of glucose 1518 bp for isoform a and 1416 bp for isoform b metabolism. from the start codon to the stop codon, although the More information about PFKFB2 protein can be immature transcript forms contain 7904 bp and found in Uniprot O60825. 3494 bp, respectively. Multiple alternatively spliced transcript variants have been described for this gene Expression (Ensembl: OTTHUMG00000036033). PFKFB2 protein is expressed mainly in heart, Pseudogene although expression is also found in other tissues at lesser extent (Minchenko et al., 2002). Moreover, it No pseudogene of PFKFB2 is known. is expressed in different cancer cell lines such as T- lymph Jurkat, K562 erythroleukemia, liver HepG2, Protein lung A549, colon RKO, bone U2OS, brain GAMG, prostate LnCap, cervix HeLa and breast MCF7. All Description this information can be found in GeneCards PFKFB2 is a homodimeric protein of 505 amino (sections proteins and expression). acids for isoform a and 471 for isoform b with a According to the RNAseq database, this gene can deduced molecular mass of 58 kDa and 54 kDa, also be expressed in thyroid, brain, kidney, skeletal respectively. muscle, ovary, testis and others.

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Figure 3. PFKFB2 activities and function in the glycolytic pathway in heart during hypoxia.

Localisation with adenosine monophosphate (AMP), inhibiting fructose 1,6-bisphosphatase (Fru-1,6-Pase) (Van PFKFB2 protein is active in the cytosol. Schaftingen, 1987). These properties confer to this Function metabolite a key role in the control of Fru-6-P/Fru- This enzyme regulates the concentration of Fru-2,6- 1,6-P2 substrate cycle and hence critically regulates P2 through the two catalytic domains. PFK-2 carbohydrate metabolism (Fig. 3). domain catalyzes the synthesis of Fru-2,6-P2, using In vertebrates, there are four different PFKFB genes fructose-6-phosphate (Fru-6-P) and adenosine-5- (PFKFB1, PFKFB2, PFKFB3 and PFKFB4), which triphosphate (ATP) as substrates; FBPase-2 domain encode the PFK-2/FBPase-2 isoenzymes. Each of catalyzes the degradation of Fru-2,6-P2 into Fru-6-P these enzymes has been originally identified in and inorganic phosphate (Pi). These two mutually different mammalian tissues: PFKFB1 in liver and opposing catalytic activities are controlled by skeletal muscle, PFKFB2 in heart, PFKFB3 in different mechanisms such that each activity is brain, adipose tissue and proliferating cells, and predominant in a given physiological condition. In PFKFB4 in testis (Okar et al., 2004; Rider et al., detail, the reactions catalyzed are: 2004). However, all four are widely expressed Kinase catalytic activity: ATP + D-fructose-6- throughout the adult organism. These isoenzymes phosphate ⇔ ADP + beta-D-fructose-2,6- show differences in their distribution and kinetic bisphosphate properties in response to allosteric effectors, Phosphatase catalytic activity: Beta-D-fructose-2,6- hormonal, and growth factors signals (Okar et al., bisphosphate + H 2O ⇔ D-fructose-6-phosphate + 2001). PFKFB2 enzyme is overexpressed in phosphate different cancer cells like melanoma, prostate, The rate of glycolytic flux is controlled at different pancreatic, gastric and mammary gland cells levels and by different mechanisms: substrate (Minchenko et al., 2005a; Minchenko et al., 2005b; availability, enzyme concentrations, allosteric Bobarykina et al., 2006). For more information effectors and covalent modifications on regulatory about PFKFB genes see: PFKFB3 (6- enzymes. One of the critically modulated steps is phosphofructo-2-kinase/fructose-2,6-biphosphatase that catalyzed by 6-phosphofructo-1-kinase (PFK- 3) and PFKFB4 (6-phosphofructo-2- 1), in which Fru-2,6-P2 is the most powerful kinase/fructose-2,6-biphosphatase 4). allosteric activator (Van Schaftingen, 1987; Okar Regulation and Lange, 1999; Rider et al., 2004). Fru-2,6-P2 PFKFB2 is an essential enzyme for the regulation relieves ATP inhibition and acts synergistically of glycolysis in heart. PFKFB2 is multisite-

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phosphorylated, integrating signaling from many Glycolysis in heart also increases in response to pathways via protein kinase cascades to a single increased the workload (Depre et al., 1993; molecule, Fru-2,6-P2, to stimulate glycolysis. Beauloye et al., 2002), rising Fru-2.6-P2 due to the The human PFKFB2 protein contains the Ser 29, activation of PFKFB2. The increase on workload Ser 466, Thr 475 and Ser 483 residues that regulate activates PKB but not p70 S6K and this increase is the activity of the enzyme. These residues are blocked by wortmannin and is rapamycin- located in its C-terminal domain and can be insensitive. Ca/CAMK (Ca 2+ /calmodulin-activated phosphorylated by protein kinases such as AMPK, protein kinase) is which phosphorylates and 3-phosphoinositide-dependent kinase-1 (PDK-1), activates PFKFB2 secondarily to a rise in cAMP-dependent protein kinase (protein kinase A; cytoplasmatic Ca2 2+ (Depre et al., 1993; Beauloye PKA), protein kinase B (PKB; also known as Akt), et al., 2002). p70 ribosomal S6 kinase (S6K1), and mitogen- Adrenalin administration in perfused rat hearts activated protein kinase 1 (MAPK-1). suggests that PKA may be responsible for the Phosphorylation of PFKFB2 results in the activation of PFKFB2, which accounts for the activation of the enzyme, increasing V max of PFK-2 increased Fru-2,6-P2 levels (Narabayashi et activity. The variations in PFK-2 activity, however, al.,1985). This hormone promotes PFKFB2 appear to be different with the phosphorylation by phosphorylation by PKA in the residues already the different kinases (Marsin et al., 2000; Rider et described in vitro, which are Ser 466 and Ser 483. al., 2004). These phosphorylations have an impact on PFK-2 In perfused rat hearts, it has been shown that the activity, decreasing the K m for Fru-6-P (Kitamura et concentration of Fru-2,6-P2 is raised by increasing al., 1988; Rider et al., 1992a; Rider et al., 1992b). the work load, after hypoxia or stimulation with PFKFB2 mRNA is induced in organs exposed to adrenalin or insulin (Hue et al., 1982; Rider and hypoxic conditions. Activation of the AMP- Hue, 1984; Depre et al., 1993; Deprez et al., 1997). activated protein kinase (AMPK) during ischemia This activation is probably mediated by the or hypoxia leads to phosphorylation of PFKFB2 at phosphorylation of three conserved residues (Ser Ser 466 which increases the levels of Fru-2,6-P2 466, Thr 475 and Ser 483) by specific protein and stimulates glycolysis. PFKFB2 phosphorylation kinases (Depre et al., 1993; Deprez et al., 1997). leads to an increase in V max with no change in K m Insulin stimulates glycolysis in heart by a for Fru-6-P (Marsin et al., 2000). Other studies combination of an increase in glucose transport and have described PFKFB2 as a hypoxia-responsive activation of PFKFB2 (Depré et al., 1998; Hue et gene in vivo but the regulation of its expression al., 2002). Two serine residues, Ser 466 ad Ser 483 following hypoxic treatments appears to occur in a can be phosphorylated in vitro by PKB in response cell-specific manner. The mechanism underlying to insulin resulting from a 2-fold increase in both the expression of each isoform in different tissues Vmax and affinity for Fru-6-P, one of the substrates remains unclear (Minchenko et al., 2002). of PFK-2 (Lefebvre et al., 1996; Deprez et al., Moreover, amino acids increase the synthesis of 1997). Fru-2,6-P2 in HeLa and MCF7 cell lines by Rat heart PFKFB2 is activated by insulin in vivo phosphorylation at PFKFB2 at Ser 483. This through a 2-fold increase in V max with no change in activation is mediated by PI3K and PKB. Similar Km for Fru-6-P (Rider and Hue, 1984). Moreover, it effects on Fru-2,6-P2 metabolism were observed in has been shown that the insulin-induced activation freshly isolated rat cardiomyocytes treated with of PFKFB2 was blocked by wortmannin, a PI3K amino acids, which indicates that these effects are inhibitor, but was insensitive to rapamycin or not restricted to human cancer cells. In these PD098059, which prevent the activation of p70 S6K cardiomyocytes, PFKFB2 phosphorylation and the MAPK cascade, respectively (Lefebvre et increases glucose consumption and the production al., 1996). These results suggest that PI3K, but not of lactate and ATP (Novellasdemunt et al., 2013). p70 S6K , is involved in the activation of PFKFB2 in PFKFB2 is also a substrate of PKC which response to insulin. New in vitro and in vivo phosphorylates Ser 84, Ser 466 and Thr 475 (Rider experiments show that SGK3 is not required for and Hue, 1986; Kitamura et al., 1988; Rider et al., insulin-induced heart PFK-2 activation and this 1992a; Rider et al., 1992b). However, the effect is likely mediated by PKB α (Mouton et al., physiological significance of phosphorylation of 2010). Moreover, it has been proposed that 14-3-3s, Ser 84, Ser 466 and Thr 475 of PFKFB2 by PKC is that have been implicated in promoting cell survival not completely understood. It seems that (Masters et al., 2002), bind to PFK-2 at Ser 483 phosphorylation of Ser 466 or Thr 475 does not when it is phosphorylated by PKB in vitro in change the enzyme activity. This might be due to response to insulin or in cells that are stimulated the fact that the phosphorylation at Ser 84 possibly with IGF-1 or transfected with active forms of counteracts the effects of phosphorylation at the PKB, mediating the stimulation of glycolysis by activating C-terminal sites (Kitamura et al., 1988; growth factors (Pozuelo et al., 2003). Rider et al., 1992b).

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The mechanism of control of PFKFB2 isoenzyme database. by phosphorylation is also difficult to explain in the absence of a crystal structure of the phosphorylated Homology isoenzyme. Phosphorylation of Ser 466 and Ser 483 Location in the mouse: chromosome 1, 56,89 cM, at the C-terminal end of the bovine heart isoenzyme cytoband E4, 130689043-130729253 bp, by PKA (Kitamura et al., 1988; Rider et al., 1992a; complement strand (MGI). Rider et al., 1992b; Deprez et al., 1997) and insulin- For a comparison of the gene from Homo sapiens, stimulated protein kinases (Deprez et al., 1997) mouse, rat, cattle, chimpanzee, chicken, zebrafish, activates PFK-2 by decreasing K m for Fru-6-P and rhesus macaque and dog see MGI. by increasing the V max without affecting FBPase-2. Also for all species known gene tree, see Treefam Ser 466 phosphorylation is responsible for the database. increase in V max whereas both phosphorylations are It appears that the use of Fru-2,6-P2 as a regulatory necessary to decrease the K m for Fru-6-P (Bertrand metabolite is a specifically eukaryotic phenomenon. et al., 1999). The most plausible hypothesis for the origin of the Regulatory sequences that account for some of the PFK-2/FBPase-2 would be the fusion of two mechanisms involved in the long-term hormonal ancestral genes coding for a kinase functional unit control and tissue-specific expression of PFKFB2 and a phosphohydrolase/mutase unit, respectively. have been identified. The 5' flanking sequence of From protein sequence alignments, it is clear that PFKFB2 contains regions that are conserved the bisphosphatase activity located in the C- between the human, bovine and rat genes. In these terminal domain of the PFK-2/FBPase-2, the regions, several potential binding sites for the Sp1, phosphoglycerate mutases (PGAMs) and the acid HNF-1 and BHLH (helix-loop-helix) (E boxes) phosphatase family diverged from a common transcription factors and for the GR have been ancestor (Jedrzejas, 2000; Okar et al., 2001). described (Tsuchiya and Uyeda, 1994; Chikri and Alignments of the bisphosphatase domain with Rousseau, 1995; Heine-Suñer et al., 1998), but a PGM and acid phosphatase can be obtained at EBI. factor binding to these sites has not been reported. On the other hand, PFK-2 domain is related to a Chromosomal rearrangements: copy number superfamily of mononucleotide binding proteins variants including adenylate kinase (AK) of E. coli., p21 There are three alterations affecting PFKFB2 Ras, EF-tu, the mitochondrial ATPase- β-subunits genome region described in patients. One of them, and myosin ATPase, all of them contain the Walker the gain of 1:195266734-216326885, shows A and B motifs and have a similar fold (Rider et al., phenotypic effects such us visual impairment, low- 2004). set ears, iris coloboma, intellectual disability, defect Orthologs (from BLAST Local Alignment Tool) in the atrial septum, ventricular septal defect and Results from BLAST Local Alignment Tool are vertical nystagmus. For more information see shown in Figure 5. Only the annotated proteins are DECIPHER. reported, the predicted proteins appearing in the No syndrome or disease was found in OMIM local alignment were excluded.

Figure 4. Domain organization and phosphorylation of PFKFB2 isoenzyme. The N-terminal PFK-2 domain is shown in violet, the C-terminal FBPase-2 domain is shown in red and the regulatory domains are shown in blue. Phosphorylation sites, the stimuli and the kinases responsible of their phosphorylation are indicated.

