Number 6 June 2014

Atlas of Genetics and Cytogenetics in Oncology and Haematology

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Volume 18 - Number 6 June 2014

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

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Scope

The Atlas of Genetics and Cytogenetics in Oncology and Haematology is a peer reviewed on-line journal in open access, devoted to genes, cytogenetics, and clinical entities in cancer, and cancer-prone diseases. It presents structured review articles (“cards”) on genes, leukaemias, solid tumours, cancer-prone diseases, 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.

Editorial correspondance

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

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

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

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

http://AtlasGeneticsOncology.org

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Editor Jean-Loup Huret (Poitiers, France) Editorial Board

Sreeparna Banerjee (Ankara, Turkey) Solid Tumours Section Alessandro Beghini (Milan, Italy) Genes Section Anne von Bergh (Rotterdam, The Netherlands) Genes / Leukaemia Sections Judith Bovée (Leiden, The Netherlands) Solid Tumours Section Vasantha Brito-Babapulle (London, UK) Leukaemia Section Charles Buys (Groningen, The Netherlands) Deep Insights Section Anne Marie Capodano (Marseille, France) Solid Tumours Section Fei Chen (Morgantown, West Virginia) Genes / Deep Insights Sections Antonio Cuneo (Ferrara, Italy) Leukaemia Section Paola Dal Cin (Boston, Massachussetts) Genes / Solid Tumours Section 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(6) Atlas of Genetics and Cytogenetics in Oncology and Haematology

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Volume 18, Number 6, June 2014

Table of contents

Gene Section

ARID1A (AT rich interactive domain 1A (SWI-like)) 368 Yohan Suryo Rahmanto, Tian-Li Wang CASP8 (Caspase 8, Apoptosis-Related Cysteine Peptidase) 372 Selcen Öztürk, Kolja Schleich, Inna N Lavrik IGF1 (Insulin-Like Growth Factor 1 (Somatomedin C)) 378 Nicholas D Panayi, Randy Burd S100A4 (S100 Calcium Binding Protein A4) 381 Gajanan V Sherbet TGM2 (transglutaminase 2) 397 Lisa Dyer THRB (Thyroid Hormone Receptor, Beta) 400 Adam Master, Alicja Nauman

Leukaemia Section t(7;9)(q11.2;p13.2) PAX5/AUTS2 434 Dagmar Denk t(8;12)(q13;p13) ETV6/NCOA2 436 Jean-Loup Huret t(9;14)(q33;q32) IGH/LHX2 438 Nathalie Nadal, Elise Chapiro t(5;12)(q13;p13) ?/ETV6 441 Jean-Loup Huret

Solid Tumour Section

Soft Tissue Tumors: Extraskeletal osteosarcoma 443 Andreas F Mavrogenis, Panayiotis J Papagelopoulos

Deep Insight Section

Angiogenic factors and cancer therapy 447 Yihai Cao

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) Atlas of Genetics and Cytogenetics in Oncology and Haematology

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Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) Atlas of Genetics and Cytogenetics in Oncology and Haematology

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Gene Section Review

ARID1A (AT rich interactive domain 1A (SWI- like)) Yohan Suryo Rahmanto, Tian-Li Wang Departments of Gynecology/Obstetrics and Oncology Johns Hopkins Medical Institutions CRBII, Rm: 306, 1550 Orleans Street Baltimore, MD 21231, USA (YSR, TLW)

Published in Atlas Database: November 2013 Online updated version : http://AtlasGeneticsOncology.org/Genes/ARID1AID44231ch1p36.html DOI: 10.4267/2042/53764 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 Human ARID1A has 2 transcript variants. The long Review on ARID1A, with data on DNA/RNA, on variant (isoform 1) transcribed into 8585 bp the protein encoded and where the gene is mRNA, the coding sequence is from 374 bp - 7231 implicated. bp. The short variant (isoform 2) transcribed into 7934 Identity bp mRNA, the coding sequence is from 374 bp - Other names: B120, BAF250, BAF250a, BM029, 6580 bp. Isoform 2 has a shorter exon 18 compared C1orf4, ELD, MRD14, OSA1, P270, SMARCF1, to isoform 1. hELD, hOSA1 HGNC (Hugo): ARID1A Protein Location: 1p36.11 Note Local order: Gene orientation: telomere-3' The longer isoform of ARID1A consists of 2285 ARID1A 5'-centromere. amino acids (pI: 6.24), with predicted molecular mass of 242,04 kDa. The shorter isoform of DNA/RNA ARID1A consists of 2068 amino acids (pI: 6.08), with predicted molecular mass of 218,33 kDa. Both Description isoforms contain a single "ARID" DNA binding ARID1A gene is encoded by 20 exons spanning domain and four "LXXLL" nuclear receptor 86,08 Mb. coactivator motifs.

DNA organization of ARID1A.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 368 ARID1A (AT rich interactive domain 1A (SWI-like)) Rahmanto YS, Wang TL

ARID1A protein.

Description developmental delay and abnormities in 5 th fingers or toes (Tsurusaki et al., 2012; Santen et al., 2013; ARID1A is a member of the SWI/SNF family that Wieczorek et al., 2013). can regulate genes transcription by chromatin structure alteration through its helicase and ATPase Somatic activities. The encoded ARID1A nuclear protein is ARID1A is located at chromosome 1p that is part of the BRG/BRM chromatin remodeling frequently deleted in tumours. ARID1A sequence complex that has been shown to play an integral mutations, deletions, and rearrangements were role in controlling gene expression. identified in ovarian, kidney, breast, lung, Expression pancreatic and stomach cancer. Ubiquitously expressed in various normal tissues, with the highest expression seen in brain, blood and Implicated in female tissues. Ovarian clear cell carcinoma Localisation Oncogenesis Mainly located at cell nucleus but not at nucleolus. ARID1A somatic mutations were identified in 57% Function of the 42 ovarian clear cell carcinomas (Jones et al., 2010). Maeda et al. (2010) has demonstrated that ARID1A contains a conserved DNA-binding ARID1a genetic mutations resulted in loss of domain (ARID) that could be important for its ARID1A protein expression in 59% of the 149 function and can specifically bind an AT-rich DNA ovarian clear cell carcinomas. sequence. ARID1A is part of the large ATP- Study with a larger cohort of 210 patient samples dependent chromatin remodeling complex has also demonstrated that ARID1A somatic SNF/SWI, which is required for transcriptional mutation were found in 46% of patients with activation of genes. Changes in this SNF/SWI ovarian clear-cell carcinoma and 30% of patients complex has been implicated in many cellular ovarian endometrioid carcinoma (Wiegand et al., processes, including development, differentiation, 2010). proliferation, DNA repair, and tumor suppression. ARID1A inactivation by ARID1A mutations has ARID1A has been reported to act as tumor been suggested as an early molecular event that can suppressor in gynecological cancers (Guan et al., lead to tumor progression from benign ovarian 2011b). Molecular studies using over-expression endometroid cysts into an aggressive ovarian clear and RNAi silencing models have demonstrated that cell and endometrioid carcinoma (Ayhan et al., ARID1A negatively regulates cellular proliferation 2012). and tumorigenicity. This negative regulation is achieved through molecular collaboration between Uterine endometroid carcinoma ARID1A/BRG1 and p53, to regulate tumor- Oncogenesis inhibiting p53-downstream target genes such as ARID1A mutations were observed in 40% of CDKN1A and SMAD3. Using mutational studies, uterine endometrioid carcinoma with none Guan et al. (2012) have further confirmed ARID1A presented in uterine serous carcinomas (Guan et al., role as tumor suppressor, with all of the in-frame 2011a). All of the mutations in endometrioid indel mutants have lost their ability to inhibit carcinomas were the nonsense or insertion/deletion cellular proliferation. types (Guan et al., 2011a) and expected to result in complete loss or clonal loss of ARID1A expression. Mutations Immunostaining confirmed the relatively significant frequency of loss of ARID1A protein expression Germinal with 25-26% and 44% in uterine low-grade and ARID1A mutations have been implicated in Coffin- high-grade endometrioid carcinomas, respectively Siris syndrome, a rare genetic disorder that causes (Guan et al., 2011a; Werner et al., 2013; Mao et al.,

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 369 ARID1A (AT rich interactive domain 1A (SWI-like)) Rahmanto YS, Wang TL

2013). Hence, mutation-related loss of ARID1A ARID1A protein (Tsurusaki et al., 2012; Santen et expression has also been hypothesized as an early al., 2013; Wieczorek et al., 2013). As a result, event and played an important role in tumor affected individuals developed abnormalities, such progression of uterine endometrioid carcinoma as missing the fifth fingers or toes and coarse (Ayhan et al., 2012; Werner et al., 2013; Mao et al., characteristic of facial features. Moreover, cancer 2013). was not detected in any of the individual with Cervical cancer ARID1A mutation, reported in this study. Oncogenesis Loss of ARID1A protein expression was observed References in 31% (14/45) of cervical Jones S, Wang TL, Shih IeM, Mao TL, Nakayama K, adenocarcinomas/adenosquamous carcinomas with Roden R, Glas R, Slamon D, Diaz LA Jr, Vogelstein B, Kinzler KW, Velculescu VE, Papadopoulos N. Frequent no correlation to any clinicopathological features mutations of chromatin remodeling gene ARID1A in (Katagiri et al., 2012). Later studies using a large ovarian clear cell carcinoma. Science. 2010 Oct series of cervical cancer tissue specimens, ARID1A 8;330(6001):228-31 expression was found to be significantly decreased Maeda D, Mao TL, Fukayama M, Nakagawa S, Yano T, in cervical cancer tissues than in non-adjacent Taketani Y, Shih IeM. Clinicopathological significance of normal cervical epithelial tissues (Cho et al., 2013). loss of ARID1A immunoreactivity in ovarian clear cell The decrease of ARID1A expression was also carcinoma. Int J Mol Sci. 2010;11(12):5120-8 found to be associated with transition from normal Wiegand KC, Shah SP, Al-Agha OM, Zhao Y, Tse K, Zeng cells to cervical carcinoma and a more aggressive T, Senz J, McConechy MK, Anglesio MS, Kalloger SE, tumor phenotype (Cho et al., 2013). Overall Yang W, Heravi-Moussavi A, Giuliany R, Chow C, Fee J, Zayed A, Prentice L, Melnyk N, Turashvili G, Delaney AD, survival was also found to be reduced in cervical Madore J, Yip S, McPherson AW, Ha G, Bell L, Fereday S, cancer patients with loss of ARID1A (Cho et al., Tam A, Galletta L, Tonin PN, Provencher D, Miller D, 2013). Jones SJ, Moore RA, Morin GB, Oloumi A, Boyd N, Aparicio SA, Shih IeM, Mes-Masson AM, Bowtell DD, Hirst Breast cancer M, Gilks B, Marra MA, Huntsman DG. ARID1A mutations in endometriosis-associated ovarian carcinomas. N Engl J Oncogenesis Med. 2010 Oct 14;363(16):1532-43 Low ARID1A expression was observed in 56% Guan B, Mao TL, Panuganti PK, Kuhn E, Kurman RJ, (63/112) of the breast cancers samples and was Maeda D, Chen E, Jeng YM, Wang TL, Shih IeM. Mutation significantly associated with advanced tumor stage, and loss of expression of ARID1A in uterine low-grade higher P53 expression, increase Ki-67 and triple endometrioid carcinoma. Am J Surg Pathol. 2011a negative (ER -/PR -/Her-2-) molecular subtype May;35(5):625-32 (Zhang et al., 2012; Mamo et al., 2012). Low Guan B, Wang TL, Shih IeM. ARID1A, a factor that ARID1A expression was a predictor of poor overall promotes formation of SWI/SNF-mediated chromatin survival of breast cancer patients (Zhang et al., remodeling, is a tumor suppressor in gynecologic cancers. Cancer Res. 2011b Nov 1;71(21):6718-27 2012; Mamo et al., 2012). Breast cancer also exhibited a low rate (3-4%) of ARID1A mutations Wang K, Kan J, Yuen ST, Shi ST, Chu KM, Law S, Chan TL, Kan Z, Chan AS, Tsui WY, Lee SP, Ho SL, Chan AK, (Jones et al., 2010; Cornen et al., 2012). Cheng GH, Roberts PC, Rejto PA, Gibson NW, Pocalyko Gastric cancer DJ, Mao M, Xu J, Leung SY. Exome sequencing identifies frequent mutation of ARID1A in molecular subtypes of Oncogenesis gastric cancer. Nat Genet. 2011 Oct 30;43(12):1219-23 Inactivating mutation of ARID1A has also been Abe H, Maeda D, Hino R, Otake Y, Isogai M, Ushiku AS, identified gastric cancer (Wang et al., 2011; Abe et Matsusaka K, Kunita A, Ushiku T, Uozaki H, Tateishi Y, al., 2012; Zhang et al., 2012). Loss of ARID1A Hishima T, Iwasaki Y, Ishikawa S, Fukayama M. ARID1A expression has been correlated with increasing expression loss in gastric cancer: pathway-dependent roles with and without Epstein-Barr virus infection and depth of tumor infiltration, higher tumor grade, and microsatellite instability. Virchows Arch. 2012 poor overall patient survival (Abe et al., 2012; Oct;461(4):367-77 Wang et al., 2012). Moreover, ARID1A expression Ayhan A, Mao TL, Seckin T, Wu CH, Guan B, Ogawa H, has been shown as an independent prognostic factor Futagami M, Mizukami H, Yokoyama Y, Kurman RJ, Shih of overall survival in multiple studies (Abe et al., IeM. Loss of ARID1A expression is an early molecular 2012; Wang et al., 2012). event in tumor progression from ovarian endometriotic cyst to clear cell and endometrioid carcinoma. Int J Gynecol Coffin-Siris syndrome Cancer. 2012 Oct;22(8):1310-5 Prognosis Cornen S, Adelaide J, Bertucci F, Finetti P, Guille A, Hepatoblastoma and multiple congenital anomalies. Birnbaum DJ, Birnbaum D, Chaffanet M. Mutations and deletions of ARID1A in breast tumors. Oncogene. 2012 Oncogenesis Sep 20;31(38):4255-6 ARID1A mutations in individual with Coffin-Siris Guan B, Gao M, Wu CH, Wang TL, Shih IeM. Functional syndrome lead to a truncation and nonfunctional analysis of in-frame indel ARID1A mutations reveals new

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 370 ARID1A (AT rich interactive domain 1A (SWI-like)) Rahmanto YS, Wang TL

regulatory mechanisms of its tumor suppressor functions. Cho H, Kim JS, Chung H, Perry C, Lee H, Kim JH. Loss of Neoplasia. 2012 Oct;14(10):986-93 ARID1A/BAF250a expression is linked to tumor progression and adverse prognosis in cervical cancer. Katagiri A, Nakayama K, Rahman MT, Rahman M, Katagiri Hum Pathol. 2013 Jul;44(7):1365-74 H, Ishikawa M, Ishibashi T, Iida K, Otsuki Y, Nakayama S, Miyazaki K. Frequent loss of tumor suppressor ARID1A Mao TL, Ardighieri L, Ayhan A, Kuo KT, Wu CH, Wang TL, protein expression in adenocarcinomas/adenosquamous Shih IeM. Loss of ARID1A expression correlates with carcinomas of the uterine cervix. Int J Gynecol Cancer. stages of tumor progression in uterine endometrioid 2012 Feb;22(2):208-12 carcinoma. Am J Surg Pathol. 2013 Sep;37(9):1342-8 Mamo A, Cavallone L, Tuzmen S, Chabot C, Ferrario C, Santen GW, Aten E, Vulto-van Silfhout AT, Pottinger C, Hassan S, Edgren H, Kallioniemi O, Aleynikova O, van Bon BW, van Minderhout IJ, Snowdowne R, van der Przybytkowski E, Malcolm K, Mousses S, Tonin PN, Basik Lans CA, Boogaard M, Linssen MM, Vijfhuizen L, van der M. An integrated genomic approach identifies ARID1A as a Wielen MJ, Vollebregt MJ, Breuning MH, Kriek M, van candidate tumor-suppressor gene in breast cancer. Haeringen A, den Dunnen JT, Hoischen A, Clayton-Smith Oncogene. 2012 Apr 19;31(16):2090-100 J, de Vries BB, Hennekam RC, van Belzen MJ. Coffin-Siris syndrome and the BAF complex: genotype-phenotype Tsurusaki Y, Okamoto N, Ohashi H, Kosho T, Imai Y, Hibi- study in 63 patients. Hum Mutat. 2013 Nov;34(11):1519-28 Ko Y, Kaname T, Naritomi K, Kawame H, Wakui K, Fukushima Y, Homma T, Kato M, Hiraki Y, Yamagata T, Werner HM, Berg A, Wik E, Birkeland E, Krakstad C, Yano S, Mizuno S, Sakazume S, Ishii T, Nagai T, Shiina Kusonmano K, Petersen K, Kalland KH, Oyan AM, Akslen M, Ogata K, Ohta T, Niikawa N, Miyatake S, Okada I, LA, Trovik J, Salvesen HB. ARID1A loss is prevalent in Mizuguchi T, Doi H, Saitsu H, Miyake N, Matsumoto N. endometrial hyperplasia with atypia and low-grade Mutations affecting components of the SWI/SNF complex endometrioid carcinomas. Mod Pathol. 2013 cause Coffin-Siris syndrome. Nat Genet. 2012 Mar Mar;26(3):428-34 18;44(4):376-8 Wieczorek D, Bögershausen N, Beleggia F, Steiner- Wang DD, Chen YB, Pan K, Wang W, Chen SP, Chen JG, Haldenstätt S, Pohl E, Li Y, Milz E, Martin M, Thiele H, Zhao JJ, Lv L, Pan QZ, Li YQ, Wang QJ, Huang LX, Ke Altmüller J, Alanay Y, Kayserili H, Klein-Hitpass L, ML, He J, Xia JC. Decreased expression of the ARID1A Böhringer S, Wollstein A, Albrecht B, Boduroglu K, Caliebe gene is associated with poor prognosis in primary gastric A, Chrzanowska K, Cogulu O, Cristofoli F, Czeschik JC, cancer. PLoS One. 2012;7(7):e40364 Devriendt K, Dotti MT, Elcioglu N, Gener B, Goecke TO, Krajewska-Walasek M, Guillén-Navarro E, Hayek J, Houge Zang ZJ, Cutcutache I, Poon SL, Zhang SL, McPherson G, Kilic E, Simsek-Kiper PÖ, López-González V, Kuechler JR, Tao J, Rajasegaran V, Heng HL, Deng N, Gan A, Lim A, Lyonnet S, Mari F, Marozza A, Mathieu Dramard M, KH, Ong CK, Huang D, Chin SY, Tan IB, Ng CC, Yu W, Mikat B, Morin G, Morice-Picard F, Ozkinay F, Rauch A, Wu Y, Lee M, Wu J, Poh D, Wan WK, Rha SY, So J, Renieri A, Tinschert S, Utine GE, Vilain C, Vivarelli R, Salto-Tellez M, Yeoh KG, Wong WK, Zhu YJ, Futreal PA, Zweier C, Nürnberg P, Rahmann S, Vermeesch J, Pang B, Ruan Y, Hillmer AM, Bertrand D, Nagarajan N, Lüdecke HJ, Zeschnigk M, Wollnik B. A comprehensive Rozen S, Teh BT, Tan P. Exome sequencing of gastric molecular study on Coffin-Siris and Nicolaides-Baraitser adenocarcinoma identifies recurrent somatic mutations in syndromes identifies a broad molecular and clinical cell adhesion and chromatin remodeling genes. Nat Genet. spectrum converging on altered chromatin remodeling. 2012 May;44(5):570-4 Hum Mol Genet. 2013 Dec 20;22(25):5121-35 Zhang X, Zhang Y, Yang Y, Niu M, Sun S, Ji H, Ma Y, Yao G, Jiang Y, Shan M, Zhang G, Pang D. Frequent low This article should be referenced as such: expression of chromatin remodeling gene ARID1A in Rahmanto YS, Wang TL. ARID1A (AT rich interactive breast cancer and its clinical significance. Cancer domain 1A (SWI-like)). Atlas Genet Cytogenet Oncol Epidemiol. 2012 Jun;36(3):288-93 Haematol. 2014; 18(6):368-371.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 371 Atlas of Genetics and Cytogenetics in Oncology and Haematology

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Gene Section Review

CASP8 (Caspase 8, Apoptosis-Related Cysteine Peptidase) Selcen Öztürk, Kolja Schleich, Inna N Lavrik Division of Immunogenetics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany (SÖ, KS, INL), Department of Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University, 39120 Magdeburg, Germany (INL)

Published in Atlas Database: November 2013 Online updated version : http://AtlasGeneticsOncology.org/Genes/CASP8ID925ch2q33.html DOI: 10.4267/2042/53765 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 they are described in Table 1. Review on CASP8, with data on DNA/RNA, on the Protein protein encoded and where the gene is implicated. Note Identity Caspases are a family of cysteinyl aspartate specific proteases which are synthesized as zymogens. Other names: ALPS2B, CAP4, Casp-8, FLICE, Caspase-8 was discovered as a component of the MACH, MCH5 CD95 (Fas/APO-1) death-inducing signaling HGNC (Hugo): CASP8 complex (DISC) (Muzio et al., 1996). Location: 2q33.1 Description Local order: CASP8 is located on chromosome 2 on the long arm (positive strand), and lies between Caspase-8 has a number of isoforms, including the CASP10 and STRADB genes. procaspase-8a (496 aa), procaspase-8b (479 aa), procaspase-8c (464 aa), procaspase-8e (235 aa), DNA/RNA procaspase-8g or caspase-8L (538 aa) and caspase- 8 short (108 aa). Description Only two isoforms are predominantly expressed in 54269 bases with 11 exons. many different tissues and cell lines: procaspase-8a and procaspase-8b (Scaffidi et al., 1997). Transcription They act as the main initiator caspases in death There are 6 transcriptional variants of CASP8 and receptor-induced apoptosis.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 372 CASP8 (Caspase 8, Apoptosis-Related Cysteine Peptidase) Öztürk S, et al.

Caspase-8L was reported to be expressed in human caspase-6 and caspase-7 by procaspase-9 (Scaffidi peripheral blood lymphocytes as a truncated et al., 1998; Scaffidi et al., 1999). protein, which lacks the C-terminal protease In addition to apoptosis, caspase-8 has a role in domain (Horiuchi et al., 2000). programmed necrosis (necroptosis) as well. Therefore, it is suggested that caspase-8L is Caspase-8 can be recruited to the necroptotic recruited into the DISC but remains proteolytically complexes (necrosome or ripoptosome) together inert, interfering with the transduction of the signal with RIP1, RIP3 and FADD. from the DISC (Himeji et al., 2002). An isoform that is detected in bone marrow mononuclear cells is named caspase-8 short. Although it only contains the first DED and a part of the second DED, overexpression of caspase-8 short is reported to increase sensitivity to apoptosis (Xu et al., 2009). In addition to caspase-8 isoforms, there is a number of cleavage products described, which are formed in the course of apoptosis. Apoptotic processing of procaspase-8a/b involves generation of the cleavage products p43/p41, p30, the prodomains p26/p24, p18 and p10 (Hoffmann et al., 2009). The latter two cleavage products form the active caspase-8 heterotetramer p10 2-p18 2 that triggers apoptosis (Lavrik and Krammer, 2012). Expression Caspase-8 is expressed in almost all kind of tissues, with the highest expression in the immune system and lowest in the nervous system (McCall et al., 2011). Localisation Procaspase-8 mainly localizes to the cytosol, in close proximity to the membrane (Medema et al., 1997). It may also localize to mitochondria (Qin et al., 2001; Chandra et al., 2004) or centrosomes (Mielgo et al., 2009). Caspase-8 cleavage products are reported to localize to the nucleus as well as the cytosol (Benchoua et al., 2002; Yao et al., 2007). Function Caspase-8 is the main initiator caspase in death receptor-induced apoptosis. Upon stimulation, procaspase-8 is recruited to the CD95 or TRAIL DISC, or TNF complex II. Procaspase-8 activation involves dimerization, oligomerization and cleavage (Schleich et al., Chromosomal location of CASP8 and nearby genes. 2012). The cleavage of procaspase-8 involves several It would then cleave RIP1 and RIP3, and therefore steps, leading to the generation of the active block necroptosis (Stupack et al., 2006; Feoktistova caspase-8 heterotetramer p10 2-p18 2 (Lavrik and et al., 2011; Tenev et al., 2011). Krammer, 2012). Caspase-8 also has an essential role for NF-kB Depending on the cell type, active caspase-8 either signaling via many different stimuli including directly cleaves effector caspases (caspase-3 and CD95, TRAIL, TCR and TLR stimulations caspase-7) or cleaves Bid, which eventually leads to (Kataoka and Tschopp, 2004; Dohrman et al., 2005; release of cytochrome C and apoptosome formation Su et al., 2005; Lemmers et al., 2007; Grunert et al., followed by cleavage of effector caspase-3, 2012).

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 373 CASP8 (Caspase 8, Apoptosis-Related Cysteine Peptidase) Öztürk S, et al.

Schematic representation of the structure of the 54kb CASP8 gene, which contains 10 exons and can be transcribed into 6 alternative splice variants.

Interestingly, although the activation of procaspase- has a shorter half-life than wild-type caspase-8 and 8 to p10 2-p18 2 heterotetramer is necessary for cannot interact with caspase-8 or FADD; therefore MAPK signaling (Kober et al., 2011), it is not lost its proapoptotic activity (Liu et al., 2002). necessary for NF-kB signaling upon CD95 - Head and neck carcinoma: A mutation was stimulation (Neumann et al., 2010). detected in the tumor cells from head and neck Caspase-8 has been also reported to affect carcinoma that modifies the stop codon and metastasis. Interestingly, although loss of caspase-8 lengthening the protein by 88 amino acids potentiates metastasis, under conditions where (Mandruzzato et al., 1997). apoptosis is compromised, caspase-8 can promote - Neuroblastoma : An Alanine to Valine missense tumor cell migration and metastasis (Stupack et al., mutation was detected at codon 96 in a 2006; Barbero et al., 2009). neuroblastoma sample which lacks CASP8 mRNA Homology expression (Takita et al., 2001). Caspase-10, FADD, c-FLIP. Implicated in Mutations Hepatocellular carcinoma Germinal Disease A homozygous C to T mutation at residue 248 leads Hepatocellular carcinoma accounts for the majority to familial autoimmune lymphoproliferative of liver cancers. Mostly, it is secondary to cirrhosis, syndrome type 2B. which is caused mainly by alcohol abuse or hepatitis B/C infections. A somatic mutation in Somatic CASP8 leading to deletion of the bases 1225-1226 Various somatic mutations of CASP8 are identified was detected in 9 out of 69 hepatocellular in different carcinomas. Mutations are observed at carcinoma samples from unrelated patients. This different parts of caspase-8, but they all lead to a deletion results in a frameshift and therefore catalytically inactive form. premature termination of amino-acid synthesis in - Hepatocellular carcinoma: CASP8 is frequently the p10 protease subunit, consequently inactivating inactivated by the frameshift somatic mutation caspase-8 (Soung et al., 2005b). 1225-1226delTG in hepatocellular carcinomas, resulting in a premature termination of amino-acid Gastric carcinoma synthesis in the p10 protease subunit (Soung et al., Disease 2005b). Gastric carcinomas arise from the epithelium of the - Gastric carcinoma: Inactivating CASP8 mutations stomach. In a study by Soung and colleagues are detected at different sites in about 10% of (Soung et al., 2005a), 122 advanced gastric advanced gastric carcinomas (Soung et al., 2005a). carcinoma samples were analyzed for mutations in - Colorectal carcinoma: Inactivating CASP8 the coding region and the exon-intron junctions of mutations are detected at different sites in about 5% CASP8 gene by PCR-SSCP analysis. In 13 of invasive colorectal carcinomas (Kim et al., samples, mutations in caspase-8 were found. All 2003). mutants were still expressed at a similar level - Vulvar squamous carcinoma: Deletion of leucine compared to wild-type caspase-8, however, when 62 ( ∆Leu62casp-8) is detected in A431 human transfected into cell lines, all mutants except one vulvar squamous carcinoma cells. ∆Leu62casp-8 showed defects in apoptosis.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 374 CASP8 (Caspase 8, Apoptosis-Related Cysteine Peptidase) Öztürk S, et al.

Involvement of procaspase-8 in death-receptor signaling. (A) Procaspase-8 is recruited to the CD95 or TRAIL DISC through the adaptor protein FADD. Upon activation of TNF, procaspase-8 is recruited through FADD and TRADD. For activation, procaspase-8 requires dimerization and internal cleavage. The major function of procaspase-8 in DR signaling is induction of apoptosis, but it also regulates necroptosis or NF-kB via RIP1/RIP3 and c-FLIP(L). (B) Procaspase-8 consists of a prodomain harboring tandem death effector domains (DED) followed by one large (p18) and one small (p10) catalytic subunit. Cleavage between p18 and p10 generates the intermediate p43/p41 which is further processed to the fully active form by cleavage between the prodomain and p18.

Colorectal carcinoma Head and neck carcinoma Disease Disease Colorectal cancer arises from colon, rectum or Head and neck carcinomas start in the lip, oral appendix. In the analysis of 82 colorectal adenomas cavity, nasal cavity, paranasal sinuses, pharynx, or and 98 invasive colorectal carcinomas, 5 mutations larynx and the majority (90%) originates from the were detected only in the colorectal carcinomas but epithelium, therefore named squamous cell not in the adenomas. 3 out of 5 of these mutations carcinomas. A mutation in CASP8 was detected in acted in a dominant negative manner and the cells from a tumor relapse resected from the oral suppressed apoptosis (Kim et al., 2003). cavity of a late-stage squamous cell carcinoma patient. This mutation was found to modify the stop Vulvar squamous carcinoma codon and add an Alu repeat to the coding region. Disease Therefore, the mutant protein is 88 amino acids In the analysis of A431 human vulvar squamous longer than the wild type and cannot efficiently act carcinoma cells, a mutation in CASP8 leading to as an apoptotic protein (Mandruzzato et al., 1997). deletion of leucine 62 was detected. This mutant Neuroblastoma version of caspase-8 retained its enzymatic activity, however, it lost the ability to interact with itself, Disease wild-type caspase-8 or FADD and therefore lost its Neuroblastoma arises from immature nerve cells. It proapoptotic activity (Liu et al., 2002). is mainly localized to adrenal medulla. In a study

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 375 CASP8 (Caspase 8, Apoptosis-Related Cysteine Peptidase) Öztürk S, et al.

where human neuroblastoma cells were transferred Autoimmune lymphoproliferative to chick chorioallantoic membrane, tumor syndrome type 2B development was monitored. Although presence or lack of caspase-8 did not Disease change primary tumor growth, metastasis was A homozygous C to T mutation in caspase-8 at highly promoted in the tumors lacking caspase-8 residue 248 in the p18 protease subunit leads to due to impaired programmed cell death (Stupack et autoimmune lymphoproliferative syndrome type 2B al., 2006). (Chun et al., 2002). In a similar study, tumor cells additionally lacking Autoimmune Lymphoproliferative Syndrome Type caspase-3 were used to test non-apoptotic effects of 2B is an autosomal dominant disease where there caspase-8 on neuroblastoma metastasis. are defects in activation of T cells, B cells, and Interestingly, tumors lacking only caspase-3 natural killer cells of the patients as well as in metastasized more efficiently than tumors lacking CD95-mediated apoptosis. Patients have both caspases, pointing out that caspase-8 also lymphoproliferation and thus lymphadenopathy, shows non-apoptotic properties such as enhancing splenomegaly and autoimmunity. cell migration (Barbero et al., 2009). Silencing of caspase-8 was also observed in human References neuroblastoma samples. In two studies by Takita Muzio M, Chinnaiyan AM, Kischkel FC et al.. FLICE, a and colleagues, 11 out of 15 and 19 out of 27 novel FADD-homologous ICE/CED-3-like protease, is neuroblastoma samples did not express caspase-8, recruited to the CD95 (Fas/APO-1) death--inducing detected by real-time PCR (Takita et al., 2000; signaling complex. Cell. 1996 Jun 14;85(6):817-27 Takita et al., 2001). Mandruzzato S, Brasseur F, Andry G, Boon T, van der Furthermore, a missense mutation was detected at Bruggen P. A CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma. J Exp codon 96 in one of the samples lacking caspase-8 Med. 1997 Aug 29;186(5):785-93 expression (Takita et al., 2000). Furthermore, silencing of CASP8 in neuroblastoma Medema JP, Scaffidi C, Kischkel FC, Shevchenko A, Mann M, Krammer PH, Peter ME. FLICE is activated by was found to be associated with MYCN association with the CD95 death-inducing signaling amplification. 10 out of 16 patients with MYCN complex (DISC). EMBO J. 1997 May 15;16(10):2794-804 amplification had completely methylated CASP8 Scaffidi C, Medema JP, Krammer PH, Peter ME. FLICE is alleles opposed to only 1 out of 26 patients without predominantly expressed as two functionally active MYCN amplification. isoforms, caspase-8/a and caspase-8/b. J Biol Chem. Interestingly, one patient among these 42 patients 1997 Oct 24;272(43):26953-8 had a deletion of the CASP8 gene (Teitz et al., Scaffidi C, Fulda S, Srinivasan A et al.. Two CD95 (APO- 2000). 1/Fas) signaling pathways. EMBO J. 1998 Mar 16;17(6):1675-87 Medulloblastoma Scaffidi C, Schmitz I, Zha J, Korsmeyer SJ, Krammer PH, Disease Peter ME. Differential modulation of apoptosis sensitivity in Medulloblastoma is a tumor of the brain that CD95 type I and type II cells. J Biol Chem. 1999 Aug originates in the cerebellum or posterior fossa. In 6;274(32):22532-8 one study of medulloblastoma, 14 out of 27 tumors Horiuchi T, Himeji D, Tsukamoto H et al.. Dominant were identified to have lost CASP8 mRNA expression of a novel splice variant of caspase-8 in human peripheral blood lymphocytes. Biochem Biophys Res expression (Zuzak et al., 2002). Furthermore, Commun. 2000 Jun 16;272(3):877-81 another study showed that CASP8 expression was reversely correlating with the disease. The 5-year Takita J, Yang HW, Bessho F, Hanada R, Yamamoto K, Kidd V, Teitz T, Wei T, Hayashi Y. Absent or reduced cumulative progression-free survival rate of the expression of the caspase 8 gene occurs frequently in patients with weak CASP8 expression was 31%, neuroblastoma, but not commonly in Ewing sarcoma or and with moderate/strong caspase-8 expression rhabdomyosarcoma. Med Pediatr Oncol. 2000 73% (Pingoud-Meier et al., 2003). Dec;35(6):541-3 Teitz T, Wei T, Valentine MB, Vanin EF et al.. Caspase 8 Small cell lung carcinoma is deleted or silenced preferentially in childhood Disease neuroblastomas with amplification of MYCN. Nat Med. Small cell lung carcinomas commonly originate 2000 May;6(5):529-35 from the lung, although rarely can originate also Qin ZH, Wang Y, Kikly KK, Sapp E, Kegel KB, Aronin N, from cervix, prostate or gastrointesinal tract. In this DiFiglia M. Pro-caspase-8 is predominantly localized in mitochondria and released into cytoplasm upon apoptotic carcinoma, tumor cells are much smaller than stimulation. J Biol Chem. 2001 Mar 16;276(11):8079-86 normal cells with almost no cytoplasm. In one study, 13 out of 25 small cell lung carcinoma Takita J, Yang HW, Chen YY, Hanada R et al.. Allelic imbalance on chromosome 2q and alterations of the samples showed silencing of CASP8 due to caspase 8 gene in neuroblastoma. Oncogene. 2001 Jul methylation (Hopkins-Donaldson et al., 2003). 19;20(32):4424-32

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 376 CASP8 (Caspase 8, Apoptosis-Related Cysteine Peptidase) Öztürk S, et al.