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Figure 5. Orthologs for PFKFB2 gene from BLAST Local Alignment Tool.

Comparison of the PFKFB2 cDNA sequence with in patient tumor samples are collected in the the bovine and rat 6-phosphofructo-2- COSMIC database. kinase/fructose-2,6-bisphosphatase (PFK- Coding silent substitutions: 20, which represent 2/FBPase-2) heart isoforms shows 87-90% 40.8% of the mutations described among all nucleotide and 92-95% amino acid identity (Sakata patients. and Uyeda, 1990; Darville et al., 1991). Two of them have been found in two patients: c.1008C>G (p.T336T) and c.1419G>A (p.S473S). Mutations Nonsense substitutions: 1, located in c.1051C>T Note (p.R351*). Genomic variants Missense substitutions: 23, which represent 46.9% There are 647 SNP variants described in PFKFB2 of the mutations described among all patients. (see GeneCards). Deletions frameshift: 1, located in c.1044delT The most SNP are found in non coding regions: 418 (p.F348fs*66). are presented in introns, 3 in splice donor variant, Insertion frameshift: 1, located in c.703_407insT 107 in 3' UTR and 25 variant within a half kb of the (p.Q235fs*37). end of gene and others. Deletion inframe: 2, located in c.28_30delAAC Furthermore, 61 SNP are presented with the coding (p.N12delN) and in in c.82_84delTGT regions. The most of them are missense (31 (p.C28delC). variants) and also synonymous variants (19 Unknown mutation: 2, one of them located in variants) and only one frameshift. c.376-2A>T and the other in c.840+1G>A. No synonymous substitutions or chromosomal Somatic fusions in PFKFB2 gene have been described in 49 somatic mutations in the PFKFB2 gene detected any tumor sample.

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Figure 6. Histogram of mutations found among the amino acid sequence of PFKFB2 protein. The maximum number of substitutions at any specific genomic region is represented in Y axis. 6-phosphofructo-2-kinase and histidine phosphatase superfamily domains are represented in green and red respectively. From: COSMIC Database.

factor frequently deregulated in cancer cells that Implicated in induces the expression of glycolytic genes (Bartrons and Caro, 2007). Various cancers In culture cells, hypoxia induces PFKFB2 in HeLa Oncogenesis and MCF7 cells. These data demonstrate that Cancer cells energy metabolism is characterized by PFKFB2 is one of the responsive to hypoxia in a high glycolytic rate, which is maintained under vivo, indicating a physiological role in the aerobic conditions, when compared to non- adaptation of the organism to environmental or malignant cells. The concentration of Fru-2,6-P2 is localized hypoxia/ischemia. Marsin et al. (2000) generally increased due to overexpression and showed that AMPK phosphorylates PFKFB2 at Ser activation of PFK-2. Adrenaline, insulin, hypoxia 466 in hypoxia conditions and this could contribute and workload stimulate heart glycolysis by to maintain the high glycolytic rate that is a activating PFKFB2, hence producing a subsequent characteristic feature of many tumors. rise in Fru-2,6-P2 concentration (Marsin et al., Acute lymphoblastic leukemia 2000; Rider et al., 2004). Hypoxia is an important component of the tumor Note microenvironment. One key mediator of the Alterations in glucose metabolism have been hypoxic response in animal cells is the hypoxia- implicated in cell death and survival decisions, inducible factor (HIF) complex, a transcription particularly in the lymphoid lineage (Plas et al.,

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2002) and in transformed cells (Tennant et al., character of the induction of expression of the 2010). PFKFB2 (Bobarykina et al., 2006). PFKFB2 was identified by microarray analysis of Hepatocellular cancer lymphoblasts isolated from glucocorticoid-treated children suffering from ALL (acute lymphoblastic Note leukemia) as one of the most promising candidate In immuhistochemistry samples of hepatocellular genes as a glucocorticoid (GC)-response gene, since carcinoma, it has been recently found that high it was regulated in the majority of patients. Its expression of MACC1 (metastasis associated in deregulation was proposed to entail disturbances in colon cancer 1), a key regulator of the HGF/Met- glucose metabolism which, in turn, have been pathway, correlates with high expression of implicated in cell death induction (Schmidt et al., PFKFB2. This correlation has an effect on TNM 2006). These data suggest that cellular metabolism stage (classification of malignant tumors), overall and apoptosis might be intertwined with survival and Edmondson-Steier classification (Ji et connections between regulation of cellular al., 2014). bioenergetics and apoptosis. Carlet et al. (2010) Papillary thyroid cancer demonstrated that both splice variants of PFKFB2 Note are expressed and specifically induced by GC in malignant lymphoid cells, however, functional The extent and presentation of papillary thyroid analysis of this gene in the human T-ALL cell line cancer (PTC) in adolescents and young adults model CCRF-CEM revealed that its over- (AYAs) is different than in older patients. This expression does not explain the anti-leukemic difference may be due to several candidate genes effects of GC. that are differentially expressed and which may have important roles in tumor cell biology. One of Prostate cancer these genes is PFKFB2 but future functional Note genomics studies are needed to shed further light on In the early stages of prostate cancer, the androgen whether a biologic basis exists to account for the receptor (AR) is one of the key regulators that disparity in AYA cancer incidence and outcome mediates tumor growth, promoting glucose uptake (Vriens et al., 2011). and anabolic metabolism, and modulates gene Heart diseases expression. Massie et al. (2011), using multiple metabolomic approaches, demonstrated that Note PFKFB2 is up-regulated as a consequence of the In the heart, acute ischemia induces rapid activation transcriptional changes by AR, with possible of AMPK which phosphorylates Ser 466 leading to control through the AR-CAMKII-AMPK signaling a two-fold increase in the V max of PFKFB2 (Hue et pathway. al., 2002). mRNA analysis indicated that PFKFB2 Other studies performing microarray analysis, using is expressed at high levels not only in the heart but total RNA isolated from LNCaP cells treated with also in the brain and lungs. However, in vivo or without R1881 (methyltreinolone), a synthetic experiments showed that hypoxia induce moderate androgen, showed that androgens induce PFKFB2 expression in the lung and liver and very strong expression in LNCaP cells (androgen-sensitive stimulation in the testis. No induction or even mild human prostate adenocarcinoma cells) by the direct inhibition was found in the heart, kidney, brain and recruitment of the ligand-activated AR to the skeletal muscle. Myocardial ischemia induces a PFKFB2 promoter. Moreover, depletion of shift to anaerobic metabolism, with a rapid PFKFB2 expression using siRNA (small interfering stimulation of glycolysis (Wang et al., 2008). RNA) or inhibiting the PFK-2 activity with Tetralogy of Fallot (TOF) is a heart defect in LY294002 (inhibitor of PI3K) treatment resulted in children that results in chronic progressive right a reduced glucose uptake and lipogenesis, ventricular pressure overload and shunt hypoxemia. suggesting that the induction of de novo lipid Western blot, RT-qPCR (real time PCR) and synthesis by androgens requires the transcriptional immunohystochemical analysis revealed that up-regulation of PFKFB2 in prostate cancer cells PKFB2 expression and mRNA of PFKFB2 (Moon et al., 2011). increased significantly in TOF patients. Like tumors, under pathological stress conditions, Gastric cancer cardiomyocytes gradually come to rely on Note glycolysis to satisfy their main energy PFKFB2 mRNA expression is increased in requirements. malignant gastric tumors as well as the expression That is why these results suggest that PFKFB2 of known HIF-1-dependent genes, Glut1 (glucose plays an important role in certain biological transporter 1) and VEGF (vascular endothelial processes related to cardiac remodeling, which growth factor), supporting the HIF-dependent occurs in response to chronic hypoxia and long-

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term pressure overload in TOF patients (Xia et al., 2013). References Glycolysis increases in cognitive heart failure Neely JR, Whitmer JT, Rovetto MJ. Effect of coronary (CHF), cardiac hypertrophy and cardiac ischemia blood flow on glycolytic flux and intracellular pH in isolated (Neely et al., 1975). rat hearts. Circ Res. 1975 Dec;37(6):733-41 Some studies producing mice with chronic and El-Maghrabi MR, Claus TH, Pilkis J, Fox E, Pilkis SJ. stable elevation of cardiac Fru-2,6-P showed Regulation of rat liver fructose 2,6-bisphosphatase. J Biol 2 Chem. 1982 Jul 10;257(13):7603-7 significant change in cardiac metabolite concentrations, increased glycolysis, reduced Hue L, Blackmore PF, Shikama H, Robinson-Steiner A, palmitate oxidation and protection of isolated Exton JH. Regulation of fructose-2,6-bisphosphate content in rat hepatocytes, perfused hearts, and perfused myocytes from hypoxia. hindlimbs. J Biol Chem. 1982 Apr 25;257(8):4308-13 Taken together, these results show that PFKFB2 is Rider MH, Hue L. Activation of rat heart one of the enzymes that control cardiac glycolysis, phosphofructokinase-2 by insulin in vivo. FEBS Lett. 1984 producing an increase in Fru-2,6-P2, causing Oct 29;176(2):484-8 detrimental effects and suggesting that the elevation Narabayashi H, Lawson JW, Uyeda K. Regulation of of glycolysis in failing hearts could be injurious to phosphofructokinase in perfused rat heart. Requirement an already compromised heart (Wang et al., 2008). for fructose 2,6-bisphosphate and a covalent modification. Inflammation J Biol Chem. 1985 Aug 15;260(17):9750-8 Van Schaftingen E. Fructose 2,6-bisphosphate. Adv Note Enzymol Relat Areas Mol Biol. 1987;59:315-95 It has been shown that purified human CD3+ T Kitamura K, Kangawa K, Matsuo H, Uyeda K. cells express PFKFB2 (Telang et al., 2012). Phosphorylation of myocardial fructose-6-phosphate,2- CCL5 (proinflammatory chemokine) treatment of kinase: fructose-2,6-bisphosphatase by cAMP-dependent ex vivo activated human CD3+ T cells induced the protein kinase and protein kinase C. Activation by activation of the nutrient-sensing kinase AMPK and phosphorylation and amino acid sequences of the phosphorylation sites. J Biol Chem. 1988 Nov downstream substrates like PFKFB2, suggesting 15;263(32):16796-801 that both glycolysis and AMPK signaling are required for efficient T cell migration in response to Sakata J, Uyeda K. Bovine heart fructose-6-phosphate 2- kinase/fructose-2,6-bisphosphatase: complete amino acid CCL5, relating therefore PFKFB2 with T-cell sequence and localization of phosphorylation sites. Proc activation and migration (Chan et al., 2012). Natl Acad Sci U S A. 1990 Jul;87(13):4951-5 Mental disorders Darville MI, Chikri M, Lebeau E, Hue L, Rousseau GG. A rat gene encoding heart 6-phosphofructo-2- Note kinase/fructose-2,6-bisphosphatase. FEBS Lett. 1991 Aug Schizophrenia presents impaired glucose 19;288(1-2):91-4 regulation. Stone et al. (2004), using a genome Rider MH, Vandamme J, Lebeau E, Vertommen D, Vidal scan, found that PFKFB2 shows linkage with H, Rousseau GG, Vandekerckhove J, Hue L. The two schizophrenia in a multiple sample of subjects forms of bovine heart 6-phosphofructo-2-kinase/fructose- (European-American samples). 2,6-bisphosphatase result from alternative splicing. Biochem J. 1992 Jul 15;285 ( Pt 2):405-11 However, it is necessary to replicate these results with other samples and if PFKFB2 contributes on Rider MH, van Damme J, Vertommen D, Michel A, Vandekerckhove J, Hue L. Evidence for new the liability for schizophrenia, its influence is likely phosphorylation sites for protein kinase C and cyclic AMP- to be modest, as most cases of schizophrenia are dependent protein kinase in bovine heart 6-phosphofructo- likely to result from multiple factors. 2-kinase/fructose-2,6-bisphosphatase. FEBS Lett. 1992 Sep 28;310(2):139-42 Growth restriction and development Depre C, Rider MH, Veitch K, Hue L. Role of fructose 2,6- Note bisphosphate in the control of heart glycolysis. J Biol Infants with intrauterine growth restriction (IUGR) Chem. 1993 Jun 25;268(18):13274-9 have a low weight at birth as a result of pathologic Tsuchiya Y, Uyeda K. Bovine heart fructose 6-P,2- restriction of fetal growth (Wollmann, 1998). kinase:fructose 2,6-bisphosphatase mRNA and gene cDNA microarrays, RT-qPCR and Western blot structure. Arch Biochem Biophys. 1994 May 1;310(2):467- analysis revealed that PFKFB2 expression increases 74 in placentas from pregnancies with IUGR causing Chikri M, Rousseau GG. Rat gene coding for heart 6- hypoglycemia. phosphofructo-2-kinase/fructose-2,6-bisphosphatase: characterization of an unusual promoter region and However, further studies have to be performed in identification of four mRNAs. Biochemistry. 1995 Jul order to elucidate the role of PFKFB2 in glucose 11;34(27):8876-84 metabolism on IUGR placenta (Lee et al., 2010). Pilkis SJ, Claus TH, Kurland IJ, Lange AJ. 6-