Benchoua A, Couriaud C, Guégan C, Tartier L et al.. caspase-8. Nature. 2006 Jan 5;439(7072):95-9 Active caspase-8 translocates into the nucleus of apoptotic cells to inactivate poly(ADP-ribose) polymerase-2. J Biol Lemmers B, Salmena L, Bidère N, Su H et al.. Essential Chem. 2002 Sep 13;277(37):34217-22 role for caspase-8 in Toll-like receptors and NFkappaB signaling. J Biol Chem. 2007 Mar 9;282(10):7416-23 Chun HJ, Zheng L, Ahmad M, Wang J et al.. Pleiotropic defects in lymphocyte activation caused by caspase-8 Yao Z, Duan S, Hou D, Heese K, Wu M. Death effector mutations lead to human immunodeficiency. Nature. 2002 domain DEDa, a self-cleaved product of caspase-8/Mch5, Sep 26;419(6905):395-9 translocates to the nucleus by binding to ERK1/2 and upregulates procaspase-8 expression via a p53-dependent Himeji D, Horiuchi T, Tsukamoto H, Hayashi K, Watanabe mechanism. EMBO J. 2007 Feb 21;26(4):1068-80 T, Harada M. Characterization of caspase-8L: a novel isoform of caspase-8 that behaves as an inhibitor of the Barbero S, Mielgo A, Torres V, Teitz T et al.. Caspase-8 caspase cascade. Blood. 2002 Jun 1;99(11):4070-8 association with the focal adhesion complex promotes tumor cell migration and metastasis. Cancer Res. 2009 Liu B, Peng D, Lu Y, Jin W, Fan Z. A novel single amino May 1;69(9):3755-63 acid deletion caspase-8 mutant in cancer cells that lost proapoptotic activity. J Biol Chem. 2002 Aug Hoffmann JC, Pappa A, Krammer PH, Lavrik IN. A new C- 16;277(33):30159-64 terminal cleavage product of procaspase-8, p30, defines an alternative pathway of procaspase-8 activation. Mol Cell Zuzak TJ, Steinhoff DF, Sutton LN, Phillips PC, Eggert A, Biol. 2009 Aug;29(16):4431-40 Grotzer MA. Loss of caspase-8 mRNA expression is common in childhood primitive neuroectodermal brain Mielgo A, Torres VA, Clair K, Barbero S, Stupack DG. tumour/medulloblastoma. Eur J Cancer. 2002 Paclitaxel promotes a caspase 8-mediated apoptosis Jan;38(1):83-91 through death effector domain association with microtubules. Oncogene. 2009 Oct 8;28(40):3551-62 Hopkins-Donaldson S, Ziegler A, Kurtz S et al.. Silencing of death receptor and caspase-8 expression in small cell Xu Z, Tang K, Wang M, Rao Q, Liu B, Wang J. A new lung carcinoma cell lines and tumors by DNA methylation. caspase-8 isoform caspase-8s increased sensitivity to Cell Death Differ. 2003 Mar;10(3):356-64 apoptosis in Jurkat cells. J Biomed Biotechnol. 2009;2009:930462 Kim HS, Lee JW, Soung YH, Park WS, Kim SY et al.. Inactivating mutations of caspase-8 gene in colorectal Neumann L, Pforr C, Beaudouin J, Pappa A, Fricker N, carcinomas. Gastroenterology. 2003 Sep;125(3):708-15 Krammer PH, Lavrik IN, Eils R. Dynamics within the CD95 death-inducing signaling complex decide life and death of Pingoud-Meier C, Lang D, Janss AJ, Rorke LB et al.. Loss cells. Mol Syst Biol. 2010;6:352 of caspase-8 protein expression correlates with unfavorable survival outcome in childhood Feoktistova M, Geserick P, Kellert B et al.. cIAPs block medulloblastoma. Clin Cancer Res. 2003 Dec Ripoptosome formation, a RIP1/caspase-8 containing 15;9(17):6401-9 intracellular cell death complex differentially regulated by cFLIP isoforms. Mol Cell. 2011 Aug 5;43(3):449-63 Chandra D, Choy G, Deng X, Bhatia B, Daniel P, Tang DG. Association of active caspase 8 with the mitochondrial Kober AM, Legewie S, Pforr C, Fricker N, Eils R, Krammer membrane during apoptosis: potential roles in cleaving PH, Lavrik IN. Caspase-8 activity has an essential role in BAP31 and caspase 3 and mediating mitochondrion- CD95/Fas-mediated MAPK activation. Cell Death Dis. endoplasmic reticulum cross talk in etoposide-induced cell 2011 Oct 6;2:e212 death. Mol Cell Biol. 2004 Aug;24(15):6592-607 McCall MN, Uppal K, Jaffee HA, Zilliox MJ, Irizarry RA. Kataoka T, Tschopp J. N-terminal fragment of c-FLIP(L) The Gene Expression Barcode: leveraging public data processed by caspase 8 specifically interacts with TRAF2 repositories to begin cataloging the human and murine and induces activation of the NF-kappaB signaling transcriptomes. Nucleic Acids Res. 2011 Jan;39(Database pathway. Mol Cell Biol. 2004 Apr;24(7):2627-36 issue):D1011-5 Dohrman A, Kataoka T, Cuenin S, Russell JQ, Tschopp J, Tenev T, Bianchi K, Darding M, Broemer M et al.. The Budd RC. Cellular FLIP (long form) regulates CD8+ T cell Ripoptosome, a signaling platform that assembles in activation through caspase-8-dependent NF-kappa B response to genotoxic stress and loss of IAPs. Mol Cell. activation. J Immunol. 2005 May 1;174(9):5270-8 2011 Aug 5;43(3):432-48 Soung YH, Lee JW, Kim SY, Jang J, Park YG et al.. Grunert M, Gottschalk K, Kapahnke J et al.. The adaptor CASPASE-8 gene is inactivated by somatic mutations in protein FADD and the initiator caspase-8 mediate gastric carcinomas. Cancer Res. 2005a Feb 1;65(3):815- activation of NF-κB by TRAIL. Cell Death Dis. 2012 Oct 21 25;3:e414 Soung YH, Lee JW, Kim SY, Sung YJ, Park WS, Nam SW, Lavrik IN, Krammer PH. Regulation of CD95/Fas signaling Kim SH, Lee JY, Yoo NJ, Lee SH. Caspase-8 gene is at the DISC. Cell Death Differ. 2012 Jan;19(1):36-41 frequently inactivated by the frameshift somatic mutation Schleich K, Warnken U, Fricker N, Oztürk S et al.. 1225_1226delTG in hepatocellular carcinomas. Oncogene. Stoichiometry of the CD95 death-inducing signaling 2005b Jan 6;24(1):141-7 complex: experimental and modeling evidence for a death Su H, Bidère N, Zheng L, Cubre A, Sakai K, Dale J, effector domain chain model. Mol Cell. 2012 Jul Salmena L, Hakem R, Straus S, Lenardo M. Requirement 27;47(2):306-19 for caspase-8 in NF-kappaB activation by antigen receptor. Science. 2005 Mar 4;307(5714):1465-8 This article should be referenced as such: Stupack DG, Teitz T, Potter MD, Mikolon D et al.. Öztürk S, Schleich K, Lavrik IN. CASP8 (Caspase 8, Potentiation of neuroblastoma metastasis by loss of Apoptosis-Related Cysteine Peptidase). Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6):372-377.

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Gene Section Short Communication

IGF1 (Insulin-Like Growth Factor 1 (Somatomedin C)) Nicholas D Panayi, Randy Burd Department of Nutritional Sciences and College of Medicine, University of Arizona, Tucson, AZ 85721, USA (NDP, RB)

Published in Atlas Database: November 2013 Online updated version : http://AtlasGeneticsOncology.org/Genes/IGF1ID40927ch12q23.html DOI: 10.4267/2042/53766 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

composed of 6 different exons. Abstract Exons 1 and 2 determine the class of the protein Short Communication on IGF1, with data on and functionally represent the signal peptide for DNA/RNA, on the protein encoded and where the cellular localization post-translation. gene is implicated. Exons 3 and 4 will primarily encode the IGF-1 mature peptide; ultimately becoming the receptor Identity binding ligand. Exons 5 and 6 will primarily represent the E Other names: IGF-I, IGF1A, IGFI domain peptide; with exon six providing the HGNC (Hugo): IGF1 different polyadenylating signals. Location: 12q23.2 These parts of the transcript give a functional distinction to the 6 isoforms produced (although DNA/RNA such distinctions have yet to be definitively identified) (Adapted from Mills et al., 2007; Description Philippou et al., 2007). Genomic size: 84779 bp. The IGF-1 Gene is

Illustrates IGF-1 splice sites and isoform variants (adapted from Mills et al., 2007).

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 378 IGF1 (Insulin-Like Growth Factor 1 (Somatomedin C)) Panayi ND, Burd R

Transcription clearance and inactivation. IGFBP (1,3,4,6) are growth promoting; IGFBP-(2,5) bind IGF-1 and Six different heterogeneous mRNA are transcribed limit IGFR/IGF-1 interaction; a growth inhibiting using alternate promoters, alternate splice sites and effect. varying polyadenylation signals. Class one and two are derived from the exon one and two promoter Function respectively; both are differentially spliced to the Important for growth/development in children and common three exon. Each class can be variably adults. Vital role in anabolic processes in general. spliced to the fifth and sixth exons, producing a Important functions in osteogenesis, axonal total of six different isoforms (Philippou et al., generation in nerves, nerve regeneration after 2007). ischemic insult, muscle repair and hypertrophy after Class one isoforms predominate in the extrahepatic trauma or exercise (Cheng et al., 2006). Although tissues and are secreted in a paracrine/autocrine still under investigation, studies suggest that fashion. individual isotype/binding protein combinations Class two isoforms predominate in the liver and are manifest in response to specific environmental secreted in an endocrine fashion. They are also interactions or physiological demands. In addition, more sensitive or responsive to growth hormone the binding proteins are critical for maintaining the relative to class 1 (Mills et al., 2007). bioavailability of the IGF-1. The unique IGF- Individual isoforms may be more favorably 1/IGF-1 receptor complex will then signal the translated depending on the tissue type, the protein cascade necessary for tissue metabolism or available binding proteins and the physiological regeneration. Ischemic damage to the brain context. illustrates how this concept materializes. Cytotoxic Researchers are discovering evidence that suggest edema and inflammatory markers induce the certain isoforms may be preferentially expressed transcription of a specific isotype; which then binds under varying amounts of mechanical pressure in to a tissue specific binding protein; protecting the skeletal muscle (Philippou et al., 2007). The integrity of the protein and preventing its renal advantages of one isoform or another, in varying clearance. The distinctive IGF-1/IGF-1R complex contexts of stress, inflammation, regeneration and will then activate the protein kinase cascade hypertrophy are yet to be elucidated. necessary for axonal regeneration. Pseudogene Cellular/molecular effects: Upon binding, the Not reported. tyrosine kinase receptor, IGF-1/IGF-1R complex activates the PI3K/AKT/mTOR and Protein RAS/RAF/MAPK protein cascades. Both interfere with apoptosis and are pro-cell survival; the latter Description additionally promotes cellular differentiation, Single polypeptide chain protein consisting of 70 metabolism, growth and repair. Given its integral amino acids and three disulfide bridges. utility in tissue growth, repair and cell cycle regulation, IGF-1 receptors are found ubiquitously Expression throughout the body and include: muscle, bone, It is primarily produced and secreted in endocrine cartilage, kidney, liver, lung and nervous tissue fashion by the liver. It is also produced and secreted (Schiaffino et al., 2011). in autocrine or paracrine fashion in a wide range of It is speculated that binding proteins may not only extra-hepatic tissues. Tissues produce IGF-1 protein enhance or subdue IGF-1/IGF-1R interaction but in response to growth hormone during periods of help specify which isoform should predominate. pre/post-natal development, exercise and injury. Biotech and Clinical Application: Recombinant Inhibited in undernourished states, low protein, IGF-1 expressed in e. Coli is being tested either for growth hormone deficiency and growth hormone symptomatic relief, tissue regeneration or receptor insensitivity (Cheng et al., 2006). penetrance reduction in the following diseases or Localisation conditions: - Laron's Dwarfism The protein is post-translationally modified by - Duchenne's Muscular Dystrophy protease cleavage of the signal and E-peptide. The - Amyotrophic Lateral Sclerosis mature protein subsequently binds to one of six - Post ischemic damage to brain (stroke) binding proteins and is then secreted form the tissue - Diabetes and Insulin Insensitivity of origin (IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6). IGFBP3 predominates, binding Homology to 80% of the available IGF-1. The binding proteins Shares some sequence homology to insulin and has increase the half life of IGF, preventing renal a relatively weak affinity to insulin receptors.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 379 IGF1 (Insulin-Like Growth Factor 1 (Somatomedin C)) Panayi ND, Burd R

IGF-1 mediated signal transduction (adapted from Schiaffino, S. et. al, 2011).

enhancing genes. Signaling through these pathways Implicated in results in increased cellular proliferation and anti- Various cancers apoptotic effects; promoting a favorable environment for tumor growth. Note In General, high levels of IGF-1 are found in solid No specific mutations of IGF-1 have been tumors (particularly breast and prostate). Certain connected to genetically acquired diseases. Despite SNP's and IGF-1 haplotypes have been associated this fact, certain haplotypes of IGF-1 have been with increased risk of colon, pancreatic, prostate implicated in the survival of solid tumors. It is and breast cancer. postulated that IGF-1 has tumor promoting effects It is likely that specific haplotypes in combination when its cellular growth/anti-apoptotic functions with other variables can create a more favorable become dysregulated (see oncogenesis below). tumorigenic environment. i.e. Individuals with a Additionally it plays a critical role in the clinical BMI>25, containing specific IGF-1 haplotypes may sequelae of Laron's dwarfism (see below). have a greater risk of developing pancreatic cancer Disease (Cheng et al., 2006). High levels implicated in the survival of solid tumors and Acromegaly. Low levels of IGF- References 1/IGFBP3 consistently found in Laron's Dwarfism; Cheng I, Stram DO, Penney KL, Pike M, Le Marchand L, An Autosomal recessive disease caused by a Kolonel LN, Hirschhorn J, Altshuler D, Henderson BE, mutation in the growth hormone receptor; causing Freedman ML. Common genetic variation in IGF1 and poor ligand interaction with growth hormone and prostate cancer risk in the Multiethnic Cohort. J Natl subsequent low levels of IGF-1. Cancer Inst. 2006 Jan 18;98(2):123-34 Prognosis Mills P, Dominique JC, Lafrenière JF, Bouchentouf M, Tremblay JP. A synthetic mechano growth factor E Peptide Individuals with Laron's are dwarfs with enhances myogenic precursor cell transplantation characteristic facial and anatomical anomalies (flat success. Am J Transplant. 2007 Oct;7(10):2247-59 nasal bridge, prominent forehead, obesity, small Philippou A, Maridaki M, Halapas A, Koutsilieris M. The mandible and phallus). They also exhibit seizures role of the insulin-like growth factor 1 (IGF-1) in skeletal secondary to hypoglycemia. Unlike muscle physiology. In Vivo. 2007 Jan-Feb;21(1):45-54 Achondroplasia, Laron's Dwarfism does not Melnik BC, John SM, Schmitz G. Over-stimulation of respond to GH. Laron Dwarfs have a greater insulin/IGF-1 signaling by western diet may promote resistance to diabetes, cancer and age progression; diseases of civilization: lessons learnt from laron underscoring the function of IGF-1 and its critical syndrome. Nutr Metab (Lond). 2011 Jun 24;8:41 role in cellular metabolism and cell cycle Schiaffino S, Mammucari C. Regulation of skeletal muscle maintenance (Melnik et al., 2011). growth by the IGF1-Akt/PKB pathway: insights from genetic models. Skelet Muscle. 2011 Jan 24;1(1):4 Oncogenesis IGF-1 signaling through RAS/RAF/MAPK and has This article should be referenced as such: been demonstrated to promote breast cancer and Panayi ND, Burd R. IGF1 (Insulin-Like Growth Factor 1 prostate tumorigenesis. IGF-1/IGF-R interaction (Somatomedin C)). Atlas Genet Cytogenet Oncol induces the transcription of survival/ growth Haematol. 2014; 18(6):378-380.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 380 Atlas of Genetics and Cytogenetics in Oncology and Haematology

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Gene Section Review

S100A4 (S100 Calcium Binding Protein A4) Gajanan V Sherbet School of Electrical, Electronic and Computer Engineering, University of Newcastle upon Tyne, UK and Institute for Molecular Medicine, Huntington Beach, CA, USA (GVS)

Published in Atlas Database: November 2013 Online updated version : http://AtlasGeneticsOncology.org/Genes/S100A4ID42192ch1q21.html DOI: 10.4267/2042/53767 This article is an update of : Sherbet GV. S100A4 (S100 calcium binding protein A4). Atlas Genet Cytogenet Oncol Haematol 2011;15(10):877-886.

its structure, translation product and tissue Abstract distribution were described by Jackson-Grusby and Review on S100A4, with data on DNA/RNA, on colleagues. The sequence of 18A2 was similar to the protein encoded and where the gene is that of the 2A9 clone described by Calabretta and implicated. collegues. Identity DNA/RNA Other names: 18A2, 42A, CAPL, FSP1, MTS1, Note P9KA, PEL98 Starts 153516089 bp from pter; ends 153522612 bp HGNC (Hugo): S100A4 from pter; 6524 bases; orientation minus strand. Location: 1q21.3 Homo sapiens chromosome 1, GRCh37 primary reference assembly. Local order NCBI reference sequence: NC_000001.10, The 1q21 locus harbours the epidermal NT_004487.19. differentiation complex (EDC) encompassing a 2.05 Mbp of human genomic DNA. The S100 Description family genes except the S100beta are arranged in The human S100A4 gene has four exons. Exon 1 is the following order: 1 cen-S100A10-S100A11- non-coding and exons 2 and 3 are coding exons. THH (trychohyalin)-FLG (filaggrin)-IVL Exon 2 with the start codon and encodes N-terminal (involucrin)-LOR (loricrin)-S100A9-S100A12- EF hand and exon 3 encodes the C-terminal EF- S100A8-S100A7A-S100A7P1-S100A7L2- hand. The fourth non-coding exon occurs in the 5'- S100A7P2-S100A7-S100A6-S100A5-S100A4 - UTR. S100A3-S100A2-S100A16-S100A14-S100A13- S100A1-1qtel. S100beta is located on 21q22.3 Transcription (Schäfer et al., 1995; Marenholz et al., 1996; Two variant RNA transcripts from Source Search. Mischke et al., 1996; GeneLoc version 2.41: The NM_019554 is a longer transcript. pseudogenes S100A7P1: HGNC 21654, NM_002961 possesses alternate 5'-UTR; both S100A7P2: HGNC 21656). S100A11 pseudogenes encode the same protein isoform. have been listed (Pseudogenes.org); they are not NCBI reference sequence NM_019554 ver 01954.2 shown here. 564 bp mRNA; NM 002961 ver 002961.2; 512 bp The 18A2 cDNA/mRNA (S100A4) was cloned and mRNA.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 381 S100A4 (S100 Calcium Binding Protein A4) Sherbet GV

Figure 1. A. Chromosome 1 ideogram showing the location of S100A4 based on National Library of Medicine Handbook. B. The position of S100A4 is based on Entrez Gene ID 6275 (not drawn to scale).

Splice variants have been reported of S100A4. In al.. One would note nonetheless that Alternative human osteosarcoma, alternative splicing within the Splicing and Transcript Diversity (ASTD) have 5'-untranslated region (UTR) generates the two listed 12 variant transcripts. variants. Both variants, hu-mts1 and hu-mts1 (var), Regulation of transcription contain one open reading frame, differ slightly in Binding sites for several transcription factors have translational capacity; possess similar stability. The been identified in the promoter of S100A4. hu-mts1 and hu-mts1 (var) splice variants with SABiosciences ChIP-qPCR Assay database lists 19 exons 1, 2, 3, and 4 and another with exons 1, 3 and p53 binding sites. 4, may be differentially expressed. The hu-mts1 Multiple NFAT (nuclear factor of activated T cells) (var) is expressed in the colon but not in the liver; it transcription factor consensus binding sites; NF- was not found in leukocytes, neutrophils, kappaB related binding site (Tulchinsky et al., macrophages and lymphocytes. The hu-mts1 1997). Much evidence is also available regarding variant predominated in human breast carcinoma activation of NF-kappaB by S100A4. S100A4 can (SK-BR-3) and lung carcinoma (A549) is activate NF-kappaB via the classical pathway predominant (Ambartsumian et al., 1995). The mediated by MEKK/IKK β; S100A6 and S100P also differential association of the variants has been are capable of exerting pro-metastatic effects again described in gastric cancers, which seems to relate by activating the NF-kappaB pathway. to disease state possibly relates to progression; Experimentally induced expression of S100A4 is however the expression status of the variants in the inhibited by NF-kappaB inhibitors. Aside from lymph node metastases is not known. A splice these, several other regulatory pathways may be transcript with loss of non-coding exon 1a/1b, but identified, e.g. the Wnt/ β-catenin/TCF, HIF/HER exons 2 and 3 present has been described in among others, as evidenced by the established infiltrating carcinoma of the breast by Albertazzi et phenotypic expression induced by the gene.

Figure 2. Based on USCS GRCh37/hg19 Feb. 2009. Not drawn to scale.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 382 S100A4 (S100 Calcium Binding Protein A4) Sherbet GV

Figure 3. Sequence.

S100A4 has been postulated to signal via RAGE configuration. (receptor for advanced glycation end products) NCBI sequence: NM_002961; NP_002952; which is known to activate NF-kappaB. NM_019554; NP_062427; UniProtKB/Swiss-Prot: A composite enhancer consisting of 6 cis-elements P26447. has been identified in the first intron of murine Features S100A4. This interacts with Sp1 and AP-1 family EF-hand domains: members and CBF (core binding factor alpha) and EF hand 1: length 36; position 12-47, KRC (zinc finger transcription factor kappa EF hand 2: length 36; position 50-84. recognition component) transcription factors. Target protein interaction domains: in the active Pseudogene state S100A4 interacts with many target proteins e.g. p53 family proteins, HDM2, Annexin II, F- None reported. actin, tropomyosin, and heavy chain of non-muscle myosin IIA, among others. In a closed Protein conformational state S100A4 is inactive, but the Description protein assumes an open conformation upon calcium binding. In the altered configuration Human S100A4 (also mouse and rat S100A4) S100A4 can interact with target proteins. These contains 101 aminoacid residues and is approx 12 target proteins interact with specific binding kDa in size. In common with most S100 family domains of S100A4, which are accessible upon members, S100A4 is an antiparallel homodimer conformational change of the apoprotein upon Ca 2+ stabilised by noncovalent interactions between two binding. The Rudland/Barraclough group has helices from each subunit forming an X-type four- shown that specific mutations that inhibit self- helix bundle. Each subunit has two calcium-binding association of S100A4 markedly reduce its EF-hands linked by the intermediate hinge region metastasis promoting effects. The mutations reduce and a distinctive C-terminal extension. A pseudo- self-association and reduce the affinity of S100A4 EF hand formed by helices 1 and 2 and the pseudo- to two target proteins viz. p53 and non-muscle EF-hand and a canonical EF-hand that are brought myosin heavy chain isoform A. The interaction into proximity by a small two-stranded antiparallel between S100A4 and target proteins can possibly beta-sheet. The hinge region and the C-terminal also be disrupted by the packaging of S100A4 in loop of S100 proteins are involved in target protein such a way as to sequester S100A4 dimers. binding. Calcium binding produces a Inhibition of S100A4 polymerisation by conformational change, which leads to the exposure suppressing TG2 (tissue transglutaminase 2) of hydrophobic pocket of residues in helices 3 and function has resulted in the inhibition of cell 5, the hinge region and the C-terminal loop. This migration in vitro. This is inspired by the fact that conformational change is required for target protein TG2 is a cross-linking protein. Treatment of cells in binding. S100A4 might be post-translationally vitro with EGF seems to up regulate the expression modified. Charged variants conceivably resulting of EGFR and TG2 accompanied by enhanced cell from post-translational changes have been migration. S100A4 over expressing tumours not described in one report, but no confirmation of infrequently tend to be EGFR postive; so tissue these findings has been forthcoming. However, transglutaminase could be promoting EGFR calculations from the predicted isoelectric point of dimerisation and facilitate EGF/EGFR signalling. S100A4 and separation of the charged variants from the major spot would suggest that two variants Expression may have displayed 17.4 and 26.1% and a third S100A4 is distributed ubiquitously in normal variant with a possible highly extended form and tissues (Mazzucchelli, 2002). nearly 2.6-fold increase in net negative charge. For expression profile: Human Protein Atlas Alterations in net molecular charge of this (HPA): CAB002618 and Human Protein Reference magnitude and charge distribution can alter protein Database HPRD.

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Figure 4. S100A4 undergoes a calcium-dependent conformational rearrangement that exposes the protein target binding cleft. Ribbon diagrams showing the NMR solution structure of apo-S100A4 (PDB code 1M31) and the X-ray structure of calcium- bound S100A4 (PDB code 2Q91). Following the addition of calcium (yellow spheres), helix 3 (green helix in dark blue monomer) moves to expose the target bind cleft. This conformational rearrangement is required for S100A4 binding to protein targets. The author is grateful to Anne R. Bresnick, Ph.D., Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, for providing this illustration and the brief legend.

S100A4 occurs in many forms of human cancer, relate to aggressive tumour behaviour and poor e.g. breast, colorectal, liver, lung, head and neck, prognosis. Translocation to the nucleus has been ovarian, endometrial, pancreatic, renal, testicular, associated with EMT induced by TGF-β/Smad and prostate cancers, and melanoma; also in many signalling. IL-induced translocation seems to cell lines of myeloid, lymphoid, lung and brain require sumoylation of specific lysine residues and origin and cell lines derived from many forms of in this way conceivably regulating target gene leukaemias. The expression of the gene is regulated expression. Expression patterns need to be explored by methylation. Over expression correlates with in more than one tumour system. This might be hypomethylation and the frequency of crucial in the development of strategies of treatment hypomethylation relates to tumour progression, e.g. targeting S100A4, especially with the postulated in ovarian cancers. There is no implication at link of S100A4 expression with chemoresistance. present that the degree of methylation is related to Function expression. S100A4 has been implicated in other human diseases, e.g. Crohn's disease and S100A4 protein promotes metastasis, functions as a rheumatoid arthritis. counter point to metastasis suppressor nm23, and is implicated in the regulation of the cell cycle, cell Localisation proliferation, motility, invasion, tubulin S100A4 occurs extracellulary and also in polymerisation, and angiogenesis. S100A4 might cytoplasmic and nuclear location. Differential suppress expression of other suppressor genes e.g. distribution has been reported between stromal PRDM2 and VASH1. PRDM2 (PR domain components of primary and metastatic tumour. containing 2, with ZNF domain) is a tumour Patterns of distribution could vary between tissues suppressor gene encoding a zinc finger protein. and between species. No firm functional link has VASH1 (vasohibin 1) inhibits cell migration, been made with the site/s of localisation. proliferation and tumour growth and angiogenesis. Intracellular distribution is an important factor in S100A4 promotes metastatic spread of cancer as determining genetic activity. demonstrated by gene transfer studies. Its It may be noted here that S100A4 is often expression has shown clear correlation with tumour expressed in component inflammatory cells of spread to lymph nodes and with prognosis. tumour stroma. It has been postulated that Cell cycle, cell proliferation, tumour growth and interactions between the stroma and tumour cells apoptosis. lead to the expression of the protein and modulate S100A4 binds to and forms complexes with p53 to its function in either or both. However, both the regulate cell cycle progression. P53 has been postulate and its potential influence in tumour confirmed as a target of S100A4, which stabilises progression are yet to be established. p53. There is conclusive evidence that S100A4 The pattern of intracellular distribution of many binds to C-terminal regulatory region of p53. genetic determinants has been found to be highly S100A4 and certain other members of the S100 relevant to invasion and metastasis. Nuclear family bind to TAD transactivation domain location of S100A4 was shown some while ago to (residues 1-57) of p53.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 384 S100A4 (S100 Calcium Binding Protein A4) Sherbet GV

Figure 5.

They may also affect p53 function by binding to the regulator of E-cadherin. The TGF-β family receptor tetramerization domain of p53 (residues 325-355) Activin involvement has been implicated. and interfering with intracellular translocation and Invasion, motility, and intercellular adhesion. subcellular localisation. One of the targets of S100A4 involved in cell This interaction is suggested to be linked with p53 motility is myosin filaments. Myosin II consists of function. Nineteen p53 binding sites have been two heavy chains (MHC) with globular domains identified in the promoter of S100A4 which interact with F-actin. The tail domains of (SABiosciences ChIP-qPCR Assay). S100A4 also heavy chains form a coiled-coil tail that participates influences waf1 ID: 139> and mdm2, a regulator of in the assembly of myosin filaments. Wrapped p53 function and the apoptosis family bax gene. It round the neck region of each heavy chain are the binds to N-terminal domain of mdm2. Signalling essential and the regulatory light chains. pathways include P53-Rb/stathmin/p53 down Phosphorylation of the regulatory light chain and stream effectors, e.g. p21 waf , p16 etc. P53/stathmin also of MHC plays an important part in the signalling modulates microtubule dynamics and cell assesmbly of myosin II monomers into filaments. division. Furthermore, p53 and down stream target S100A4 inhibits CK2-mediated phosphorylation of apoptosis family genes such as BNIP3, caspases; MHC, inhibits the assembly of myosin monomers calpain/Fas (?) are postulated as important into filaments. The affinity of S100A4 for the pathways in S100A4 signalling. Knockdown of myosin-IIA can be reduced by CK2-mediated S100A4 has been reported to lead to apoptosis. The phosphorylation. S100A4 destabilises MHCIIA transcription factor NF-kappaB which involved in filaments phosphorylated by PKC and inhibits the anti-apoptosis has been implicated in S100A4 assembly of monomers. PKC and CK2 can signalling. phosphorylate distinct serine residues but yet be S100A4 proliferative signalling seems to involve additive in their effect. The outcome is that S100A4 epidermal growth factor receptors (EGFR). EGFR promotes dissociation of the filaments and prevents expression correlates with S100A4 expression. self assembly of monomers resulting in enhanced Interactive signalling with HER2 might be migration. Thus S100A4 seems to provide a postulated with the finding that S100A4 stimulates mechanistic link between the actomyosin EGFR/HER2 receptor signalling and on the cytoskeletal and migration. identification in human S100A4 promoter of an Signalling systems include modulation of HER2 response element 1099-1487 bp up stream of cytoskeletal dynamics; cadherin/catenin complex the transcription start site. The interaction of cytoskeletal linkage and significantly a TCF, a S100A4 with the TGF-beta system via Smad has component of the canonical Wnt signalling system, also been reported. S100A4 seems able to bind to binding site has been identified in the S100A4 the N-ter region of Smad3. TGF-beta is an promoter and S100A4 directly binds heterodimeric important activator of epithelial mesenchymal beta-catenin/TCF complexes; CD44/cytoskeletal transition leading to acquisition of invasive ability. linkage; ECM associated proteolytic enzyme The interaction between S100A4 and Smad thus system/ECM remodelling, affects tubulin falls in place with the metastasis-promoting polymerisation. S100A4 and tumour suppressor function of the former. Some of these pathways are nm23 exert opposite effects on tubulin dynamics. pictorially represented above (figure 5). S100A4 Two C-terminal lysine residues are required for activates EMT via up regulation of Snail, a negative enhanced motility and invasion and interaction with

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target proteins. The connective tissue growth factor found to reduce tumour growth and tumour (CTGF) has been reported to up regulate S100A4 associated microvessel density. expression and inhibition of S100A4 blocks CTGF- Osteopontin was identified as a metastasis- induced cell motility. associated protein some time ago. Many strands of S100A4 seems to function via the MMP/TIMP evidence suggest that osteopontin is an system in promoting invasion as well as induction intermediary in S100A4 signalling pathway. In of angiogenesis. S100A4 is over expressed in breast cancer expression of osteopontin in the invasive glioma cell lines together with down background of S100A4 has generally correlated regulation of TIMP-2, indicating a close linkup of with poor patient survival. S100A4 with the MMP system in the promotion of Osteopontin is associated with several activated invasion. NF-kappaB pathways. S100A4 induces the Angiogenesis signalling occurs via activation of expression and secretion of osteopontin in some MMP/TIMP; activation of angiogenic factors osteosarcoma cell lines in an NF-kappaB-dependent VEGF/endothelial cell proliferation; MetAP2/p53- fashion. Inhibition of osteopontin inhibits tumour mediated inhibition of endothelial cell proliferation. development and angiogenesis; inhibition of both S100A4 stimulates angiogenic signalling in breast might result in synergistic suppression of tumour cancer. An indirect link is suggested by the progression. inhibition of S100A4 by Interferon-gamma which Shown below are the potential pathways of S100A4 might inhibit angiogenesis by down regulating signalling in cell motility/invasion and VEGF expression. Hypoxia is a major regulator of angiogenesis, emphasising the possibility that angiogenesis. HIF-1α (hypoxia-inducible factor-1α) S100A4 seems able to influence many significant is a transcription regulator in hypoxia. It can systems leading to angiogenesis. activate VEGF to induce angiogenesis and TGF-α and promotes cell survival. Exposure to hypoxia Homology has been correlated with reduced methylation of the Sequence homology to protein from Pan hypoxia response element in S100A4's promoter troglodytes (Chimpanzee) (Gene ID: 457320; region and enhanced HIF binding to the promoter Protein NCBI RefSeq: XP_001138744.1). and increased transcription of the gene together Bos taurus (Bovine) (Gene ID: 282343). with increased cell proliferation and invasion. Canis lupus familiaris (Gene ID: 403787; NCBI Given that HIF also promotes VEGF expression reference sequence: NP_001003161.1; protein: one can see a potential two pronged approach to NP_777020.1). control tumour growth with HIF inhibition. Some Sequence homology 93% to murine S100A4 clinical studies are underway to study the effects of (Entrez Gene ID 20198; NP_035441). Sorafenib-mediated inhibition of HIF-1α and Sequence homology 91% to rat protein (Entrez VEGF. In laboratory studies Sorafenib has been Gene ID 24615; NP_036750).