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Horm Res. 1998;49 Suppl 2:1-6 family overexpression in human lung tumor. Ukr Biokhim Bertrand L, Alessi DR, Deprez J, Deak M, Viaene E, Rider Zh. 2005;77(6):46-50 MH, Hue L. Heart 6-phosphofructo-2-kinase activation by Minchenko OH, Opentanova IL, Ogura T, Minchenko DO, insulin results from Ser-466 and Ser-483 phosphorylation Komisarenko SV, Caro J, Esumi H. Expression and and requires 3-phosphoinositide-dependent kinase-1, but hypoxia-responsiveness of 6-phosphofructo-2- not protein kinase B. J Biol Chem. 1999 Oct kinase/fructose-2,6-bisphosphatase 4 in mammary gland 22;274(43):30927-33 malignant cell lines. Acta Biochim Pol. 2005;52(4):881-8 Okar DA, Lange AJ. Fructose-2,6-bisphosphate and Bobarykina AY, Minchenko DO, Opentanova IL, Moenner control of carbohydrate metabolism in eukaryotes. M, Caro J, Esumi H, Minchenko OH. Hypoxic regulation of Biofactors. 1999;10(1):1-14 PFKFB-3 and PFKFB-4 gene expression in gastric and Jedrzejas MJ. 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(protein kinase B), but not SGK3 (serum- and CCL5 regulates glucose uptake and AMP kinase signaling glucocorticoid-induced protein kinase 3). Biochem J. 2010 in activated T cells to facilitate chemotaxis. J Biol Chem. Oct 15;431(2):267-75 2012 Aug 24;287(35):29406-16 Tennant DA, Durán RV, Gottlieb E. Targeting metabolic Telang S, Clem BF, Klarer AC, Clem AL, Trent JO, Bucala transformation for cancer therapy. Nat Rev Cancer. 2010 R, Chesney J. Small molecule inhibition of 6- Apr;10(4):267-77 phosphofructo-2-kinase suppresses t cell activation. J Transl Med. 2012 May 16;10:95 Massie CE, Lynch A, Ramos-Montoya A, Boren J, Stark R, Fazli L, Warren A, Scott H, Madhu B, Sharma N, Bon H, Novellasdemunt L, Tato I, Navarro-Sabate A, Ruiz-Meana Zecchini V, Smith DM, Denicola GM, Mathews N, Osborne M, Méndez-Lucas A, Perales JC, Garcia-Dorado D, M, Hadfield J, Macarthur S, Adryan B, Lyons SK, Brindle Ventura F, Bartrons R, Rosa JL. Akt-dependent activation KM, Griffiths J, Gleave ME, Rennie PS, Neal DE, Mills IG. of the heart 6-phosphofructo-2-kinase/fructose-2,6- The androgen receptor fuels prostate cancer by regulating bisphosphatase (PFKFB2) isoenzyme by amino acids. J central metabolism and biosynthesis. EMBO J. 2011 May Biol Chem. 2013 Apr 12;288(15):10640-51 20;30(13):2719-33 Xia Y, Hong H, Ye L, Wang Y, Chen H, Liu J. Label-free Moon JS, Jin WJ, Kwak JH, Kim HJ, Yun MJ, Kim JW, quantitative proteomic analysis of right ventricular Park SW, Kim KS. Androgen stimulates glycolysis for de remodeling in infant Tetralogy of Fallot patients. J novo lipid synthesis by increasing the activities of Proteomics. 2013 Jun 12;84:78-91 hexokinase 2 and 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase 2 in prostate cancer cells. Biochem J. Ji D, Lu ZT, Li YQ, Liang ZY, Zhang PF, Li C, Zhang JL, 2011 Jan 1;433(1):225-33 Zheng X, Yao YM. MACC1 expression correlates with PFKFB2 and survival in hepatocellular carcinoma. Asian Vriens MR, Moses W, Weng J, Peng M, Griffin A, Bleyer A, Pac J Cancer Prev. 2014;15(2):999-1003 Pollock BH, Indelicato DJ, Hwang J, Kebebew E. Clinical and molecular features of papillary thyroid cancer in This article should be referenced as such: adolescents and young adults. Cancer. 2011 Jan 15;117(2):259-67 Rodríguez-García A, Fontova P, Simon H, Manzano A, Bartrons R, Navarro-Sabaté À. PFKFB2 (6-phosphofructo- Chan O, Burke JD, Gao DF, Fish EN. The chemokine 2-kinase/fructose-2,6-biphosphatase 2). Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11):838-848.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 848

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

WWTR1 (WW domain containing transcription regulator 1) Yulei Zhao, Xiaolong Yang Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada (YZ, XY)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/WWTR1ID44545ch3q25.html DOI: 10.4267/2042/54169 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Identity Other names: TAZ WWTR1 (also called TAZ in publications. Therefore, TAZ is used in the following HGNC (Hugo): WWTR1 description) is a WW domaing-containing Location: 3q25.1 transcriptional coactivator, which was first Local order identified as a 14-3-3 binding protein. TM4SF4-WWTR1-COMMD2-ANKUB1. TAZ is the downstream component in the Hippo pathway, and also has been found to interact with DNA/RNA different pathways, such as Wnt, TGFbeta, etc. TAZ is involved in mesenchymal stem cell Description differentiation as well as tumorigenesis. TAZ maps to NC_000003.12, in the region between High level of TAZ has been found in different 149235022 to 149454501 and spans 220 kilobases. cancers, such as breast cancer, colon cancer, lung TAZ has 7 exons, ranging in size from 112 bp to cancer, etc. 3754 bp. Keywords Transcription Oncogene, cell differentiation, transcriptional The mRNA transcript spans 5135 bp with 1202 bp coactivator. open reading frame.

TAZ structure domain. TB: TEAD binding domain; WW: WW domain; TA: Transactivation domain, which contains a Gln-rich region (194-241 aa) and Coiled-coil region (225-259 aa); PDZB: PDZ-binding domain; S89-LATS phosphorylation site.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 849 WWTR1 (WW domain containing transcription regulator 1) Zhao Y, Yang X

TAZ relates to tissue homeostasis and development Protein as well. TAZ knockout mice develop Polycystic Description Kidney Disease (PKD) and emphysema, suggesting an important role of TAZ in renal and lung TAZ is a downstream transcriptional coactivator in development (Liu et al., 2011). the Hippo pathway (Kanai et al., 2000; Lei et al., TAZ also plays a role in mechanotransduction. 2008; Hong and Guan, 2012). TAZ has one WW Extracellular matrix stiffness or confined domain which allows its interaction with PPxY adhesiveness can cause TAZ retention in nuclear, motif-containing proteins such as LATS kinases in which, therefore contributes to cell proliferation, the Hippo pathway as well as other transcription mesenchymal stem cell differentiation as well as factors (TFs). Its N-termini contains a Tead-binding cancer malignant progression (Dupont et al., 2011). (TB) domain, through which TAZ can bind to TEAD, which is a well-known TF involved in cell Homology proliferation and anti-apoptosis. In its C-termini, TAZ gene is conserved across species. Homologous there is a Transcriptional Activation (TA) domain proteins have been found in chimpanzee, dog, cow, which contains a Gln-rich region (amino acid (aa.) mouse, rate, chicken and zebrafish. 194-241) and Coiled-coil region (aa. 225-259). From 394 to aa. 400 of TAZ, there is a PDZ- Mutations binding domain, which has been found important for transcriptional coactivating function of TAZ Note (Wang et al., 2009; Liu et al., 2011). TAZ has a missense mutation (F299V), which was detected at 7% and 10% in primary mammary Expression tumor and xenograft respectively, as well as 28% TAZ is expressed in various tissues, and high mutant allele frequency in metastatic breast cancers expression of TAZ has been found in thyroid, (Ding et al., 2010). kidney, heart, placenta and lung. Localisation Implicated in TAZ localizes in both cytoplasm and nucleus. Non-small cell lung cancer Normally, in the nucleus, TAZ can possess its Note transcription-activating function and help initiate target genes' expressions through binding with High level of TAZ has been found in different non- related transcriptional factors. And the localization small cell lung cancer (NSCLC) cell lines. of TAZ can be regulated by cell-cell contact. Once TAZ overexpression in immortalized non- cells get confluent (high cell-cell contact), the tumorigenic lung epithelial cells causes increased Hippo pathway will be activated (Zhao et al., cell proliferation and transformation, whereas TAZ 2011). As a result, TAZ will be phosphorylated on knockdown in NSCLC cells significantly reduces S89, initiating its binding with 14-3-3 (Lei et al., tumor cell proliferation and tumor growth in nude 2008; Zhao et al., 2008), which will anchor TAZ in mice (Zhou et al., 2011). the cytoplasm. Besides, the interaction with some Significantly, TAZ expression was found associated proteins, such as AMOT and ZO-1, can also with lung adenocarcinoma, metastasis, poorer localize TAZ to cell membrane (Chan et al., 2011; differentiation and poor prognosis (Xie et al., Remue et al., 2010). 2012). Lung cancer patients with negative TAZ expression have prolonged overall survival (Lau et Function al., 2014). TAZ functions as an oncogene. Over-expression of TAZ induces increased cell proliferation, epithelial- Colorectal cancer mesenchymal transition (EMT), cell migration and Note transformation (Chan et al., 2009; Lai et al., 2011). High levels of TAZ mRNA are significantly In addition, enhanced levels of TAZ causes drug correlated with shorter survival in colorectal cancer resistance by activating CTGF and Cyr61 (Lai et patients. This was due to the increased levels of al., 2011). TAZ downstream target genes CTGF and AXL, TAZ is also involved in mesenchymal stem cell which are involved in colorectal cancer differentiation. TAZ can activate TF RUNX2 to development (Yuen et al., 2013). induce osteoblast differentiation, while TAZ binds and inhibits PPARG TF, which further blocks Breast cancer adipocyte differentiation. Besides, TAZ also Note regulates myoblast differentiation by enhancing TF TAZ has been found correlated with breast cancers. MyoD-dependent myogenic gene expression (Hong The breast cancer cell lines have high expression of et al., 2005; Jung et al., 2009; Cho HH et al., 2010). TAZ and 20% of breast cancer samples have TAZ

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 850 WWTR1 (WW domain containing transcription regulator 1) Zhao Y, Yang X