Figure 6.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 386 S100A4 (S100 Calcium Binding Protein A4) Sherbet GV

Also indicative of poor prognosis is high S100A4 Mutations expression coupled with reduced E-cadherin Note expression in pancreatic, oral squamous cell Many SNPs have been identified; 7 shown in NCBI carcinoma and in melanoma. S100A4 expression is and 26 in Applied Biosystems data source. The inversely related with expression of metastasis NCBI Entrez SNP database lists 19 submissions. suppressor nm23 and with prognosis of breast Chromosomal rearrangements cancer. The locus 1q21 is a hotspot for chromosomal Cytogenetics rearrangements, microdeletions and duplications; No cytogenetic data are available. significance uncertain and there are no clear Abnormal protein implications for metastasis. No translocations No fusion proteins or hybrid genes involving leading to hybrid S100A4 have been recorded. S100A4 are known. There are 11 common and 1 rare fragile sites on chromosome 1. The common FRA1F occurs in Breast cancer 1q21. Chromosome 1 is prone to sister chromatid Note recombination (SCR) and >70% SCRs occur at the Both tumour and serum levels are reportedly fragile sites or in the same band as the fragile sites, enhanced in breast cancer patients. S100A4 but no link with S100A4 established. expression is inversely related to that of the Germinal metastasis suppressor nm23 in breast cancers. None reported. Tumour levels might correlate with proliferative state and shown to be linked with p53 dysfunction. Somatic S100A4 proliferative signalling seems to involve No simple mutations, gene fusions, or structural epidermal growth factor receptors (EGFR). Breast variants detected in breast and colorectal cancers that are high S100A4s expressers tend to be carcinomas and in gliomas (Cosmic: Catalogue Of oestrogen (ER)/progesterone receptor (PR) Somatic Mutations In Cancer, Welcome Trust negative. Sanger Institute). Given that ER/PR expression is inversely related to No mutations have been found coding regions in the expression of epidermal growth factor receptors, human, canine and feline S100A4. Mutating ER/PR status together with S100A4/nm23 phenylalanine 72 to alanine reduces functional expression status could provide significant leads to effectiveness. Toombak (tobacco rich in tobacco- the prediction of prognosis. S100A4 signalling specific nitrosamine) dipping (placing between the could interact with HER2 function; this is lower lip and gums) has been indirectly linked with suggested by the finding that S100A4 stimulates S100A4 mutations in oral squamous cell EGFR/HER2. carcinoma, but mutations have been described also Up regulated expression was associated with in non-dippers. The carcinoma from dippers had 4 increased tumour angiogenesis and this would be mutations (one transition, 3 transversions) and non- expected to contribute to the invasive spread of snuff-dippers showed 3 mutations each (one breast cancer. transition, 2 transversions). The suggestion is that Of some interest is the suggestion that S100A4 mutations could be complementing the phosphosulindac might target and induce apoptosis effects of more frequent mutations of p53 and of breast cancer stem cells. waf1 p21 . Prognosis S100A4 may be regarded as an independent Implicated in predictor of prognosis. General notes on association with Colorectal cancer human cancer Note Note Primary cancers show enhanced S100A4 S100A4 has been implicated in the progression and expression and associated with metastatic disease in prognosis of several forms of human cancer, e.g. the lymph nodes. breast, colorectal, gastric, pancreatic and bladder Up regulation of its expression has been correlated cancer, SCLC and oesophageal squamous cell with enhanced invasion and nodal dissemination. carcinoma, among others. Poor prognosis Nuclear expression has been reported to be a associated with high S100A4 expression is prognostic indicator. accompanied by clear signs of disease progression, As in the case of breast cancer there are indications e.g. high histological and clinical grades and that S100A4 might interact with and abrogate p53 involvement of lymph nodes. function. The implied association with aggressive

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disease is underscored by the emergence of Perineural invasion has been associated with correlated expression of S100A4 with the enhanced S100A4 expression. Worthy of note is extracellular matrix metalloproteinase inducer that perineural invasion is a feature linked with CD147/Basigin/EMMPRIN but a causal link is yet tumour spread and poor prognosis and its to be established. One should note in this context correlation with S100A4 might have implications that S100A9 can also bind CD147. for disease management. Efforts are being made to inhibit Wnt/ β-catenin Prognosis mediated targeting of S100A4 using sulindac. Some preliminary evidence is available indicating Bladder cancer that S100A4 expression levels relate to shorter Note overall survival of patients with pancreatic cancer. Higher expression S100A4 has been observed and Gastric cancer this might be associated with muscle invasion. Note Prognosis Higher expression of S100A4 has been noted in High expression has been related to decreased gastric cancer and correlated with the presence of survival. the tumour in lymph node and the occurrence of Oesophageal squamous cell distant metastases, and with poor prognosis. Consistent with the situation in certain other forms carcinoma of cancer, in gastric cancer S100A4 levels inversely Note relate to E-cadherin expression. Indeed, down S100A4 expression levels negatively corresponded regulation of E-cadherin has been found to occur in with E-cadherin expression in ESCCs patients with parallel with hypomethylation of S100A4. metastatic disease. In vitro studies of migration of Melanoma cells with experimentally enhanced S100A4 expression have lent support to the perceived Note relationship. Transfection of gall bladder carcinoma A marked inverse relationship has been described cell lines E-cadherin has led to the suppression of between S100A4 and E-cadherin in these tumours. S100A4. Gliomas Ovarian cancer Note Note S100A4 is over expressed in invasive glioma cell The expression of nuclear S100A4 expression is lines together with down regulation of TIMP-2, associated with more aggressive disease in primary indicating a close linkup of S100A4 with the MMP carcinoma where the level of expression has been system in the promotion of invasion. This ability to reported to be higher in solid tumours than in induce angiogenesis and metastatic dissemination effusions. could be complemented and indeed augmented by Lung cancer its postulated ability to enhance endothelial permeability. Occludin is a transmembrane tight- Note junction protein essential for maintaining integrity Higher expression of S100A4 has been encountered of both epithelia and endothelia. S100A4 is said to in squamous cell but not adenocarcinoma of the reduce occludin expression and could compromise lung. in this way the integrity of the vascular Prognosis endothelium. This might enhance endothelial A large study of the expression levels has revealed permeability. Indeed the ability of tumours to form S100A4 to be significantly predictive of survival in metastatic deposits in the brain has been attrributed squamous cell but not adenocarcinoma of the lung. to possible dysfunction of the blood brain barrier. If S100A4 was significantly associated with patients' future work provides substantive evidence for this, poor prognosis in lung squamous cell carcinoma this might have potential implication for the but not lung adenocarcinoma. management of metastatic disease. But then it ought Pancreatic cancer to be recognised here that gliomas do not normally metastasise to extracranial sites. Glioma cell lines Note do over express S100A4, but there is little S100A4 up regulation might be accompanied by information concerning the tumours. S100A8 and reduced E-cadherin expression. This inverse S100A9 have been implicated to impede diapedesis relationship has also been encountered in melanoma with up regulated expression of the adhesion and oral squamous cell carcinoma cell lines. This proteins ICAM-1 and VCAM-1. S100A4 and A9 might generate an additive effect on tumour can form heterodimers in vivo and the heterodimers aggression. carry features that resemble S100A9 homodimers in

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respect of the ability to bind pro-inflammatory number of viable cardiac myocytes. The receptors. It is not known if S100A4 and A9 ERK1/ERK2 signalling system has been found to heterodimers and A9 homodimers differ with be activated in these processes. regard to effects on endothelial permeability. Two putative neurotropic motifs have been Crohn's disease (a form of irritable identified in S100A4 and neuroprotective function has been attributed to the protein. bowel disease) S100A4 might be associated with PAH (pulmonary Note arterial hypertension) and putatively linked with S100A4 expression is increased in structure 17 β-oestradiol modulated S100A4/RAGE fibroblasts of fibrostenosing Crohn's disease signalling. A clinical trial is underway at present to promoting intestinal fibroblast migration. study S100A4 as a marker in PAH treatment Rheumatoid arthritis (NCT01305252). Note Breakpoints Increase S100A4 mRNA found in proliferating synovial fibroblasts. Also protein expression up Note regulated in rheumatoid arthritis synovial tissues A 1q21 breakpoint was described some time ago in and linked with joint invasion. IL-7 and S100A4 renal cell carcinoma (RCC)-associated occurs in cartilage osteoarthritis and can lead to (X;1)(p11;q21) translocation. This has been increased MMP-13 production by chondrocytes. mapped to the S100 gene cluster, but its link with The JAK/STAT/RAGE signalling has been S100A4 is uncertain. No translocations involving implicated here. S100A4 have been recorded. No fusion proteins or Psoriasis hybrid genes involving S100A4 are known. There are 11 common and 1 rare fragile sites on Note chromosome 1. The common FRA1F occurs in Many S100 proteins are found in the dermis. 1q21. Chromosome 1 is prone to sister chromatid S100A4 is up regulated in the dermis and colossal recombination (SCR) and >70% SCRs occur at the release of the protein has been reported. Enhanced fragile sites or in the same band as the fragile sites, stabilisation of p53 near cells expressing S100A4 but no link with S100A4 has been established. has been noticed. It appears to affect cell proliferation and induce angiogenesis. Suppression To be noted of S100A4 using antibodies seems to suppress vascularisation of psoriatic skin xenografts, Note together with diminution of both number and size S100A4 expression has been linked with of blood vessels. There are no suggestions of co- chemoresistance, but the mechanisms involved operative or interactive function of S100A4 with remain to be elucidated. Whether this occurs via S100A7 (Psoriasin). engagement of RAGE by S100A4 and activation of Cardio-vascular, nervous and RAGE signalling leading up to chemoresistance is an avenue yet to be explored. pulmonary systems S100A4 activates interacting and multi-functional Note signalling systems, including EMT signalling The focus in this review is on the role of S100A4 in systems such as Wnt/ β-catenin, NF-kappaB, and E- the disease process. Indeed, S100A4 is expressed in cadherin among others, highly relevant in the normal as well as in pathological conditions, context of cancer. Mediation of its function by subserves several physiological functions such as osteopontin and potential function of S100 family regulating macrophage motility, and participates in proteins to act as RAGE ligands are imporatnt fibrosis and tissue remodeling in several diseased considerations. These would make S100A4 an and damaged states, e.g. fibrosis of the kidney and eminently valuable chemotherapeutic target. Some loss of renal function, cardiac fibrosis, tissue repair downstream effectors of the S100A4 pathways and regeneration and wound healing; central might also lend themselves as targets of interest. nervous system injury, and pulmonary vascular The diversity of biological effects flowing from the disease. inappropriate expression of S100A4 and S100A4 may be involved in disorders of these interactions of S100A4 signalling with several systems, but data currently available are somewhat systems which modulate biological response would fragmentary. Both S100A4 mRNA and protein are merit investigations into suppressing S100A4 said to be up regulated in the hypertrophic hearts. expression as a possible therapeutic approach. Up regulation is associated with hypertrophy S100A4 function is allied with growth factor and induced by aortic stenosis or myocardial infarction. steroid hormone receptors and osteopontin which is In vitro, recombinant S100A4 protein increases the itself subject to regulation by Wnt, NF-kappaB

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among others, so here is ample provision of options motility. Of this RhoA has been linked with available for therapeutic studies. Also the perceived membrane ruffling and cell motility. Recently link up between EMT and S100A4 expression S100A4 has been shown to bind to Rhotekin, a affords fresh avenues of approach. RhoA interacting scaffold protein, via the RBD The antihelminth drug Niclosamide which targets (Rho binding domain), but Rho and S1004 seem to the Wnt/ β-catenin pathway can suppress S100A4 bind to different residues of RBD. The Rho- expression with parallel inhibition of cell Rhotekin-S100A4 complex generates the invasive proliferation, migration, promotion of apoptosis, phenotype. At the practical level it is worthy of note and metastatic spread in vitro and xenograft tumour and future pursuit that Paclitaxel at low doses well models. below therapeutic levels has been shown to inhibit Xanthohumol is a prenylflavonoid antioxidant, S100A4 in the nuclear compartment and in parallel derived from the female flowers of the hops plant reduce cell migration and invasion in vitro of (Humulus lupulus). It was identified to possess human cholangiocarcinoma cells in a Rho-GTPase anticancer properties some while ago. Recent work mediated manner. has shown its ability to suppress cell proliferation, Membrane bound mucins such as MUC16 (CA125) invasion and tumour progression. The flavonoid has and MUC2, MUC4, MUC13 have been associated been found to inhibit proliferation and invasion in with cancer malignancy. MUC16 is associated with many breast cancer cell lines, including the triple the formation of peritoneal metastases in ovarian negative breast cancers MDA-MB-231 cells. It cancer. MUCs 2 and 13 are over expressed in induces apoptosis and might be functioning by pancreatic cancer. MUC4 is overexpressed in inhibiting Akt and NF-kappaB activation. The oesophageal, lung and colon cancer. Its expression postulated suppression of tumour progression by correlates with progression of pancreatic cancer. Xanthohumol is based on assays purportedly Experimental suppression of MUC4 in oesophageal resembling intravasation of tumour emboli through cancer cells by shRNA reduces S100A4 expression defects in the endothelial barrier, so needs much and reduces cell proliferation and tumorigenic further scrutiny. It would be necessary to perform ability as compared with MUC4 expressing parent in vivo studies, ethically undesirable they might be, cells. and ascertain these claims by using stringent criteria The causal linkup is not established; however being to evaluate intravasation of tumour cells and integral membrane glycoproteins possibly they formation of metastases. anchor S100A4 to the cell membrane and interferes It may be noted here that Xanthohumol is said to with activation of S100A4 signalling pathways. inhibit cell motility and EMT activation and this is Growth factors are known to induce accompanied by inhibition of S100A4 among other phosphoryalation of the cytoplasmic domain of determinants. Also of interest is that Xanthohumol MUC1 and activate nuclear localization of MUC1 induces apoptosis which is caspase-mediated and and β-catenin and participate in growth factor requires Annexin I. This might be of particular signalling. interest since S100A4 as well as other S100A Little is known about the involvement of the proteins bind to and regulate the function of many immune system in relation to S100A4 function, but target proteins which include annexins. Disruption there are indications that it may be recruited to NK of Annexin/S100A11 alters the phenotypic cell immune synapses and possibly contribute to behaviour. Annexins display divergent effects on immune synapse formation. Whether S100A4 cell proliferation, apoptosis and invasion. The participates in restraining NK lytic function or effects may relate to whether the specific S100A is promotes the formation and function of inhibitory a tumour promoter or suppressor. It would be synapses is uncertain. The cytoplasmic Src kinases needless iteration that further investigation is regulate the activating or inhibitory pathways and it warranted. A clinical study of its pharmacokinetics has been suggested S100A4 might be capable of has been undertaken (NCT01367431). influencing the operation of these pathways. CTLs Phenanthrenes are a class of compounds originally and NK cells induce apoptosis via the pore forming obtained from various members of Orchidaceae and protein perforin and granzyme B pathway. S100A4 described to possess cytotoxic, antimicrobial and may not be involved in this apoptosis pathway, but anti-inflammatory activity. They may be could be involved in the FasL pathway. therapeutically important in preventing metastasis The non-steroidal anti-inflammatory agent sulindac by inhibiting the interaction of S100A4 with its has been found to interfere with Wnt signalling. target molecules (see Bresnick AR patent The β-catenin/TCF transcription complex targets WO2011146101 A1). and regulates S100A4. Using an in vivo model The induction of motility response to S100A4 involving intrasplenic xenografting of colon cancer seems to be mediated by Rho signalling. Rho, Rac cells, sulidac has been shown to down regulate and Cdc42 are most prominent players in S100A4 promoter activity and expression together cytoskeletal reorganization and modulation of cell with inhibition of Wnt/ β-catenin signalling.

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increased in idiopathic inflammatory myopathies. confers neuroprotection in brain injury. Nat Commun. Rheumatology (Oxford). 2011 Oct;50(10):1766-72. doi: 2012;3:1197. doi: 10.1038/ncomms2202. 10.1093/rheumatology/ker218. Epub 2011 Jun 28. Du M, Wang G, Ismail TM, Gross S, Fernig DG, Dempsie Y, Nilsen M, White K, Mair KM, Loughlin L, Barraclough R, Rudland PS.. S100P dissociates myosin Ambartsumian N, Rabinovitch M, Maclean MR.. IIA filaments and focal adhesion sites to reduce cell Development of pulmonary arterial hypertension in mice adhesion and enhance cell migration. J Biol Chem. 2012 over-expressing S100A4/Mts1 is specific to females. May 4;287(19):15330-44. doi: 10.1074/jbc.M112.349787. Respir Res. 2011 Dec 20;12:159. doi: 10.1186/1465-9921- Epub 2012 Mar 6. 12-159. Fabris L, Cadamuro M, Sambado L, Beretta I, Spirli C, Lu W, Lin C, Roberts MJ, Waud WR, Piazza GA, Li Y.. IndraccoloS , Strazzabosco M.. Selective reduction in Niclosamide suppresses cancer cell growth by inducing S100A4 nuclear expression by low dose paclitaxel halts Wnt co-receptor LRP6 degradation and inhibiting the invasiveness of human cholangiocarcinoma cells through a Wnt/b-catenin pathway. PLoS One. 2011;6(12):e29290. Rho-A/Cdc42 dependent mechanism. J Hepatol 2012 doi: 10.1371/journal.pone.0029290. Epub 2011 Dec 16. April; 56, Suppl 2, S112. Mace EM, Orange JS.. Multiple distinct NK-cell synapses. Horiuchi A, Hayashi T, Kikuchi N, Hayashi A, Fuseya C, Blood. 2011 Dec 15;118(25):6475-6. doi: 10.1182/blood- Shiozawa T, Konishi I.. Hypoxia upregulates ovarian 2011-10-381392. cancer invasiveness via the binding of HIF-1? to a hypoxia-induced, methylation-free hypoxia response Nishioku T, Furusho K, Tomita A, Ohishi H, Dohgu S, element of S100A4 gene. Int J Cancer. 2012 Oct Shuto H, Yamauchi A, Kataoka Y.. Potential role for 15;131(8):1755-67. doi: 10.1002/ijc.27448. Epub 2012 Mar S100A4 in the disruption of the blood-brain barrier in 14. collagen-induced arthritic mice, an animal model of rheumatoid arthritis. Neuroscience. 2011 Aug 25;189:286- Li X, Baskin JG, Mangan EK, Su K, Gibson AW, Ji C, 92. doi: 10.1016/j.neuroscience.2011.05.044. Epub 2011 Edberg JC, Kimberly RP.. The unique cytoplasmic domain May 26. of human Fc?RIIIA regulates receptor-mediated function. J Immunol. 2012 Nov 1;189(9):4284-94. doi: Osada T, Chen M, Yang XY, Spasojevic I, Vandeusen JB, 10.4049/jimmunol.1200704. Epub 2012 Sep 28. Hsu D, Clary BM, Clay TM, Chen W, Morse MA, Lyerly HK.. Antihelminth compound niclosamide downregulates Liu LP, Ho RL, Chen GG, Lai PB.. Sorafenib inhibits Wnt signaling and elicits antitumor responses in tumors hypoxia-inducible factor-1? synthesis: implications for with activating APC mutations. Cancer Res. 2011 Jun antiangiogenic activity in hepatocellular carcinoma. Clin 15;71(12):4172-82. doi: 10.1158/0008-5472.CAN-10-3978. Cancer Res. 2012 Oct 15;18(20):5662-71. doi: Epub 2011 Apr 29. 10.1158/1078-0432.CCR-12-0552. Epub 2012 Aug 28. Sack U, Walther W, Scudiero D, Selby M, Kobelt D, Lemm Zhu C, Cheng KW, Ouyang N, Huang L, Sun Y, M, Fichtner I, Schlag PM, Shoemaker RH, Stein U.. Novel Constantinides P, Rigas B.. Phosphosulindac (OXT-328) effect of antihelminthic Niclosamide on S100A4-mediated selectively targets breast cancer stem cells in vitro and in metastatic progression in colon cancer. J Natl Cancer Inst. human breast cancer xenografts. Stem Cells. 2012 2011 Jul 6;103(13):1018-36. doi: 10.1093/jnci/djr190. Epub Oct;30(10):2065-75. doi: 10.1002/stem.1139. 2011 Jun 17. Bresnick AR.. Preventing or inhibiting tumor metastasis in Sherbet GV.. Growth factors and their receptors in cell subject involves administering aromatic compound differentiation, cancer and cancer therapy. E-book; 2011 including 9,10-dihydro-phenanthrene derivative, (1,4) July 12; Elsevier naphthoquinone derivative, quinoline-5,8-dione derivative, to the subject. US Patent WO2011146101-A1; Stein U, Arlt F, Smith J, Sack U, Herrmann P, Walther W, US2013090355-A1, 2013 Apr 11. Lemm M, Fichtner I, Shoemaker RH, Schlag PM.. Intervening in beta-catenin signaling by sulindac inhibits Chen M, Bresnick AR, O'Connor KL.. Coupling S100A4 to S100A4-dependent colon cancer metastasis. Neoplasia. Rhotekin alters Rho signaling output in breast cancer cells. 2011 Feb;13(2):131-44. Oncogene. 2013 Aug 8;32(32):3754-64. doi: 10.1038/onc.2012.383. Epub 2012 Sep 10. Boye K, Nesland JM, Sandstad B, Haugland Haugen M, Maelandsmo GM, Flatmark K.. EMMPRIN is associated Kawakita T, Espana EM, Higa K, Kato N, Li W, Tseng SC.. with S100A4 and predicts patient outcome in colorectal Activation of Smad-mediated TGF-? signaling triggers cancer. Br J Cancer. 2012 Aug 7;107(4):667-74. doi: epithelial-mesenchymal transitions in murine cloned 10.1038/bjc.2012.293. Epub 2012 Jul 10. corneal progenitor cells. J Cell Physiol. 2013 Jan;228(1):225-34. doi: 10.1002/jcp.24126. Hibino T, Sakaguchi M, Miyamoto S, Yamamoto M, Motoyama A, Hosoi J, Shimokata T, Ito T, Tsuboi R, Huh Nomura M, Tanaka K, Wang L, Goto Y, Mukasa C, Ashida NH.. S100A9 is a novel ligand of EMMPRIN that promotes K, Takayanagi R.. Activin type IB receptor signaling in melanoma metastasis. Cancer Res. 2013 Jan 1;73(1):172- prostate cancer cells promotes lymph node metastasis in a 83. doi: 10.1158/0008-5472.CAN-11-3843. Epub 2012 Nov xenograft model. Biochem Biophys Res Commun. 2013 7. Jan 4;430(1):340-6. doi: 10.1016/j.bbrc.2012.11.011. Epub 2012 Nov 15. Chen D, Zheng XF, Yang ZY, Liu DX, Zhang GY, Jiao XL, Zhao H.. S100A4 silencing blocks invasive ability of Scheiter M, Lau U, van Ham M, Bulitta B, Grobe L, esophageal squamous cell carcinoma cells. World J Garritsen H, Klawonn F, Konig S, Jansch L.. Proteome Gastroenterol. 2012 Mar 7;18(9):915-22. doi: analysis of distinct developmental stages of human natural 10.3748/wjg.v18.i9.915. killer (NK) cells. Mol Cell Proteomics. 2013 May;12(5):1099-114. doi: 10.1074/mcp.M112.024596. Dmytriyeva O, Pankratova S, Owczarek S, Sonn K, Soroka Epub 2013 Jan 13. V, Ridley CM, Marsolais A, Lopez-Hoyos M, Ambartsumian N, Lukanidin E, Bock E, Berezin V, Sherbet GV.. Therapeutic strategies in cancer biology and Kiryushko D.. The metastasis-promoting S100A4 protein pathology. E-book 2013 Aug; Elsevier.

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Tamaki Y, Iwanaga Y, Niizuma S, Kawashima T, Kato T, Wang Z, Griffin M.. The role of TG2 in regulating S100A4- Inuzuka Y, Horie T, Morooka H, Takase T, Akahashi Y, mediated mammary tumour cell migration. PLoS One. Kobuke K, Ono K, Shioi T, Sheikh SP, Ambartsumian N, 2013;8(3):e57017. doi: 10.1371/journal.pone.0057017. Lukanidin E, Koshimizu TA, Miyazaki S, Kimura T.. Epub 2013 Mar 1. Metastasis-associated protein, S100A4 mediates cardiac fibrosis potentially through the modulation of p53 in cardiac Yu CC, Tsai CH, Hsu HI, Chang YC.. Elevation of S100A4 fibroblasts. J Mol Cell Cardiol. 2013 Apr;57:72-81. doi: expression in buccal mucosal fibroblasts by arecoline: 10.1016/j.yjmcc.2013.01.007. Epub 2013 Jan 23. involvement in the pathogenesis of oral submucous fibrosis. PLoS One. 2013;8(1):e55122. doi: Tsukamoto N, Egawa S, Akada M, Abe K, Saiki Y, Kaneko 10.1371/journal.pone.0055122. Epub 2013 Jan 31. N, Yokoyama S, Shima K, Yamamura A, Motoi F, Abe H, Hayashi H, Ishida K, Moriya T, Tabata T, Kondo E, Kanai Zhang J, Antonyak MA, Singh G, Cerione RA.. A N, Gu Z, Sunamura M, Unno M, Horii A.. The expression mechanism for the upregulation of EGF receptor levels in of S100A4 in human pancreatic cancer is associated with glioblastomas. Cell Rep. 2013 Jun 27;3(6):2008-20. doi: invasion. Pancreas. 2013 Aug;42(6):1027-33. doi: 10.1016/j.celrep.2013.05.021. Epub 2013 Jun 13. 10.1097/MPA.0b013e31828804e7. Zheng X, Gai X, Wu Z, Liu Q, Yao Y.. Metastasin leads to Viola K, Kopf S, Rarova L, Jarukamjorn K, Kretschy N, poor prognosis of hepatocellular carcinoma through partly Teichmann M, Vonach C, Atanasov AG, Giessrigl B, inducing EMT. Oncol Rep. 2013 May;29(5):1811-8. doi: Huttary N, Raab I, Krieger S, Strnad M, de Martin R, Saiko 10.3892/or.2013.2341. Epub 2013 Mar 12. P, Szekeres T, Knasmuller S, Dirsch VM, Jager W, Grusch M, Dolznig H, Mikulits W, Krupitza G.. Xanthohumol This article should be referenced as such: attenuates tumour cell-mediated breaching of the Sherbet GV. S100A4 (S100 Calcium Binding Protein A4). lymphendothelial barrier and prevents intravasation and Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6):381- metastasis. Arch Toxicol. 2013 Jul;87(7):1301-12. doi: 396. 10.1007/s00204-013-1028-2. Epub 2013 Mar 17.

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Gene Section Short Communication

TGM2 (transglutaminase 2) Lisa Dyer Departments of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA (LD)

Published in Atlas Database: November 2013 Online updated version : http://AtlasGeneticsOncology.org/Genes/TGM2ID44078ch20q11.html DOI: 10.4267/2042/53768 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 Localisation Short Communication on TGM2, with data on Mostly intracellular (cytosol, nucleus and cell DNA/RNA, on the protein encoded and where the membrane); extracellular. gene is implicated. Function Identity Multifunctional enzyme with transglutaminase crosslinking activity (catalyze covalant bonds Other names: G-ALPHA-h, GNAH, TG2, TGC between the ε-amino group of a lysine and the γ- HGNC (Hugo): TGM2 carboxyl group of a glutamine residue) stabilizing Location: 20q11.23 the extracellular matrix (ECM) and cell-ECM interaction; require Ca2+ for catalytic activity; DNA/RNA inhibited by GTP; under stress (loss of Ca2+) may play a role in apoptosis; involved in wound healing Description and inflammation; aberrent overexpression of TG2 The gene encompasses 36838bp; 13 exons; a dense is linked to chemotherapeutic drug resistance. CpG island engulfs exon 1 and the transcriptional start site; negative strand. Mutations Transcription Germinal Two transcript variants 1) 3937bp; 2) 2326bp (C- No germinal mutations have been identified; low terminal truncation exons 11-13); induced by frequency of SNPs. NFkB, retinoic acid, IL-6, TGF-beta1, HRE, AP-1 and GRE. Somatic No somatic mutations have been identified in Protein cancer; can be transcriptionally silenced by CpG Description methylation in cancer (breast and brain); overexpression in many cancers; 3 missense 687 amino acids; 78 kilodaltons; monomer. mutations identified in early-onset type 2 diabetes Expression patients (c.989T>G, c.992T>A, c.998A>G).

Ubiquitously expressed.

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Implicated in References Breast cancer Bernassola F, Federici M, Corazzari M, Terrinoni A, Hribal ML, De Laurenzi V, Ranalli M, Massa O, Sesti G, McLean Oncogenesis WH, Citro G, Barbetti F, Melino G. Role of Overexpression renders culture mammary epithelial transglutaminase 2 in glucose tolerance: knockout mice studies and a putative mutation in a MODY patient. FASEB cells resistant to doxorubicin, associated with J. 2002 Sep;16(11):1371-8 invasive phenotype and epithelial-to-mesenchymal transition; epigenetic silencing has been Mehta K, Fok J, Miller FR, Koul D, Sahin AA. Prognostic significance of tissue transglutaminase in drug resistant documented in many breast cancer cell lines and and metastatic breast cancer. Clin Cancer Res. 2004 Dec patient samples. In 30 patients, TG2 overexpression 1;10(23):8068-76 was found more often in lymph node metastases Porzio O, Massa O, Cunsolo V, Colombo C, Malaponti M, than primary tumors. Bertuzzi F, Hansen T, Johansen A, Pedersen O, Meschi F, Terrinoni A, Melino G, Federici M, Decarlo N, Menicagli M, Ovarian cancer Campani D, Marchetti P, Ferdaoussi M, Froguel P, Prognosis Federici G, Vaxillaire M, Barbetti F. Missense mutations in the TGM2 gene encoding transglutaminase 2 are found in Overexpression of TG2 is associated with negative patients with early-onset type 2 diabetes. Mutation in brief survival in 93 patients (p=0.007). no. 982. Online. Hum Mutat. 2007 Nov;28(11):1150 Oncogenesis Satpathy M, Cao L, Pincheira R, Emerson R, Bigsby R, Overexpression renders ovarian cancer cells Nakshatri H, Matei D. Enhanced peritoneal ovarian tumor resistant to cisplatin and is associated with higher dissemination by tissue transglutaminase. Cancer Res. 2007 Aug 1;67(15):7194-202 tumor stage. Verma A, Mehta K. Tissue transglutaminase-mediated Pancreatic cancer chemoresistance in cancer cells. Drug Resist Updat. 2007 Prognosis Aug-Oct;10(4-5):144-51 Overall survival of stage II pancreatic ductal Ai L, Kim WJ, Demircan B, Dyer LM, Bray KJ, Skehan RR, adenocarcinoma (PDAC) patients with TG2- Massoll NA, Brown KD. The transglutaminase 2 gene (TGM2), a potential molecular marker for mediated loss of PTEN was poor (20.7 months) chemotherapeutic drug sensitivity, is epigenetically compared to 68.6 months for TG2 negative and silenced in breast cancer. Carcinogenesis. 2008 PTEN positive patients. Mar;29(3):510-8 Oncogenesis Cao L, Petrusca DN, Satpathy M, Nakshatri H, Petrache I, Overexpression; resistance to gemcitabine; in 51 Matei D. Tissue transglutaminase protects epithelial ovarian cancer cells from cisplatin-induced apoptosis by PDAC tumor samples overexpression of TG2 was promoting cell survival signaling. Carcinogenesis. 2008 associated with loss of PTEN expression. Oct;29(10):1893-900 Non-small cell lung cancer (NSCLC) Hwang JY, Mangala LS, Fok JY, Lin YG, Merritt WM, Spannuth WA, Nick AM, Fiterman DJ, Vivas-Mejia PE, Prognosis Deavers MT, Coleman RL, Lopez-Berestein G, Mehta K, In 429 Korean patients, TG2 expression was Sood AK. Clinical and biological significance of tissue associated with shorter disease free survival and transglutaminase in ovarian carcinoma. Cancer Res. 2008 correlated with recurrence. Jul 15;68(14):5849-58 Oncogenesis Verma A, Guha S, Diagaradjane P, Kunnumakkara AB, Sanguino AM, Lopez-Berestein G, Sood AK, Aggarwal BB, Overexpression ; cisplatin resistance. Krishnan S, Gelovani JG, Mehta K. Therapeutic significance of elevated tissue transglutaminase Celiac disease expression in pancreatic cancer. Clin Cancer Res. 2008 Autoantibodies against TG2; autoantibodies are Apr 15;14(8):2476-83 deposited in the small-bowl mucosa; TG2 Shao M, Cao L, Shen C, Satpathy M, Chelladurai B, crosslinks gliadin peptides (gluten) derived. Bigsby RM, Nakshatri H, Matei D. Epithelial-to- mesenchymal transition and ovarian tumor progression

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 398 TGM2 (transglutaminase 2) Dyer L

induced by tissue transglutaminase. Cancer Res. 2009 Ai L, Skehan RR, Saydi J, Lin T, Brown KD. Ataxia- Dec 15;69(24):9192-201 Telangiectasia, Mutated (ATM)/Nuclear Factor κ light chain enhancer of activated B cells (NF κB) signaling controls Lindfors K, Mäki M, Kaukinen K. Transglutaminase 2- basal and DNA damage-induced transglutaminase 2 targeted autoantibodies in celiac disease: Pathogenetic expression. J Biol Chem. 2012 May 25;287(22):18330-41 players in addition to diagnostic tools? Autoimmun Rev. 2010 Sep;9(11):744-9 Király R, Barta E, Fésüs L. Polymorphism of transglutaminase 2: unusually low frequency of genomic Park KS, Kim HK, Lee JH, Choi YB, Park SY, Yang SH, variants with deficient functions. Amino Acids. 2013 Kim SY, Hong KM. Transglutaminase 2 as a cisplatin Jan;44(1):215-25 resistance marker in non-small cell lung cancer. J Cancer Res Clin Oncol. 2010 Apr;136(4):493-502 This article should be referenced as such: Choi CM, Jang SJ, Park SY, Choi YB, Jeong JH, Kim DS, Dyer L. TGM2 (transglutaminase 2). Atlas Genet Kim HK, Park KS, Nam BH, Kim HR, Kim SY, Hong KM. Cytogenet Oncol Haematol. 2014; 18(6):397-399. Transglutaminase 2 as an independent prognostic marker for survival of patients with non-adenocarcinoma subtype of non-small cell lung cancer. Mol Cancer. 2011 Sep 24;10:119

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Gene Section Review

THRB (Thyroid Hormone Receptor, Beta) Adam Master, Alicja Nauman The Centre of Postgraduate Medical Education, Department of Biochemistry and Molecular Biology, Marymoncka 99/103, 01-813 Warsaw, Poland (AM, AN)

Published in Atlas Database: November 2013 Online updated version : http://AtlasGeneticsOncology.org/Genes/THRBID42554ch3p24.html DOI: 10.4267/2042/53769 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 LOC101927874), RNA5SP125 (RNA, 5S ribosomal pseudogene 125), RARB (retinoic acid Review on THRB, with data on DNA/RNA, on the receptor, beta), CFL1P7 (cofilin 1 (non-muscle) protein encoded and where the gene is implicated. pseudogene 7), RNA5SP126 (RNA, 5S ribosomal pseudogene 126), LOC100505947 (uncharacterized Identity LOC100505947), TOP2B (topoisomerase (DNA) II Other names: C-ERBA-2, C-ERBA-BETA, beta 180kDa), MIR4442 (microRNA 4442), ERBA2, GRTH, NR1A2, PRTH, THR1, THRB1, CRIP1P2 (cysteine-rich protein 1 (intestinal) THRB2 pseudogene 2), NGLY1 (N-glycanase 1), RPL32P11 (ribosomal protein L32 pseudogene 11), HGNC (Hugo): THRB TAF9BP1 (TAF9B RNA polymerase II, TATA box Location: 3p24.2 binding protein (TBP)-associated factor, 31kDa Local order: According to the NCBI map viewer pseudogene 1), OXSM (3-oxoacyl-ACP synthase, genes flanking THRB (3p24.2) from telomere to mitochondrial), LINC00692 (long intergenic non- centromere are: UBE2E2-AS1 (UBE2E2-AS1 protein coding RNA 692), RPEP2 (ribulose-5- UBE2E2 antisense RNA 1 (head to head)), phosphate-3-epimerase pseudogene 2), HMGB3P12 UBE2E2 (ubiquitin-protein ligase E2), (high mobility group box 3 pseudogene 12), LOC100420471 (ADP-ribosylation factor-like 4A VENTXP4 (VENT homeobox pseudogene 4), see pseudogene), UBE2E1 (ubiquitin carrier protein diagram 1. E1), NKIRAS1 (NFKB inhibitor interacting Ras- Note like 1), RPL15 (ribosomal protein L15), NR1D2 The human thyroid hormone receptor beta (THRB), (nuclear receptor subfamily 1, group D, member 2), a member of several nuclear receptors for thyroid NPM1P23 (LOC100422256, nucleophosmin 1 hormone, has been shown to mediate the biological (nucleolar phosphoprotein B23, numatrin) activities of triiodothyronine (T3). This gene pseudogene 23), LINC00691 (LOC152024, long encodes 3 protein isoforms, the TR β1, TR β2, and intergenic non-protein coding RNA 691), and intra- TR β4 differentially expressed in developmental and THRB: LOC101927854 (uncharacterized tissue-specific patterns and implicated in regulation LOC101927854) sharing some exons with two of transcription of target genes affecting multiple transcript variants (GeneBank: CB994391.1, physiological processes, including cell growth, AW950510.1) present in ACE View description of differentiation, apoptosis, and maintenance of NR1D2 gene locus, RPL31P20 (ribosomal protein metabolic homeostasis. The gene controls thyroid L31 pseudogene 20), THRB-IT1 (THRB intronic hormone levels, liver and kidney metabolism and is transcript 1), THRB-AS1 (LOC644990, THRB critical for normal development of auditory and antisense RNA 1), at 5' side of THRB: MIR4792 visual systems. The THRB has been also implicated (microRNA 4792), EIF3KP2 (eukaryotic in the pathology of numerous diseases including translation initiation factor 3, subunit K pseudogene thyroid hormone resistance syndrome (RTH), 2), LOC101927874 (uncharacterized obesity, neurodegenerative disorders and cancer.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 400 THRB (Thyroid Hormone Receptor, Beta) Master A, Nauman A

Diagram 1. Schematic representation of human chromosome 3 indicating the position of THRB locus (red bar) . The official symbols, NCBI IDs and relative transcriptional orientation of genes in the 3p24.2 locus (+ plus or - minus DNA strand) are shown with respect to the centromere. The genes located at 5'- and 3'-side of the THRB as well as the genes positioned at least in part inside the THRB sequence (intra-THRB genes) are highlighted by three coloured rectangles. The diagram was drawn on the basis of the standard ideogram taken from the NCBI Map Viewer and NCBI Human Genome Resources.