overexpression (Chan et al., 2009). TAZ causes a transcription factor implicated in Holt-Oram syndrome. increased cell migration through activation of Proc Natl Acad Sci U S A. 2005 Dec 13;102(50):18034-9 BMP4, and resistance to chemotherapeutic drug Tian Y, Kolb R, Hong JH, Carroll J, Li D, You J, Bronson Taxol through downstream Cyr61 and CTGF (Lai R, Yaffe MB, Zhou J, Benjamin T. TAZ promotes PC2 degradation through a SCFbeta-Trcp E3 ligase complex. et al., 2011; Lai and Yang, 2013). TAZ can also Mol Cell Biol. 2007 Sep;27(18):6383-95 cause increased cell proliferation and tumorigenesis by activating KLF5 through inhibition of KLF5 Lei QY, Zhang H, Zhao B, Zha ZY, Bai F, Pei XH, Zhao S, Xiong Y, Guan KL. TAZ promotes cell proliferation and degradation (Zhao et al., 2012). Also, TAZ has epithelial-mesenchymal transition and is inhibited by the been suggested to play a role in breast cancer stem hippo pathway. Mol Cell Biol. 2008 Apr;28(7):2426-36 cell self-renewal and tumor-initiation capabilities Zhao B, Lei QY, Guan KL. The Hippo-YAP pathway: new (Cordenonsi et al., 2011; Bartucci et al., 2014). connections between regulation of organ size and cancer. Moreover, TAZ is also found amplified in 44% Curr Opin Cell Biol. 2008 Dec;20(6):638-46 basal-like and 30% luminal breast cancer (Skibinski Chan SW, Lim CJ, Loo LS, Chong YF, Huang C, Hong W. et al., 2014). TEADs mediate nuclear retention of TAZ to promote oncogenic transformation. J Biol Chem. 2009 May Tongue squamous cell carcinoma 22;284(21):14347-58 (TSCC) Jung H, Lee MS, Jang EJ, Ahn JH, Kang NS, Yoo SE, Bae Note MA, Hong JH, Hwang ES. Augmentation of PPARgamma- TSCC cells and specimens have significantly higher TAZ interaction contributes to the anti-adipogenic activity of KR62980. Biochem Pharmacol. 2009 Nov expression of TAZ than those in non-cancerous 15;78(10):1323-9 cells and normal tongue mucosa. Overexpression of Wang K, Degerny C, Xu M, Yang XJ. YAP, TAZ, and TAZ in TSCC was significantly associated with Yorkie: a conserved family of signal-responsive tumor size, clinical stage and reduced overall and transcriptional coregulators in animal development and disease-free survival (Wei et al., 2013). human disease. Biochem Cell Biol. 2009 Feb;87(1):77-91 Polycystic kidney disease (PKD) Cho HH, Shin KK, Kim YJ, Song JS, Kim JM, Bae YC, Kim CD, Jung JS. NF-kappaB activation stimulates osteogenic Note differentiation of mesenchymal stem cells derived from TAZ knockout mice develop PKD during human adipose tissue by increasing TAZ expression. J development. NEK1 kinase can phosphorylate Cell Physiol. 2010 Apr;223(1):168-77 TAZ, which can disable TAZ's role in promoting Ding L, Ellis MJ, Li S, Larson DE, Chen K, Wallis JW, the degradation of PC2, a protein involved in Harris CC, McLellan MD, Fulton RS, Fulton LL, Abbott RM, ciliogenesis. The proper balance of NEK1 and TAZ Hoog J, Dooling DJ, Koboldt DC, Schmidt H, Kalicki J, Zhang Q, Chen L, Lin L, Wendl MC, McMichael JF, will help keep a good level of PC2, which will Magrini VJ, Cook L, McGrath SD, Vickery TL, Appelbaum protect kidney from PKD (Tian et al., 2007; Yim et E, Deschryver K, Davies S, Guintoli T, Lin L, Crowder R, al., 2011). Tao Y, Snider JE, Smith SM, Dukes AF, Sanderson GE, Pohl CS, Delehaunty KD, Fronick CC, Pape KA, Reed JS, Holt-Oram syndrome Robinson JS, Hodges JS, Schierding W, Dees ND, Shen D, Locke DP, Wiechert ME, Eldred JM, Peck JB, Oberkfell Note BJ, Lolofie JT, Du F, Hawkins AE, O'Laughlin MD, Bernard TAZ can interact with and activate transcription KE, Cunningham M, Elliott G, Mason MD, Thompson DM Jr, Ivanovich JL, Goodfellow PJ, Perou CM, Weinstock factor TBX5, which is essential in cardiac and limb GM, Aft R, Watson M, Ley TJ, Wilson RK, Mardis ER. development. In Holt-Oram syndrome, TBX5 has a Genome remodelling in a basal-like breast cancer truncated mutation, which will lose its interaction metastasis and xenograft. Nature. 2010 Apr with TAZ and therefore, fail to activate genes 15;464(7291):999-1005 involved in cardiac and limb development Remue E, Meerschaert K, Oka T, Boucherie C, (Murakami et al., 2005). Vandekerckhove J, Sudol M, Gettemans J. TAZ interacts with zonula occludens-1 and -2 proteins in a PDZ-1 dependent manner. FEBS Lett. 2010 Oct 8;584(19):4175- References 80 Kanai F, Marignani PA, Sarbassova D, Yagi R, Hall RA, Chan SW, Lim CJ, Chong YF, Pobbati AV, Huang C, Hong Donowitz M, Hisaminato A, Fujiwara T, Ito Y, Cantley LC, W. Hippo pathway-independent restriction of TAZ and YAP Yaffe MB. TAZ: a novel transcriptional co-activator by angiomotin. J Biol Chem. 2011 Mar 4;286(9):7018-26 regulated by interactions with 14-3-3 and PDZ domain proteins. EMBO J. 2000 Dec 15;19(24):6778-91 Cordenonsi M, Zanconato F, Azzolin L, Forcato M, Rosato A, Frasson C, Inui M, Montagner M, Parenti AR, Poletti A, Hong JH, Hwang ES, McManus MT, Amsterdam A, Tian Y, Daidone MG, Dupont S, Basso G, Bicciato S, Piccolo S. Kalmukova R, Mueller E, Benjamin T, Spiegelman BM, The Hippo transducer TAZ confers cancer stem cell- Sharp PA, Hopkins N, Yaffe MB. TAZ, a transcriptional related traits on breast cancer cells. Cell. 2011 Nov modulator of mesenchymal stem cell differentiation. 11;147(4):759-72 Science. 2005 Aug 12;309(5737):1074-8 Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Murakami M, Nakagawa M, Olson EN, Nakagawa O. A Cordenonsi M, Zanconato F, Le Digabel J, Forcato M, WW domain protein TAZ is a critical coactivator for TBX5, Bicciato S, Elvassore N, Piccolo S. Role of YAP/TAZ in

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 851 WWTR1 (WW domain containing transcription regulator 1) Zhao Y, Yang X

mechanotransduction. Nature. 2011 Jun 8;474(7350):179- mediator of mammary cell migration downstream of the 83 Hippo pathway component TAZ. Cell Signal. 2013 Aug;25(8):1720-8 Lai D, Ho KC, Hao Y, Yang X. Taxol resistance in breast cancer cells is mediated by the hippo pathway component Wei Z, Wang Y, Li Z, Yuan C, Zhang W, Wang D, Ye J, TAZ and its downstream transcriptional targets Cyr61 and Jiang H, Wu Y, Cheng J. Overexpression of Hippo CTGF. Cancer Res. 2011 Apr 1;71(7):2728-38 pathway effector TAZ in tongue squamous cell carcinoma: correlation with clinicopathological features and patients' Liu C, Huang W, Lei Q. Regulation and function of the TAZ prognosis. J Oral Pathol Med. 2013 Nov;42(10):747-54 transcription co-activator. Int J Biochem Mol Biol. 2011;2(3):247-56 Yuen HF, McCrudden CM, Huang YH, Tham JM, Zhang X, Zeng Q, Zhang SD, Hong W. TAZ expression as a Yim H, Sung CK, You J, Tian Y, Benjamin T. Nek1 and prognostic indicator in colorectal cancer. PLoS One. TAZ interact to maintain normal levels of polycystin 2. J 2013;8(1):e54211 Am Soc Nephrol. 2011 May;22(5):832-7 Bartucci M, Dattilo R, Moriconi C, Pagliuca A, Mottolese M, Zhao B, Tumaneng K, Guan KL. The Hippo pathway in Federici G, Benedetto AD, Todaro M, Stassi G, Sperati F, organ size control, tissue regeneration and stem cell self- Amabile MI, Pilozzi E, Patrizii M, Biffoni M, Maugeri-Saccà renewal. Nat Cell Biol. 2011 Aug 1;13(8):877-83 M, Piccolo S, De Maria R. TAZ is required for metastatic Zhou Z, Hao Y, Liu N, Raptis L, Tsao MS, Yang X. TAZ is activity and chemoresistance of breast cancer stem cells. a novel oncogene in non-small cell lung cancer. Oncogene. 2014 Feb 17; Oncogene. 2011 May 5;30(18):2181-6 Lau AN, Curtis SJ, Fillmore CM, Rowbotham SP, Mohseni Hong W, Guan KL. The YAP and TAZ transcription co- M, Wagner DE, Beede AM, Montoro DT, Sinkevicius KW, activators: key downstream effectors of the mammalian Walton ZE, Barrios J, Weiss DJ, Camargo FD, Wong KK, Hippo pathway. Semin Cell Dev Biol. 2012 Sep;23(7):785- Kim CF. Tumor-propagating cells and Yap/Taz activity 93 contribute to lung tumor progression and metastasis. EMBO J. 2014 Mar 3;33(5):468-81 Xie M, Zhang L, He CS, Hou JH, Lin SX, Hu ZH, Xu F, Zhao HY. Prognostic significance of TAZ expression in Skibinski A, Breindel JL, Prat A, Galván P, Smith E, Rolfs resected non-small cell lung cancer. J Thorac Oncol. 2012 A, Gupta PB, Labaer J, Kuperwasser C. The Hippo May;7(5):799-807 transducer TAZ interacts with the SWI/SNF complex to regulate breast epithelial lineage commitment. Cell Rep. Zhao D, Zhi X, Zhou Z, Chen C. TAZ antagonizes the 2014 Mar 27;6(6):1059-72 WWP1-mediated KLF5 degradation and promotes breast cell proliferation and tumorigenesis. Carcinogenesis. 2012 This article should be referenced as such: Jan;33(1):59-67 Zhao Y, Yang X. WWTR1 (WW domain containing Lai D, Yang X. BMP4 is a novel transcriptional target and transcription regulator 1). Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11):849-852.

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Leukaemia Section Short Communication t(5;11)(q35;q12) NSD1/FEN1 Nathalie Douet-Guilbert, Etienne De Braekeleer, Corinne Tous, Nadia Guéganic, Audrey Basinko, Marie-Josée Le Bris, Frédéric Morel, Marc De Braekeleer Cytogenetics Laboratory, Faculty of Medicine, University of Brest, France (NDG, EDB, CT, NG, AB, MJLB, FM, MDB)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0511q35q12ID1679.html DOI: 10.4267/2042/54170 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Cytogenetics Review on t(5;11)(q35;q12) NSD1/FEN1, with data Note on clinics, and the genes implicated. The t(5;11)(q35;q12) involves two genes of which one, the NSD1 gene, has been already shown to Clinics and pathology form a fusion gene with NUP98 in the t(5;11)(q35;p15.1) (Jaju et al., 2001). Disease Acute monocytic leukemia (AML-M5b) Epidemiology Four cases of acute myeloid leukemia with t(5;11)(q35;q12-13) are reported in the literature: two acute myeloblastic leukemia with differentiation (AML-M2) (Wang et al., 2006; de Oliveira et al., 2007), one acute myelomonocytic leukemia (AML-M4) (Itoh et al., 1999) and one acute monoblastic leukemia (AML-M5) (Leverger et al., 1988). No molecular characterization was performed in these cases but the NSD1 gene was shown not to be involved by fluorescent in situ hybridization in the AML-M2 case reported by Wang et al. (2006). Clinics A 37-year-old man seen because of throat infection resistant to antibiotics, persistent fever and dyspnea. Treatment Induction therapy and several salvage therapies failed to achieve complete remission followed by RHG banding showing chromosomes 5 and 11 and the bone marrow transplantation. derivatives der(5) and der(11). Evolution Cytogenetics morphological Patient alive in complete remission 35 months t(5;11)(q35;q12) is identified by banding following bone marrow transplantation. cytogenetics.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 853 t(5;11)(q35;q12) NSD1/FEN1 Douet-Guilbert N, et al.

FISH with BACs RP11-99N22 (spectrum orange, located in 5q35 and containing NSD1) and RP11-467L20 (spectrum green, located in 11q12 and containing FEN1) showing co-hybridization.

Cytogenetics molecular Protein To determine the position of the breakpoints on The protein has 2696 amino acids and localizes to chromosomes 5 and 11, BACs located in the bands the nucleus. It contains a SET domain, 2 LXXLL of interest were used as probes in FISH motifs, 3 nuclear translocation signals, 4 plant experiments. homeodomain (PHD) finger regions, and a proline- Analysis with RP11-99N22 showed that one signal rich region. The protein acts as a basic hybridized to the normal chromosome 5, and the transcriptional factor and also as a bifunctional other split and hybridized to both der(5) and transcriptional regulator, capable of both negatively der(11). or positively influencing transcription, depending FISH with overlapping BACs identified a very on the cellular context (Huang et al., 1998; small region of breakage in RP11-467L20. Analysis Kurotaki et al., 2001). with RP11-467L20 showed that one signal FEN1 hybridized to the normal chromosome 11, and the Location other split and hybridized to both der(11) and 11q12.2 der(5). Co-hybridization with both BAC clones showed DNA/RNA two fusion signals. RP11-99N22 contains the NSD1 The FEN1 gene contains 2 exons, of which a sole is gene and RP11-467L20 the FEN1 gene. coding, spanning 4 kb (Hiraoka et al., 1995). Protein Genes involved and The protein has 380 amino acids and localizes to the nucleus. It is a structure-specific nuclease with proteins 5'-flap endonuclease and 5'-3' exonuclease activities involved in DNA replication and repair. It acts as a NSD1 genome stabilization factor that prevents flaps from Location equilibrating into structures that lead to duplications 5q35.3 and deletions and participates in telomere DNA/RNA maintenance (Saharia et al., 2008; Zheng et al., The NSD1 gene contains 24 exons, of which 23 are 2011; Tsutakawa et al., 2011). It has been coding, spanning 167 kb. Two alternative suggested that FEN1 is a tumor suppressor gene transcripts are known (Kurotaki et al., 2001). (Henneke et al., 2003).

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 854 t(5;11)(q35;q12) NSD1/FEN1 Douet-Guilbert N, et al.