This gene may function as a tumor suppressor and transcripts were shown to be expressed in the disturbances of the THRB expression are frequent THRB locus, in which may affect the expression of findings in various cancers. However, the genomic the thyroid hormone receptors (see diagram 2). The actions of the nuclear receptor can interface with THRB gene is one of two thyroid hormone receptor nongenomically initiated and TR β -mediated effects genes in the human genome located on of thyroid hormone on angiogenesis and cancer cell chromosome 3. The other gene, THRA located on proliferation (Davis et al., 2009; Puzianowska- chromosome 17, encodes the related thyroid Kuznicka et al., 2013). hormone receptors TR α1, TR α2 and TR ∆α 1 (p28 and p43). These genes were initially identified by DNA/RNA their homology to the avian retroviral oncogene v- erbA encoding a mutated variant of chicken TR α1. The human THRB gene spans a region of 376609 bp and is divided into 20 different exons inlcuding Description 16 separated by large introns which may give rise According to the NCBI Gene, the THRB maps to to 19 various TR β1 transcripts mostly differing in 5' NC_000003.11 (24158644..24536772, untranslated region (5'UTR), 1 truncated variant complement) and spans a region over 376 kilo TR β4 (TR β1 isoform) and 1 TR β2 transcript with bases. Within the sequence, some other differential promoter usage (see diagram 2 and 3). transcriptionally active genes have been identified Note that several sense and antisense non-TR β (see diagram 1 and 2).

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 401 THRB (Thyroid Hormone Receptor, Beta) Master A, Nauman A

Diagram 2. Human THRB Gene Structure, a transcript and protein. Multiple transcript variants and 3 protein isoforms (TR β1 TR β2 and truncated variant TR β4) are generated by transcription of pre-mRNA using two THRB promoters (P1 and P2 located between exon 4 and 5), alternative splicing of exons marked with numbers 1-10 (horizontal bars) and separated by large introns (shown by distances between exons) as well as protein translation, which is differentially regulated by the 5'UTR variants. A. Genes localized within the THRB sequence are marked with orange or blue arrows. The orange one (THRB-IT1, THRB intronic transcript 1) is transcribed in the same orientation (-) to the THRB, whereas blue arrows indicate the genes in opposite transcriptional orientation (+): THRB-AS1, RPL31P20, LOC101927854, and two transcript variants (GeneBank: CB994391.1, AW950510.1) described in NR1D2 gene locus (see local order). Expression of the genes may result in production of long naturally occurring antisense transcripts (long NATs) that may bind complementary target strands of THRB DNA or newly synthesized pre-mRNA. These sense-antisense pairs may affect the expression of the THRB gene (for more details see diagram 1). Large CpG islands identified as methylated within THRB sequence in human cerebellum, sperm and bone marrow cells are marked with small green ovals on double stranded DNA, whereas the green/orange ovals represent CpG islands identified as methylated in human colon mucosa and colorectal tumor (drawn on the basis of the NCBI Epigenomics database). B. An example of the multiple mRNA variants, wherein blue boxes represent alternatively spliced exons of 5' untranslated region (5'UTR), red - protein coding sequence, green - TR β1 3'UTR (exon 10). C. One of two TR β proteins (here TR β1) and its functional domains: N-terminal AF1 domain (A/B), DNA binding domain (C), hinge region (D), ligand (T3) binding domain (E) and C-terminal AF2 domain (F), all presented in the context of subsequent amino acids (aa) encoded by the TR β1 mRNA (CDS). Amino acid ranges of the NCBI reference conserved domains: C and E are indicated by gray arrows. For protein function see the text below.

Two precursor mRNA (pre-mRNA) are transcribed diagram 3). using two THRB promoters: P1 (GeneBank: The alternative TR β1 splicing results in expression S37458.1), updated according to the sequence of of multiple mRNAs encoding the same protein, chromosome 3 genomic contig (GenBank: however its translation is differentially regulated by NT_022517.18) allowing for expression of TR β1 the 5'UTR variants. and P2 promoter of TR β2 isoform (see diagram 3), These transcripts consist of 10 - 14 different exons located between exon 4 and 5 (GeneBank: forming a 5'UTR region (exons 1, 2 and the NG_009159.1; 330058..334363), however the beginning of exon 3), protein coding region (the sequence range of P2 has not been exactly end of exon 3, 4-9, first 242nt of exon 10) and long established. 3'UTR (last part of exon 10) (Frankton et al., 2004). The THRB gene consists of 20 different exons The exons 5-10 are common to the most of including 16 separated by large introns. transcripts including TR β2 mRNA that contains one The exons are described in details above (see more, first exon named "a" (see diagram 2 and 3).

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Diagram 3. THRB exons distribution and alternatively spliced 5'UTR variants of TR β1 TR β2 and TR β4 mRNAs. At least 19 mRNA variants of TR β1 and one of TR β2 are synthesized using two THRB promoters: P1 and internal promoter P2 located between exon 4 and 5. The human variant TR β4 is a carboxyl-terminal splicing variant of TR β1 (using P1 promoter), which contains a stop codon due to the presence of a 137-bp insertion located between exon 7 and 8.The third known, non-functional in humans promoter (P3) is shown as well. This locus is located upstream of the exon 5 but downstream of the P2 promoter and allows for transcription of two additional isoforms: TR β3 and TR ∆β 3, expressed ubiquitously only in rats, however, the regulatory elements present in this region may interfere with transcription of the TR β2 in humans. The mRNA variants are shown in the context of alternative and constitutive exons and their distribution within the DNA sequence. Blue boxes represent alternatively spliced exons of 5' untranslated region (5'UTR), red - protein coding sequence, green -3' untranslated region (3'UTR, the longest 3' part of exon 10). Most of exons are separated by large introns, which are marked at the beginning and the end by numbers representing their relative position on DNA. The diagram shows a region of 378,609 kb (24133709..24516317; Ref.seq. : NC_000003.11). The exact size (nt) of each exon is given below the exon boxes. The 5'UTR region of TR β1 may include up to 10 alternatively spliced exons and 44 bp of exon 3, whereas 5'UTR of TR β2 contains only 105 of 434 nucleotides of exon 1 named "a". The exons 5-10 are mostly constitutive and common with almost all transcripts including TR β2 mRNA. The most of identified transcript variants (shown at the middle of the diagram) differ only in the 5' untranslated region, which can influence the protein synthesis. 3'UTR variant H lacks 123 nucleotides (miRHS fragment). This region contains a putative binding site for miRNA-204, located between nucleotides 2313-2319 of TR β 3'UTR. Variants IVS4A and IVS4B contain alternatively spliced exons of 5'UTR, exon 3, 4 and a fragment of intron with a stop codon located downstream of the exon 4 that may result in translation of truncated protein (28 amino acids) of unknown function. The GeneBank accession numbers for each reference sequence are given next to the adjacent transcript variant.

The variant TR β4 contains additional 137-bp reading frames (uORFs) and internal ribosome insertion (exon) between exons 7 th and 8 th shown in entry site (IRES) predicted in the variant A diagram 3 (intron 5 th according to Tagami et al., (GeneBank: AY286465.1). These regulatory 2010) that results in synthesis of a truncated protein sequences may be organized in secondary and lacking the ligand binding domain. tertiary RNA structures that are recognized by This isoform may modulate T3 action as an trans-acting factors such as protein translation endogenous antagonist (according to Tagami et al., factors and naturally occurring small RNAs. 2010). Moreover, the most of TR β mRNA variants contain The multiple 5'UTR splice variants of the TR β1 can long 3'UTR (see diagram 3) with multiple differently regulate translation of the TR β1 coding microRNA binding sites that may affect the sequence. The 5'UTRs vary in length, GC-content, expression of these receptors. Disturbances in the secondary structures, number of upstream open expression of the TR β mRNA variants have been

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reported in various cancers. Some of the metabolism. disturbances seem to be a cancer specific. For Description example, the loss of transcript variants F1 (GQ456950) and IVS4B (GQ919288.1) has been There are three human TR β isoforms TR β1, TR β2 observed in clear cell renal cell cancer (ccRCC). In and TR β4, which are differentially expressed in the same tissues, over 70% reduction in TR β1 various tissues. The TR β1 and TR β2 receptors have mRNA (coding sequence) has been reported. the typical domain structure of a nuclear receptor Simultaneously, expression of 5'UTR variants A with an N-terminal domain (A/B), central DNA and F (AY286470.1) has been reduced in ccRCC binding domain (C) consisting of a double C4-type by 75% and 62%, respectively, compared to control zinc fingers, hinge region (D), C-terminal ligand samples (Master et al., 2010). binding domain (E), and AF2 domain (F). These Note also that several sense and antisense non-TR β receptors have a variable N-terminal domain (A/B) transcripts have been shown to be expressed in the that differs between TR β1 and TR β1 isoforms (see THRB locus, in which may affect the expression of diagram 4). The N-terminus is responsible for the thyroid hormone receptors (see diagram 2 and cofactor and regulatory protein binding, T3- 3). independent transactivation, receptor dimerization and DNA recognition. Local dimerization sites Transcription have been found in domain C, E, corepressor Two precursor mRNAs (transcribed from two interaction sites in domain D, E and coactivator different promoters of the THRB) undergo interaction sites in domain A/B, E and F. The hinge extensive co- and post-transcriptional modification region (D) is also found to be required for TR β in the nucleus that includes tissue specific, dependent suppression of ras-mediated responses. alternative splicing of the pre-mRNAs. The TR β1 can bind to target DNA sites regardless of T3 binding status as a homodimer or heterodimer Pseudogene with retinoid X receptors (RXR) or as a monomer No pseudogene has been reported for the THRB (more weakly). gene. Nevertheless, the THRB may be regulated by TR β can bind to DNA in the absence of ligand and pseudogenes identified within the THRB gene therefore is thought to have the potential to mediate sequence or in the same locus (see diagram 1 and both T3-dependent and T3-independent regulation 2). The pseudogenes may produce long naturally of target gene transcription. The protein occurring antisense transcripts (cis-NATs) forming phosphorylation has been shown to enhance its sense-antisense pairs (see Homology). These sense- cytoplasmic-nuclear import (Maruvada et al., antisense pairs may activate numerous mechanisms, 2003). Phosphorylated T3 receptors can exhibit similar to those observed in a pseudogene-mediated increased TRE binding as a homodimer, but not as regulation of a target gene via pseudogene-derived heterodimer or monomer. Moreover, integrin- small interfering RNAs or on the level of RNA- mediated non-genomic action of T4 (Davis et al., directed DNA methylation, pre-mRNA 2009) may result in phosphorylation of TR β1 transcription, alternative splicing as well as RNA Ser142 leading to dissociation of the corepressors editing, transport and localized protein translation. and transactivation of target genes (Davis et al., However, this regulation remains to be established 2000). In case of typical positively regulated genes for the THRB gene. such as DIO1 encoding iodothyronine deiodinase type I and GH1 of human growth hormone 1, T3 Protein binding stimulates a conformational changes in the TR β allowing for dissociation of corepressors Note followed by recruitment of coactivators to form an The human THRB encodes three protein isoforms, activating complex stimulating the transcription. the TR β1 and TR β2 that are T3-dependent There is also identified a group of negatively receptors mediating genomic and nongenomic regulated genes that includes hypothalamic actions of the thyroid hormone, and TR β4 isoform, thyrotropin-releasing hormone (TRH) and pituitary which is a carboxyl-terminal splicing variant of thyroid stimulating hormone (TSH) encoded by TR β1 that lacks the ligand binding domain and TSHB and controlling the hypothalamic-pituitary thus, may modulate T3 action as an endogenous thyroid (HPT) axis. In this regulation liganded antagonist in the tissue or cellular context (Tagami nuclear receptors down-regulate target gene et al., 2011). The TR β proteins are implicated in transcription, with the cooperative binding of regulation of transcription of target genes and various transcription factors to multiple regulatory control key cellular processes including elements on DNA (for more see genomic actions of differentiation, proliferation, apoptosis and T3 mediated by TR β receptors).

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Diagram 4. Structural and functional organization of TR β proteins. A. Three thyroid hormone receptor beta isoforms: TR β1 TR β2 and truncated variant TR β4 are encoded by the human THRB gene. Functional domains of the transcription factors are divided as follows: N-terminal AF1 domain (A/B) responsible for hormone independent transactivation and regulatory proteins binding; DNA binding domain (C) containing two C4-type zinc fingers; hinge region (D) with nuclear localization signal allowing for nuclear transport (regardless of T3 binding status); ligand (T3) binding domain (E) that allows for dimerization (usually with RXR α receptor) as well; C-terminal AF2 domain responsible for T3-dependent transactivation of target genes (F). The domains E and F bind corepressors or coactivators regulating the activity of the TR β receptors. The TR β1 and TR β2 are functional receptors of T3 differing only in the length of the A/B domain, however their expression is tissue specific. The human variant TR β4 is a truncated variant of TR β1, lacks T3 -binding ability and acts as an endogenous dominant-negative isoform. B. Crystallographic structure of heterodimer formed by the human thyroid hormone receptor DNA-binding domain and retinoid X receptor DNA-binding domain (rainbow colored) complexed with double strained DNA (green). Zinc atoms are depicted as blue/pink dots. The structure is presented in the context of the thyroid hormone response elements (TREs) that are specific DNA sequences recognized by the receptors. The TRE-DR4 is formed by consensus core recognition motif (AGGTCA) that is positioned as direct repeats separated by 4 nucleotides. The other TREs including palindrome (TRE-P0) or inverted palindrome (TRE-IP6) are shown as well. C. Crystallographic structure of the ligand binding domain of the human thyroid hormone (T3) receptor beta (rainbow colored, N-terminus in blue, C-terminus in red) complexed with triiodothyronine (T3). Binding of unliganded receptor alone to DNA usually leads to recruitment of corepressors (CoR) and inhibition of target gene basal transcription, whereas binding of T3 in hormone binding pocket is thought to cause conformational changes leading to dissociation of corepressors followed by recruitment of coactivators (CoA) and activation of the transcription. Mutations in C- terminal α-helix domain (red) that can close the hormone binding pocket and the α-helix shown here in green are frequent findings in the thyroid hormone resistance syndromes (RTHs) and cancers. The conserved domains were visualized using PyMOL 1.3 Molecular Graphics System, on the basis of crystallographic structure files (PDB: 2NLL, 1XZX) of The RCSB Protein Data Bank and The NCBI Conserved Domains Database (CDD, ref.c.d.: cd06961 NR_DBD_TR, cd06935: NR_LBD_TR).

The TR β4 is a C-terminal splicing variant of TR β1 bind thyroid hormone, thus may modulate T3 action which contains a stop codon in an 137-bp insertion as an endogenous antagonist in the tissue or cellular (exon) insertion that results in synthesis of a context. The TR β4 cannot mediate T3-dependent truncated protein lacking the ligand binding domain gene regulation but may inhibit the negative (see diagram 4). The TR β4 contains full A/B, C, D regulation of TSH mediated by TR β1 or TR β2, that domain of TR β1 and a fragment of domain E (is was shown in TSA-201 cells, a clone of human identical for the first 246 amino acids) but the embryonic kidney 293 cells (according to Tagami et carboxyl-terminal 215 amino acids of TR β1 are al., 2010 and Tagami et al., 2011). These findings replaced by an entirely distinct sequence of 13 are consistent with current model for T3-dependent residues, which results from an insertion (see negative regulation of TSHB gene (see genomic RNA). Therefore, this truncated protein is unable to actions below).

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The levels of TR β proteins depend on the protein for regulation of thyroid hormone levels through stability and transcription/translation efficiency that the hypothalamic-pituitary-thyroid (HPT) axis, a is tissue specific and differentially regulated in negative feedback loop, which includes auto- various mRNA variants. The frequently reported control of hypothalamic Thyrotropin-Releasing lack of correlation between the mRNA and protein Hormone (TRH) and pituitary Thyroid-Stimulating suggests that apart from transcriptional control the Hormone (TSH) by the thyroid hormones (TH). In expression of TR β receptors is accurately controlled this regulation, the TR β receptors mediate TRH- at the level of translation. In fact, multiple 5'UTR and TSH- lowering effects of TH. variants of TR β1 have been shown in vitro to TR β2 is restricted to the hypothalamus, anterior differentially regulate the protein translation. The pituitary, developing brain, cochlea (inner ear) and major renal TR β1 transcript contains a 5'UTR retina, wherein TR β2 alone is crucial for relevant to variant A (GeneBank: AY286465.1). development of mid-wavelength (MW) cones Analysis of the effects of 5'UTR variants on protein photoreceptors, which play a significant role in expression in JEG-3 choriocarcinoma cells circadian clock light entrainment and in phase (Francton et al., 2004) and Caki-2 renal cancer cells shifting of the circadian oscillator (Dkhissi- (Master et al., 2010) indicated that the weakly Benyahya et al., 2007). This isoform is therefore folded variant A permitted also the highest level of important for visual and auditory function. Down protein expression. In contrast, the strongly folded regulation of hypothalamic TRH is TR β2 specific. 5'UTR variants: F (AY286470.1) or F1 TR β2 mRNA has been also identified in situ in (GQ456950) was identified to be transcribed and human chondrocytes and osteoclasts (Abu et al., translated at the lowest levels. Although, structured 2000). 5'UTR such as variant F treated in vitro with a TR β4 isoform is expressed in various human tissues trans-acting factor (an antisense oligonucleotide) but is highly abundant in testis and skeletal muscle. significantly up-regulated the translation efficiency This isoform lacks T3 -binding domain and may act of a downstream sequence up to the level of the as a dominant-negative protein. It has been variant A. This may estimate the potential of the identified in a TSH-secreting pituitary adenoma 5'UTR-mediated translational control during (TSHoma) as well (Tagami et al., 2011). expression of TR β receptors. The translation of the In contrast, TR α mediates T3 actions during TR β can be also modulated by multiple regulatory development of heart, bone, intestinal and is ORFs that exist upstream of the primary ORF. On responsible for body temperature and basal heart the other side, the protein synthesis may be rate in adults. TR α1 and TR α2 isoforms are highly controlled by long TR β 3'UTR through binding of expressed in the brain, with lower abundance in the various microRNAs including miR-21 and miR- kidneys, skeletal muscle, lungs, heart, and testes. 146a. The microRNA can trigger the RNA The TR α1, TR β1, and TR β2 isoforms can bind interference (RNAi) phenomenon that may lead to DNA and T3 acting as functional thyroid hormone translational repression or even degradation of TR β receptors, whereas TR α2 and TR α3 do not bind T3 mRNAs (Jazdzewski et al., 2011). Since UTR- due to the presence of the longer AF2 (F) domain, mediated translation initiation is a key rate-limiting and act as antagonists. There are some truncated phase affecting efficiency of the protein synthesis, isomers of TR α with specific mitochondrial the translational control mediated by the multiple functions (p28, p43) or may act as dominant- UTR variants is emerging to be a major regulator of negative receptors (TR ∆α 1, TR ∆α 2). There are also the final protein levels in cells. The TR β receptors known two additional TR β receptors expressing have been also reported to be affected by aberrant only in rats: functional T3 receptor - TR β3 and its promoter methylation, alternative splicing and dominant negative isoform -TR ∆β 3 (Williams et impaired cell signaling. al., 2000). Both, TR β and TR α isoforms are involved in Expression circadian cycle that was demonstrated in an in vivo The thyroid hormone receptor isoforms are mouse model, in different metabolic tissues products of both the THRB and THRA genes. including white adipose tissue (WAT), brown During development, TR β and TR α isoforms are adipose tissue (BAT), liver, and skeletal muscle differentially expressed in a temporospatial and (Yang et al., 2007). While TH levels are generally tissue-specific patterns and in adult tissues are constant, the TR β and TR α along with their key present in distinct ratios (Williams, 2000; Francton target genes dramatically cycle in a coordinated et al., 2004). manner that is in agreement with known cyclic TR β1 is widely expressed in all human tissues, but behaviour of lipid and glucose metabolism. TR β is prominent in brain, thyroid, kidney and liver. has been shown to cycle in WAT, whereas TR α in TR β1 controls liver and kidney metabolism and WAT, BAT and liver. Analysis of TR β mRNA mediates cholesterol lowering effects of thyroid expression revealed a unique rhythmic pattern in hormones. Both the TR β1 and TR β2 are essential which their transcripts spike at ZT4 (Zeitgeber

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time, 4h after lights were turned on) followed by a presence of a nuclear localization signal (NLS) in precipitous decline in the next 4h, and remain at the TR β hinge region (D). The TR β nuclear import low levels through the rest of the day. Interestingly, is ATP-dependent and can be regulated by mRNA levels of heme-liganded nuclear receptor nongenomic actions of TH through 1) T4- Rev-erb β encoded by NR1D2 gene, which may dependent activation of plasma membrane integrin interact with THRB expression (see diagram 1 and αvβ3 (Davis et al., 2009) followed by activation of 2), dramatically cycle in the all examined metabolic downstream pathways leading to phosphorylation tissues. of ERK1/ERK2 and TR β1 proteins and/or 2) via Although precisely how the circadian clock acts to T3-dependent formation of cytoplasmic TR β1 control metabolic rhythms is not clear, it is known complexes with p85 subunit of PI3K that may that the ratio of TR α1/TR α2 isoforms is closely activate downstream pathways. The TR β complexes determined by co-expression of Rev-erb α (NR1D1) with p85, ERK1/2 or nuclear receptor coactivators - a key component of the circadian core oscillator may facilitate nuclear import as well. TR β rapidly complex. shuttles between the nuclear and the cytoplasmic The Rev-erb α represses the expression of BMAL compartments. Energy-dependent blockade (ATP changing the expression of other genes of the cycle depletion) enhances TR β nuclear export to including CLOCK, PER1, PER2, PER3 and cytoplasm. Nevertheless, coexpression of nuclear cryptochrome genes CRY1 and CRY2 responsible corepressors (NCoRs) and/or retinoid X receptors for blue light photoreceptor activity (see specific (RXRs) can markedly decrease the shuttling by functions of TR β2). maintaining unliganded TR β within the nucleus. A The cell -autonomous feedback loop allows for TR β mutant defective in DNA binding has a cyclic expression of these oscillator genes at slightly altered nuclear-cytoplasmic distribution various phases with the same period length of when compared with wild-type TR β. TR β mutants approximately 24r (Zhang et al., 2009). that abrogate its interaction with the NCoRs All this findings suggest a role for TR β, TR α and accumulates within the cytoplasm due to an heme receptors (Rev-erb β, α) in coupling the increase in the rate of nuclear export when peripheral circadian rhythm to divergent metabolic compared with nuclear import. Nuclear-cytoplasmic outputs. shuttling has been proposed as a mechanism for Since the expression of TR β is prominent in brain modulating TR β-mediated regulation of and metabolic tissues, it is expected that these transcription (Maruvada et al., 2003). receptors may serve peripheral clock input Subcellular localization of TR β4 is unknown; pathways that can integrate signals from the light- however the lack of T3 binding domain might sensing central clock in the suprachiasmatic nuclei suggest an altered nuclear/cytoplasmic distribution. (SCN) and other cues including xenobiotic Function metabolism pathways. Interplay between the circadian clock and TH TR β proteins are high affinity receptors for thyroid receptors may be a part of a large -scale signaling hormone (TH) functioning as ligand-dependent network that links biological timing to metabolic (T3) and sequence-specific DNA binding (TRE) physiology (Yang et al., 2006). transcription factors (see genomic actions below) that regulate expression of target genes affecting Localisation cell growth, development, proliferation, Subcellular localization and changes in expression differentiation, apoptosis, organ morphogenesis, of TR β receptors can vary depending on the cell heart rate, body fat distribution, bone density. These cycle phase, cell density, cellular stress, signaling receptors are required for the development of the events, tissue types, metabolic rate or even auditory system and of the cone photoreceptors that circadian cycle (Maruvada et al., 2003). mediate colour visual function. TR β1 isoform is Typically, TR β1 and TR β2 isoforms are expressed in most tissues, whereas TR β2 is predominantly localized to the nucleus and retained restricted to the hypothalamus, pituitary, cochlea, in the nucleus regardless of the ligand binding and retina that may indicate the functional status (in the absence and presence of T3). specificity of the isoforms. However, T3 can induce a nuclear reorganization of The TR β1 control the major responses of the liver TR β receptors. The nuclear localization is essential and kidney to T3 and play a critical role in for TR β-mediated genomic actions of T3. In mediating changes in metabolism and standard conditions, a minor fraction of TR β thermogenesis. The T3 receptors are able to proteins resides in the cytoplasm, wherein the increase metabolic rate by accelerating fuel receptors are thought to be mediators of oxidation in most of tissues wherein they may nongenomic actions of thyroid hormone (TH). The activate lipolysis, glucose metabolism and protein cytoplasmic-nuclear shuttling is facilitated by the synthesis.

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Diagram 5. Schematic representation of selected TR β-mediated, genomic and non-genomic actions of TH. The thyroid gland, in response to TSH, produces thyroxine (T4) and 3,5,3'-triiodo-L-thyronine (T3), however greater amounts of T4 are produced than T3. The thyroid hormone (TH) in the circulation are bound to protein -transporters that deliver TH to peripheral tissues wherein TH triggers TR β-mediated effects including negative regulation of the hypothalamic-pituitary thyroid (HPT) axis. For genomic actions, T4 needs to be converted to T3 by DIO1 or DIO2 present in peripheral tissues such as liver, brain or kidney. G. Classical model of TH actions in the nucleus. The model is based on the action of triiodothryonine (T3, ligand) on positively regulated genes. The regulation requires: thyroid hormone response elements (TREs) on specific genes, complexes of nuclear TH receptors (TRs) and T3, coactivator (CoA) or corepressor (CoR) nucleoproteins, and histone acetyl transferase (HAT) or deacetylase (HDAC). G1. Ligand-bound state. TR β can bind to DNA as heterodimer with RXR and regardless of T3 binding status. However, T3 binding results in release of corepressor complex, recruitment of a coactivator complex (e.g. SRC-1, CBP, p300, pCAF, TRAP-DRIP, mediator/integrator components) and HAT. The HAT activity allows for reducing chromatin compaction and permitting general transcriptional factors (GTFs) to interact with DNA and activate transcription of a target gene. G2. Ligand-free state. In absence of ligand, the unliganded TH receptors interact with a copressor complex that may include NCoR, SMRT and histone deacetylase 1 (HDAC1). The recruitment of this complex may result in reduced histone acetylation (shown as Ac), which in turn compacts chromatin structure and represses the gene transcription. The transactivation domain of the T3-free receptor, as a heterodimer with RXR, assumes a conformation that promotes interaction with a group of transcriptional corepressor molecules. N. Nongenomic actions of TH mediated by TR β1. These actions are fast (within 10-40 min), frequently reported to result in pro-proliferative, pro-angiogenic, anti-apoptotic effects. These actions may be simplified into two main signaling cascades: N4) extracellular-T4/ αvβ3-integrin/ PLC/ PKC α/ ERK1/2/ TR β1-Ser 142 phosphorylation that among others can result in specific gene transactivation or transrepression (N4); N1) cytoplasmic-T3/ TR β1/ CSH 2-p85 α-p110(PI3K)/ Akt/ mTOR phosphorylation leading to transcription of PI3K-specific genes such as HIF1A and GLUT1 (SLC2A1) (N1, N1b, N1c). Specific inhibitors of these pathways are shown in red font. N1. Nongenomic effects of T3 may be initiated in cytoplasm by TR β- dependent activation of PI3K that leads to sequential activation of Akt/PKB/mTOR-p70 S6K as well as the other mTOR targets including upregulation PI3K-dependent genes. A fraction of TR β1 present in the cytosol forms a complex with p85 α subunit (regulatory subunit of PI3K) in a ligand independent manner that activate PI3K. The kinase generates phosphatidyl inositol-3,4,5- triphosphate (PIP 3) from PtdIns(4,5)P2 (PIP 2) activating downstream pathways via Akt/PKB (N1b) or through phosphorylation of the TR β1 followed by its nuclear import. Wild-type TR β1 competes with corepressor NCoR or mutant TR βPV for binding to the CSH 2 domain of p85 α (N1c). This PI3K activity is blocked by specific inhibitors such as Wortmannin or LY294002. N2. Signal transduction via plasma membrane receptor αvβ3 by T3 binding to the extracellular part of the receptor. The binding domain includes a receptor site (S1) exclusively for T3 that activates phosphatidylinositol 3-kinase (PI3K) and leads to shuttling of cytoplasmic TR β to the nucleus followed by transcription of specific target genes such as HIF1A. N3. Cytosolic TR β1 and PI3K are involved in T3-stimulated activation of Na +/K +-ATPase and other features of the sodium pump (gene expression, plasma membrane insertion). Besides, TH is known through αvβ3 to modulate the activity of several other ion transport systems including Na +/H + exchanger NHE (SLC9C1). N4. T4-induced activation of ERK1/2 through plasma membrane receptor αvβ3 (site S2). This action is relevant to: intracellular trafficking of proteins, including TR β1, serine phosphorylation (P) and acetylation (Ac) of this nuclear receptor, assembly within the nucleus of complexes of coactivators or corepressors and transcription of specific genes, including that for TR β1. The action includes T4 binding to the extracellular part of the receptor, activation of PLC, PKC, ERK1/2 (MAPK) pathway, phosphorylation on TR β (Ser 142 ), derepression and enhancement of transcription. Among the consequences of ERK1/2 activation are specific serine phosphorylation of the cytoplasmic/nuclear TR β1 (Ser 142 ) and estrogene receptor α ER α, phosphorylation of signal transducers and activators of transcription STAT1 α, STAT3 as well as p53, wich were found to be co-immunoprecipitated with the activated ERK1/2. Cytoplasmic fraction of TR β are shuttled to the nucleus, wherein the proteins are transcriptionally active and can modulate the actions of certain cytokines and growth factors including those involved in tumor cell proliferation and on angiogenesis. TETRAC is an analog of T4 that can inhibit this nongenomic action of T4, however, showing thyromimetic properties it can also affect gene expression in the cells, regulating transcription of target genes such as THBS1, CASP2 and CBY1. For details see text.

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TH receptors are responsible for T3-dependent several T4- and T3- analogues (see ligands) have homeostasis and maintenance of a steady body been also reported to induce TR β-specific response. temperature that is realized via an auto-regulatory Genomic and nongenomic actions negative feedback loop controlling the Genomic actions of TR β are initiated with nuclear hypothalamic-pituitary thyroid (HPT) axis. Its translocation of the newly synthesized receptors regulation is mainly determined by local T3 (Maruvada et al., 2003) that is facilitated by nuclear concentration in the anterior pituitary as well as localization signal (NLS) found in the hinge region paraventricular nucleus (PVN) of the hypothalamus (D) of the receptors (see diagram 2 and 3). The TR β wherein low levels of T3 abolish TR β-mediated proteins are classified as type II nuclear receptors, transcriptional repression of pituitary thyroid- which are retained in the nucleus regardless of the stimulating hormone (TSH) promoter and ligand binding status (free or occupied by T3) and hypothalamic thyrotropin-releasing hormone (TRH) bind to TREs (thyroid hormone response elements) promoter. However, the local tissue T3-levels are as heterodimers with retinoid X receptors (RXR), dependent on the availibility of circulating thyroid rarely as homodimers or monomers. The TREs are prohormone - thyroxine (T4), its intracellular specific DNA sequences of the consensus core transport and cytoplasmic T3 production, catalyzed recognition motif AGGTCA, AGGACA or by iodothyronine deiodinases. Since TRs can form (A/G)GGT(C/A/G)A hexamers (half-site →), in heteroduplexes with retinoid X receptors, TH is which two or more motifs are positioned as direct found to modulate the skin response to retinoids. repeats separated by 4, 0 or 6-nucleotide: (TRE- Genomic effects of TR β1 have been recognized as DR4, →n4→), palindromes (TRE-P0, →← ) or tumor suppressive and disturbances of the TR β1 inverted palindromes (TRE-IP6, →n6←). The expression have been found in different cancers analysis of the response elements formed by direct (Martínez-Iglesias et al., 2009b; Kim at al., 2013). repeats of the half-sites demonstrated that a spacer Description of 4-nucleotides can provide maximal TR β is involved in many processes that compose transactivation by TR β in TR β-RXR heterodimers thyroid hormone (TH) actions and gene expression. but the transactivation efficiency may depends on This T3-dependent receptor act as a transcription sequence context of the TRE, tissue specific trans- factor regulating expression of genes involved in acting factors and ligand concentration. It has been the cell cycle progression, differentiation, apoptosis also shown that for an efficient genomic action of and cellular metabolic rate. TH exerts a pleiotropic the heterodimers, the presence of TR β and RXR effect on development and homeostasis. This effect ligands: triiodothyronine (T3) and 9-cis retinoic results from genomic and nongenomic TH actions acid (9-cis RA) may be necessary. However mediated by both, the TR β and TR α receptors synergistic effects of the RXR ligand and T3 on the (TRs) that regulate hundreds of genes responding to heterodimers (RXR/TR)-mediated transcription T3-liganded or unliganded receptors. A large have been reported for specific promoters, the other number of genes have been identified to respond to studies suggest that RXR ligands may inhibit T3- T3 stimulation (~10% of all expressed genes) and dependent transactivation, possibly by promoting the divergence between T3-treated and untreated the formation of RXR homodimers. The biological cells can grow rapidly over time. The initial studies effects of TRE binding by the unoccupied versus of TRs actions revealed near complete overlaps in the T3-occupied receptor are quite different. In their effects (TR α and TR β can regulate similar many cases, binding of the unliganded or gene sets). However, gene-specific differential TR α antagonist-liganded receptor alone to DNA may and TR β actions are reported as well. These lead to repression of transcription, whereas binding differences appear to result from the 1) differences of the agonist (T3)- liganded receptor complex in tissue-specific expression patterns, 2) diurnal activates transcription. However, the receptor rhythm (see expression and specific functions), 3) activity is mainly regulated by ligand-dependent various time-courses of actions with different interactions with corepressor (CoR) and coactivator kinetics 4) target gene-specific variations in pattern (CoA) proteins. In the absence of ligand (T3), the of response to T3 concentration. For instance, TR β TR β receptors are often complexed with has been shown to exhibit gene-specific corepressor proteins (CoRs, NCoR, SMRT) and requirements for higher T3 levels (compared to histone deacetylases (HDACs) allowing the TR α) for regulation of HR, MYH6, ALPI and histones to wrap the DNA more tightly. T3 binding FURIN genes, whereas HIF1A is identified to have to these nuclear receptors causes conformational lower T3 requirements during TR β-mediated changes leading to dissociation of corepressors and regulation. ANGPTL4 encoding a PPARG recruitment of coactivator proteins (CoAs, SRC3, angiopoietin related protein is a verified in parental CBP, p300, pCAF), as well as mediators (TRAP- HepG2 cells direct TR β target. Prolonged T3 DRIP multi-subunit complex) that are thought to treatment selectively augments TR β action in the target the entire complex to a liganded receptor context of the TR β-dependent genes. Moreover, through a single subunit, TRAP220. The thyroid

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hormone (TH) receptors, similarly as vitamin D3 promoter, and this activation was repressed by Zn- receptors (VDRs), and peroxisome proliferator- finger region of liganded TR β. Upon T3 binding, activated receptors (PPAR), exhibit a strong the T3/TR β complex is thought to be released from activation function 2 (AF2)-dependent preference nTRE, favoring its translocation and interaction for receptor binding domain 2 (RBD-2) of the with GATA-2 zinc finger region (GATA2-Zf) on TRAP220 protein. It has been also demonstrated GATA-RE located next to the nTRE. The that RXR receptor (in TR β/RXR heterodimer) T3/TR β/GATA2-Zf/DNA complex formation is the displays a weak yet specific AF2-dependent principal complex responsible for the TSHB gene preference for another TRAP220 RBD-1 domain. down regulation. This interaction occurs via the Addition of ligand for the RXR receptor (9-cis RA), highly conserved zinc-finger in DNA binding in addition to T3 for TR β partner, might further domains that may resemble the trans-repression- strengthen the RXR-RBD-1 interaction and like mechanism in which direct binding of the presumably stabilize the overall association of receptor to another transcription factor occurs in a TRAP220 with the heterodimer. After formation of DNA-independent manner. In the absence of T3 the coactivator multi-subunit complex, general TR β prefers nTRE element, allowing for the gene transcription factors (TAFs, TFIIA, TBP) and RNA transcription. This negative regulation shows that polymerase II are recruited to the complex that T3 weakens TR β binding to the regulatory element initiates transcription DNA into pre-RNA. The pre- (nTRE) in the TSHB promoter. The displacement mRNA undergoes further modifications including of the liganded TR β from nTRE to another site alternative splicing, mRNA editiing and translation (GATA-RE/GATA2-Zf) seems to be critical for the into protein that may result in a change in cell mechanism used by the cell for silencing the TSHB function. The nuclear receptors including TR β gene transcription (Figueira et al., 2010). Down- proteins can regulate gene expression by binding regulation of hypothalamic thyrotropin-releasing directly or indirectly (via other proteins) to specific hormone TRH gene by T3/TR β complexes involves sequences in the promoters of target genes acetylation and methylation of specific residues of (Puzianowska-Kuznicka, 2013). Apart from the histone tails in the TRH promoter region and relies current model of HRE/TRE - dependent on changing amount of the TR β receptors on TRH transcriptional control, it has been proposed that nTRE after T3 binding (Umezawa et al., 2009). one transcription factor may repress the activity of a Prolonged administration of T3 causes second transcription factor through a protein- demethylation of specific histones and subsequently protein interaction, without the requirement of two the release of TR β receptors from the gene to different DNA binding sites. These proteins, suppress it. Both negative regulations may require notably AP1 and NF-kB, can act by interfering with T3/TR β to be removed from its nTRE to down- transcriptional complex formation in a DNA- regulate the genes, although the mechanism used by independent manner and may lead to a ligand-bound TR β leading to repression of transrepression of target gene transcription, what transcription is still a subject of contention. The has been also shown in TR β-mediated negative classical mechanism of TR β activity suggests that regulation of the hypothalamic-pituitary thyroid receptors typically complexed with RXR bind more (HPT) axis. TH responsive genes can be both strongly to DR4 in a clearly cooperative binding positively and negatively regulated by T3-liganded between the transcription factors and DNA (see TR β receptors. Iodothyronine deiodinase type I diagram 5). Nevertheless, coexpression of RXR and (DIO1) or GH1 of human growth hormone 1 are TR β2 has been also shown to slightly reduce both up-regulated in the presence of T3 (see positive the transactivation and transrepression by liganded regulation model - diagram 5). In contrast, pituitary TR β2 that may act more strongly as homodimer or TSH encoded by TSHB gene is down-regulated by monomer, depending on the architecture of the T3 and rises during T3 deprivation. In this TRE. Thus, the mechanism responsible for TR β- regulation, liganded nuclear receptors down- mediated genomic action of T3 is still unclear. regulate target gene transcription, with the Several recent studies indicate that at the genomic cooperative binding of various transcription factors level TR β may act as a tumor suppressor. For to multiple regulatory elements on DNA. It has instance, the gain-of-function approach by stably been proposed that the negative regulation of the expressing TR β in a human breast cancer cell line TSHB gene may require at least two response MCF-7 (MCF-7-TR β), which normally lacks the elements on promoter DNA (nTRE-"negative" TRE TR expression, has been reported to inhibit the and GATA-RE) as well as dimerization of liganded growth of the MCF-7 cell tumors in xenograft TR β with a GATA transcription factor (e.g. models (Park et al., 2013). These estrogen (E2) GATA2), that can repress the activity of the GATA. dependent cells show elevated JAK2-STAT3-cyclin In fact, the nTRE in the promoter of TSHB gene D signal that is repressed by the TR β expression at contains a single half site-like sequence the level of transcription. Interestingly, other (GGGTCA). The GATA2 alone can activate TSHB studies indicate that TH has anti-apoptotic and pro-

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proliferative effects in ER α-positive human breast observed as early as in 10 min and persisted for up cancer cells (Perri et al., 2013). However, these to 90 min. The increase in nuclear TR β1 was opposite effects are recognized as a result of the assumed to be originated from the pool of cytosolic αvβ3 integrin -dependent nongenomic actions of fraction of the TR β1. Interestingly, the nongenomic TH (see below). action of T4 has been shown to block the p53- Nongenomically initiated, TR β -mediated thyroid mediated proapoptotic activity of resveratrol, a hormone (TH) actions occur at the plasma polyphenolic compound found in grapes and wine membrane or in cytoplasm and may also culminate (SIRT1 activator), by disrupting an ERK1/2- in complex, nucleus-mediated cellular events nucleoprotein complex. Although, inhibition of T4 leading to the transcription of specific genes such as binding at the cell surface receptor can restore the HIF1A, MCL1 or GLUT1 (Moeller et al., 2006; apoptotic action of the resveratrol (Lin et al., 2002). Davis et al., 2009). The nongenomic actions of TH The action of T4 on cellular signal transduction is can interfere with genomic effects of T3, hence, a initiated at a cell-surface by activation of an clear distinction between nongenomic and genomic integrin receptor - αvβ3 (identified in 2005). The effects may no longer be practical. However, TH plasma-membrane receptor was previously initiation events leading to these effects are quite reported as a putative G protein-coupled receptor different and may have different kinetics or timing. (GPCR) that preferentially binds T4 and 3,5,3',5'- For instance, enhancement of the antiviral activity tetraiodothyroacetic acid (TETRAC), an antagonist of interferon-γ by TH is achieved nongenomically of the nongenomic actions of TH (Lin et al., 1998; and genomically by T4-dependent activation of the Lin et al.,1999). Extracellular binding of T4 to its mitogen activated protein kinase /extracellular transmembrane receptor was demonstrated to signal-regulated kinase 1/2 (MAPK/ ERK1/2) activate the signal transduction cascade which which can phosphorylate serine (Ser142) located on included G-proteins, PLC, PKC, Ras, Raf-1, DNA binding domain of TR β1 - a nuclear receptor MAPK kinase (MEK), the MAPK (ERK1/2) and of T3. This phosphorylation leads to dissociation of downstream pathways (Davis et al., 2000). TR β1 from corepressor proteins, the NCoR (nuclear The current model of nongenomic actions of TH is receptor co-repressor) and SMRT (silencing based on transduction of the hormone signal mediator of retinoid and TH receptors) allowing for through the membrane by the integrin αvβ3, which transactivation of T3-specific target genes. can preferentially bind T3 to S1 and T4 to S2 sites TETRAC and TRIAC (see ligands), which can of the integrin (Davis et al., 2009). The signal inhibit nongenomic actions of TH, can also block transduction may result in pro-proliferative and pro- the T4 potentiation of the antiviral and angiogenic effects achieved in a ligand -dependent immunomodulatory actions of the interferon-γ, manner via the αvβ3/ERK1/2 cascade or by the even though these analogues have no direct effect hormone activated phosphatidylinositol 3-kinase on the interferon-γ action. There is increasing (PI3K), respectively. The αvβ3 integrin is evidence that nongenomic and genomic actions of concentrated largely in plasma membranes of TH may overlap with genomic and nongenomic endothelial cells, vascular smooth muscle cells, effects of estrogens and testosterone in tumor cells. various cancer cells, osteoclasts and platelets. The thyroid hormone, estrogene and Hormone-binding domain of the integrin receptors dihydrotestosterone have similar ERK1/2- includes two recognition sites that are capable of dependent proliferative actions on the estrogen binding T3 (S1) or T4 and T3 (S2). S2 site can bind receptor α (ER α)-positive human breast cancer both T4 and T3, though the affinity for T3 binding cells. TH and steroids also have interacting to the S2 site is lower than that for T4. Binding T4 nongenomic and genomic actions in heart and brain to integrin αvβ3 (without cell entry) may mediate cells. The binding affinity of T4 to its plasma- nongenomic actions of TH by activation of membrane receptor modulates intracellular protein extracellular-regulated kinases 1/2 (ERK1/2), which trafficking of the ER α and TR β1 receptors from the transduces the hormone signal into complex cellular cytoplasm to nucleus. T4 -transduced activation of and nuclear events including angiogenesis and the ERK1/2 promotes its nuclear uptake and tumor cell proliferation. This T4-induced pathway ERK1/2-dependent phosphorylation of the TR β1, can stimulate shuttling of TR β1 receptor from the ER α and signal transducers and activators of cytoplasm to the nucleus and increase the TR β- transcription 3 and 1 α (STAT1 α, STAT3). In the mediated transactivation of specific genes. nucleus of T4-treated cells, the TR β1, ER α, Moreover, phosphorylation of serine 142 located on STAT1 α and STAT3 transcription factors were DNA binding domain (DBD) of the found to be co-immunoprecipitated with the cytoplasmic/nuclear TR β1 is thought to facilitate activated ERK1/2. The complexing of TR β1 and transcription derepression by dissociation of ERK1/2 was relatively rapid and detected with 1.4- corepressors (NCoR and SMRT) and recruiting and 7.8-fold increases in TR β1 in 30 and 40 min of coactivators and mediators (SRC-1, CBP, p300, T4 treatment, respectively. This effect of T4 was pCAF, TRAP-DRIP). The nongenomic action via

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integrin receptors may be specifically inhibited by (ATPase, Na+/K+-ATPase) and insertion of the TETRAC, a deaminated T4 analogue that can gene product into the plasma membrane (Davis et displace T4 and T3 from both sites (S1, S2) but al., 2009). does not mimic the agonist functions of the Regardless of TR β1, also TR α1 can interact with hormones through the integrin receptors. This T4- the p85 α subunit of PI3K in a T3 -dependent analogue blocks thyroid hormone effects on manner, leading to phosphorylation of Akt and angiogenesis and cancer cell proliferation and activation of downstream signaling pathways. The might have some benefits in cancer treatment. In other nongenomic actions of the TH have been also the chick chorioallantoic membrane (CAM) model, shown to modulate cellular ion fluxes, sodium the cells treated with TETRAC - an agonist for TRs current (I(Na)), inward rectifying potassium current (in genomic action), significantly enhance the (IKir), sodium pump (Na, K-ATPase) and ERK1/2- expression of thrombospondin, an antiangiogenic regulated Na/H exchanger (NHE) encoded by gene. This effect has been shown to complement SLC9C1 - solute carrier family 9 subfamily C the anti-VEGF and anti-bFGF actions of TETRAC. (Na+-transporting carboxylic acid decarboxylase), In contrast, protein ligands present in extracellular member 1. Both, the genomic and nongenomic matrix (ECM) and containing an Arg-Gly-Asp actions of TH have been shown to proceed the (RGD) motif fully inhibits T3 actions initiated at S1 transcription and activity of the sarcoplasmic site (PI3K pathway) and does not affect T3 actions reticulum Ca(2+)-ATPase (calcium pump). T4 and initiated at S2 site and ERK1/2-dependent cell rT3 but not T3 may act through a truncated form of proliferation. The S1 site can bind T3 exclusively TR α1 (TR ∆α 1) located in cytoplasm, wherein the and rapidly transduces the hormone signal via PI3K liganded TR ∆α 1 may take part in conversion of leading to cytoplasm-nucleus shuttling of TR α1 and soluble actin to fibrous (F) actin that is important to expression of HIF1A gene encoding the hypoxia- cell motility. Certain of these actions appear to inducible factor-1α, alpha subunit (HIF-1α) (Davis interfere with genomic function of the TR β et al., 2009). receptors. The nongenomic effects of TR β ligands The similar activation of the HIF-1α transcription occur rapidly and are unaffected by inhibitors of may be also initiated intracellularly by interaction transcription or translation processes. of T3 with cytoplasmic fraction of TR β1 (see THs exert important physiological actions by both diagram 5).The liganded TR β1 mediates action of genomic and nongenomic effects in mitochondria. T3 on expression of specific genes, including pro- T3 and T2 - a thyroid hormone metabolite, regulate angiogenic genes through binding to the regulatory mitochondrial genome transcription and subunit p85 α of PI3K (see diagram 5) followed by nongenomically initiated mitochondrial processes activation of downstream cascade of protein kinase such as cellular respiration and thermogenesis. T2 Akt/PKB, mammalian target of rapamycin (mTOR) metabolite binds and activates the mitochondrial and its substrate - p70S6K that is involved in cytochrome-c-oxidase Va, whereas T3 binds to two cellular protein synthesis (Kenessey and Ojamaa, truncated TR α1 isoform (p28 and p43). Whereas 2006; Moeller et al., 2006). Activation of mTOR is the role of p28 remains unknown, p43 protein is a rapid, with detectable phosphorylation within the T3-dependent transcription factor of the minutes after T3 treatment. This pathway may lead mitochondrial genome, acting via dimeric to up-regulation of specific genes including: HIF1A complexes involving two other truncated forms of and HIF-1α target genes; SLC2A1 (GLUT1) - nuclear receptors: mtRXR and mtPPAR. All these solute carrier family 2 (facilitated glucose mitochondrial actions as well as expression of other transporter), member 1; PFKP - nuclear TH receptors (e.g. TR α1, TR α2) may have phosphofructokinase, platelet; SLC16A4 (MCT4) - an impact on thyroid hormone availability and the solute carrier familymonocarboxylate transporter 4; TR β function in the cell. RCAN2 regulator of calcineurin 2 (ZAKI-4). Concluding this section, the nongenomic actions of Rapamycin - an inhibitor of mTOR, abrogates the TH that are mediated by TR β is fast (10-40min), thyroid hormone-dependent induction of the ZAKI- frequently reported to result in pro-proliferative, 4α, suggesting the role of sequential activation of pro-angiogenic, anti-apoptotic effects and may be the kinases in the PI3K pathway. Treatment of simplified into two main signaling cascades: 1) endothelial cells with T3 leads to activation of the extracellular-T4/ αvβ3-integrin/ PLC/ PKC α/ Akt/PKB and endothelial nitric oxide synthase ERK1/2/ TR β1-Ser 142 phosphorylation that among (eNOS) that is abolished by PI3-kinase inhibitors, others can result in specific gene transactivation or but not by the inhibitors of transcription. The PI3K- transrepression (see diagram 5N4); 2) cytoplasmic- mediated effects are important to angiogenesis and T3/ TR β1/ CSH 2-p85 α-p110(PI3K)/ Akt/ mTOR other recently appreciated cell functions but not to phosphorylation leading to transcription of PI3K- tumor cell division. The T3 liganded TR β1 in specific genes such as HIF1A and GLUT1 (diagram cytoplasm may also nongenomically initiate a 5N1, 5N1b, 5N1c). Specific inhibitors of these process that result in transcription of the ATP1A1 pathways are shown on diagram 5.

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Interaction DNA target) receptors. The members of the thyroid TR β has been shown to interact with: BRD8, hormone receptor-like subfamily are classified as CCND1, NCOA1, NCOA6, NCOR2, NR2F6, type II nuclear receptors, which are retained in the PPARGC1A and RXRA. nucleus and can bind to DNA regardless of the Crosstalk signaling with proteins of nuclear ligand binding status and usually form heterodimers hormone receptor superfamily. with Retinoid X Receptor-like subfamily Apart from TR β proteins, the other T3 regulated transcription factors, which includes the retinoid X nuclear receptors may affect the thyroid hormone receptor (RXR), hepatocyte nuclear factor-4 levels and the TR β function in cells. T3 and T4 (HNF4), testicular receptor (TR2, TR4) and exert a pleiotropic effect on cellular homeostasis photoreceptor cell-specific nuclear receptor (PNR). and are mediated by protein products of both the The class II receptors include the members of the THRB and THRA genes. The THRA encodes estrogen receptor-like subfamily as well. TR α1, TR α2, TR α3 and some truncated variants Interactions in the nuclear superfamily could be TR ∆α 1, TR ∆α 2, p28 and p43. The TR α1 can illustrated on the basis of crosstalk signaling display both a nuclear and cytoplasmic location, between TRs and PPARs. This interaction appears and is the only thyroid hormone receptor that is to be important in TRs -mediated adipogenesis and imported into the mitochondrial matrix as p28 and carcinogenesis. Majority of the effects of PPARs p43 truncated variants. The thyroid hormone and TRs were found to be opposing during the receptor beta gene may produce TR β1, TR β2, TR β4 crosstalk, however the cooperative effects were (in humans) and two additional isoforms expressed reported as well (Lu and Cheng, 2009). PPARs in rats: TR β3 and TR ∆β 3. The TR α1, TR β1, TR β2 liganded by prostaglandins, prostacyclins or a as well as TR β3 can bind T3 and mediate T3- conjugated linoleic acids (CLAs) and T3 liganded dependent actions, thus, the proteins are bona fide TRs can reciprocally affect the target gene receptors, whereas the TR α2, TR α3 or TR ∆α 1 expression. In humans, the DBDs of TRs and TR ∆α 2, TR ∆β 3 and TR β4 do not bind the hormone PPARs are highly homologous and can bind to the and their function remains to be elucidated. The same half-site sequence that is present in both, TRE TR α2 and TR α3 have longer carboxy-terminal and PPRE elements. The canonical DR1 PPRE domains (AF 2) that does not bind T3 and weakly consists of two direct repeats of AGGTCA with a 1 binds DNA, thus, the variants act as dominant bp spacer, whereas the DR4 TRE is built by the negative antagonists of T3 signalling. The truncated same direct repeats, separated with a 4 bp spacer. variant TR ∆β 3 lacks the DNA-binding domain but TRs can competitively bind to PPREs of PPAR retains T3 binding activity and acts as a dominant- target genes. Moreover, either TRs or PPARs negative antagonist. The human variant TR β4 that compete with the other receptor for binding to RXR is a C-terminal spliced variant of TR Β1 lacks T3 - that may result in decreased availability of RXR binding ability and acts as an endogenous and reduced transcriptional activity of the PPAR dominant-negative isoform. The TR β4 weakly but target genes. Consequently, the PPAR γ agonist significantly inhibits transcription mediated by rosiglitazone is able to reverse the effects of functional T3 receptors. dexamethasone and to increase serum T3 and T4 The function of the TR β proteins may be influenced levels. Interestingly, in rats with a high-fat diet and by the other members of the receptor superfamily in a hyperthyroid state, administration of the that may lead to either synergistic or antagonistic PPAR α agonist Wy14,643 restores glucose effects. The nuclear receptor superfamily includes tolerance by enhancing glucose-stimulated insulin the estrogen receptor-like subfamily liganded by secretion and relieves the effect of hyperthyroidism. estrogene (ER) or 3-ketosteroids: glucocorticoid These data suggest that PPAR α activity may restore (GR), progesterone (PR), androgen (AR), the pancreatic islet function affected by abnormal mineralocortycoid (MR) and the thyroid hormone T3/TR signaling. Furthermore, the genomic receptor-like subfamily that consists of the nuclear crosstalk of these two receptors may occur also via receptors for the thyroid hormone (TR α, TR β), liver nongenomic actions of the receptors (Lu and Cheng X receptor-like proteins (LXR, FXR), vitamin D 2009). receptor-like proteins (VDR, PXR, CAR), retinoic Ligands and metabolites acid receptors (RARs) and peroxisome proliferator- L-thyroxine (T4), a major secretory product of activated receptors (PPARs). This subfamily also thyroid gland, and 3, 5, 3'-triiodo-L-thyronine (T3), include the heme receptors: Rev-ErbA α encoded by the most active form of thyroid hormone are NR1D1 gene that regulates various cellular function naturally occurring ligands for TR β receptors. T3 is including circadian cycle and the ratio of a tyrosine-based derivative of T4 that is produced TR α1/TR α2 isoforms as well as Rev-erb β (NR1D2) by the thyroid gland in response to thyroid- identified in the THRB locus (3p24.2, see diagram stimulating hormone (TSH) from the anterior 2). These heme binding receptors were identified pituitary. The TSH is released by thyrotropin- previously as "orphan" (unknown ligand and/or releasing hormone (TRH) from the paraventricular

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nucleus (PVN) of the hypothalamus and T4 transduced, αvβ3-integrin-mediated pathway), synthesis is controlled by negative feedback loop: DITPA (3,5-diiodothyropropionic acid), DIMIT hypothalamic-pituitary thyroid (HPT) axis. Free (3,5-dimethyl-3'-isopropylthyronine), GC-1 (3,5- thyroid hormones in the circulation act negatively dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)- on the pituitary and hypothalamus, thus reducing phenoxy acetic acid (sobetirome), a TR β-selective), the release of TRH, TSH and finally T4 and T3 KB-141 (3,5-dichloro-4-[(4-hydroxy-3- concentration in plasma. In the nucleus, the thyroid isopropylphenoxy) phenyl] acetic acid), SKF hormones bind TR β with high affinity and 94901(3,5-dibromo-3'-pyridazinone-L-thyronine). specificity with K d values in the nM range. Most T4 TETRAC expresses nucleus-mediated and T3 in the circulation are bound to proteins thyromimetic activity, though current studies including Thyroxine-binding globulin (TBG), demonstrated its role in suppression of T4-induced transthyretin and albumin. This proteins are αvβ3-integrin/ERK1/2 pathway (Davis et al., 2009). responsible for carrying the thyroid hormones in the Covalently bonded to a nanoparticle, TETRAC acts bloodstream. exclusively at the cell surface TH αvβ3 integrin Under normal conditions only a small fraction of receptor without the thyromimetic activity and TR- T3 is generated by the thyroid gland, the remainder mediated effects in nucleus. of T3, which is available for binding sites in the TRs agonist relative potencies: GC- plasma and body cells, is synthesized by mono- 1~T3~TRIAC~T4>>rT3 have been determined for deiodination of T4, that is inactive and needs to be both coactivator recruitment and corepressor converted to T3 what occurs in peripheral tissues. dissociation. The reaction is catalyzed by type 1 (DIO1, The TR β and liver selective thyromimetics EC1.97.1.10) or type 2 (DIO2, EC 1.97.1.11) (STRMs) such as GC-1 (sobetirome), KB2115 iodothyronine deiodinases (selenoproteins), the first (eproterome), KB141 and MB07811 have been is abundant in kidney, liver and thyroid whereas the developed to selectively lower serum total and LDL last one is mainly present in brown adipose tissue, cholesterol without affecting HDL cholesterol pituitary and central nervous system. DIO1 is levels and induce weight loss without deleterious sensitive to inhibition by the anti-thyroid drug effects on heart and combat other aspects of propylthiouracil (PTU). The enzyme activity of the metabolic disease. GC-1 is the first TR β agonist. kidney and liver is responsive to the nutritional KB141 is a selective TR β agonist that can bind with status of an organism and is found to be more active an affinity 14-fold higher than that for TR α and is a during states of accelerated glucose metabolism. useful candidate for attenuating the features of Most T3 molecules are produced by enzymatic metabolic syndrome. This agonist lowers outer ring deiodination (ORD) of T4. Alternative, cholesterol, causes significant weight reduction in inner ring deiodination (IRD) of T4 yields the primates and has a 10-fold window in which metabolite rT3. ORD is regarded as an activating therapeutic increases in metabolic rate are seen pathway and IRD as an inactivating pathway. DIO1 without cardiac hypertrophy or tachycardia shows the ORD and IRD activity, DIO2 only ORD therefore is a promising candidate for treating activity and the third iodothyronine deiodinase - obesity, hyperlipidemia and diabetes. KB2115 has DIO3 (expressed above all in brain tissue) mediates been described as a TR β-selective agonist that is only the degradation of thyroid hormone since it preferentially taken up by the liver and is also has only IRD activity. T3 and rT3 undergo further effective in patients on statin therapy. MB07811 is deiodination to the common metabolite 3,3'- a liver-selective pro-drug that after hepatic diiodothyronine (3,3'T2), which is generated by activation through enzymatic cleavage is converted IRD of T3 and by ORD of rT3. Recent evidence for to the active form MB07344 - a strong TR β ligand binding of T2 by a subunit of mitochondrial binding the other TRs with significantly lower cytochrome c oxidase and its stimulation appears to affinity. Selective targeting with TR β agonists may be of a receptor/effector nature, showing as well present an innovative strategy for searching for that the T2 metabolite may have an important TR β-specific effects. biological role that influences cellular respiration. The most commonly studied antagonists are: Apart from T4, T3, rT3 and 3,3'T2, the other DIBRT (3,5-dibromo-4-(3'5'-diisopropyl-4'- thyroid hormone derivatives have been shown to hydroxyphenoxy)benzoic acid) and NH-3 (4-(4- take part in iodothyronine-like endogenous hydroxy-3-isopropyl-5-(4-nitrophenylethynyl) signaling, which may includes T2 (3,5-diiodo-L- benzyl)-3,5-dimethylphenoxy) acetic acid (NH-3, a thyronine), TAMs (thyronamines), and sulfate or low affinity TR antagonist). The TR β-selective (but glucuronic acid derivatives of thyroid hormones. not TR α-selective) agonists and antagonists are The most commonly studied TRs agonists are: expected to alter gene expression in a TR β biased TRIAC (3,5,3'-triiodothyroacetic acid showing manner that can differ from T3, which binds the thyromimetic activity, TR β selective), TETRAC two TRs with similar affinity. Amiodarone (Amio) (tetraiodothyroacetic acid, an inhibitor of T4- and dronedarone (Dron) are drugs used to

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discriminate between TR α1 or TR β1 regulated 90% of DNA identity between the genes), located genes in central and peripheral TH metabolism. A in THRB introns: downstream of exon 1 (4x); 2a major metabolite of Amio, desethylamiodarone, (2x), 2b, 3 (2x) and 4. These Alu-like elements acts as a TR α1 and TR β1 antagonist, whereas the were found in 5'-end of THRA sequence as well as major metabolite of Dron -debutyldronedarone acts in NR1D1 and NR1D2 genes. Moreover, two full- as a selective TR α1 antagonist that allows the TR β1 length of ADAR-binding sequence motif flanking effects to become apparent. A-to-I RNA editing sites (Ramaswami et al., 2012) are present in introns before exon 2a and 2c of the Homology THRB. This consensus sequence is also present in Human THRB gene is reported to be conserved in THRA and NR1D1 genes. Euteleostomi and has orthologs identified in: Pan The other reported paralogs for THRB gene are: troglodytes (Chr3, NCBI protein reference VDR, NR4A3, NR4A1, NR1I2, RARG, NR1I3, sequence: XP_001163850.1), Macaca mulatta RARA, NR1H2, NR4A2, NR1H4, RARB, NR1H3. (Chr2, XP_001090554.1), Canis lupus (Chr23, Interestingly, the THRB has close structural and XP_862690.2), Bos taurus (Chr27, functional relatives in NR1D2 (in the same locus), XP_002698801.1), Mus musculus (Chr14 7.08 cM, which encodes Rev-erb β as well as THRA NP_001106888.1), Rattus norvegicus (Chr15 p16, encoding TR α1 and TR α2 isoforms and NR1D1 NP_036804.2), Danio rerio (Chr19, NP_571415.1) gene expressing Rev-erA α. The THRB and NR1D2 and Gallus gallus (Chr2, erythroblastic leukemia genes are also linked to the RARB encoding RAR β. viral (v-erb-a) oncogene homolog 2, avian, Since it has been shown that the THRA/NR1D1 NP_001239150.1); according to the NCBI locus is also linked to the RARA gene, these data HomoloGene. Since the THRB shows multiple suggest that the two receptor gene clusters alignment, pairwise similarity scores and RARA/THRA/NR1D1 and RARB/THRB/NR1D2 evolutionary distances, additional putative were generated by a single large-scale duplication. orthologs are likely in a variety of different species Moreover, the THRA gene shares a partial overlap and can be viewed using alignment tools (BLAST, with the NR1D1 gene that influences the NCBI Resources). TR α1/TR α2 ratio. Similar regulation may occur in This gene is highly homologous to ERBB2 (v-erb- case of the TR β transcripts and the products of the b2 erythroblastic leukemia viral oncogene homolog genes sharing the same DNA sequence with the 2, neuro/glioblastoma derived oncogene homolog THRB (see diagram 1 and 2). The genes located in (avian)). A connection between TR β1 and cancer the same locus may produce long naturally became evident when the chicken TR α1 ortholog occurring antisense transcripts (cis-NATs) forming was characterized as the c-erbA proto-oncogene, sense-antisense pairs with a single stranded DNA or the cellular counterpart of the retroviral v-erbA transcripts of the THRB. These sense-antisense oncogene. However, a growing number of pairs may activate a pseudogene-mediated evidences suggest that TR β could serve as a tumor regulation (see Pseudogene). suppressor. Reference conserved domains on TR β1 protein THRB is a member of a large superfamily of (Homo sapiens, NP_001121649.1): 1) cd06961: nuclear hormone receptor genes (see interactions) NR_DBD_TR superfamily, DNA-binding domain and displays extensive structural and functional of thyroid hormone receptors, Heterodimer similarity with the paralogous gene - THRA, which interface of human Thyroid Hormone; 2) cd06935: evolved from a common ancestor gene, duplicated NR_LBD_TR superfamily, The ligand binding 500 Mya. The gene is duplicated at least in domain of thyroid hormone receptor, coactivator vertebrates, birds and amphibians. Divergent recognition site (polypeptide binding site), dimer evolution of the genes increased the variety of interface (polypeptide binding site). cellular responses to TH in vertebrates. The Reference conserved domains on TR β2 (Homo paralogous THRB and THRA show 77% of mRNA sapiens): 1) cd06929: NR_LBD_F1 (aa : 281-454), coding sequence (CDS) identity and 84% of protein Ligand-binding domain of nuclear receptor family identity. The similarities were determined based on 1; 2) cd06916 : NR_DBD_like (aa : 122- 194) the NCBI BLAST protein seq.: TR β1 DNA-binding domain of nuclear receptors is (NP_000452.2) ver. TR α1 (NP_955366.1); results: composed of two C4-type zinc fingers. identities: 310/367(84%); positives: 342/367(93%); Animal models gaps: 0/367(0%); method: Compositional matrix Animal models imply close associations between adjust. NCBI BLAST mRNA CDS seq.: TR β1 aberrant expression of TR β or TR β mutants and (NM_000461.4) ver. TR α1 (NM_199334.3); pathogenesis of some diseases, such as dominant or results: identities: 854/1113(77%); score 814 reccessice Generalized Resistance to Thyroid bits(902); gaps: 16/1113(1%). Comparative DNA Hormone (GRTH) and Follicular Thyroid analysis of the THRB and THRA revealed 10 Carcinoma (FTC). Creation of a mouse model that conserved fragments of at least 261bp in length (73- harbors a knockin mutation of TR β has facilitated