References Trends Biochem Sci. 2003 Jul;28(7):384-90 Wang TF, Horsley SW, Lee KF, Chu SC, Li CC, Kao RH. Leverger G, Bernheim A, Daniel MT, Flandrin G, Schaison Translocation between chromosome 5q35 and G, Berger R. Cytogenetic study of 130 childhood acute chromosome 11q13-- an unusual cytogenetic finding in a nonlymphocytic leukemias. Med Pediatr Oncol. primary refractory acute myeloid leukemia. Clin Lab 1988;16(4):227-32 Haematol. 2006 Jun;28(3):160-3 Hiraoka LR, Harrington JJ, Gerhard DS, Lieber MR, Hsieh de Oliveira FM, Tone LG, Simões BP, Falcão RP, CL. Sequence of human FEN-1, a structure-specific Brassesco MS, Sakamoto-Hojo ET, dos Santos GA, endonuclease, and chromosomal localization of the gene Marinato AF, Jácomo RH, Rego EM. Acute myeloid (FEN1) in mouse and human. Genomics. 1995 Jan leukemia (AML-M2) with t(5;11)(q35;q13) and normal 1;25(1):220-5 expression of cyclin D1. Cancer Genet Cytogenet. 2007 Jan 15;172(2):154-7 Huang N, vom Baur E, Garnier JM, Lerouge T, Vonesch JL, Lutz Y, Chambon P, Losson R. Two distinct nuclear Saharia A, Guittat L, Crocker S, Lim A, Steffen M, Kulkarni receptor interaction domains in NSD1, a novel SET protein S, Stewart SA. Flap endonuclease 1 contributes to that exhibits characteristics of both corepressors and telomere stability. Curr Biol. 2008 Apr 8;18(7):496-500 coactivators. EMBO J. 1998 Jun 15;17(12):3398-412 Tsutakawa SE, Classen S, Chapados BR, Arvai AS, Itoh M, Okazaki T, Tashima M, Sawada H, Uchiyama T. Finger LD, Guenther G, Tomlinson CG, Thompson P, Acute myeloid leukemia with t(5;11): two case reports. Sarker AH, Shen B, Cooper PK, Grasby JA, Tainer JA. Leuk Res. 1999 Jul;23(7):677-80 Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 Jaju RJ, Fidler C, Haas OA, Strickson AJ, Watkins F, Clark superfamily. Cell. 2011 Apr 15;145(2):198-211 K, Cross NC, Cheng JF, Aplan PD, Kearney L, Boultwood J, Wainscoat JS. A novel gene, NSD1, is fused to NUP98 Zheng L, Jia J, Finger LD, Guo Z, Zer C, Shen B. in the t(5;11)(q35;p15.5) in de novo childhood acute Functional regulation of FEN1 nuclease and its link to myeloid leukemia. Blood. 2001 Aug 15;98(4):1264-7 cancer. Nucleic Acids Res. 2011 Feb;39(3):781-94

Kurotaki N, Harada N, Yoshiura K, Sugano S, Niikawa N, This article should be referenced as such: Matsumoto N. Molecular characterization of NSD1, a human homologue of the mouse Nsd1 gene. Gene. 2001 Douet-Guilbert N, De Braekeleer E, Tous C, Guéganic N, Nov 28;279(2):197-204 Basinko A, Le Bris MJ, Morel F, De Braekeleer M. t(5;11)(q35;q12) NSD1/FEN1. Atlas Genet Cytogenet Henneke G, Friedrich-Heineken E, Hübscher U. Flap Oncol Haematol. 2014; 18(11):853-855. endonuclease 1: a novel tumour suppresser protein.

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Leukaemia Section Short Communication t(7;9)(q11;p12) PAX5/POM121 Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0709q11p12ID1558.html DOI: 10.4267/2042/54171 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

binding region: aa 137-265; a Pro-rich region: aa Abstract 144-214; a Ser-rich region: aa 378-445; a Thr-rich Review on t(7;9)(q11;p12) PAX5/POM121, with region: aa 751-947; there are also phosphoserines data on clinics, and the genes implicated. aa: 345; 351; 371; 393; 5 nuclear localization signals (NLS): aa 321-323 (KKR); 339-342 Clinics and pathology (KRRR); 387-390 (KRSR); 453-456 (KKIR); 504- Disease 507 (RKRK); and xFxFG motifs (repeats based on cores containing Phenylalanine and Glycine (X B-cell acute lymphoblastic leukemia (B-ALL) denotes any amino acid) separated by linkers rich in Phenotype/cell stem origin serines and threonines): aa 703-707; 835-839; 861- Pre-B ALL (C µ). 865; 881-885; 916-922; 994-998; 1096-1100; 1126- 1130. These FG repeats are thought to bind Epidemiology transport receptors such as importin beta and Only two cases to date, 2 boys aged 1 and 2 years transportin. POM121 anchors the nuclear pore (Nebral et al., 2009; Coyaud et al., 2010). complex (NPC) to the nuclear envelope; the N- terminal domain required for nuclear targeting, the Prognosis N-terminal and transmembrane domain are required One case was noted as high risk ALL. for NPC targeting; POM121 makes complexes with The patient remains in complete remission 20 NUP210 (nucleoporin 210 kDa, also called GP210, months after diagnosis. 3p25.1). Nuclear pore complexes (NPCs) assemble 1) at the end of mitosis during nuclear envelope Genes involved and reformation (where the nuclear envelope has not fully formed, and NPCs are inserted into chromatin- proteins bound ER sheets), and 2) into an intact nuclear POM121 envelope during interphase when the nuclear envelope is completely closed and the process has Location to be coordinated across the highly organized 7q11.23 structure with separated inner nuclear membrane Protein and outer nuclear membrane. 1249 amino acids (aa); from N-term to C-term, Interphase NPC formation is initiated by POM121 is made of a cisternal side domain (aa 1- recruitment of POM121 followed by the 34), a transmembrane (Helical) domain (aa 35-55), incorporation of the Nup107-160 complex and and a pore side domain (aa 56-1249), and contains: POM121 is required for interphase NPC formation a region for inner nuclear membrane - outer nuclear (the order is reverse in post-mitotic NPC formation, membrane fusion: aa 1-129, a nuclear envelope and POM121 is dispensable).

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 856 t(7;9)(q11;p12) PAX5/POM121 Huret JL

PAX5/POM121 protein.

POM121 and SUN1 (Sad1 and UNC84 domain The chromatin-binding protein AHCTF1 (AT hook containing 1, 7p22.3) promote early steps of containing transcription factor 1, also called ELYS, interphase NPC assembly, prior to the incorporation 1q44) targets nuclear pore assembly to the surface of the Nup107-160 complex (made of NUP160, of chromosomes as nuclei form at the end of NUP133, NUP107, NUP98, NUP85, NUP43, mitosis (Koser et al., 2005; Funakoshi et al., 2011; NUP37, SEC13, and SEH1L), which interacts with Talamas and Hetzer, 2011). POM121. PAX5 POM121 is required for the inner nuclear membrane (INM) - outer nuclear membrane fusion Location (ONM) and NPC formation. 9p13.2 POM121 region aa 1-129 is sufficient to induce Protein bending of the ONM and INM toward each other. 391 amino acids; from N-term to C-term, PAX5 POM121 localized to the inner nuclear membrane. contains: a paired domain (aa: 16-142); an The 5 nuclear localization signals of POM121 have octapeptide (aa: 179-186); a partial homeodomain an active role in its targeting to nuclear pore (aa: 228-254); a transactivation domain (aa: 304- complexes during interphase. 359); and an inhibitory domain (aa: 359-391). Interaction of POM121 with importin beta is Lineage-specific transcription factor; recognizes the dependent on direct binding of importin alpha to the concensus recognition sequence nuclear localization signals of POM121. GNCCANTGAAGCGTGAC, where N is any POM121 is transported into the nucleus through nucleotide. Involved in B-cell differentiation. Entry nuclear pores by a mechanism involving RAN of common lymphoid progenitors into the B cell (12q24.33) and importins and subsequently binds to lineage depends on E2A, EBF1, and PAX5; inner nuclear membrane proteins prior to its activates B-cell specific genes and repress genes incorporation into the nuclear pore complexes. involved in other lineage commitments. Activates POM121 contains a nuclear envelope-binding the surface cell receptor CD19 and repress FLT3. region involved in nuclear pore complex targeting. Pax5 physically interacts with the RAG1/RAG2 The nuclear envelope-binding domain is involved complex, and removes the inhibitory signal of the in postmitotic nuclear pore complex assembly, The lysine-9-methylated histone H3, and induces V-to- nuclear envelope-binding region of POM121 DJ rearrangements. Genes repressed by PAX5 interacts with LBR (lamin B receptor, 1q42.12), a expression in early B cells are restored in their component of the inner nuclear membrane. function in mature B cells and plasma cells, and

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 857 t(7;9)(q11;p12) PAX5/POM121 Huret JL

PAX5 repressed (Fuxa et al., 2004; Johnson et al., GS, Burrows PD, Hagman J, Feeney AJ, Cooper MD.. 2004; Zhang et al., 2006; Cobaleda et al., 2007; Transcription factor Pax5 (BSAP) transactivates the RAG- mediated V(H)-to-DJ(H) rearrangement of immunoglobulin Medvedovic et al., 2011). genes. Nat Immunol. 2006 Jun;7(6):616-24. Epub 2006 May 7. Result of the chromosomal Cobaleda C, Schebesta A, Delogu A, Busslinger M.. Pax5: the guardian of B cell identity and function. Nat Immunol. anomaly 2007 May;8(5):463-70. Hybrid gene Nebral K, Denk D, Attarbaschi A, Konig M, Mann G, Haas OA, Strehl S.. Incidence and diversity of PAX5 fusion Description genes in childhood acute lymphoblastic leukemia. Fusion of PAX5 exon 5 to POM121 non coding Leukemia. 2009 Jan;23(1):134-43. doi: exon 4. 10.1038/leu.2008.306. Epub 2008 Nov 20. Coyaud E, Struski S, Prade N, Familiades J, Eichner R, Fusion protein Quelen C, Bousquet M, Mugneret F, Talmant P, Pages Description MP, Lefebvre C, Penther D, Lippert E, Nadal N, Taviaux S, 1288 amino acids. The predicted fusion protein Poppe B, Luquet I, Baranger L, Eclache V, Radford I, Barin C, Mozziconacci MJ, Lafage-Pochitaloff M, Antoine-Poirel contains the DNA binding paired domain of PAX5 H, Charrin C, Perot C, Terre C, Brousset P, Dastugue N, (the 201 N-term aa) and the entire 999 amino acids Broccardo C.. Wide diversity of PAX5 alterations in B-ALL: splice variant of POM121, containing most of the a Groupe Francophone de Cytogenetique Hematologique pore region of POM121. study. Blood. 2010 Apr 15;115(15):3089-97. doi: 10.1182/blood-2009-07-234229. Epub 2010 Feb 16. References Funakoshi T, Clever M, Watanabe A, Imamoto N.. Localization of Pom121 to the inner nuclear membrane is Fuxa M, Skok J, Souabni A, Salvagiotto G, Roldan E, required for an early step of interphase nuclear pore Busslinger M. Pax5 induces V-to-DJ rearrangements and complex assembly. Mol Biol Cell. 2011 Apr;22(7):1058-69. locus contraction of the immunoglobulin heavy-chain gene. doi: 10.1091/mbc.E10-07-0641. Epub 2011 Feb 2. Genes Dev. 2004 Feb 15;18(4):411-22 Medvedovic J, Ebert A, Tagoh H, Busslinger M.. Pax5: a Johnson K, Pflugh DL, Yu D, Hesslein DG, Lin KI, Bothwell master regulator of B cell development and AL, Thomas-Tikhonenko A, Schatz DG, Calame K. B cell- leukemogenesis. Adv Immunol. 2011;111:179-206. doi: specific loss of histone 3 lysine 9 methylation in the V(H) 10.1016/B978-0-12-385991-4.00005-2. (REVIEW) locus depends on Pax5. Nat Immunol. 2004 Aug;5(8):853- Talamas JA, Hetzer MW.. POM121 and Sun1 play a role in 61 early steps of interphase NPC assembly. J Cell Biol. 2011 Koser J, Maco B, Aebi U, Fahrenkrog B.. The nuclear pore Jul 11;194(1):27-37. doi: 10.1083/jcb.201012154. Epub complex becomes alive: new insights into its dynamics and 2011 Jul 4. involvement in different cellular processes. Atlas Genet Cytogenet Oncol Haematol. 2005; 9(2):191-209. This article should be referenced as such: http://atlasgeneticsoncology.org/Deep/NuclearPoreComplI Huret JL. t(7;9)(q11;p12) PAX5/POM121. Atlas Genet D20048.html Cytogenet Oncol Haematol. 2014; 18(11):856-858. Zhang Z, Espinoza CR, Yu Z, Stephan R, He T, Williams

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Leukaemia Section Short Communication t(9;12)(q34;p13) ETV6/ABL1 Etienne De Braekeleer, Nathalie Douet-Guilbert, Marc De Braekeleer Cytogenetics Laboratory, Faculty of Medicine, University of Brest, France (EDB, NDG, MDB)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t912ID1080.html DOI: 10.4267/2042/54172 This article is an update of : Heerema NA. t(9;12)(q34;p13). Atlas Genet Cytogenet Oncol Haematol 2001;5(1):42-43.

fusion gene affecting ETV6 and the partner gene, Abstract activation of a proto-oncogene in the vicinity of a Review on t(9;12)(q34;p13) ETV6/ABL1, with data chromosomal translocation and dominant negative on clinics, and the genes implicated. effect of the fusion protein over transcriptional repression mediated by wild-type ETV6). Clinics and pathology Cytogenetics Disease Malignant hemopathies (26 cases reported) Note t(9;12)(q34;p13) as the sole abnormality or Phenotype/cell stem origin associated with other abnormalities. AML (3 cases), B-cell ALL (8 cases), T-cell ALL Cytogenetics morphological (1 case), RAEB evolving into AML (1 case), chronic myeloproliferative neoplasm (2 cases), t(9;12)(q34;p13) is very difficult to be identified by Philadelphia chromosome-negative CML (11 conventional cytogenetics. cases). Cytogenetics molecular Epidemiology t(9;12)(q34;p13) usually requires FISH analysis Gender: 17 males, 8 females; age at diagnosis: 8 with ETV6 and ABL1 probes to be detected months to 81 years. (cryptic translocation). Insertions are also frequently identified. Clinics Eosinophilia appears to be a common feature of Additional anomalies malignancies associated with the ETV6-ABL1 Additional anomalies are frequent but show no fusion gene (15/20 cases). consistent features (trisomies and monosomies of various chromosomes, structural rearrangements Genetics including deletions and translocations). Note Variants The t(9;12)(q34;p13) involves the ETV6 gene t(9;12;14)(q34;p13;q22) (seen in conventional (12p13), a transcription factor frequently rearranged cytogenetics), in myeloid and lymphoid leukemias. More than 30 t(8;9;12)(p12;q34;p13) (seen in conventional ETV6 fusion gene partners have been described. cytogenetics), Most translocations involving ETV6 generate ins(9;12)(q34;p13p13) (seen by molecular fusion genes that lead to the activation of cytogenetics), transcription factors or kinases but other ins(12;9)(p13;q34q34) (seen by molecular mechanisms are also known (loss of function of the cytogenetics).