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the study of the molecular actions of TR β mutants elements, whereas TR ∆β 3 retains T3-binding in vivo. Knock-out studies in mice suggest that the activity but lacks a functional DNA-binding domain different TH receptors may mediate different and does not activate target gene transcription. functions, though several studies of TRs actions Therefore, this isoform acts as a modulator (potent revealed near complete overlaps in their effects. antagonist) of TR β3 when coexpressed at low Tissue-specific expression of TR β isoforms is concentrations. At higher concentrations, TR ∆β 3 is thought to be a major factor responsible for the a TRE-selective and cell-specific antagonist of observed differences in phenotypes, including those TR α1, Tr β1, and TR β3. Identified in humans TR β4 that are affected by TR β mutations. In mice knock- is a C-terminal variant that lacks the ligand binding outs, the TR β abnormalities may affect the domain (truncated variant of TR β1), thus may following systems: endocrine/exocrine glands function as a potent endogenous antagonist, (increased thyrotroph cell number, enlarged thyroid however its expression and function in animals is gland, abnormal pituitary gland physiology); unknown. hearing/vestibular/ear (abnormal cochlea Mice with targeted deletions in TR genes have morphology, increased or absent threshold for provided understanding of the possible roles of the auditory brainstem response, sensorineural hearing different TR β isoforms. Knockout mice that are loss); homeostasis/metabolism (increased unable to produce theTR α1 receptor show circulating thyroid-stimulating hormone level); subnormal body temperature and mild nervous system (increased thyrotroph cell number, abnormalities in cardiac function, whereas mice abnormal pituitary gland physiology, decreased which lack expression of both TR α isoforms were cochlear outer hair cell number, abnormal retinal severely hypothyroid and die within the first few cone cell morphology); skeleton (spiral ligament weeks of life. Mice with disruptions of the entire degeneration), vision/eye (abnormal retinal cone beta gene (TR β1 and TR β2) exhibit elevated TSH cell morphology). levels and deafness suggesting its role in auditory THRB gene, like mice Thrb ortholog, encodes TH system, while mice with mutations disturbing only receptor isoforms TR β1 TR β2 and TR β4. TR β2 had elevated TSH, but normal hearing. These Moreover, function of these receptors may be mutants allow determination of which functions of influenced by two additional isoforms: TR β3 and the different receptor isoforms are redundant and TR ∆β 3, expressed in rat models (Williams et al., which are not. TRs play important roles in the 2000). TR β1, TR β2, and TR β3 are bona fide T3 pathogenesis of thyroid cancers and hepatocellular receptors that bind DNA, T3 and regulate carcinoma (HCC). For instance, v-erbA, a mutant expression of T3-responsive target genes. Studies form of TR lacking ligand-binding ability, triggers of Tr β and Tr β2 knockout mice indicated that Tr β1 HCC development in transgenic mice. Similarly, is essential for development of auditory function, TR βPV (Kaneshige et al., 2000) mutation harmoring whereas Tr β2 is not required, but that Tr β2 alone is mice develope thyroid cancers (see exemplary essential for development of mid-wavelength (MW) mutants below). cones photoreceptors. In contrast, both Tr β1 and There are various mouse knock-outs for THRB: Tr β2 are required for regulation of hypothalamic- - TR βPV/PV ; mutant thyroid hormone receptor pituitary-thyroid axis. The Tr β2 deletion in mice kindred PV (Kaneshige et al., 2000); Synonyms: induces a complete and selective loss of MW-cone TR βPV; Allelic composition: homozygous opsin without significant changes in total cone TR βPV/PV and heterozygous TR βPV/+ ; Mutation numbers. TR β3 and TR ∆β 3 variants are transcribed details: PV has an unusual mutation in exon 10, a using third promoter (P3) positioned upstream of C-insertion at codon 448, which produces a human exon 5 and downstream of second promoter frameshift of the carboxyl-terminal 14 amino acids (P2) of TR β2 (see diagram 3). TR ∆β 3 mRNA lacks of TR β1. PV was derived from a patient (called PV) rat exon B (315 nt) encoding a fragment of DNA- with severe RTH characterized by elevated thyroid binding domain, present in TR β3, which contains hormone levels accompanied by normal TSH, short both exon A (342 nt) and B of rat TR β. Start codons stature, goiter, and tachycardia. This naturally of TR β3 has been identified in frame in various occurring mutation shows lost T3-binding, animal Thrb sequences including mouse, dog, transactivation activities, and displays dominant chicken but not in human, chimpanzee and negative activity. Moreover, PV strongly interferes macaque. Nevertheless, none of these ATG codons with the transactivation activity of wild-type TRs in are positioned within a favorable Kozak translation vitro and unlike the missense mutations or single initiation sequence context and the lack of murine amino acid deletion of TR β found in other patients, TR β3 or TR ∆β 3 expressed sequence tags (NCBI this unique frame-shifted mutated sequence is EST) suggests that in rats, expression from P3 immunogenic, for which high-affinity specific promoter is differentially regulated. TR β3 is a antibodies have been developed. TR β PV mutant functional T3 receptor and the most potent isoform, has been obtained by using homologous but dependent on the sequence context of TRE recombination and the CreyloxP System. Affected

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systems: endocrine/exocrine glands, endocrine/exocrine glands, hearing/vestibular/ear, homeostasis/metabolism; Human disease model: 1) homeostasis/metabolism, nervous system but not Thyroid Hormone Resistance (NCBI OMIM: behavior/neurological phenotype; Human disease 274300, 188570); TR β PV/+ and TR β PV/PV mice model : Thyroid Hormone Resistance, Generalized, faithfully reproduce human RTH - the TR βPV Autosomal Recessive - GRTH (OMIM: 274300). mutation was initially identified in a patient who - Thrb tm3Few ; thyroid hormone receptor beta; has the syndrome of resistance to thyroid hormone targeted mutation 3, Frederic E Wondisford; (RTH). Mice expressing a single PV allele showed Synonyms: GS125 KI, TR-BetaGS; Allelic the typical abnormalities of thyroid function found composition: homozygous, Thrb tm3Few /Thrb tm3Few , in heterozygous humans (mutation of one copy of involves: 129* C57BL/6; Mutation details: human THRB) with RTH that is characterized by a Missense mutations were introduced at codons 125 reduced sensitivity of tissues to the action of and 126 (exon 3), resulting in Glu to Gly and Gly to thyroid hormones. 2) Thyroid Carcinoma (OMIM: Ser substitutions. 188470), pituitary tumors; TR βPV/PV mice but not The substitutions were within the P-box of the first TR βPV/+ , spontaneously develop follicular thyroid zinc finger and were shown, in vitro, to abolish carcinoma and pituitary tumors with tumor DNA-binding while retaining the ability to interact progression similar to human cancer. The with T3 and cofactors. Western blot analysis homozygous PV mice exhibit severe dysfunction of showed endogenous levels of protein in the pituitary-thyroid axis, abnormal bone homozygous mutant mice; Affected systems: development and impaired weight gains. This endocrine/exocrine glands, hearing/vestibular/ear, phenotype is distinct from that seen in mice with a homeostasis/metabolism, nervous system; null mutation in the TR β gene (TR β-/-) vision/eye; Human disease model: Thyroid demonstrating the interference of the mutant TR β Hormone Resistance, Generalized, Autosomal with the functions of the wild-type TR β. TR βPV/PV Recessive, GRTH (OMIM: 274300). mutants may serve also as molecular model for - Thrb tm2Few ; thyroid hormone receptor beta; nongenomic actions of T3 condributing to thyroid targeted mutation 2, Frederic E Wondisford; carcinogenesis. The mutant mice allows the Synonyms: TRbeta delta337T ; Allelic composition: elucidation of oncogenic activity of the TR β(PV) homozygous, Thrb tm2Few /Thrb tm2Few , involves: through cytoplasmic effects of T3 (see diagram 5 129X1/SvJ * C57BL/6; Mutation details: The N1). This nongenomic action is mediated by deletion of 3 base pairs in exon 6, corresponding to interaction of PV with p85 α regulatory subunit of a deletion that results in thyroid horomone PI3K to activate the downstream AKT/mTOR, resistance in humans, was introduced via site- p70 S6K and PI3K-integrin-linked kinase-matrix directed mutagenesis along with a neomycin metalloproteinase-2 signaling pathways. The PV- selection cassette inserted into intron 5. The mediated PI3K activation leads to increased cell mutation in exon 6 affects the ligand-binding proliferation, motility, migration, and metastasis. In domain which is common to both isoforms this regulation, a nuclear receptors corepressor produced from this locus; Affected systems: NCoR competes with PV for binding to the p85 α behavior/neurological, homeostasis/metabolism, that result in reduction of the AKT-mTOR-p70 S6K nervous system; Human disease model: Thyroid signaling. The NCoR protein levels are Hormone Resistance, Generalized, Autosomal significantly lower in thyroid tumor cells than in Dominant, GRTH (OMIM: 188570). wild type thyrocytes, allowing more effective - Thrb tm2Few/+ ; thyroid hormone receptor beta; binding of PV to p85 α to activate the PI3K targeted mutation 2, Frederic E Wondisford; pathway, thereby contributing to tumor progression Synonyms: TRbeta delta337T ; Allelic composition: in the TR βPV/PV mutant mice (Guigon and Cheng, heterozygous, Thrb tm2Few /Thrb +, involves: 2009). 129X1/SvJ * C57BL/6; Mutation details: (see - Thrb tm1Df ; thyroid hormone receptor beta; targeted Thrb tm2Few /Thrb tm2Few ); Affected systems: mutation 1, Douglas Forrest ; Synonyms: Thrb-, behavior/neurological, homeostasis/metabolism, TRbeta- Allelic composition: homozygous, nervous system; Human disease model: Thyroid Thrb tm1Df /Thrb tm1Df involves: 129 S1/Sv * Hormone Resistance, Generalized, Autosomal C57BL/6J; Mutation details: Insertion of a Dominant, GRTH (OMIM: 188570). neomycin cassette into exon 3, disrupts both the - Thrb tm1Df/tm1.1Syc ; thyroid hormone receptor beta; beta1 and beta2 isoforms of this gene. This targeted mutation 1, Douglas Forrest; Synonyms: transcript revealed a deletion of exon 3 sequences, Thrb-, TRbeta-; Allelic composition: heterozygous, and fused beta1 exon 2 to exon 4 resulting in an Thrb tm1Df /Thrb tm1.1Syc , involves: 129S1/Sv * aberrant open reading frame, which terminates early 129S6/SvEvTac * C57BL/6 * C57BL/6J; Mutation into exon 4. No functional protein is predicted from details: see above; Affected systems: this transcript, as the essential DNA binding and T3 endocrine/exocrine glands, mortality/aging, binding domains not present; Affected systems: tumorigenesis, respiratory system; Human disease

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model: Thyroid Carcinoma, Follicular; FTC and table 1). Some of them inhibits homodimer (OMIM: 188470). formation or stabilizes homodimer ligand- - Thrb tm1Few ; thyroid hormone receptor beta; dependent conformational changes. targeted mutation 1, Fredric E Wondisford; The majority of the mutated TR β receptors lost Synonyms: TRbeta2 null; Allelic composition: their function of transactivation (e.g. DIO1, GH1) homozygous mutant mice Thrb tm1Few /Thrb tm1Few , or transrepression (e.g. TSHB, TRH) in T3- involves: 129S4/SvJae; Mutation details: A PGK- dependent manner. neo cassette replaced the transcription site, the These variants usually act as dominant-negative entire Thrb2-specific exon, and the splice donor mutants. Most of the germline, clinically associated acceptor site. RT-PCR analysis of pituitary RNA mutations of the TR β receptor have been identified confirmed the preservation of the Thrb1 isoform, as in patients with autosomal recessive or dominant, well as the absence of the Thrb2 isoform. Affected generalized thyroid hormone resistance (RTH, systems: endocrine/exocrine glands, OMIM: 188570, 274300 respectively) as well as homeostasis/metabolism but not selective pituitary thyroid hormone resistance hearing/vestibular/ear. (OMIM: 145650). - Thrb tm4Few ; thyroid hormone receptor beta; Patients with RTH have got impairment of the targeted mutation 4, Frederic E Wondisford; mechanism negatively regulating the feedback of Synonyms: TR-Beta-, TrbetaKO ; Allelic T4/T3 to the hypothalamic TRH and pituitary TSH composition: homozygous, Thrb tm4Few /Thrb tm4Few genes by the mutated TR β receptors. involves: involves: 129 * C57BL/6; Mutation There are also studies showing the mutations in details: Exon 3 was replaced with a self-excising TR β-DNA binding domain, in 5' and 3' mRNA PGK-neo cassette. The deletion of exon 3 untranslated regions (UTRs) and intronic variants putatively results in an aberrant open reading frame with potential disease association. caused by the fusion of exons 2 and 4. Using a C- These allelic variants have been identified to impair terminal mAb, protein was undetected by Western transcription, alternative splicing, translation or blot analysis of homozygous mutant mice; Affected TR β protein function such as hormone binding, systems: all mentioned at the begining of this DNA binding, ligand-dependent conformational section. changes or corepressors/coactivators More information on the Mouse Genome dissociation/association function (see references). Informatics website (Mouse Genome Database Somatic (MGD)). There are no reference variants reported as somatic Mutations in the NCBI dbSNP and ClinVar databases, however 3 allele variants (ID: nsv429603, Note nsv429566, nsv429555) are present in the NCBI 9555 human THRB variants (NCBI dbSNP) dbVar. includind 8007 single nucleotide polymorphisms Morover, several studies tested the hypothesis that (SNPs) and 326 human variants (20 studies, NCBI the functions of TR β could be impaired in various dbVar) have been recorded in the NCBI databases. cancer tissues by somatic mutations (see Moreover, 42 pathogenic variants of clinical references). significance including 31 germline SNP, 4 copy For instance, Puzianowska-Kuznicka et al. (2002) gain, 3 deletions and 3 insertions of the gene have tested this hypothesis in selected human thyroid been catalogued in the NCBI ClinVar database (see papillary cancer. Based on cancer-derived cDNAs, Table 1). These allelic variants have been identified they found that the mean expression levels of TR β1 to impair hormone binding, DNA binding or ligand- mRNA and TR α1 mRNA were significantly lower, dependent conformational changes. Some of them whereas the protein levels of both were higher in inhibits homodimer formation or stabilizes cancer tissues compared to healthy thyroid samples. homodimer ligand-dependent conformational Sequencing of TR β1 and TR α1 cDNAs, cloned changes. The majority of the mutated TR β receptors from 16 papillary cancers, revealed that mutations lost their trans-activation function and exhibited affected receptor amino acid sequences in 93.75% dominant-negative activity. and 62.5% of cases, respectively. In contrast, no mutations were identified in healthy Germinal thyroid controls, and only 11.11% and 22.22% of There are 31 annotated germline THRB allelic thyroid adenomas had such TR β1 or TR α1 variants in the NCBI dbSNP and ClinVar but only mutations, respectively. 15 reported in dbVar of the NCBI. Most of them The authors summarized that the findings suggest a are SNPs and insertions or deletions are less possible role for mutated thyroid hormone receptors frequently found. The most relevant SNPs are those in the tumorigenesis of human papillary thyroid located on hormone binding domain (see diagram 6 carcinoma (NCBI OMIM: 188550).

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Table 1. Pathogenic variants of clinical significance, according to NCBI ClinVar.

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Diagram 6. Clinically asociated amino acid variants of TR β proteins. Graphic representation of three mutational "hot spots" in the TR β ligand-binding domain, in which natural mutations have clustered. Crystallographic structure of the TR β ligand binding domain (LBD, E) complexed with triiodothyronine (T3) and C-terminal domain T3-dependent transactivation (F) are shown on all four sides (A,B,C,D) to visualize listed (on the right) reference variants, which colour corresponds to each α-helix structures of LBD (rainbow colored, N-terminus in blue, C-terminus in red). Mutations in the LBD may be associated with resistance to thyroid hormone (RTH). The most frequent mutations in RTH are bolded. Substitutions associated with pituitary-specific RTH (PRTH, R338L, R338W, R429Q) and generalized RTH (GRTH, P453S, G345S) are given. Mutations of GRTH (P453S, G345S) impair both TR β2 and TR β1 function proportionally, whereas variants of PRTH disproportionately disrupt the function of TR β2 (Wan et al., 2005). An increased inability of the mutants to properly release the nuclear corepressors is postulated to inhibit the T3- mediated transactivation or transrepression of target genes. The TR β mutants function in a dominant-negative fashion to interfere with the transcription activity of other wild-type thyroid hormone receptors (TR α) leading to resistance in peripheral tissues and dysregulation of the hypothalamic-pituitary thyroid axis (Dumitrescu and Refetoff, 2013). The conserved T3 binding domain was visualized using PyMOL 1.3 Molecular Graphics System, on the basis of crystallographic structure file (PDB: 1XZX) of the RCSB Protein Data Bank and The NCBI Conserved Domains Database (CDD, ref.c.d.: cd06961, NR_LBD_TR). For a more extensive listing of mutations, see references.

The similar conclusion can be found in studies of has been proposed to serve as a novel epigenetic Kamiya et al. (Kamiya et al., 2002), who have marker for early detection and prognosis of high cloned and sequenced 22 cDNAs obtained from the grade serous ovarian cancer (Kashuba et al., 2013) human renal clear cell carcinoma (OMIM: 144700). and identified to be frequently methylated in Somatic mutations were found in 7 TR β1 and 3 prostate cancer, breast cancer, non-small cell lung TR α1 samples. These findings are consistent with cancer and acute lymphoblastic leukemia (Dmitriev the results obtained on hepatocellular carcinoma et al., 2009; Dmitriev et al., 2012; Ling et al., 2010; HCC (Lin et al., 1999). However, some data from Vasiljevic et al., 2011). Searching the cBio Cancer direct genomic DNA sequencing provided an Genomics Portal (Cerami et al., 2012), currently evidence that somatic THRB gene mutations may providing access to data from more than 5000 not be as common in differentiated thyroid cancers, tumor samples from 20 cancer studies revealed that in which hypermethylation of the gene was shown somatic mutations were the most frequently found to be a major mechanism responsible for down- in Skin Cutaneous Melanoma (up to 5.7% cases regulation of the gene expression. Indeed, THRB altered in the database).

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The mutations were also reported in stomach sequencing data from the same individuals. These adenocarcinoma (4.1%), kidney renal clear cell preliminary findings, however obtained from non- carcinoma (3.6%), colorectal cancer (2.8%), cancerous samples, may indicate that the ADAR- pancreatic adenocarcinoma (2.4%), colon and mediated RNA editing may change the sequence of rectum adenocarcinoma (1.9%), head and neck THRB pre-mRNA post-transcriptionally squamous cell carcinoma (1.8%), lung (Ramaswami et al., 2012) and that this process may adenocarcinoma (1.6%), ovarian serous be impaired in numerous cancers (Huang et al., cystadenocarcinoma (1.6%), uterine corpus 2013). endometrioid carcinoma (1.3%), sarcoma (1.2%), lung squamous cell carcinoma (1.1%), breast Implicated in invasive carcinoma (0,9%) and acute myeloid leukemia (0,5%). Thyroid related disorders and Most of the cancer mutations were identified as substitutions, however deletions were the most cancers frequently found in renal clear cell. There were also Note reported amplifications in pancreatic THRB gene mutations (NCBI OMIN : 190160) are adenocarcinoma, kidney chromophobe renal cell known to be a cause of several disorders including carcinoma and sarcoma (Gao et al., 2013; see autosomal recessive or dominant, generalized cBioPortal in external links). It is noticeable that thyroid hormone resistance (GRTH; NCBI OMIM: the reported alteration frequency can vary in 188570, 274300 respectively) as well as selective different studies depending on the target pituitary thyroid hormone resistance (PRTH; populations, number of tested samples, analytical OMIM: 145650). Some forms of peripheral methods and histopathological classification of the resistance to TH observed in familial euthyroid tumors. hyperthyroxinemia (OMIM: 145680) also appear to Somatic mutations/sequence variants have been have a defect in the nuclear receptor for TH (Winter shown to be created post-transcriptionally in and Signorino, 2001). This gene has been also various cancer-derived transcripts (Klimek- implicated in cancers such as follicular or papillary Tomczak et al., 2006; Chen et al., 2013). Adenosine thyroid carcinoma (FTC, PTC; OMIM: 188470, (A) to inosine (I) RNA editing of AZIN1 was 188550 respectively). Disturbances of the THRB demonstrated to be increased in the hepatocellular gene are frequent findings in numerous cancers carcinoma and suggested as a potential driver in the including renal cell cancer (RCC; OMIM: 144700) pathogenesis of human cancers, particularly HCC. as well. ADAR1-mediated A-to-I RNA editing was shown TR β is a member of thyroid hormone receptors to change the RNA nucleotide sequence relative to subfamily that mediates genomic and nongenomic that of the encoding DNA that was reported to actions of thyroid hormone (TH, T4/T3) that can result in cancer development and progression influence cell growth, metabolism, apoptosis, and (Huang et al., 2013). ADAR belongs to the family metastasis. TRβ mutations are involved in the of RNA specific adenosine deaminase, which acts reduced sensitivity to TH, short stature, attention- on double-stranded RNA (dsRNA) substrats deficit hyperactivity disorder, autoimmune thyroid including those created by long naturally occurring disease, erythroleukemia, hepatocellular carcinoma, antisence transcripts (cis- and trans-NATs) such as and thyroid carcinoma (Rosen et al., 2011). The Rev-erb α/TR α2 or intra-THRB transcripts (see reduced sensitivity to TH may include defects of diagram 1). Then, inosine, which in RNA can pairs transport, metabolism and action of TH. TR β with cytosine (C) uracil (U) or adenine (A, mutations have been identified to affect some of generating wobble pairs), is recognized as these processes (Dumitrescu and Refetoff, 2013). guanosine (G) by ribosomes (translation) and Clinically, effects of TH are observed as changes in reverse transcriptase during cDNA synthesis. In metabolic rate, altered lipid metabolism, and fact, the comparison of whole genome sequences characteristic effects on cardiovascular and deeply sequenced transcriptomes of human development. Aberrations in the levels of TH can lymphoblastoid cell line GM12878 and a Han cause multiple disorders, including cardiovascular Chinese individual (YH) allowed for identification disease, diabetes mellitus, chronic liver disease and of two mismatches corresponding to A/I(G) ediging is implicated in various cancers. Interestingly, TH sites within THRB sequence (DNA ref. sec. can modulate response to interferon-γ and has position: 24167619, 24321108) as well as several potential therapeutic applications in hepatitis B and editing sites in NR1D2 gene (see diagram 1, C (Chi et al., 2013). Knowledge of the molecular genomic context, Ramaswami et al., 2012). The mechanisms involved in TH action allows the next-generation sequencing used in the study recognition of the phenotypes caused by defects of permitted comparing genomic DNA and RNA TH action including the syndromes of reduced

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sensitivity to thyroid hormone (Dumitrescu and reported to be a factor that may contribute to Refetoff, 2013). carcinogenesis in clear cell renal cell cancer (ccRCC). In this cancer, TR β1 mRNA and protein Carcinogenesis levels were reduced by 70% and 91% in ccRCC A close association of TR β mutations with human and accompanied by absent DIO1 protein (a TR β1 cancers has become apparent, however the role of target gene) and a 58% reduction in tissue T3 TR β mutants in the carcinogenesis is still not clear concentration when compared to controls obtained (Weinert et al., 2012). Besides, a growing number from the opposite pole of malignant kidneys. These of studies suggest that the THRB can function as a data provide an evidence of impaired T3 action in tumor suppressor (Guigon et al., 2013). This ccRCC that is maintained by reduced expression of putative role of the gene is consistent with findings TR β1. The observed discordance in the magnitude showing that four markers spanning the 3p24-p21.3 of the change in TR β1 mRNA level compared to region, THRB, AP20R, D3S1029, and D3S32, are protein (70/91 % reduction) together with the regularly eliminated from three human chromosome aberrant splicing of various TR β1 5'UTRs leading 3 (chr3)/mouse microcell hybrids (MCHs) during to differences in the ratios of the variants may tumor growth in SCID mice. These studies confirm that TR β1 expression is subject to complex indicated that tumor suppressor gene may be post-transcriptional regulation at least in ccRCC located in this area, as suggested by frequent loss of (Master et al., 2010). At this level, the gene is also heterozygosity (LOH) within the region containing regulated by microRNAs that are small endogenous the THRB and observed in several types of solid noncoding RNAs binding to 3'UTR of the TR β tumors (Kholodnyuk et al., 1997). The loss of mRNA and affecting its level through RNA normal expression of the THRB gene due to interpherence (RNAi) phenomenon. miR-21 and truncation or deletion has been observed in many miR-146a have been found to inhibit the expression malignancies including kidney, lung, melanoma, of the THRB by lowering the levels of both, TR β breast, head and neck, uterine cervical, ovarian, and mRNAs and proteins, suppressed down to 10-28% testicular tumors. in papillary thyroid cancer (PTC) (Jazdzewski et al., Moreover, the epigenetic silencing of the THRB 2011). gene is common in human cancers. TRs play A knock-in mouse harboring a dominant negative important roles in the pathogenesis of Tr β mutation develops metastatic thyroid cancer hepatocellular carcinoma (HCC). that suggests the involvement of TR β in PV/PV It has been shown that cloned TR α and TR β are carcinogenesis. The Thrb mice (Kaneshige et truncated or mutated at high frequencies in the al., 2000) harboring a knockin dominant negative human HCCs. TR β1 isoform is essential for PV mutation (see animal models), identified in a genomic actions of T3 in liver, wherein TH can patient with resistance to thyroid hormone, influence hepatoma cell growth, metabolism, develops the follicular thyroid carcinoma (FTC). apoptosis, and metastasis. The more aggressive thyroid tumor progression in PV/PV Therefore modulation of the TR β-mediated actions the Thrb mice results not only from the loss of of TH may have powerful therapeutic potential in tumor suppressor functions but also gain-of- clinical applications (Chi et al., 2013). Both TR α function in the oncogenic activities of the PV and TR β have been shown to mediate action of T3 variant to drive thyroid carcinogenesis. Cell-based that blocks the response to the oncogenic forms of studies with simian virus-40 (SV40)-induced the three ras isoforms (H-ras, K-ras, and N-ras). carcinogenesis demonstrated that TR β can inhibit However, the TR β isoform has stronger anti- tumorigenesis by blocking the oncogenic actions of transforming properties than the TR α isoform and SV40-Tag via protein-protein interaction. The TR β importantly can inhibit neuroblastoma was shown to compete with Rb and 53 for binding tumorigenesis even in hypothyroid mice. to SV40-Tag oncoprotein that were accompanied These results show the existence of a transcriptional by reduced cell proliferation and delayed cell entry cross talk between the TR β and the ras oncogene from the cell cycle G1 to the S phase. In another that may influence relevant processes such as cell research, estrogen (E2)-dependent growth of MCF- proliferation, transformation, or tumorigenesis 7 cells that express the estrogen receptor, but not (García-Silva and Aranda, 2004). TRs, was inhibited by the expression of TR β in the Furthermore, decreased THRB expression by presence of T3. In a xenograft mouse model, large promoter hyper-methylation has been reported in tumors rapidly developed after inoculation of MCF- human breast cancer, lung cancer, and thyroid 7 cells that lacks the TRs expression. Markedly carcinoma, whereas reactivation of the silenced smaller tumors (98% smaller) were found when thyroid hormone receptor β gene expression delays MCF-7-TR β cells were inoculated in athymic mice, thyroid tumor progression (Kim et al., 2013). indicating that TR β can inhibit the E2-dependent Aberrant TR β1 mRNA and protein levels have been cancer growth. This study provides additional in vivo evidence to support the hypothesis that TR β

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could act as a tumor suppressor in breast cancer TH in promoting the thyroid carcinogenesis of development and progression. Moreover, cell-based Thrb PV/PV mice (Guigon and Cheng, 2009). studies in T47D, a breast cancer cell line, showed Importantly, these findings suggest an anti-cancer that T3 represses STAT5 signaling in TR β- potential of anti-thyroid drugs (Lu et al., 2012). The expressing cells through decreasing STAT5- autors proposed a model in which the the TR βPV mediated transcription activity and target gene mutant directly interacts with PI3K to activate AKT expression whereas sustained STAT5 signaling was signaling pathway. Suppression of TH in these observed in TR βPV -expressing cells. The Thrb PV cells, downregulates the membrane receptor mutant increases the activity of STAT5 to increase integrin αvβ3 switching off a nongenomic action of cell proliferation and the expression of the STAT5 T4. Furthermore, PTEN was found to be activated target gene encoding β-casein in the mammary in these cells that can decrease the formation of gland. Another transcription factor - STAT3 is PIP3, repress p-AKT and its downstream β-catenin frequently found to be activated in breast cancer and GSK3 β signaling pathways, finally leading to cell lines and patients with advanced breast cancers. inhibition of cell proliferation (Lu et al., 2012). In Importantly, the STAT3 as well as STAT1 α are addition, TR βPV mutant is known to activate the found to be activated as a result of nongenomic TR βPV /PI3K/AKT signaling cascade via binding to actions of T4 via αvβ3 integrins (Davis et al., p85 α regulatory subunit of the PI3K competing 2009), which are also responsible for activation of with NCoR, which can also bind to p85 α and cytoplasmic fraction of TR β1 via ERK1/2-mediated repress this pathway (Guigon and Cheng, 2009). phosphorylation of its Ser 142 (see nongenomic Besides, elevated levels of TR β1 expression have actions of TH). This pathway does not need to be been reported to reduce cell proliferation, malignant mediated by genomic-actions of TR β receptors and phenotype and to enhance apoptosis, indicating the results in activation of the STAT3 and STAT1 α that suppressive role of the receptor, which is T3- finally may lead to pro-proliferative, pro- dependent at the genomic level. Furthermore, FTC- angiogenic and anti-apoptotic effects. The T4 action 236 cells, stably expressing TR β, exhibited lower through αvβ3 integrins can be selectively blocked cell proliferation and migration through inhibition with a T4 analogue - TETRAC, without affecting of β-catenin signaling pathways when compared to the TR β-mediated genomic actions of T3. FTC-236 without TR β. There are also studies In various reports, enhanced growth and indicating that the phenotype of tumors induced in proliferation of cancer cells are observed at low or hypothyroid hosts is more mesenchymal and their high levels of the thyroid hormone, depending on invasiveness and metastatic behaviour are the origin of cells or tissues examined. However, enhanced. These findings are in line with reports these confusing data could result from activation of documenting reduced tissue T3 in human gliomas different and cell-specific mechanisms involved in (Nauman et al., 2004). Moreover, the reduced TR β1 genomic and nongenomic actions of T4/T3. TH has expression and tissue hypothyroidism have been been shown to be a ERK1/2-dependent growth also reported in clear cell renal cell cancer factor for Human Myeloma Cells acting via αvβ3 (ccRCC). The level of T4 did not differ between Integrin (Cohen et al., 2011). Several studies have normal and ccRCC tissues, whereas the demonstrated as well that T3 promotes growth and concentration of T3 was reduced by 58% in ccRCC proliferation of cancer cells through TR β1/Oct-1- and was accompanied by 92% decrease of DIO1 mediated cyclin D1 activation that was confirmed mRNA - a TR β1 target gene (Master et al., 2010). in papillary thyroid carcinoma cell lines (Perri et These results are in agreement with genomic and al., 2013). Decreased concentration of T3 has been nongenomic actions of TH that could be executed also demonstrated to reduce proliferation of Caki-2 in ccRCC via T4-activated αvβ3-integrin/ERK1/2 cells in vitro (Poplawski and Nauman, 2008). There pathway (T4 levels were not altered) or are studies indicating that elevated levels of TH TR β1/p85/PI3K/Akt/mTOR pathway but not may initiate direct effects on proliferation including necessarily through TR β-mediated genomic actions those engaged in the regulation of cell cycle of T3 (low levels of TR β1, DIO1, T3). These progression that may at least partially reflect the disturbances are likely to be involved in the process nongenomic actions of TH. Moreover, Thrb PV/PV of carcinogenesis or in maintaining a proliferative mice (see animal models) treated with advantage to malignant cells. Indeed, propylthiouracil (PTU), wchich blocks TH tetraidothyroacetic acid (TETRAC), a thyromimetic production, have been shown to reduce thyroid agonist of TR β that can also block the T4 integrin tumor growth by 42% when compared to control (αVβ3) receptor at the cell surface, has been shown Thrb PV/PV mice (Lu et al., 2012). The tumor cell to inhibit growth of human renal cell carcinoma proliferation, invasion and metastasis was also xenografts (Yalcin at al., 2009) and human decreased and accompanied by marked attenuation medullary thyroid carcinoma (MTC) xenografts in of the TR βPV/PI3K/AKT/ β-catenin/cyclin-D2 the nude mouse (Yalcin at al., 2008). Interestingly, signaling pathway thus, showing a critical role of both the MEK/ERK- and PI3K/Akt-dependent

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pathways mediate CD74-induced tumorigenesis of genes involved in retardation of tumor growth and ccRCC and it is known that TR β1 is involved in progression. (Martínez-Iglesias et al., 2009b; Kim these signal cascades (see genomic and nongenomic at al., 2013). TR β-dependent transrepression (see actions of TH). The CD74 overexpression could not genomic actions of TH) is thought to be a significantly induce the expression of TR β target mechanism that may have an important function in genes: HIF1A or HIF2A, what is in agreement with suppression of transforming effects of at least the low levels of T3 in ccRCC. T3 is required not several oncogenes. The inhibitory action of T3 on only for genomic but also nongenomic actions ras-mediated transformation (Garcìa-Silva and mediated by TR β1 in cytoplasm (see diagram 5) Aranda, 2004) can be enhanced by over-expression contributing to expression of PI3K-dependent of corepressors and reversed by silencing of the genes, which includes the HIF1A (HIF-1α), corepressors. This shows an important functional SLC2A1 (GLUT1) and RCAN2 (ZAKI-4) genes. role of endogenous corepressors in suppression of At the same time, TR β1 nuclear import transformation and tumorigenesis by TR β1. All (cytoplasmic/nuclear localization) and its these findings raise the possibility that TR β could transcription factor activity depend on act as a tumor suppressor in tumorigenesis. phosphorylation of TR β1 Ser 142 by ERK1/2. However, the presence of several TR isoforms, Simultaneously, TR βPV mutant has been various TH metabolites, multiple transcription demonstrated to activate cytoplasmic actions of T3 cofactors as well as simultaneous activation of the via binding to CSH 2 domain of p85 α (see diagram 5 genomic and nongenomic actions of TH make its N1c) (Furuya et al., 2009). This nongenomic action final effect more pleiotropic and less clear. is mediated by direct protein-protein interaction of Thyroid carcinoma, papillary (PTC) TR βPV with p85 α regulatory subunit increasing the catalytic activity of p110 of phosphatidylinositol 3- OMIM: 188550, MedGen UID: 66773. kinase (PI3K) to activate the downstream Synthesis and release of TH by follicular cells in AKT/mTOR, p70 S6K and PI3K-integrin-linked the thyroid gland is regulated through the kinase-matrix metalloproteinase-2 signaling hypothalamic-pituitary thyroid (HPT) axis, a pathways. The TR βPV -mediated PI3K activation negative feedback loop controlled by both, the leads to increased cell proliferation, motility, TR β1 and TR β2 isoformes. Nonmedullary thyroid migration, and metastasis (Furuya et al., 2009), but cancer (NMTC) includes thyroid cancers of these effects are TH-dependent (Lu et al., 2012). In follicular cell origin and accounts for more than addition, a nuclear receptor corepressor (NCoR) as 95% of all thyroid cancer cases (Vriens et al., well as wild-type TR β1 competes with TR βPV for 2009). The remaining cancers originate from parafollicular cells - medullary thyroid cancer binding to the C-terminal SH 2 domain (CSH 2) of p85 α. Up-regulation of NCoR in thyroid tumor (MTC). NMTC is classified into: follicular, S6K papillary, Hurthle cell, and anaplastic carcinoma. cells reduces AKT-mTOR-p70 signaling. In PV contrast, lowering cellular NCoR by siRNA Dominant-negative TR β mutant (Kaneshige et al., knockdown in tumor cells results in over-activation 2000) which lacks the C-terminus of the receptor of PI3K-AKT signaling. Importantly, NCoR protein (see animal models), causes severe disruption of the levels are significantly lower in thyroid tumor cells HPT axis, goiter, TSHomas, and metastatic than in wild type thyrocytes that allows for more follicular thyroid carcinoma (FTC). A double mice effective binding of PV to p85 α to activate the knockout of both TR α and TR β results in a higher PI3K pathway, thereby contributing to tumor incidence of follicular thyroid carcinoma and progression (Furuya et al., 2009). Furthermore, the increased aggressiveness in a skin cancer model. suppressive role of TR β has been demonstrated These animal models indicate the meaning of TRs using MCF-7 cell line in xenograft models of in the pathogenesis of FTC. estrogen-dependent tumorigenesis. The TR β- Disease mediated inhibition of tumor growth has been Papillary thyroid cancer (PTC) is the most common elucidated via down-regulation of JAK-STAT- subtype of FNMTC (familial NMTC), accounting cyclin D pathways (Park et al., 2013). Tumor for 72- 85% of cases. PTC occurs more frequently suppressor function of TR β has been demonstrated in women and in the 20-55 year age group. PTC in a mouse model of metastatic follicular thyroid appears as an irregular solid or cystic mass in a carcinoma as well (Zhu et al., 2010). normal thyroid parenchyma and is characterized by According to the findings mentioned above, it could distinctive nuclear alterations including grooves, be hypothesized that TH may act as a growth, pro- pseudoinclusions, and chromatin clearing. PTCs angiogenic, pro-proliferative and anti-apoptotic that are smaller than 1 cm are referred to as factor when initiated at the nongenomic level papillary microcarcinomas. These tumors have been (Cohen et al. 2011; Davis, 2009), whereas in identified in up to 35% of individuals at autopsy, nucleus, TR β1 could serve as a suppressor itself or suggesting that they may be extremely common mediating some genomic actions of T3 on specific although rarely clinically relevant. PTC can also be

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multifocal but is typically slow growing with a The overall 5-year survival rate for FTC is 91%, tendency to spread to lymph nodes and usually has whereas 10-year - 85% (Biersack and Grünwald, an excellent prognosis. Activation of the mitogen- 2005). activated protein kinase (MAPK) pathway as a Cytogenetics result of mutations or somatic recombination is FTCs are known to harbor RAS mutation, PAX8/ found in the majority of PTCs (Bonora et al., 2010). PPAR γ rearrangement and activation of the PTEN/ Prognosis AKT pathway. These mutations are also mutually Depending on source, the overall 5-year survival exclusive and identified in 70% of follicular rate for PTC is 96-97%, whereas a 10-year survival carcinomas. Molecular classifiers measure the rate is 93%. expression of a large number of genes on a For younger patients, the prognosis is better than microarray chip providing a substantial negative for patients older than 45 years (Ito et al., 2013; predictive value pending further validation. Biersack and Grünwald, 2005). Aberrant THRB gene expression is thought to be Cytogenetics implicated in FTCs (see Mutations and Animal Germline mutations were found in approximately models of FTC). 5% of NMTC, occurring as a primary feature Thyroid hormone resistance, FNMTC or as a minor component of a familial cancer syndrome (familial adenomatous polyposis, generalized, autosomal dominant Carney complex) that are hereditary. (GRTH) Moreover, several cases of PTC including Note differentiated PTC have been reported to be OMIM: 188570, MedGen UID: 424846. associated with the RTH syndrome (Ramos-Prol et Resistance to TH (RTH), a syndrome of reduced al., 2013). Furthermore, TR β has been found to be a end-organ responsiveness to TH, was identified in major target gene for microRNAs in PTC 1967 (Refetoff et al., 1967) but linkage between a (Jazdzewski et al., 2011). Both, miR-21 and miR- TR β locus on chromosome 3 and the RTH 146a have been reported to inhibit the expression of phenotype was demonstrated in 1988 (Usala et al., the TR β mRNA and protein, lowered down to 10- 1988). Recent discoveries of genetic defects that 28% in PTC (Jazdzewski et al., 2011). In addition, reduce the effectiveness of TH through altered cell 70% of PTCs have been shown to harbor point membrane transport and metabolism broadened the mutations of the BRAF and RAS genes or definition of reduced TH sensitivity to include all RET/PTC rearrangements, all of which can activate defects that interfere with the biological activity of the mitogen-activated protein kinase pathways TH secreted in normal amounts (Dumitrescu and (Witt et al., 2013). For more information see Refetoff, 2013). A number of humans with a Mutations. syndrome of TH resistance have been identified to have mutations in the THRB gene (Dumitrescu and Thyroid carcinoma, follicular (FTC) Refetoff, 2013). Clinically, such individuals show a Note type of hypothyroidism characterized by goiter, OMIM: 188470, MedGen UID: 64630. elevated serum concentrations of T3, T4 and near TRs have been shown to serve as tumor suppressors normal serum concentrations of TSH. More than in a mouse model of metastatic follicular thyroid half of affected children show attention-deficit carcinoma (Zhu et al., 2010). See Animal models. disorder, which shows the role of thyroid hormones Disease in brain development. THRB gene mutations Follicular thyroid cancer (FTC) accounts for produce two forms of generalized resistance to TH approximately 15% of NMTC and occurs more (GRTH) - autosomal recessive and dominant. The commonly in women over 50 years of age. FTC is first one is less common and described in a family defined by invasive features that result in containing deletion of all coding sequences of the infiltration of blood vessels and full penetration of THRB gene that is inherited as an autosomal the tumor capsule as well as the absence of the recessive trait (Takeda et al., 1992). The more nuclear alterations, which characterize papillary common form of RTH is inherited in a dominant carcinoma. FTC is rarely multifocal and usually mode and is characterized by defects in only one does not metastasize to the regional lymph nodes allele of THRB, usually a missense mutation. The but tends to spread via the bloodstream to the lung mutant THRB allele produces mutant TR β protein and bones. The Oncocytic follicular carcinoma that cannot mediate effects of T3 and acts by (Hurthle cell, oxyphilic) is an important histologic interfering with the function of the wild-type TRs variant of FTC, composed of eosinophilic cells (wt-TRs), finally contributing to a dominant replete with mitochondria (Bonora et al., 2010). negative effects (DNEs). The majority of the mutated TR β receptors abolish ligand binding, lost Prognosis their trans-activation function, and exhibited

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dominant-negative activity. The TR β mutants may emotional disturbances 60%, hyperkinetic disturb the wt-TRs binding to TREs or preserve the behaviour 33-68%, attention deficit hyperactivity ability to dimerize with a partner (e.g. RXR). The disorder 40-60, learning disability 30%, mental DNE can be exerted also through reduced retardation (IQ 2 SD 29-47, recurrent ear and throat association with cofactors (CoAs) or increased infections 55% (Dumitrescu and Refetoff, 2013). affinity for corepressors (CoRs), which have been Diagnosis is based on the clinical findings and found to play a role in the autosomal dominant standard laboratory tests including searching for RTH. Indeed, mutants that fail to interact with germline mutations by sequencing of THRB exons. coactivators or are defective in T3-induced release Nevertheless, a new role of of corepressors have been identified in RTH mutations/polymorphisms within intronic patients. Importantly, the presence in a TRβ mutant sequences is recognised to affect alternative of an additional mutation that abolishes either DNA splicing and other events of post-transcriptional binding, dimerization or the association with a CoR processing of TR β RNA (Alberobello et al., 2011). can result in the abrogation of the DNE Thus, the further association studies involving (Dumitrescu and Refetoff, 2013). Moreover, RTH whole THRB sequencing (376 609 bp) would be has been found to be modulated in vivo by the needed to be carried out in patients with TRH corepressor - NCoR1 (Fozzatti et al., 2011). Thus, symptoms who have no mutations in the THRB full and potent dominant-negative activity of TR β exons. Recently, the alternative splicing have been mutant requires functional DBD to retain the ability shown to produce human TR β4 isoform, a to bind DNA and to form homodimers and carboxyl-terminal splicing variant of TR β1 that RXR/TR heterodimers. contains a stop codon due to the presence of an The findings reveal that dominant-negative activity intronic 137-bp insertion located between exon 7 in RTH is mediated by transcriptionally inactive and 8. TR β4 lacks the ligand binding domain and complexes containing TR mutants bound to TREs. thus, may modulate T3 action as an endogenous TRH is estimated to occur in approximately 1 per dominant-negative protein. This variant is 40000 newborns (Refetoff and Dumitrescu, 2007). expressed in various human tissues regardless of The gene defect remains unknown in 15% of mutation status in the coding sequence and may subjects with RTH. interfere with the function of wild-type TR Familial occurrence of RTH has been documented isoforms (Tagami et al., 2011). in about 75% of cases, whereas incidence of Prognosis sporadic cases has been reported in 21.0% of cases RTH affected individuals have elevated serum TH that is in agreement with estimate of the frequency levels and normal or elevated TSH but are usually of de novo mutations of 20.8%. RTH has been clinically euthyroid and require no treatment. found with equal frequency in both male and However, the clinical presentation of RTH is female gender. The prevalence may vary among variable and requires differential diagnosis different ethnic groups however appears to have excluding all other possible causes of wide geographic distribution among Caucasians, hyperthyroxinemia. Most patients have normal Africans, Asians and Amerindians (Dumitrescu and growth and development, and lead a normal life at Refetoff, 2013). the expense of high TH levels and a small goiter. In Disease some cases, abnormalities may be found in: The majority of patients with RTH are identified by connective tissue, head and neck, their persistent elevation of circulating free TH metabolism/homeostasis, abdomen, cardiovascular levels association with non-suppressed serum TSH system, ear, eye, endocrine system, integument, and higher doses of exogenous TH are required to musculature, nervous system, respiratory system, obtain appropriate secretion of pituitary TSH as skeletal system and increased upper to lower well as the metabolic responses in peripheral segment ratio. Goiter has recurred in every patient tissues. The apparent resistance to TH may vary in who underwent thyroid surgery. As a consequence, severity and the magnitude of the hormonal some patients have been submitted to several resistance is mainly dependent on the nature of TR β thyroidectomies or treatments with radioiodide mutations. RTH shows a variable clinical (Dumitrescu and Refetoff, 2013). presentation, however the common features of the Cytogenetics RTH syndrome may include: elevated levels of free Mutations in THRB gene have been identified in T4 and to a lesser degree T3, normal or slightly approximately 85% cases of the RTH, however increased level of TSH responding to TRH, goiter other genes such as MCT8 and SECISBP2 are and the absence of the metabolic consequences of believed to be associated with the disease as well TH excess. The frequency of the most frequently (Bottcher et al., 2007). TR β dominant negative observed manifestations are as follows: thyroid mutants have been shown not only to fail its gland: goiter 66-95%, tachycardia 33-75%, function in a transcriptional response to T3 but also

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to interfere with wild-type TR α and TR β actions. transition, codon 311) resulting in novel Glu-311- Haplotyping of intragenic polymorphic markers Lys (p.E311K) substitution has been reported as showed that, in most instances, identical mutations well. The homozygous patient was characterized by have developed independently in different families severe symptoms of RTH. Both parents were (Dumitrescu and Refetoff, 2013). Among 457 heterozygous, suggesting autosomal recessive mode families 170 different mutations have been of the inheritance (Slezak et al., 2012). identified and 78 of the mutations were shared by more than one family. Thyroid hormone resistance, Majority of the families (430) were found to have selective pituitary (PRTH) single nucleotide substitutions (SNP) resulting in a Note single amino acid substitution (419), stop codons OMIM: 145650, MedGen UID: 333543. producing truncated proteins (11). In 20 families, In contrast to GRTH, PRTH is characterized by deletions, insertions and a duplication were resistance in the pituitary gland but not in identified. peripheral tissues. Note that the anterior pituitary Most mutations were found in exon 9 and 10, (secreting TSH) and in particular hypothalamus however they were present in exon 6, 7, 8 as well. (releasing TRH) are brain structures wherein TR β2 Unrelated families (33) shared the R338W is predominantly expressed. The presence of the mutation. Variants: R243Q, A317T, R338W, TR β2 isoform in cochlea, retina is extremely R423H and P453T were found in more than 15 important during development (see Expression and families. specific functions). Due to the tissue-specific All TR β gene mutations were located in the expression and function of the TR isoforms all functionally relevant domain of T3-binding and its tissues other than the pituitary have been grouped adjacent hinge region (see diagram 6). Three together under the term peripheral tissues. mutational clusters containing CpG hot spots have Disease been identified (Dumitrescu and Refetoff, 2013). For more disease information, see references. This form of resistance to thyroid hormone is pituitary-selective and is characterized by Thyroid hormone resistance, hyperthyroidism and TRH-stimulated, inappropriate generalized, autosomal recessive secretion of TSH (Gershengorn et al., 1975). (GRTH) Subjects with PRTH may have equally high levels of serum TH and non-suppressed TSH. These Note individuals may appear to be hypermetabolic, OMIM: 274300, MedGen UID: 333543. restless and may have sinus tachycardia or other Recessively inherited resistance to thyroid hormone thyrotoxic effects. In GRTH, the TH response of (RTH) is a rare autosomal disorder usually caused both the pituitary and peripheral tissues is by mutations in the THRB gene. The loss of both disrupted, whereas in PRTH (designated also THRB alleles may result in severe abnormalities central), the ability of the pituitary to sense (and reflecting unresponsiveness to TH. down-regulate) elevated TH is selectively impaired. Disease Simultaneously, the peripheral tissues remains A family with deletion of all coding sequences of relatively TH-responsive that results in peripheral the THRB gene has been reported to be inherited as thyrotoxicity (Wan et al., 2005). It has been an autosomal recessive trait (Takeda et al., 1992). proposed that PRTH syndrome is associated with The complete lack of TR β in this family produces T3 receptor mutants that selectively impair β2 severe deafness, contributing to mutism and isoform function in pituitary and hypothalamic cells monochromatic vision (see Animal Models). (Wan et al., 2005). The wild-type TR β2 isoform has Heterozygous individuals that express a single TR β been reported to display an enhanced T3 response gene have no clinical or laboratory abnormalities. It relative to the TR β1, expressed broadly in almost has been demonstrated that this is not due to all tissues. In the normal subjects, and in GRTH, compensatory overexpression of a single normal TR β2 in the pituitary can sense rising T3 levels in allele of the THRB nor that of the THRA gene. advance of TR β1 in the peripherial tissues, However, normally expressed TR α1 is capable of preventing the thyrotoxicity. In contrast, the THRB partially substituting for the TR β function mutations associated with pituitary RTH (see (Dumitrescu and Refetoff, 2013). diagram 6) disproportionately disrupt the pituitary's Cytogenetics ability to sense and suppress elevated T3 levels in advance of the peripheral tissues, producing The following homozygous mutations in the THRB symptoms of the thyrotoxicity (Wan et al., 2005). have been identified: THRBdel (deletion of both alleles of THRB), T337del, I280S, G347, R316C Prognosis (Ferrara et al., 2012). A novel rare homozygous Prognosis depends on clinical manifestations and mutation in the gene in position 1216 (G to A laboratory testing (see GRTH). There are some

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difficulties in differentiating thyrotropin secreting normal or low serum T4 and autoimmune thyroid pituitary microadenoma from pituitary-selective disease (AITD) that was confounded with initially thyroid hormone resistance accompanied by diagnosed RTH. This variant would not have an pituitary incidentaloma (Akiyoshi et al., 1996). effect on the hypothalamic-pituitary-thyroid axis as Importantly, THRB mutations have been found to determined by thyroid hormone binding in vitro and be similar in both diseases (Dumitrescu and thyroid function tests in vivo (Larsen et al., 2013). Refetoff, 2013). Somatic mutations in the THRB have been Cytogenetics identified in some TSH-secreting pituitary tumors Both forms, PRTH and GRTH are linked to (e.g. TSHomas). These mutations can be identical mutations in THRB expressing TR β1 TR β2 and to those occurring in the germline. However, TR β4 isoforms in tissue-specific pattern (see because their expression is limited to thyrotrophs, Diagram 2 and 3). Despite striking differences the phenotypes that of TSH induced thyrotoxicosis. among the clinical presentations between these It is postulated that defective TR interfering with forms of RTH, there is a lack of direct genotype- the negative regulation of TSH by TH is phenotype correlation and almost identical THRB responsible for the development of the pituitary gene mutations have been observed in PRTH or tumor (Refetoff and Dumitrescu, 2007). GRTH patients. Nevertheless, there are several Interestingly, TR β4, a dominant negative variant of association studies showing such correlations. TR β1, has been proposed to affect the function of Germline mutations associated with GRTH (P453S, wild type TRs in the TSHomas (Tagami et al., G345S) have been reported to impair both TR β2 2011). and TR β1 function proportionally, whereas mutations associated with PRTH (R338L, R338W, To be noted R429Q) have been demonstrated to Note disproportionately disrupt TR β2 function (Wan et This work was supported by the National Science al., 2005). Moreover, TR β mutants R383H and Centre (NCN) research grants no.: NN 401611940. R429Q have been shown to have greater The authors have declared that no competing impairment of transactivation on negatively than interests exist. positively regulated promoters. These two mutants Contributor Adam Master is the author of all the are candidates for predominantly PRTH, even figures which have been created especially for this though they have been clinically described as review. generating both, GRTH and PRTH. It has been proposed that the substitution of these charged References amino-acids could disrupt the unique property of TR β2 to bind coactivators through multiple contact Refetoff S, DeWind LT, DeGroot LJ. Familial syndrome surfaces. This may result in a decrease in T3- combining deaf-mutism, stuppled epiphyses, goiter and abnormally high PBI: possible target organ refractoriness mediated feedback suppression. Consequently, the to thyroid hormone. J Clin Endocrinol Metab. 1967 mutation affects predominantly TR β2 mediated Feb;27(2):279-94 action of TH (Dumitrescu and Refetoff, 2013). Gershengorn MC, Weintraub BD. Thyrotropin-induced Another proposed mechanism for PRTH is a hyperthyroidism caused by selective pituitary resistance to "double-hit" combining a SNP and the mutant thyroid hormone. A new syndrome of "inappropriate R338W (Alberobello et al., 2011). Recent studies secretion of TSH". J Clin Invest. 1975 Sep;56(3):633-42 have demonstrated that an intron enhancer region Gharib H, Klee GG. Familial euthyroid hyperthyroxinemia may play a critical role in the pituitary expression secondary to pituitary and peripheral resistance to thyroid of the TR β2 isoform. It has been hypothesized that hormones. Mayo Clin Proc. 1985 Jan;60(1):9-15 intronic polymorphisms in the intronic region could Usala SJ, Bale AE, Gesundheit N, Weinberger C, Lash modulate the pituitary expression of the mutated RW, Wondisford FE, McBride OW, Weintraub BD. Tight gene contributing to the clinical presentation of linkage between the syndrome of generalized thyroid hormone resistance and the human c-erbA beta gene. Mol RTH. The combined coding mutation such as Endocrinol. 1988 Dec;2(12):1217-20 missense R338W and two common SNPs Fein HG, Burman KD, Djuh YY, Usala SJ, Bale AE, (rs2596623T, rs2596622C) located in the intron Weintraub BD, Smallridge RC. Tight linkage of the human enhancer region of the THRB gene can generate a c-erbA beta gene with the syndrome of generalized thyroid tissue-specific dominant-negative conditions for hormone resistance is present in multiple kindreds. J development of the pituitary-selective RTH. Endocrinol Invest. 1991 Mar;14(3):219-23 Moreover, the results suggest that rs2596623T may Takeda K, Sakurai A, DeGroot LJ, Refetoff S. Recessive lead to pituitary over-expression of the mutant inheritance of thyroid hormone resistance caused by allele (Alberobello et al., 2011). complete deletion of the protein-coding region of the thyroid hormone receptor-beta gene. J Clin Endocrinol A novel TR β variant - G339S has been found in Metab. 1992 Jan;74(1):49-55 several members of a family with elevated TSH,

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Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, Jacobsen A, Byrne CJ, Heuer ML, Larsson E, Chen L, Li Y, Lin CH, Chan TH, Chow RK, Song Y, Liu M, Antipin Y, Reva B, Goldberg AP, Sander C, Schultz N.. Yuan YF, Fu L, Kong KL, Qi L, Li Y, Zhang N, Tong AH, The cBio cancer genomics portal: an open platform for Kwong DL, Man K, Lo CM, Lok S, Tenen DG, Guan XY.. exploring multidimensional cancer genomics data. Cancer Recoding RNA editing of AZIN1 predisposes to Discov. 2012 May;2(5):401-4. doi: 10.1158/2159-8290.CD- hepatocellular carcinoma. Nat Med. 2013 Feb;19(2):209- 12-0095. 16. doi: 10.1038/nm.3043. Epub 2013 Jan 6. Dmitriev AA, Kashuba VI, Haraldson K, Senchenko VN, Davis PJ, Lin HY, Tang HY, Davis FB, Mousa SA.. Pavlova TV, Kudryavtseva AV, Anedchenko EA, Krasnov Adjunctive input to the nuclear thyroid hormone receptor GS, Pronina IV, Loginov VI, Kondratieva TT, Kazubskaya from the cell surface receptor for the hormone. Thyroid. TP, Braga EA, Yenamandra SP, Ignatjev I, Ernberg I, Klein 2013a Dec;23(12):1503-9. doi: 10.1089/thy.2013.0280. G, Lerman MI, Zabarovsky ER.. Genetic and epigenetic Epub 2013 Nov 5. analysis of non-small cell lung cancer with NotI- Davis PJ, Mousa SA, Cody V, Tang HY, Lin HY.. Small microarrays. Epigenetics. 2012 May;7(5):502-13. doi: molecule hormone or hormone-like ligands of integrin 10.4161/epi.19801. Epub 2012 May 1. αVβ3: implications for cancer cell behavior. Horm Cancer. Ferrara AM, Onigata K, Ercan O, Woodhead H, Weiss RE, 2013b Dec;4(6):335-42. doi: 10.1007/s12672-013-0156-8. Refetoff S.. Homozygous thyroid hormone receptor β-gene Epub 2013 Aug 14. mutations in resistance to thyroid hormone: three new Dumitrescu AM, Refetoff S.. The syndromes of reduced cases and review of the literature. J Clin Endocrinol Metab. sensitivity to thyroid hormone. Biochim Biophys Acta. 2013 2012 Apr;97(4):1328-36. doi: 10.1210/jc.2011-2642. Epub Jul;1830(7):3987-4003. doi: 2012 Feb 8. (REVIEW) 10.1016/j.bbagen.2012.08.005. Epub 2012 Aug 16. Kashuba V, Dmitriev AA, Krasnov GS, Pavlova T, Ignatjev (REVIEW) I, Gordiyuk VV, Gerashchenko AV, Braga EA, Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Yenamandra SP, Lerman M, Senchenko VN, Zabarovsky Sumer SO, Sun Y, Jacobsen A, Sinha R, Larsson E, E.. NotI Microarrays: Novel Epigenetic Markers for Early Cerami E, Sander C, Schultz N.. Integrative analysis of

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complex cancer genomics and clinical profiles using the β in xenograft models. Am J Cancer Res. 2013 Jun cBioPortal. Sci Signal. 2013 Apr 2;6(269):pl1. doi: 20;3(3):302-11. Print 2013. 10.1126/scisignal.2004088. Puzianowska-Kuznicka M, Pawlik-Pachucka E, Owczarz Huang WH, Tseng CN, Tang JY, Yang CH, Liang SS, M, Budzin'ska M, Polosak J.. Small-molecule hormones: Chang HW.. RNA editing and drug discovery for cancer molecular mechanisms of action. Int J Endocrinol. therapy. ScientificWorldJournal. 2013 Apr 2013;2013:601246. doi: 10.1155/2013/601246. Epub 2013 24;2013:804505. doi: 10.1155/2013/804505. Print 2013. Feb 28. (REVIEW) Ramos-Prol A, Antonia Perez-Lazaro M, Isabel del Olmo- Ito Y, Miyauchi A, Kobayashi K, Miya A.. Prognosis and Garcia M, Leon-de Zayas B, Moreno-Macian F, Navas-de growth activity depend on patient age in clinical and Solis S, Merino-Torres JF.. Differentiated thyroid subclinical papillary thyroid carcinoma [Review]. Endocr J. carcinoma in a girl with resistance to thyroid hormone 2013 Nov 9. [Epub ahead of print] management with triiodothyroacetic acid. J Pediatr Endocrinol Metab. 2013;26(1-2):133-6. doi: 10.1515/jpem- Kim WG, Zhu X, Kim DW, Zhang L, Kebebew E, Cheng 2012-0230. SY.. Reactivation of the silenced thyroid hormone receptor β gene expression delays thyroid tumor progression. Witt RL, Ferris RL, Pribitkin EA, Sherman SI, Steward DL, Endocrinology. 2013 Jan;154(1):25-35. doi: Nikiforov YE.. Diagnosis and management of differentiated 10.1210/en.2012-1728. Epub 2012 Nov 26. thyroid cancer using molecular biology. Laryngoscope. 2013 Apr;123(4):1059-64. doi: 10.1002/lary.23838. Epub Larsen CC, Dumitrescu A, Guerra-Arguero LM, Gallego- 2013 Feb 12. (REVIEW) Suarez C, Vazquez-Mellado A, Vinogradova M, Fletterick R, Refetoff S, Weiss RE.. Incidental Identification of a Kim WG, Zhao L, Kim DW, Willingham MC, Cheng SY.. Thyroid Hormone Receptor Beta (THRB) Gene Variant in a Inhibition of Tumorigenesis by the Thyroid Hormone Family with Autoimmune Thyroid Disease. Thyroid. 2013 Receptor β in Xenograft Models. Thyroid. 2014 Dec;23(12):1638-43. doi: 10.1089/thy.2013.0174. Epub Feb;24(2):260-9. doi: 10.1089/thy.2013.0054. Epub 2013 2013 Sep 13. Sep 4. Lin HY, Su YF, Hsieh MT, Lin S, Meng R, London D, Lin C, Perri A, Catalano S, Bonofiglio D, Vizza D, Rovito D, Qi H, Tang HY, Hwang J, Davis FB, Mousa SA, Davis PJ.. Aquila S, Panza S, Rizza P, Lanzino M, Ando S.. T3 Nuclear monomeric integrin αv in cancer cells is a enhances thyroid cancer cell proliferation through coactivator regulated by thyroid hormone. FASEB J. 2013 TR β/Oct-1-mediated cyclin D1 activation. Mol Cell Aug;27(8):3209-16. doi: 10.1096/fj.12-227132. Epub 2013 Endocrinol. 2014 Jan 25;382(1):205-17. doi: May 2. 10.1016/j.mce.2013.10.001. Epub 2013 Oct 9.

Mottis A, Mouchiroud L, Auwerx J.. Emerging roles of the This article should be referenced as such: corepressors NCoR1 and SMRT in homeostasis. Genes Dev. 2013 Apr 15;27(8):819-35. doi: Master A, Nauman A. THRB (Thyroid Hormone Receptor, 10.1101/gad.214023.113. (REVIEW) Beta). Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6):400-433. Park JW, Zhao L, Cheng SY.. Inhibition of estrogen- dependent tumorigenesis by the thyroid hormone receptor

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Leukaemia Section Short Communication t(7;9)(q11.2;p13.2) PAX5/AUTS2 Dagmar Denk CCRI, Children's Cancer Research Institute, St. Anna Kinderkrebsforschung e.V., Zimmermannplatz 10, 1090 Vienna, Austria (DD)

Published in Atlas Database: November 2013 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0709q11p13ID1633.html DOI: 10.4267/2042/53770 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 10 exons, alternatively spliced transcript variants Short Communication on t(7;9)(q11.2;p13.2) encoding different isoforms. PAX5/AUTS2, with data on clinics, and the genes Protein implicated. PAX5 is a transcription factor harboring a conserved paired box DNA-binding domain. Clinics and pathology It is a master regulator of B-cell commitment and Disease maintenance and within the hematopoietic system is expressed in B-cells from the pro-B cell to the B-cell precursor acute lymphoblastic leukemia mature B-cell stage and repressed upon plasma cell (BCP-ALL) differentiation (Cobaleda et al., 2007; Medvedovic Epidemiology et al., 2011). In BCP-ALL PAX5 is a frequent target of somatic This unbalanced chromosomal rearrangement was mutations, comprising deletions, point mutations, found in three pediatric patients with B-cell and structural rearrangements resulting in the precursor acute lymphoblastic leukemia (Kawamata expression of fusion transcripts (Mullighan et al., et al., 2008; Coyaud et al., 2010a; Denk et al., 2007). 2012). To date, 16 different in-frame PAX5 fusions genes Clinics have been reported in B-ALL (Cazzaniga et al., All three patients achieved a complete remission 2001; Bousquet et al., 2007; Mullighan et al., 2007; (CR) after completion of induction therapy; Nebral et al., 2007; Kawamata et al., 2008; Nebral however, two of the patients experienced an early et al., 2009; Coyaud et al., 2010b; Lee et al., 2012). relapse and both patients died, one from an The PAX5 fusion partners comprise a infectious complication in second CR and one from heterogeneous group of genes that encode progressive leukemia after a further relapse. transcription factors, structural proteins, kinases, as The third patient remains in first CR for more than well as several genes with so far unknown two years after diagnosis (Denk et al., 2012). functions. AUTS2 Genes involved and Location proteins 7q11.22 PAX5 DNA/RNA 19 exons, alternatively spliced transcript variants Location encoding different isoforms. 9p13.2

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 434 t(7;9)(q11.2;p13.2) PAX5/AUTS2 Denk D

Figure 1. Schematic representation of the structure of AUTS2 (top) and PAX5 (bottom) wild-type proteins as well as the putative consensus chimeric protein (middle). PD: paired domain; OP: octapeptide; HD: partial homeodomain; TA: transactivation domain; I: inhibitory domain; H: histidine-rich regions; P: proline-rich region; arrows indicate nuclear localization signals (NLS); filled lollipops represent serine phosphorylation sites.

Protein 2007 May;8(5):463-70 AUTS2, is a highly conserved nuclear protein with Mullighan CG, Goorha S, Radtke I et al.. Genome-wide so far unknown function, contains several putative analysis of genetic alterations in acute lymphoblastic N-terminal nuclear localization signals (NLS), two leukaemia. Nature. 2007 Apr 12;446(7137):758-64 proline alternating with two histidine-rich regions, Nebral K, König M, Harder L, Siebert R, Haas OA, Strehl and two potential serine phosphorylation sites. It is S. Identification of PML as novel PAX5 fusion partner in strongly expressed in fetal and adult brain, childhood acute lymphoblastic leukaemia. Br J Haematol. 2007 Oct;139(2):269-74 particularly in the frontal, parietal, and temporal lobes. Mutations in the gene have been associated Kawamata N, Ogawa S, Zimmermann M et al.. Cloning of genes involved in chromosomal translocations by high- with autism and mental retardation (Oksenberg and resolution single nucleotide polymorphism genomic Ahituv, 2013). microarray. Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11921-6 Result of the chromosomal Nebral K, Denk D, Attarbaschi A et al.. Incidence and diversity of PAX5 fusion genes in childhood acute anomaly lymphoblastic leukemia. Leukemia. 2009 Jan;23(1):134-43 Hybrid gene Coyaud E, Struski S, Dastugue N, Brousset P, Broccardo C, Bradtke J. PAX5-AUTS2 fusion resulting from Transcript t(7;9)(q11.2;p13.2) can now be classified as recurrent in B In-frame fusions between PAX5 exon 6 and cell acute lymphoblastic leukemia. Leuk Res. 2010a AUTS2 exon 4, 5 or 6 have been described Dec;34(12):e323-5 (Kawamata et al., 2008; Coyaud et al., 2010; Denk Coyaud E, Struski S, Prade N, Familiades J et al.. Wide et al., 2012). diversity of PAX5 alterations in B-ALL: a Groupe Francophone de Cytogenetique Hematologique study. Fusion protein Blood. 2010b Apr 15;115(15):3089-97 Description Medvedovic J, Ebert A, Tagoh H, Busslinger M. Pax5: a The putative consensus chimeric protein contains master regulator of B cell development and leukemogenesis. Adv Immunol. 2011;111:179-206 the DNA-binding paired domain, the octapeptide, and the partial homeodomain of PAX5 fused to the Denk D, Nebral K, Bradtke J, Pass G, Möricke A, Attarbaschi A, Strehl S. PAX5-AUTS2: a recurrent fusion C-terminal regions of AUTS2. gene in childhood B-cell precursor acute lymphoblastic leukemia. Leuk Res. 2012 Aug;36(8):e178-81 References Lee ST, Ji Y, Kim HJ, Ki CS, Jung CW, Kim JW, Kim SH. Cazzaniga G, Daniotti M, Tosi S et al.. The paired box Sequential array comparative genomic hybridization domain gene PAX5 is fused to ETV6/TEL in an acute analysis identifies copy number changes during blastic lymphoblastic leukemia case. Cancer Res. 2001 Jun transformation of chronic myeloid leukemia. Leuk Res. 15;61(12):4666-70 2012 Apr;36(4):418-21 Bousquet M, Broccardo C, Quelen C et al.. A novel PAX5- Oksenberg N, Ahituv N. The role of AUTS2 in ELN fusion protein identified in B-cell acute lymphoblastic neurodevelopment and human evolution. Trends Genet. leukemia acts as a dominant negative on wild-type PAX5. 2013 Oct;29(10):600-8 Blood. 2007 Apr 15;109(8):3417-23 This article should be referenced as such: Cobaleda C, Schebesta A, Delogu A, Busslinger M. Pax5: the guardian of B cell identity and function. Nat Immunol. Denk D. t(7;9)(q11.2;p13.2) PAX5/AUTS2. Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6):434-435.

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Leukaemia Section Short Communication t(8;12)(q13;p13) ETV6/NCOA2 Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: November 2013 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0812q13p13ID1503.html DOI: 10.4267/2042/53771 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

conventional cytogenetics, with cases described Abstract with a t(8;12)(q10;p10), a t(8;12)(q11;p11), a Short Communication on t(8;12)(q13;p13) t(8;12)(q12;p12), a t(8;12)(q12;p13), or a ETV6/NCOA2, with data on clinics, and the genes t(8;12)(q13;p13), but all exhibiting an implicated. ETV6/NCOA2 fusion transcript (Strehl et al., 2008). The t(8;12) was the sole anomaly in five Clinics and pathology cases (four ALLs and one M1-AML/BAL), and was accompanied with a del(5q) in two ALL cases, a Disease del(6q), a +9, and a del(11q) in an ALL each, a T-cell acute lymphoblastic leukemia (T-ALL), B- t(11;19)(q23;p13) in the M4-AML, and a +22 in a cell acute lymphoblastic leukemia (B-ALL), M1-AML/BAL. biphenotypic acute leukemia (BAL), and acute myeloid leukemia (AML). Genes involved and Phenotype/cell stem origin proteins This entity most often appears as a childhood Note leukemia expressing both T-lymphoid and myeloid Heterozygous NOTCH1 mutations were found in antigens. Eleven cases are available to date: one five of six samples where they were studied (Strehl case of common B-ALL, 6 cases of T-ALL, 3 cases et al., 2008; Zhou et al., 2012). of BAL, and one M4-AML (Pui et al., 1987; Schneider et al., 2000; Yamamoto et al., 2002; NCOA2 Strehl et al., 2008; Zhou et al., 2012). Location Epidemiology 8q13.3 There were 5 male and 6 female patients, aged 2, 2, Protein 2, 7, 8, 11, 14, 23, 75 years, and two additional 1464 amino acids. NCOA2 is composed of a basic cases came from a series of "childhood leukemia". helix-loop-helix (HLH), a PAS (Per/Arnt/Sim) region, four LXXLL motifs (L=leucine, X=any Prognosis amino acid) that are critical for interaction with Although the sample is far too small for definitive nuclear receptors, a LLXXLXXXL motif is conclusions, the prognosis does not seem bad (see involved in transcriptional coactivation and Figure). CREBBP/CBP binding, and a polyglutamine tract. Transcriptional coactivator for steroid receptors and Cytogenetics nuclear receptors. Involved in skeletal muscle differentiation (Chen et al., 2000). Acts as a tumor Cytogenetics morphological suppressor in liver cancer (O'Donnell et al., 2012). Breakpoints were difficult to assign accurately by

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 436 t(8;12)(q13;p13) ETV6/NCOA2 Huret JL

Overall survival in patients with t(8;12)(q13;p13) ETV6/NCOA9 (n=9).