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 859 t(9;12)(q34;p13) ETV6/ABL1 De Braekeleer E, et al.

Schematic diagram of the ETV6, ABL1 and ETV6-ABL1 proteins.

9q34, includes the 5' alternative first exons 1b and Genes involved and 1a and ten common exons numbered from 2 to 11. proteins Alternative splicing using exons 1b and 1a gives rise to mRNA of 7 and 6 kb, respectively. Note Protein As both genes have opposite orientation in relation The ABL1 protein has three SRC homology (SH) to the centromeres, an in frame ETV6-ABL1 fusion domains called SH1, SH2 and SH3, of which SH1 gene requires at least three chromosomal breaks to that has a tyrosine kinase function. be generated. The SH2 and SH3 domains are involved in protein- ETV6 protein interactions, which regulate the tyrosine Location kinase activity; they are necessary for signal 12p13 transduction function. The ABL1 protein has also three nuclear Note localization signal domains and three DNA binding The ETV6 gene encodes a transcription factor regions and an F-actin binding domain. frequently rearranged in myeloid and lymphoid leukemias. Result of the chromosomal DNA/RNA The ETV6 gene spans a region of less than 250 kb anomaly at band 12p13.1 and consists of 8 exons. There are two start codons, one (exon 1a starting at Hybrid gene codon 1) located at the beginning of the gene and Transcript another alternative (exon 1b starting at codon 43) Two ETV6-ABL1 transcripts were identified in upstream of exon 3. most of the patients, one joining exon 5 of ETV6 to Protein exon 2 of ABL1, the other, usually found at very The ETV6 protein (452 amino acids) contains two low levels, joining ETV6 exon 4 to ABL1 exon 2. major domains, the HLH (helix-loop-helix) and Fusion protein ETS domains. The HLH domain, also referred to as the pointed or Description sterile alpha motif domain, is encoded by exons 3 The fusion protein retains all three SH domains, and 4 and functions as a homo-oligodimerization including the tyrosine kinase domain, of ABL1, domain. The ETS domain, encoded by exons 6 which make these patients sensitive to tyrosine through 8, is responsible for sequence specific kinase inhibitors. DNA-binding and protein-protein interaction. The retained N-terminal part of the ETV6 protein contains the helix-loop-helix domain necessary for ABL1 oligomerization of the protein, which is required for Location tyrosine kinase activation, cytoskeletal localization 9q34 and neoplastic transformation. DNA/RNA Oncogenesis The ABL1 gene, spanning a 230-kb region at band Constitutive tyrosine kinase activation of ABL1.

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References Cytogenet. 2005 Feb;157(1):74-7. Mozziconacci MJ, Sainty D, Chabannon C.. A fifteen-year Papadopoulos P, Ridge SA, Boucher CA, Stocking C, cytogenetic remission following interferon treatment in a Wiedemann LM. The novel activation of ABL by fusion to patient with an indolent ETV6-ABL positive an ets-related gene, TEL. Cancer Res. 1995 Jan myeloproliferative syndrome. Am J Hematol. 2007 1;55(1):34-8 Jul;82(7):688-9. Baens M, Peeters P, Guo C, Aerssens J, Marynen P. Baeumler J, Szuhai K, Falkenburg JH, van Schie ML, Genomic organization of TEL: the human ETS-variant Ottmann OG, Nijmeijer BA.. Establishment and cytogenetic gene 6. Genome Res. 1996 May;6(5):404-13 characterization of a human acute lymphoblastic leukemia cell line (ALL-VG) with ETV6/ABL1 rearrangement. Cancer Brunel V, Sainty D, Carbuccia N, Mozzicconacci M, Genet Cytogenet. 2008 Aug;185(1):37-42. doi: Fernandez F, Simonetti J, Gabert J, Dubreuil P, Lafage- 10.1016/j.cancergencyto.2008.05.001. Pochitaloff M, Birg F.. A TEL/ABL fusion gene on chromosome 12p13 in a case of Ph-, BCR-atypical CML. Kawamata N, Dashti A, Lu D, Miller B, Koeffler HP, Leukemia 1996; 10: 2003. Schreck R, Moore S, Ogawa S.. Chronic phase of ETV6- ABL1 positive CML responds to imatinib. Genes Golub TR, Goga A, Barker GF, Afar DE, McLaughlin J, Chromosomes Cancer. 2008 Oct;47(10):919-21. doi: Bohlander SK, Rowley JD, Witte ON, Gilliland DG.. 10.1002/gcc.20593. Oligomerization of the ABL tyrosine kinase by the Ets protein TEL in human leukemia. Mol Cell Biol. 1996 Kelly JC, Shahbazi N, Scheerle J, Jahn J, Suchen S, Aug;16(8):4107-16. Christacos NC, Mowrey PN, Witt MH, Hostetter A, Meloni- Ehrig AM.. Insertion (12;9)(p13;q34q34): a cryptic Andreasson P, Johansson B, Carlsson M, Jarlsfelt I, rearrangement involving ABL1/ETV6 fusion in a patient Fioretos T, Mitelman F, Hoglund M.. BCR/ABL-negative with Philadelphia-negative chronic myeloid leukemia. chronic myeloid leukemia with ETV6/ABL fusion. 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Br J Haematol. 2010 Keung YK, Beaty M, Steward W, Jackle B, Pettnati M.. Oct;151(1):101-2. doi: 10.1111/j.1365-2141.2010.08323.x. Chronic myelocytic leukemia with eosinophilia, Epub 2010 Jul 7. t(9;12)(q34;p13), and ETV6-ABL gene rearrangement: Zuna J, Zaliova M, Muzikova K, Meyer C, Lizcova L, case report and review of the literature. Cancer Genet Zemanova Z, Brezinova J, Votava F, Marschalek R, Stary Cytogenet. 2002 Oct 15;138(2):139-42. (REVIEW) J, Trka J.. Acute leukemias with ETV6/ABL1 (TEL/ABL) La Starza R, Trubia M, Testoni N, Ottaviani E, Belloni E, fusion: poor prognosis and prenatal origin. Genes Crescenzi B, Martelli M, Flandrin G, Pelicci PG, Mecucci Chromosomes Cancer. 2010 Oct;49(10):873-84. doi: C.. Clonal eosinophils are a morphologic hallmark of 10.1002/gcc.20796. ETV6/ABL1 positive acute myeloid leukemia. De Braekeleer E1, Douet-Guilbert N, Rowe D, Bown N, Haematologica. 2002 Aug;87(8):789-94. Morel F, Berthou C, Ferec C, De Braekeleer M.. ABL1 Lin H, Guo JQ, Andreeff M, Arlinghaus RB.. Detection of fusion genes in hematological malignancies: a review. Eur dual TEL-ABL transcripts and a Tel-Abl protein containing J Haematol. 2011 May;86(5):361-71. doi: 10.1111/j.1600- phosphotyrosine in a chronic myeloid leukemia patient. 0609.2011.01586.x. Epub 2011 Mar 23. (REVIEW) Leukemia. 2002 Feb;16(2):294-7. Perna F, Abdel-Wahab O, Levine RL, Jhanwar SC, Imada O'Brien SG, Vieira SA, Connors S, Bown N, Chang J, K, Nimer SD.. ETV6-ABL1-positive "chronic myeloid Capdeville R, Melo JV.. Transient response to imatinib leukemia": clinical and molecular response to tyrosine mesylate (STI571) in a patient with the ETV6-ABL t(9;12) kinase inhibition. Haematologica. 2011 Feb;96(2):342-3. translocation. Blood. 2002 May 1;99(9):3465-7. doi: 10.3324/haematol.2010.036673. Epub 2010 Dec 29. Barbouti A, Ahlgren T, Johansson B, Hoglund M, Lassen De Braekeleer E, Douet-Guilbert N, Morel F, Le Bris MJ, C, Turesson I, Mitelman F, Fioretos T.. Clinical and genetic Basinko A, De Braekeleer M.. ETV6 fusion genes in studies of ETV6/ABL1-positive chronic myeloid leukaemia hematological malignancies: a review. Leuk Res. 2012 in blast crisis treated with imatinib mesylate. Br J Aug;36(8):945-61. doi: 10.1016/j.leukres.2012.04.010. Haematol. 2003 Jul;122(1):85-93. Epub 2012 May 12. (REVIEW) Meyer-Monard S, Muhlematter D, Streit A, Chase AJ, Zhou MH, Gao L, Jing Y, Xu YY, Ding Y, Wang N, Wang Gratwohl A, Cross NC, Jotterand M, Tichelli A.. Broad W, Li MY, Han XP, Sun JZ, Wang LL, Yu L.. Detection of molecular screening of an unclassifiable myeloproliferative ETV6 gene rearrangements in adult acute lymphoblastic disorder reveals an unexpected ETV6/ABL1 fusion leukemia. Ann Hematol. 2012 Aug;91(8):1235-43. doi: transcript. Leukemia. 2005 Jun;19(6):1096-9. 10.1007/s00277-012-1431-4. Epub 2012 Feb 29. Tirado CA, Sebastian S, Moore JO, Gong JZ, Goodman This article should be referenced as such: BK.. Molecular and cytogenetic characterization of a novel rearrangement involving chromosomes 9, 12, and 17 De Braekeleer E, Douet-Guilbert N, De Braekeleer M. resulting in ETV6 (TEL) and ABL fusion. Cancer Genet t(9;12)(q34;p13) ETV6/ABL1. Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11):859-861.

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Leukaemia Section Short Communication t(9;22)(p13;q13) PAX5/BRD1 Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: March 2014 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0922p13q13ID1562.html DOI: 10.4267/2042/54173 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

of common lymphoid progenitors into the B cell Abstract lineage depends on E2A, EBF1, and PAX5; Review on t(9;22)(p13;q13) PAX5/BRD1, with activates B-cell specific genes and repress genes data on clinics, and the genes implicated. involved in other lineage commitments. Activates the surface cell receptor CD19 and repress FLT3. Clinics and pathology Pax5 physically interacts with the RAG1/RAG2 complex, and removes the inhibitory signal of the Disease lysine-9-methylated histone H3, and induces V-to- B-cell acute lymphoblastic leukemia (ALL) DJ rearrangements. Genes repressed by PAX5 expression in early B cells are restored in their Epidemiology function in mature B cells and plasma cells, and Only one case to date, a 2-years old boy wit h a PAX5 repressed (Fuxa et al., 2004; Johnson et al., CD10+ ALL (Nebral et al., 2009). 2004; Zhang et al., 2006; Cobaleda et al., 2007; Prognosis Medvedovic et al., 2011). The patient was considered as at intermediate risk. BRD1 He remains in complete remission 44 months after Protein diagnosis. 1058 amino acids (aa); from N-term to C-term, BRD1 contains a zinc finger PHD-type (aa 214- Genes involved and 264); a bromo domain (aa 579-649) (recognizes acetylated lysine residues; prerequisite for protein- proteins histone association and chromatin remodeling); a PAX5 PWWP domain (aa 929-1012) (conserved Pro-Trp- Trp-Pro motif; binds histones independently of their Location acetylation, also binds DNA). Phosphoserines are at 9p13.2 aa 128, 1052, and 1055, and N6-acetyllysine at aa Protein 368, 516, 519, and 903. BN1 and BN2 (BRPF N- 391 amino acids; from N-term to C-term, PAX5 terminal conserved region 1 and 2) (aa 60-222) is contains: a paired domain (aa: 16-142); an the domain binding MOZ/MORF (Ullah et al., octapeptide (aa: 179-186); a partial homeodomain 2008). An enhancer of polycomb homology region (aa: 228-254); a transactivation domain (aa: 304- (EPL1) (EPL1 protein are members of histone 359); and an inhibitory domain (aa: 359-391). acetyltransferase complex) , which binds to ING5 Lineage-specific transcription factor; recognizes the and mediates association with MEAF6, is located concensus recognition sequence from amino acids 540 and 640 (Ullah et al., 2008). GNCCANTGAAGCGTGAC, where N is any BRD1 is a subunit of the MOZ histone acetyl nucleotide. Involved in B-cell differentiation. Entry transferase (HAT) complex.

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PAX5/BRD1 fusion protein.

The MOZ HAT functions is a multi-protein complex with BRD1, ING5 (inhibitor of growth 5, Result of the chromosomal 2q37.3), and MEAF6 (MYST/Esa1-Associated anomaly Factor 6, 1p34.3). BRD1 links the MOZ catalytic subunit to the ING5 Hybrid gene and MEAF6 subunits, and promotes MOZ HAT Description activity. Fusion of PAX5 exon 5 to BRD1 non coding exon The bromodomain of BRD1 acetylates lysine 1. residues of histones H3, H4, H2A, and H2B (H3K14ac, H4K5ac, H4K8ac, H4K12ac, and Fusion protein H2AK5ac). The PWWP domain of BRD1 is the Description binding module for trimethylation of Lys36 of 1264 amino acids, according to the authors. The histone H3 (H3K36me3) associated with the predicted fusion protein contains the DNA binding elongation phase of transcription (Vezzoli et al., paired domain and octapeptide of PAX5 (201 aa), 4 2010). amino acids, and the entire BRD1 (1058 aa). There are different splice variants, which are induced differentially in different brain regions References (Fryland et al., 2012). Fuxa M, Skok J, Souabni A, Salvagiotto G, Roldan E, The variant BRPF2 may be a negative regulatory Busslinger M. Pax5 induces V-to-DJ rearrangements and factor of the variant BRPF1. BRD1 is widely locus contraction of the immunoglobulin heavy-chain gene. expressed in various tissues, the brain in particular Genes Dev. 2004 Feb 15;18(4):411-22 (in the cell nucleus, perikaryal cytosol and proximal Johnson K, Pflugh DL, Yu D, Hesslein DG, Lin KI, Bothwell dendrites of the neurons), with high expression at AL, Thomas-Tikhonenko A, Schatz DG, Calame K. B cell- early embryonic stages (Severinsen et al., 2006; specific loss of histone 3 lysine 9 methylation in the V(H) Bjarkam et al., 2009). locus depends on Pax5. Nat Immunol. 2004 Aug;5(8):853- 61 BRD1 has a central role during embryonic development through Moz-dependent histone Severinsen JE, Bjarkam CR, Kiaer-Larsen S, Olsen IM, acetylation, to maintain expression of Hox genes Nielsen MM, Blechingberg J, Nielsen AL, Holm IE, Foldager L, Young BD, Muir WJ, Blackwood DH, Corydon (Laue et al., 2008). BRD1 forms a novel HAT TJ, Mors O, Børglum AD. Evidence implicating BRD1 with complex with KAT7 (HBO1, 17q21.33) and is brain development and susceptibility to both schizophrenia required for transcriptional activation of erythroid and bipolar affective disorder. Mol Psychiatry. 2006 developmental regulator genes (Mishima et al., Dec;11(12):1126-38 2011). BRD1 showed association with Zhang Z, Espinoza CR, Yu Z, Stephan R, He T, Williams schizophrenia and bipolar affective disorder GS, Burrows PD, Hagman J, Feeney AJ, Cooper MD. susceptibility (Bjarkam et al., 2009). Transcription factor Pax5 (BSAP) transactivates the RAG-

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mediated V(H)-to-DJ(H) rearrangement of immunoglobulin Leukemia. 2009 Jan;23(1):134-43 genes. Nat Immunol. 2006 Jun;7(6):616-24 Vezzoli A, Bonadies N, Allen MD, Freund SM, Santiveri Cobaleda C, Schebesta A, Delogu A, Busslinger M. Pax5: CM, Kvinlaug BT, Huntly BJ, Göttgens B, Bycroft M. the guardian of B cell identity and function. Nat Immunol. Molecular basis of histone H3K36me3 recognition by the 2007 May;8(5):463-70 PWWP domain of Brpf1. Nat Struct Mol Biol. 2010 May;17(5):617-9 Laue K, Daujat S, Crump JG, Plaster N, Roehl HH, Kimmel CB, Schneider R, Hammerschmidt M. The multidomain Medvedovic J, Ebert A, Tagoh H, Busslinger M. Pax5: a protein Brpf1 binds histones and is required for Hox gene master regulator of B cell development and expression and segmental identity. Development. 2008 leukemogenesis. Adv Immunol. 2011;111:179-206 Jun;135(11):1935-46 Mishima Y, Miyagi S, Saraya A, Negishi M, Endoh M, Ullah M, Pelletier N, Xiao L, Zhao SP, Wang K, Degerny C, Endo TA, Toyoda T, Shinga J, Katsumoto T, Chiba T, Tahmasebi S, Cayrou C, Doyon Y, Goh SL, Champagne Yamaguchi N, Kitabayashi I, Koseki H, Iwama A. The N, Côté J, Yang XJ. Molecular architecture of quartet Hbo1-Brd1/Brpf2 complex is responsible for global MOZ/MORF histone acetyltransferase complexes. Mol Cell acetylation of H3K14 and required for fetal liver Biol. 2008 Nov;28(22):6828-43 erythropoiesis. Blood. 2011 Sep 1;118(9):2443-53 Bjarkam CR, Corydon TJ, Olsen IM, Pallesen J, Nyegaard Fryland T, Elfving B, Christensen JH, Mors O, Wegener G, M, Fryland T, Mors O, Børglum AD. Further Børglum AD. Electroconvulsive seizures regulates the immunohistochemical characterization of BRD1 a new Brd1 gene in the frontal cortex and hippocampus of the susceptibility gene for schizophrenia and bipolar affective adult rat. Neurosci Lett. 2012 May 10;516(1):110-3 disorder. Brain Struct Funct. 2009 Dec;214(1):37-47 This article should be referenced as such: Nebral K, Denk D, Attarbaschi A, König M, Mann G, Haas OA, Strehl S. Incidence and diversity of PAX5 fusion Huret JL. t(9;22)(p13;q13) PAX5/BRD1. Atlas Genet genes in childhood acute lymphoblastic leukemia. Cytogenet Oncol Haematol. 2014; 18(11):862-864.

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Deep Insight Section

Class III beta-tubulin, drug resistance and therapeutic approaches in cancers

Roshan Karki, Cristiano Ferlini Danbury Hospital Research Institute, 06810, Danbury, CT, USA (RK, CF)

Published in Atlas Database: April 2014 Online updated version : http://AtlasGeneticsOncology.org/Deep/ClassIIIbetatubulinID20135.html DOI: 10.4267/2042/54174 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Class III beta-tubulin is one of the critical proteins associated with microtubule assembly, important to many cellular functions including mitochondrial respiration and intracellular trafficking. Widely regarded as a specific neuronal marker in developmental neurobiology and stem cell research, it is also highly expressed in a wide range of tumors of both neuronal and non-neuronal origin. The expression of class III beta-tubulin is tightly controlled at multiple levels with tissue-dependent mechanisms of regulation. For instance, class III beta-tubulin expression is under the control of estrogens in breast cancer cells but is influenced by exposure to hypoxia and poor-nutrient supply in ovarian cancer. In some but not all cancers, class III beta-tubulin expression is purely a prognostic biomarker, predicting poor outcome of patients regardless of chemotherapy treatment. Moreover, the expression of class III beta-tubulin does not confer an aggressive phenotype by itself. Instead, class III beta- tubulin functions like a cytoskeletal gateway, which enhances the incorporation of pro-survival kinases into the cytoskeleton and protects them from degradation. The associations of class III beta-tubulin with survival kinase PIM-1, RNA-binding protein HuR, microRNAs are examples highlighting the functional complexity of this protein. The utility of class III beta-tubulin as a prognostic biomarker can also greatly improve if combined with these pro-survival partners. Subsequently, pharmacogenetic approaches, designed to counteract and target these pathways and associated-factors concurrently, might lead to better therapies and prognostic tools for class III beta-tubulin expressing cancers.

1. Introduction microtubules are highly conserved among species but have complex expression pattern, which denote Microtubules (MTs) are highly dynamic, cellular and functional specificity and diversity cytoskeletal structures that are essential to a variety (Katsetos et al., 2003). of cellular functions, including cell division, There are at least seven mammalian beta-tubulin proliferation, migration, protein trafficking, isotypes known to have formed through distinct intracellular transport, and maintaining cellular gene products without having gone any splicing polarity (Kreis and Vale, 1993; Katsetos and events (Ludueña, 1998). Dráber, 2012). Microtubules are formed by the These beta-tubulin subtypes differ from one another polymerization of the heterodimers: α-tubulin mainly in a region, which is limited to the last 15 C- subunits and β-tubulin subunits, the isotypes of terminal residues. which are encoded by multiple genes (Kreis and Class III beta-tubulin differs from other β-tubulin Vale, 1993; Katsetos and Dráber, 2012). subtypes in its post-translation changes, such as Interactions with several microtubule associated differences in phosphorylation and proteins (MAPs) are crucial to diverse microtubule polyglutamination in the same terminal residues functionality. The phylogenetic analyses from (Orr et al., 2003). Furthermore, TUBB3 gene is the vertebrate species suggest α and β isotypes of most conserved subtype across vertebrate species

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(Katsetos et al., 2003). In somatic tissues, class III Fornier, 2008). Both classes of drugs target specific beta-tubulin has mostly been used as a biomarker binding sites of beta-tubulin, which disrupt the for neural stem cells and neuronal differentiation microtubules, inhibiting its assembly or during fetal and post-natal development (Caccamo disassembly, leading to cell death mostly by et al., 1989; Verdier-Pinard et al., 2009). It is also apoptosis (Morris and Fornier, 2008). However, the constitutively expressed in the Sertoli cells of the exact mechanism by which each drug works to testes, transiently expressed in fetal respiratory inhibit microtubules is still speculative and not fully epithelium, and at lower levels at other tissues explained. Taxanes are regularly administered as (Verdier-Pinard et al., 2009; Abel et al., 2009). In part of adjuvant therapy in patients with breast, cancers however, class III beta-tubulin is expressed ovarian, and non-small cell lung cancer, and recent in tumors of both neuronal and non-neuronal origin. indications suggest that it can further improve Class III beta-tubulin is by far the most investigated prognosis and therapy through combination with microtubule isotype in human cancer. A growing biologic agents (Dumontet and Jordan, 2010; Karki body of evidence suggests high expression of class et al., 2014). III beta-tubulin is associated with poor prognosis The clinical success of taxanes led to development and aggressiveness of several cancers including of drugs like epothilones, which are macrolide non-small cell lung cancer (NSCLC), breast, antibiotics and can enhance microtubule ovarian, and gastric cancers (Katsetos and Dráber, polymerization and structurally more suitable to 2012; Katsetos et al., 2003). However, there are synthetic modifications (Morris and Fornier, 2008; also reports of expression of class III beta-tubulin Dumontet and Jordan, 2010). Similar to taxanes and good outcome as reported in clear cell ovarian that bind to beta-tubulin, epothilones exert similarly cancer and estrogen-receptor negative breast cancer and are thought to compete with taxane binding (Aoki et al., 2009; Wang et al., 2013). This report sites. More importantly, early trials of epothilones will try to highlight the function of class III beta- seem to indicate that they have better efficacy in tubulin and explain the seemingly contradicting patients resistant to taxane-including regimen therapeutic results. (Cheng et al., 2008; Goodin et al., 2004). Some of 2. Microtubule targeting agents - the modified versions of epothilones include patupilone, a naturally occurring epothilone, which growing list of agents for cancer was found to be 20 times more potent than therapeutics paclitaxel in taxane-resistant cell lines. Ixabepilone, The dynamic reorganization of the microtubules a semisynthetic version, has also shown to be allows for the formation of mitotic spindle, which is effective in resistant cell lines. Yet, many crucial for the faithful segregation of chromosomes promising agents that interfere with microtubules into daughter cells during cell division (Stanton et like epothilone B and D analogues, colchicines, and al., 2011). laulimalide binding agents are also currently at Thus, disrupting microtubules to inhibit mitotic cell different stages of development (Morris and division has become an attractive pharmaceutical Fornier, 2008; Dumontet and Jordan, 2010). approach to treat many different cancers. 3. Mechanism of drug resistance Accordingly, Microtubule Targeting Agents - the role of beta III tubulin (MTAs) are some of the most widely used drugs in cancer treatment and have met significant clinical Efficacy of taxanes and other MTAs is limiting due success (Jordan and Wilson, 2004). MTAs are to the development of acquired and intrinsic natural, small molecules that interfere with resistance of tumor cells to the drugs, as well as microtubule function at low concentrations by increased hypersensitivity and neurological preventing the formation of normal mitotic spindle toxicities. The mechanistic details of such chemo- during cell division, a process that goes awry resistance are entirely not clear at present. during cancer development (Jordan and Kamath, However, earlier studies in the 90s on class III beta- 2007). Classically, MTAs being used in cancer tubulin suggested that this protein mediated treatment fall into two kinds of drugs - MT chemoresistance in response to taxanes and widely stabilizing (taxanes and epothilones) and MT regarded to be predictive biomarker to taxane-based destabilizing ( Vinca alkaloids) agents. Taxanes - therapies (Derry et al., 1997). Owing to different paclitaxel docetaxel, abraxane, and cabazitaxel, are mechanisms of action, this classical theory entailed usually administered in the treatment of a wide class III beta-tubulin to be predictive of taxane range of solid cancers, such as breast, ovarian, based chemotherapy but not of Vinca alkoloids. NSCLC, and cancers of the head and the neck. Initial studies on this protein supported this theory, Vinca alkaloids, such as vinorelbine, vinflunine and where high expression of class III beta-tubulin was vinblastine, are usually used in the treatment of associated with chemo-resistance in taxane-based hematological malignancies like the lymphoma and therapies in different cell-based models. leukemia (Jordan and Wilson, 2004; Morris and This positive correlation of chemo-resistance

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association was also seen by our group when we hormonal modulators were able to inhibit class III investigated class III beta-tubulin in the context of beta-tubulin mRNA induction suggesting class III using paclitaxel in ovarian cancer (Kavallaris et al., beta-tubulin expression is mediated through ER 1997; Mozzetti et al., 2005). More recent data on dependent pathway (Saussede-Aim et al., 2009). class III beta-tubulin and various MTA agents have The evaluation of class III beta-tubulin to predict been rather conflicting. In one of the studies, class responses and outcome in triple-negative breast III beta-tubulin gene silencing desensitized effects cancer would be interesting as this breast cancer is of both taxanes as well as other drugs, in particular devoid of effect by ER and other hormones. A cisplatin. In another study, epothilones were found recent study in Lancet has demonstrated that to be extremely active in ovarian cancer cell models exposure to high levels of oestradiol and and other gynecological cancers but not for lung progesterone found in breast and ovarian cancer cancer, even when both sets of cancers exhibited patients are linked to BRCA1/BRCA2 mutations high class III beta-tubulin content (Gan et al., 2007; (Widschwendter et al., 2013). In a recent translation Mozzetti et al., 2008; Carrara et al., 2012). To study of ER negative breast cancer, class III beta- further gain insight into this contradiction, our tubulin was found to correlate with good group recently did a comprehensive review of 59 pathogenic response to chemotherapy, suggesting translational studies assessing class III beta-tubulin the protein maybe identified differently in specific in many different types of cancers. Not subsets of breast cancer (Wang et al., 2013). It is surprisingly, our analysis refuted the notion that unclear if there is any relationship between class III beta-tubulin is predictive of taxane-based BRCA1/BRCA2 mutations, chemotherapy chemotherapy (Karki et al., 2013). In those studies sensitivity, and class III beta-tubulin expression. that claimed class III beta-tubulin to be predictive Hormones and gender can also play a role in of response to MTA-including chemotherapy, either expression of class III beta-tubulin in male cancers. the sample size was small or analyses were not In a panel of 23 colorectal cancer cell lines, the stringent. Instead, our analysis of the studies show expression of class III beta-tubulin is increased in that class III beta-tubulin is a pure prognostic response to androgens only in males. This basal biomarker in some solid malignancies, regardless of activation of class III beta-tubulin seems associated the choice of chemotherapies employed (Karki et with poor clinical outcome in male colorectal al., 2013). In order to understand the basis of the cancer patients (Mariani et al., 2012). It was further prognostic capability of class III beta-tubulin, it is found that CYP17A1, a critical enzyme regulating necessary to understand the mechanisms regulating androgen levels, have a specific allele (GG its expression and the varied functions this protein phenotype) that confers colorectal patients with may have in the context of different cells and high level of androgens (Mariani et al., 2012). In tissues (Karki et al., 2013). prostate cancer, there is also an aspect of class III 4. Regulation and expression of beta-tubulin regulation by androgen levels. In one study, class III beta-tubulin increased signficantly class III beta-tubulin - hormones in castration-resistant prostate cancer (CRPC) and gender patients after treatment with anti-androgen therapy. In cases of ovarian and breast cancers, hormones This is probably due to loss of regulation by may play a significant role in the expression and androgen receptor or CRPC association with regulation of class III beta-tubulin. In breast cancer, hypoxic conditions (Forde et al., 2012). class III beta-tubulin expression is under the control 5. Regulation of beta III tubulin - of estrogens. This was demonstrated in a study exposure to microenvironment where estradiol exposure to MCF-7 breast cancer cell line, positive for estrogen receptor (ER), and survival pathway upregulated the TUBB3 mRNA and class III beta- In many cancers, class III beta-tubulin is a part of tubulin protein. However, in cells negative for ER an adaptive function circuit, which allows cancer such as MDA-MB-231, no such induction was cells thrive in microenvironment featured with poor observed (Saussede-Aim et al., 2009). In this oxygen and low nutrient supply. Our previous work context of ER positive cells, expression of class III has demonstrated that hypoxia is able to induce beta-tubulin cannot confer an aggressive phenotype TUBB3 gene expression through HIF1 binding to and be linked to resistance of the drug. Instead, its 3' region (Raspaglio et al., 2008). In ovarian these ER positive patients with high class III beta- cancer the regulation and expression of class III tubulin expression have better outcome because beta-tubulin is more complex and involves the role they are sensitive to aromatase inhibitors and other of additional transcription factors, RNA-binding anti-hormonal therapies. This was further proteins such as HuR, microRNAs, and components confirmed in this study using ER modulators: of the survival pathway involved in the adaptation tamoxifen - a selective ER modulator and to hypoxia. In hypoxic and hypoglycemic fulvestrant - a pure antagonist of ER. These conditions, prevalent in many advanced cancers

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 867 Class III beta-tubulin, drug resistance and therapeutic approaches in cancers Karki R, Ferlini C

including ovarian cancer, RNA binding protein like 6. Utility of combined biomarkers HuR facilitates class III beta-tubulin translation to counteract glucose shortage (Raspaglio et al., with beta III tubulin to increase 2010). HuR expression is nuclear in normal tissues prognostics but is found to be cytoplasmic in advanced cancers From both our recent review of translational studies exposed to hypoxia. Cytoplasmic HuR and high of cancers and previous studies on class III beta- class III beta-tubulin in ovarian cancer patients are tubulin, it is clear that the utility of class III beta- indicators of exposure to hypoxia and are linked to tubulin as a biomarker increases significantly when poor outcome (Raspaglio et al., 2010). A more used in combination with additional biomarkers, recent work from our group and others have further proteins, or other discrete factors in different elucidated a combined regulatory mechanism that cancers. In fact, it is important to stress the concept drives class III beta-tubulin expression through that class III beta-tubulin does not mediate the HuR and miR-200C in ovarian cancer (Prislei et al., resistant phenotype alone but only in a multi- 2013). When HuR is nuclear, a condition typical of molecular concerted pathway. Therefore, it is not low stage ovarian cancer, high expression of miR- surprising that this protein, when taken as a single 200C inhibits TUBB3 expression and results in agent, can even be a hallmark of good outcome, as good prognosis. On the other hand, when mir-200C seen in estrogen positive breast cancer. In a study is associated to cytoplasmic HuR, the conversion of conducted in gastric cancer, the combination of TUBB3 mRNA into beta III tubulin is enhanced, thymidine phosphorylase (TP)-positive and class III resulting in poor outcome of patients (Prislei et al., beta-tubulin negative tumors gave a stronger 2013). This report demonstrates that the same predictive power than TP negative and class III microRNA can exert different cellular functions for beta-tubulin positive tumors alone. Both overall class III beta-tubulin based on location-specific survival (OS) and progression free survival (PFS) interaction with the RNA-binding protein HuR. reached significance when surviving class III beta- In ovarian cancer, class III beta-tubulin function is tubulin was analyzed in combination (Gao et al., linked to the adaptation to hypoxia and poor 2011). In another study in gastric cancer, nutrient supply by incorporating pro-survival inactivation of Brca1 and low class III beta-tubulin kinases like PIM1 into the cytoskeleton. The provide a better utility in predicting responses to preferential incorporation of PIM1 into cytotoxic therapy, regardless of taxane-containing microtubules facilitates the cytoskeletal increase of or taxane-free drug combinations (Moiseyenko et the GBP1 GTPase. GBP1 incorporation stabilizes al., 2013). In breast cancer, the double-negative binding of the PIM1 kinase into microtubules and expression of class III beta-tubulin and survivin protects PIM1 from rapid degradation. This responded significantly better to docetaxel and had mechanism is not specific only for PIM1 but it is longer PFS (p<0.05) when compared with double- shared by additional kinases such as NEK6, AXL, positive patients (Yuan et al., 2012). In another and others (De Donato et al., 2012). As an ensuing clinical set of breast cancer, the class III beta- mechanism, signaling of these pro-survival tubulin negative tumors when combined with the pathways is prolonged, thus enabling cancer cells to BCL-2 and ERCC1 proteins increased predictive thrive in hypoxic conditions. Such analogous potential in response to CP (carboplatin/paclitaxel) adaptation of cancer cells in harsh therapy (Chen et al., 2012). Since BCL-2 and microenvironment is seen in many cancers, but the Survivin are both important in the pathway that role of cytoskeleton in the process is only beginning regulates apoptosis; they could possibly impact to emerge with recent data from class III beta- sensitivity to chemotherapy. In ovarian cancer, high tubulin expression. In some ovarian cancer cells, expression of both class III beta-tubulin and PIM1 regulation of class III beta-tubulin is under the combination has a synergistically higher prognostic control of a transcription factor, Gli1, one of the potential (De Donato et al., 2012). In thymic drivers of epithelial-to-mesenchymal transition epithelial cancer patients, ERCC1, BRCA1, and program and also a hallmark of metastatic potential class III beta-tubulin combination strongly correlate of solid tumors. In this study, Gli1 was able to with one another and can be used to improve increase class III beta-tubulin expression under accuracy of prognostic and predictive tests against hypoxic conditions conferring a more invasive chemotherapeutic regimens (Kaira et al., 2011). In ovarian cancer phenotype (Mozzetti et al., 2012). lung cancer, several studies support the notion of improved efficacy of integrated biomarkers. In

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(11) 868 Class III beta-tubulin, drug resistance and therapeutic approaches in cancers Karki R, Ferlini C

completely resected patients with NSCLC, double useful to identify new scaffolds to generate novel negative patients with class III beta-tubulin and therapeutics that are active in drug-resistant cells. ERCC1 had even more significant improved Therefore, such an inhibitor of the GBP1:PIM1 outcome (p=0.023) than when they were used interaction might be useful in those patients, who individually (Okuda et al., 2008). In patients exhibit high level of class III beta-tubulin and relapsed and treated with platinum/taxane, double consequently, have a poor response to negative ERCC1 and class III beta-tubulin tumors chemotherapy. As opposed to the currently predicted OS significantly. The prediction was not available targeted agents capable of inhibiting one significant when class III beta-tubulin was used kinase family, this new agent is expected to inhibit alone (p=0.015 vs. 0.087) (Azuma et al., 2009). the incorporation of a wide number of kinases into 7. Therapeutic approaches with the cytoskeleton. Such an effect will have a broader but telling impact on the resistant cancer phenotype class III beta-tubulin than that achieved by inhibiting a single kinase Microtubules enriched of class III beta-tubulin are family. featured by increased sensitivity to epothilones, 8. Summary and conclusion particularly to epothilone B. The configuration of the pocket binding epothilone B (patupilone) in Class III beta-tubulin will continue to be at the class III beta-tubulin differs from the one present in forefront of cancer therapeutics, owing to its class I beta-tubulin, the most abundantly expressed expression in the majority of solid tumors and its beta-tubulin (Magnani et al., 2006; Ferlini et al., prognostic potential. 2005). Therefore, cells with high expression of The field is shifting away from the hypothesis class III beta-tubulin appears more sensitive to linking class III beta-tubulin directly to resistance patupilone (Mozzetti et al., 2008). This property to MTAs based on a single protein mechanism. explains the increased effects of epothilones noticed The better comprehension of the mechanisms in a large number of clinical trials conducted in underlying class III beta-tubulin function is patients relapsing after multiple lines of improving its potential use as a prognostic chemotherapy (Ferrandina et al., 2012). Therefore, biomarker and as a potential factor to select it seems rather attractive to use class III beta- alternative treatments for those patients, where class tubulin as a potential biomarker for selection of III beta-tubulin pathway confers an aggressive patients eligible for treatment with epothilones. In phenotype. this regard, such approach necessitates development Its role in the functional gateway of cytoskeletal of an integrated biomarker as mentioned above, to drug resistance suggests that inhibition of this ensure active status of class III beta-tubulin protein by novel targeted agents will be useful in function. the treatment of diseases, which are currently Based on mechanism of regulation and interaction refractory to standard treatments. For all these with its functional partners in the tumor reasons, we expect class III beta-tubulin will microenvironment, class III beta-tubulin can be also continue to be actively pursued as a source of new exploited to develop cancer therapeutics. The rapid diagnostics and therapeutics for aggressive cancers. evolution of technologies is providing incredible and unprecedented amount of information about References various forms of cancers. 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