ETV6 Hypodiploidy is associated with a poor prognosis in childhood acute lymphoblastic leukemia. Blood. 1987 Location Jul;70(1):247-53 12p13.2 Chen SL, Dowhan DH, Hosking BM, Muscat GE. The Protein steroid receptor coactivator, GRIP-1, is necessary for MEF-2C-dependent gene expression and skeletal muscle 452 amino acids. ETV6 is composed of a HLH differentiation. Genes Dev. 2000 May 15;14(10):1209-28 domain responsible for hetero- and homodimerization in N-term, and an ETS domain Schneider NR, Carroll AJ, Shuster JJ, Pullen DJ, Link MP, Borowitz MJ, Camitta BM, Katz JA, Amylon MD. New responsible for sequence specific DNA-binding in recurring cytogenetic abnormalities and association of C-term (binds to the DNA sequence 5'- blast cell karyotypes with prognosis in childhood T-cell CCGGAAGT-3'). Transcriptional regulator; tumor acute lymphoblastic leukemia: a pediatric oncology group suppressor. Involved in bone marrow report of 343 cases. Blood. 2000 Oct 1;96(7):2543-9 hematopoiesis. Yamamoto K, Nagata K, Tsurukubo Y, Inagaki K, Ono R, Taki T, Hayashi Y, Hamaguchi H. Translocation (8;12)(q13;p13) during disease progression in acute Result of the chromosomal myelomonocytic leukemia with t(11;19)(q23;p13.1). Cancer anomaly Genet Cytogenet. 2002 Aug;137(1):64-7 Strehl S, Nebral K, König M, Harbott J, Strobl H, Ratei R, Hybrid gene Struski S, Bielorai B, Lessard M, Zimmermann M, Haas OA, Izraeli S. ETV6-NCOA2: a novel fusion gene in acute Description leukemia associated with coexpression of T-lymphoid and There was fusion of ETV6 exon 4 with NCOA2 myeloid markers and frequent NOTCH1 mutations. Clin exon 15 in five of six cases, and fusion of ETV6 Cancer Res. 2008 Feb 15;14(4):977-83 exon 5 with NCOA2 exon 14 in one case (Strehl et O'Donnell KA, Keng VW, York B, Reineke EL, Seo D, Fan al., 2008). D, Silverstein KA, Schrum CT, Xie WR, Mularoni L, Wheelan SJ, Torbenson MS, O'Malley BW, Largaespada Fusion protein DA, Boeke JD. A Sleeping Beauty mutagenesis screen reveals a tumor suppressor role for Ncoa2/Src-2 in liver Description cancer. Proc Natl Acad Sci U S A. 2012 May Fuses the pointed or sterile alpha motif (SAM) 22;109(21):E1377-86 oligomerization domain of ETV6 to the C-term Zhou MH, Gao L, Jing Y, Xu YY, Ding Y, Wang N, Wang poly Gln of NCOA2 (with or without the W, Li MY, Han XP, Sun JZ, Wang LL, Yu L. Detection of LLXXLXXXL motif of NCOA2). ETV6 gene rearrangements in adult acute lymphoblastic leukemia. Ann Hematol. 2012 Aug;91(8):1235-43

References This article should be referenced as such: Pui CH, Williams DL, Raimondi SC, Rivera GK, Look AT, Huret JL. t(8;12)(q13;p13) ETV6/NCOA2. Atlas Genet Dodge RK, George SL, Behm FG, Crist WM, Murphy SB. Cytogenet Oncol Haematol. 2014; 18(6):436-437.

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Leukaemia Section Short Communication t(9;14)(q33;q32) IGH/LHX2 Nathalie Nadal, Elise Chapiro Laboratoire d'hematologie, CHU Hopital Nord, F-42055 St Etienne cedex 2, France (NN), Service d'Hematologie Biologique, Hopital Pitie-Salpetriere, APHP, Universite Pierre et Marie Curie-Paris 6, France (EC)

Published in Atlas Database: November 2013 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0914q33q32ID1659.html DOI: 10.4267/2042/53772 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

VP16, ifosfamide, and methotrexate, followed by Abstract an allograft). Short Communication on t(9;14)(q33;q32) IGH/LHX2, with data on clinics, and the genes Evolution implicated. After induction, minimal residual disease (MRD) detection by CMF and by molecular analysis was Clinics and pathology negative, whereas RT-PCR for BCR-ABL1 transcript was still positive. Chromosomal Disease examination showed the presence of one metaphase Chronic myeloid leukemia (CML) in B-cell out of 30 with only the t(9;22)(q34;q11), suggesting lymphoid blast crisis that the t(9;14) translocation was a secondary Phenotype/cell stem origin chromosomal abnormality. Thus, the chemotherapy had eradicated the B cell phenotype (CD19, CD10) with 2 aberrant lymphoblast cells but a CML clone persisted, myeloid markers (CD13 and CD33). further supporting the diagnosis of CML in BC. Etiology By 7 months after diagnosis, the patient underwent Unknown. allogenic stem cell transplantation from his HLA- matched sister. At 2 years post-transplantation, the Epidemiology patient was alive and well. BCR-ABL1 transcript Only one case to date, a 10-year-old male patient was undetectable (<0.001%). (Nadal et al., 2012). Clinics Cytogenetics Lymphadenopathies, enlarged spleen and liver. Additional anomalies Central nervous system involvement. The t(9;14)(q33;q32) translocation appears as a Cytology secondary abnormality occurring at acutisation of a High WBC with blast cells (44%), myelemia, CML with the usual t(9;22)(q34;q11) with a eosinophilia and basophilia. Bone marrow breakpoint in the mBCR region. The latest is aspiration showed 60% of undifferentiated blast usually observed in BCR-ABL1+ de novo acute cells with persistence of the granulocytic lineage. lymphoblastic leukemia but is rare in CML. i(7)(q10), present in 2 out of the 20 metaphases Treatment analyzed using conventional karyotype, and in The patient was treated according to the European 3/100 metaphases using FISH (7q22/7q36 Dual- protocol ESPHALL (imatinib, asparaginase, Color probe, Kreatech Diagnostics). vincristine, vindesine, daunorubicin, aracytine,

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 438 t(9;14)(q33;q32) IGH/LHX2 Nadal N, Chapiro E

A. Conventional karyotype: partial R and G-banded karyotype. The derivative chromosomes of translocations t(9;14)(q33;q32) and t(9;22)(q34;q11) are denoted by solid and dotted arrows, respectively. B. FISH: representative metaphase hybridized with dual color break-apart IGH probe (Abbott, Rungis, France). A fusion signal is seen on normal chromosome 14 (large arrows), a red signal on derivative chromosome 14 (small solid arrows) and a green signal on derivative chromosome 9 (small dotted arrows). C. FISH: representative metaphase hybridized with a BCR/ABL ES probe (Abbott). A green signal is seen on a normal chromosome 22 (large arrows), and two fusion signals on derivative chromosomes 9 and 22 (small dotted arrows), confirming the BCR-ABL1 rearrangement with a breakpoint in the mBCR region. A red signal is observed on derivative chromosome 14 (small solid arrows), indicating that the breakpoint of t(9;14) was centromeric to the ABL1 gene in chromosome 9.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 439 t(9;14)(q33;q32) IGH/LHX2 Nadal N, Chapiro E

strong over-expression of LHX2, which may have Genes involved and contributed to the rapid progression in the blastic phase. It has been shown that over-expression of proteins LHX2 in murine hematopoietic precursors leads to LHX2 the development of chronic myeloproliferative disorders (Richter et al., 2003). Thus, Location transcriptional deregulation of LHX2 plays a 9q33 recurrent role in leukemogenesis. Note LIM homeobox gene LHX2 is a member of the References LIM homeobox family of transcription factors Xu Y, Baldassare M, Fisher P, Rathbun G, Oltz EM, characterized by a DNA binding homeodomain and Yancopoulos GD, Jessell TM, Alt FW. LH-2: a a cystein-rich LIM-domain. LHX2, initially LIM/homeodomain gene expressed in developing identified as an early marker in B-lymphocyte lymphocytes and neural cells. Proc Natl Acad Sci U S A. differentiation (Xu et al., 1993), is involved in the 1993 Jan 1;90(1):227-31 neurogenesis, hair follicle, and hematopoietic Wu HK, Heng HH, Siderovski DP, Dong WF, Okuno Y, Shi development (Porter et al., 1997). XM, Tsui LC, Minden MD. Identification of a human LIM- Hox gene, hLH-2, aberrantly expressed in chronic IGH myelogenous leukaemia and located on 9q33-34.1. Oncogene. 1996 Mar 21;12(6):1205-12 Location 14q32 Porter FD, Drago J, Xu Y, Cheema SS, Wassif C, Huang SP, Lee E, Grinberg A, Massalas JS, Bodine D, Alt F, Westphal H. Lhx2, a LIM homeobox gene, is required for Result of the chromosomal eye, forebrain, and definitive erythrocyte development. Development. 1997 Aug;124(15):2935-44 anomaly Richter K, Pinto do O P, Hägglund AC, Wahlin A, Carlsson L. Lhx2 expression in hematopoietic progenitor/stem cells Hybrid gene in vivo causes a chronic myeloproliferative disorder and Note altered globin expression. Haematologica. 2003 Dec;88(12):1336-47 The translocation links sequence located 148 kb centromeric of LHX2 on chromosome 9 to JH6 Nadal N, Chapiro E, Flandrin-Gresta P, Thouvenin S, segment on chromosome 14. Vasselon C, Beldjord K, Fenneteau O, Bernard O, Campos L, Nguyen-Khac F. LHX2 deregulation by juxtaposition with Fusion protein the IGH locus in a pediatric case of chronic myeloid leukemia in B-cell lymphoid blast crisis. Leuk Res. 2012 Note Sep;36(9):e195-8 No fusion protein. This article should be referenced as such: Oncogenesis LHX2 juxtaposition with the IGH locus results in Nadal N, Chapiro E. t(9;14)(q33;q32) IGH/LHX2. Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6):438-440.

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Leukaemia Section Short Communication t(5;12)(q13;p13) ?/ETV6 Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: December 2013 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0512q13p13ID1662.html DOI: 10.4267/2042/53773 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 Prognosis Scarce data available: a M2-AML case was still Short Communication on t(5;12)(q13;p13) ?/ETV6, alive but in relapse 9 months after diagnosis (Park with data on clinics, and the genes implicated. et al., 2007), and the MDS case evolved towards a M2-AML and the patient died 4 months after Clinics and pathology diagnosis of the MDS (Yamamoto et al., 2000). Disease Cytogenetics Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), as well as acute Cytogenetics morphological lymphoblastic leukemia (ALL) The t(5;12) was the sole anomaly in two myeloid Phenotype/cell stem origin cases and part of a complex karyotype in the treatment-related myeloid case. Five cases are available to date: 3 myeloid cases: an The t(5;12) was accompanied with a marker AML not otherwise specified, a M2-AML, and a chromosome in the infant case, and with numerical RAEB transforming into M2-AML (Kobayashi et anomalies in the other lymphoid case. al., 1994; Yamamoto et al., 2000; Park et al., 2007); and 2 lymphoid cases: 2 ALLs (Heinonen et al., Genes involved and 1988; Sato et al., 1997). Is not taken into account in this review a case of proteins translocation between 5q13, 12p13, 22q11, and 3q12 with no possible fusion transcript on any Note derivative chromosome, although ETV6 (12p13) The partner(s) of ETV6 in 5q13 remain(s) and MN1 (22q12), with also sequences on 5q and unknown. on 3q are implicated in the rearrangements (Belloni Both one myeloid and one lymphoid cases were et al., 2004). tested, and it appears that the breakpoint in ETV6 is One case occurred after exposure to mutagenic not identical: the breakpoint was located in intron 1, agents: a patient experienced a renal carcinoma 3 and exon 1 was deleted, in the treatment-related years before onset of the secondary MDS myeloid case (Yamamoto et al., 2000), whereas it (Yamamoto et al., 2000). was located 3' of the ETV6 coding sequence in an ALL case (Sato et al., 1997). Epidemiology ETV6 The myeloid cases were a 58-year old female patient and two 65-year old male patients; the ALL Location cases were a 1-year old girl and a 12-year old girl. 12p13

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 441 t(5;12)(q13;p13) ?/ETV6 Huret JL

Protein in the breakpoints in balanced rearrangements involving band 12p13 in hematologic malignancies identified by 452 amino acids. ETV6 is composed of a HLH fluorescence in situ hybridization: TEL (ETV6 ) is involved domain responsible for hetero- and in only one half. Blood. 1997 Dec 15;90(12):4886-93 homodimerization in N-term, and an ETS domain Yamamoto K, Nagata K, Yagasaki F, Tsurukubo Y, responsible for sequence specific DNA-binding in Tamura A, Taniwaki M, Hamaguchi H. Interstitial deletion C-term (binds to the DNA sequence 5'- of the short arm of chromosome 12 during clonal evolution CCGGAAGT-3'). Transcriptional regulator; tumor in myelodysplastic syndrome with t(5;12)(q13;p13) suppressor. Involved in bone marrow involving the ETV6 gene. Cancer Genet Cytogenet. 2000 Jun;119(2):113-7 hematopoiesis. Belloni E, Trubia M, Mancini M, Derme V, Nanni M, References Lahortiga I, Riccioni R, Confalonieri S, Lo-Coco F, Di Fiore PP, Pelicci PG. A new complex rearrangement involving Heinonen K, Rautonen J, Siimes MA, Knuutila S. the ETV6, LOC115548, and MN1 genes in a case of acute Cytogenetic study of 105 children with acute lymphoblastic myeloid leukemia. Genes Chromosomes Cancer. 2004 leukemia. Eur J Haematol. 1988 Sep;41(3):237-42 Nov;41(3):272-7 Kobayashi H, Montgomery KT, Bohlander SK, Adra CN, Park TS, Song J, Lee KA, Lee SG, Min YH, Choi JR. Lim BL, Kucherlapati RS, Donis-Keller H, Holt MS, Le t(5;12)(q13;p13) in acute myeloid leukemia with preceding Beau MM, Rowley JD. Fluorescence in situ hybridization granulocytic sarcoma. Cancer Genet Cytogenet. 2007 mapping of translocations and deletions involving the short Sep;177(2):158-60 arm of human chromosome 12 in malignant hematologic diseases. Blood. 1994 Nov 15;84(10):3473-82 This article should be referenced as such: Sato Y, Bohlander SK, Kobayashi H, Reshmi S, Suto Y, Huret JL. t(5;12)(q13;p13) ?/ETV6. Atlas Genet Cytogenet Davis EM, Espinosa R, Hoopes R, Montgomery KT, Oncol Haematol. 2014; 18(6):441-442. Kucherlapati RS, Le Beau MM, Rowley JD. Heterogeneity

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Solid Tumour Section Short Communication

Soft Tissue Tumors: Extraskeletal osteosarcoma Andreas F Mavrogenis, Panayiotis J Papagelopoulos First Department of Orthopaedics, Athens University Medical School, 41 Ventouri Street, 15562 Holargos, Athens, Greece (AFM, PJP)

Published in Atlas Database: December 2013 Online updated version : http://AtlasGeneticsOncology.org/Tumors/ExtraskeletalOsteosarcID5200.html DOI : 10.4267/2042/53774 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

between 50 and 70 years of age. The reported male Abstract to female ratio is 1.9:1. Short communication on extraskeletal osteosarcoma Clinics with data on clinics. Most extraskeletal osteosarcomas are deep seated; Identity <10% are superficial, originating in the dermis or subcutis. The most common location is the lower Other names extremity (75%; thigh and buttock), followed by the Soft tissue osteosarcoma upper extremity (15%-23%, shoulder girdle), and Note the retroperitoneum (17%). A progressively enlarging soft tissue mass, rarely causing pain or Extraskeletal osteosarcoma is a high-grade tenderness is the most common clinical malignant mesenchymal soft tissue neoplasm presentation. composed of neoplastic cells (osteoblastic, Imaging chondroblastic and fibroblastic) that produce Plain radiographs, CT and MRI usually reveal a osteoid, neoplastic bone or chondroid matrix and large deep-seated soft tissue mass with variable has a clinically aggressive course. For a lesion to be mineralization. By definition, extraskeletal defined as extraskeletal osteosarcoma, it must arise osteosarcomas arise in the soft tissue and are not in the soft tissue and not attached to bone or attached to bone or periosteum; however, they may periosteum, have a uniform sarcomatous pattern, secondarily involve the periosteum, cortex or and produce osteoid and/or cartilage matrix. In medullary canal. contrast to the more common osteogenic sarcoma - Radiography: Shows a mass, possibly with (osteosarcoma), extraskeletal osteosarcoma is a rare peripheral, chunky or linear and mature tumor occurring mainly in adults. Often it arises at calcification similar to mature bone or heterotopic the site of prior radiation therapy. Response to ossification. treatments (mainly chemotherapy) is worse and - Computed tomography: Shows a mass with prognosis is much poorer compared to that for ossified rim. The mass is close to the bone but not primary osteosarcoma of bone. attached to it; a soft tissue mass is interposed between the bone and the ossified mass. Clinics and pathology - MR imaging: The unmineralized portion of the Epidemiology tumor shows heterogeneous and relatively hypointense signal intensity on T1-weighted images Extraskeletal osteosarcoma accounts for 1% to 2% and hyperintense signal intensity on T2. of all soft tissue sarcomas and approximately 2% to - Scintigraphy: Shows intense accumulation of 4% of all osteosarcomas. It typically affects patients radioactivity in the tumor.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 443 Soft Tissue Tumors: Extraskeletal osteosarcoma Mavrogenis AF, Papagelopoulos PJ

Figure 1. Axial T1-weighted (left) and coronal T2-weighted fat suppression (right) MR imaging show a large soft tissue mass in the postero-medial thigh. The tumor is not attached to bone. Figure 2. Tumor specimen of an extraskeletal osteosarcoma.

- Angiography: May show hypervascularity of the tumors and adheres to the surrounding structures, tumor. making dissection difficult. The tumor has a Pathology remarkable ability to infiltrate the surrounding Pathogenesis unknown. The majority develops de tissues; occasionally it can be confined to the novo; in approximately 10% extraskeletal subcutis or dermis, or ulcerate the overlying skin. osteosarcomas may be radiation induced. Invasion of blood vessels is not common. Gross pathology: Extraskeletal osteosarcomas may Micro pathology: Extraskeletal osteosarcoma is grow up to 50 cm (mean, 8-10 cm). characterized by anaplastic sprindle cell Macroscopically, the tumors are hemorrhagic and proliferation with the presence of osteoid matrix or focally necrotic, and firmly attached to the fascia immature bone formed by the neoplastic cells. The without attachment to the skeleton; rarely, it may tumor cells are spindle or polyhedral with lie in contact with the periosteum; >30% appear cytological atypia, malignant chondroid areas, grossly encapsulated, and >10% may exhibit extensive areas of necrosis, mitotic activity (>10 extensive haemorrhagic cystic changes. A tough mitoses per 10 high-power fields), and atypical connective tissue capsule usually surrounds the mitotic figures.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 444 Soft Tissue Tumors: Extraskeletal osteosarcoma Mavrogenis AF, Papagelopoulos PJ

Figure 3. Low power histological sections show calcified bone trabeculae and osteoid elaborated by malignant cells (Stain, hematoxylin and eosin; original magnification, 40x). Figure 4. High power histological sections shows pleomorphic malignant cells with extracellular matrix production (Stain, hematoxylin and eosin; original magnification, 200x).

All the major subtypes of osteosarcoma of bone Differential diagnosis: Myositis ossificans; have been reported in extraskeletal osteosarcoma. Synovial and epithelioid sarcoma; Extraskeletal Common to all variants is the presence of chondrosarcoma; Malignant fibrous histiocytoma; neoplastic bone deposited in a lacy, trabecular or Rhabdomyosarcoma; Hamartoma; Malignant sheet-like pattern. In contrast to myositis ossificans schwannoma; Malignant mesenchymoma; the bone is usually most prominent in the centre of Liposarcoma with metaplastic bone. the tumor. Cytogenetics - Osteoblastic extraskeletal osteosarcoma: the most Systematic genetic differences between common variant; the tumor cells resemble extraskeletal and bone osteosarcomas have not been malignant osteoblasts. Bone matrix is abundant. documented. Three cases with clonal chromosomal - Fibroblastic extraskeletal osteosarcoma: the aberrations have been reported; two tumors showed second in frequency; the tumor cell are spindle cells highly complex aberration patterns, whereas the arranged in a herringbone or storiform pattern. third showed a moderately hyperdiploid karyotype - Chondroid extraskeletal osteosarcoma: malignant with relatively few chromosomal abnormalities. cartilage predominates in the tumor matrix. - Telangiectatic extraskeletal osteosarcoma: it Treatment contains numerous large blood filled spaces lined Wide margin surgical resection is the treatment of by malignant cells. choice for extraskeletal osteosarcoma. Resection - Small cell extraskeletal osteosarcoma: the tumor can be with amputation of limb salvage surgery if cells are arranged in sheets of small round cells that microscopically negative margins can be achieved. mimic Ewing's sarcoma or lymphoma. Adjuvant chemotherapy and/or preoperative - Well differentiated extraskeletal osteosarcoma: radiation therapy may be useful, although rare variant; it contains abundant bone deposited in extraskeletal osteosarcoma seems relatively well formed trabeculae, surrounded by a minimally chemoresistant compared to osseous atypical spindle cell component similar to parosteal osteosarcomas. Radiation may be delivered by osteosarcoma. external beam, intraoperative, and brachytherapy. Immunohistochemistry: Several studies indicate Given the high grade malignant tumor and poor that the immunophenotype of extraskeletal prognosis, aggressive treatment with preoperative osteosarcoma is similar to osteosarcoma of bone; radiation therapy and surgical resection for local CD99 is expressed in all types of osteosarcoma; tumor control, and (neo-)adjuvant multi-agent stain for ALP is positive with a very strong chemotherapy to improve survival are current reaction; osteocalcin is the most specific antigen for treatments of choice for the patients with extraskeletal osteosarcomas expressed in the extraskeletal osteosarcomas. Aggressive malignant cells and matrix in 82% and 75% of thoracotomy and resection of the pulmonary cases, respectively. Extraskeletal osteosarcomas are metastases may be necessary in patients with lung uniformly positive for vimentin, 68% express metastases. smooth muscle actin, 25% desmin, 20% S100 Note: The role of (neo-)adjuvant chemotherapy or protein, 52% EMA, 8% keratin, and 0% PLAP. The radiation therapy is controversial. Disease-specific stain for the Ki-67 analogous MIB-1 shows high clinical trials are required to improve the outcome proliferative activity with values around 25%. of these patients.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 445 Soft Tissue Tumors: Extraskeletal osteosarcoma Mavrogenis AF, Papagelopoulos PJ

Prognosis Balcerzak S, Kempf R, Wolman SR. Cytogenetic aberrations and DNA ploidy in soft tissue sarcoma. A The prognosis of the patients with extraskeletal Southwest Oncology Group Study. Cancer Genet osteosarcoma is poor; the 5-year survival ranges Cytogenet. 1997 Nov;99(1):45-53 from 10% to 46%. More than 50% of the patients Lidang Jensen M, Schumacher B, Myhre Jensen O, Steen experience multiple local recurrences and distal Nielsen O, Keller J. Extraskeletal osteosarcomas: a metastases. Distal metastases are usually to the clinicopathologic study of 25 cases. Am J Surg Pathol. 1998 May;22(5):588-94 lungs (>80%), followed by the regional lymph nodes, bone, brain, liver and skin. Mertens F, Fletcher CD, Dal Cin P, De Wever I, Mandahl Note: Reported factors associated with a better N, Mitelman F, Rosai J, Rydholm A, Sciot R, Tallini G, Van den Berghe H, Vanni R, Willén H. Cytogenetic analysis of outcome include size <5 cm (the most important), 46 pleomorphic soft tissue sarcomas and correlation with fibroblastic or chondroblastic histological subtype, morphologic and clinical features: a report of the CHAMP and diminished proliferative activity as measured Study Group. Chromosomes and MorPhology. Genes by Ki-67 index. However, the validity of these Chromosomes Cancer. 1998 May;22(1):16-25 prognostic factors has not been confirmed in large Rosenberg AE, Heim S.. Extraskeletal osteosarcoma. In: studies. The well differentiated variant may behave Fletcher CDM, Unni KK, Mertens F (Eds), World Health Organization classification of tumours of soft tissue and in a more indolent manner; however, too few cases bone. Pathology and Genetics of Tumours of Soft Tissue have been reported to draw definitive conclusions and Bone, IARC Press, Lyon: 35-7, 2002. regarding their biologic potential. Mavrogenis AF, Papadogeorgou E, Papagelopoulos PJ.. Extraskeletal osteosarcoma: a case report. Acta Orthop References Traumatol Turc. 2012;46(3):215-9. Lorentzon R, Larsson SE, Boquist L. Extra-osseous Tan KT, Idowu OK, Chandrasekar CR, Yin Q, Helliwell osteosarcoma: a clinical and histopathological study of four TR.. Extraskeletal osteosarcoma of the hand. Hand (N Y). cases. J Bone Joint Surg Br. 1979 May;61-B(2):205-8 2012 Mar;7(1):124-6. doi: 10.1007/s11552-011-9371-3. Epub 2011 Nov 4. Sordillo PP, Hajdu SI, Magill GB, Golbey RB. Extraosseous osteogenic sarcoma. A review of 48 Cao Q, Lu M, Huebner T, Dilsizian V, Chen W.. 18F-FDG patients. Cancer. 1983 Feb 15;51(4):727-34 PET/CT in a rare malignant extraskeletal osteosarcoma. Clin Nucl Med. 2013 Sep;38(9):e367-9. doi: Mandahl N, Heim S, Willén H, Rydholm A, Eneroth M, 10.1097/RLU.0b013e3182868ace. Nilbert M, Kreicbergs A, Mitelman F. Characteristic karyotypic anomalies identify subtypes of malignant fibrous Kang K, Lee JH, Kim HG.. Contralateral referred pain in a histiocytoma. Genes Chromosomes Cancer. 1989 patient with intramedullary spinal cord metastasis from Sep;1(1):9-14 extraskeletal small cell osteosarcoma. J Spinal Cord Med. 2013 Nov;36(6):695-9. doi: Bane BL, Evans HL, Ro JY, Carrasco CH, Grignon DJ, 10.1179/2045772312Y.0000000087. Epub 2013 Apr 12. Benjamin RS, Ayala AG. Extraskeletal osteosarcoma. A clinicopathologic review of 26 cases. Cancer. 1990 Jun Llamas-Velasco M, Rutten A, Requena L, Mentzel T.. 15;65(12):2762-70 Primary cutaneous osteosarcoma of the skin: a report of 2 cases with emphasis on the differential diagnoses. Am J Hasegawa T, Hirose T, Kudo E, Hizawa K, Usui M, Ishii S. Dermatopathol. 2013 Aug;35(6):e106-13. doi: Immunophenotypic heterogeneity in osteosarcomas. Hum 10.1097/DAD.0b013e31827f0a6f. Pathol. 1991 Jun;22(6):583-90 Hu B, Liu Y, Cheng L, Li W, Cao X.. SPECT/CT imaging of Yi ES, Shmookler BM, Malawer MM, Sweet DE. Well- retroperitoneal extraskeletal osteosarcoma. Clin Nucl Med. differentiated extraskeletal osteosarcoma. A soft-tissue 2014 Feb;39(2):200-2. doi: homologue of parosteal osteosarcoma. Arch Pathol Lab 10.1097/RLU.0000000000000309. Med. 1991 Sep;115(9):906-9 Lee JS, Fetsch JF, Wasdhal DA, Lee BP, Pritchard DJ, This article should be referenced as such: Nascimento AG. A review of 40 patients with extraskeletal Mavrogenis AF, Papagelopoulos PJ. Soft Tissue Tumors: osteosarcoma. Cancer. 1995 Dec 1;76(11):2253-9 Extraskeletal osteosarcoma. Atlas Genet Cytogenet Oncol Mohamed AN, Zalupski MM, Ryan JR, Koppitch F, Haematol. 2014; 18(6):443-446.

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Deep Insight Section

Angiogenic factors and cancer therapy Yihai Cao Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 171 77 Stockholm, Sweden (YC)

Published in Atlas Database: December 2013 Online updated version : http://AtlasGeneticsOncology.org/Deep/AngiogenicFactorsID20129.html DOI: 10.4267/2042/53775 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 Tumor growth and metastasis are dependent on neovascularization, which is mainly accomplished by the process of angiogenesis-sprouting of new microvessels from the existing blood vessels. To gain its ability of growth and invasiveness, malignant cells often express high levels of angiogenic factors that stimulate tumor angiogenesis and remodel tumor vessels. In addition to malignant cells, other host cells in the tumor microenvironment, including inflammatory cells, stromal fibroblasts and perivascular cells also significantly contribute to production of angiogenic factors and cytokines. Co-existence of various angiogenic factors and cytokines will inevitably cause interplay of various signaling pathways, leading to synergistic effects of tumor angiogenesis. Thus, therapeutic development of angiogenic factor inhibitors should be aimed to block not only the vertical signaling pathway triggered by a specific factor, but the horizontal interplay of various angiogenic pathways. This review discusses the mechanisms that underlie tumor angiogenesis, provides a few examples of angiogenic pathways that are commonly seen in most tumor types, and discusses the challenges of antiangiogenic cancer therapy.

Key words validating the concept that tumors produce angiogenic factors to induce neovascularization Angiogenesis, vasculature, growth factors, cancer, (Shing et al., 1984). Simultaneous to identification metastasis of tumor angiogenic factors, Dvorak and colleagues Discovery of tumor angiogenic identified a potent vascular permeability factor factors (VPF) from tumor cells (Senger et al., 1983). These initial findings demonstrated that tumor-derived In 1971, Judah Folkman, in his hypothetical and angiogenenic factors are able to stimulate conceptual article, proposed that tumors produce angiogenesis and to modulate vascular structures. angiogenic factors and inhibition of tumor The same VPF, clones sequenced by Connolly and angiogenesis would offer a new therapeutic option colleagues, was found to be structurally related to for treatment of cancer (Folkman, 1971). Based on platelet-derived growth factor (Keck et al., 1989). this hypothesis, extensive research was initiated In the same issue of Science magazine, Dr. during early research for identification of tumor- Napoleone Ferrara and colleagues published their derived-angiogenic factors and accumulating findings on identification of vascular endothelial evidence supported the existence of tumor growth factor (VEGF) as a potent angiogenic angiogenic factors although their identities factor, which sequence identity with VPF (Leung et remained unknown at that time (Folkman et al., al., 1989). These initial findings paved new avenues 1971; Langer et al., 1976). The first angiogenic for subsequent identification and cloning of factor was isolated from the pituitary of mammals numerous other angiogenic factors related to tumor (Gospodarowicz, 1976). In 1984, Folkman and growth and invasion, including those members in colleagues isolated the first tumor angiogenic the VEGF, FGF, PDGF, angiopoietin, and notch factor, i.e., basic fibroblast growth factor (bFGF or ligand families. FGF-2), from a chondrosarcoma, and thus

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 447 Angiogenic factors and cancer therapy Cao Y

Tumor microenvironment and the they significantly contribute to the switch of tumor switch to an angiogenic angiogenesis (Figure 1). phenotype Angiogenic signaling pathways Genetic instability of malignant cells often leads to Since identification of VEGF and FGF-2 as potent accumulation of mutations of oncogenes and tumor angiogenic factors in 1980s, numerous angiogenic suppressor genes and these mutated oncogenic pathways have been discovered and their specific proteins often upregulate expression levels of roles in regulation of tumor angiogenesis and angiogenic factors (Cao et al., 2009). The fact that vascular remodeling have been defined. Most tumor tissues contain heterogenic populations of angiogenic factors trigger angiogenic signals malignant cells implies that various tumor cells through specific interaction with their cell surface even in the same tumor mass would produce receptors that often contain tyrosine kinase domains different levels of angiogeneic factors. Although the in endothelial cells. clinical significance of highly angiogenic tumor In general, various angiogenic factors appear to cells in tumor growth and invasion is not fully have distinct functions in regulation of vessel understood, it is reasonably speculated that the growth, vascular permeability and remodeling. highly angiogenic tumor cell population might VEGF is a potent angiogenic and vascular eventually dominate the entire tumor mass owing to permeability factor that induces vascular sprouting their growth advantages. In contrast to tumor cells, and vascular leakiness (Leung et al., 1989; Senger somatic cells in healthy adult tissues may only et al., 1983). The Dll4-Notch signaling system produce modest levels of proangiogenic factors, prevents excessive vascular sprouting from the which are not able to induce an angiogenic "stalk region" of blood vessels (Noguera-Troise et phenotype. Additionally, endogenous angiogenesis al., 2006; Ridgway et al., 2006) (Figure 2). The inhibitors are predominately expressed at high PDGF-BB-PDGFR-β signaling in perivascular cells levels to prevent excessive neovascularization (Cao, such as pericytes mediates recruitment of these 2008). Thus, angiogenesis rarely occurs in healthy mural cells onto the newly formed vasculatures adult tissues, except in the reproductive organs and (Lindahl et al., 1997). These distinctive functions tissue regeneration. To create an angiogenic can be divided even within the same family phenotype, tumors have to tip the balance of angiogenic factors. For example, Ang1 and Ang2 angiogenic factors over inhibitors (Figure 1). Other within the angiopoietin family seem to display cell types in the tumor microenvironment including opposing effects on vascular remodeling even inflammatory cells and stromal fibroblasts are also though they bind to the same endothelial Tie2 significant sources of tumor angiogenic factors and receptor (Maisonpierre et al., 1997).

Figure 1. Angiogenic switch in tumor tissues. Tumor cells together with other host cells including inflammatory cells and stromal fibroblasts produce high levels of proangiogenic factors and reduced levels of endogenous inhibitors, tipping the balance towards a proangiogenic phenotype.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 448 Angiogenic factors and cancer therapy Cao Y

Figure 2. Angiogenic regulators that control the precise steps of new vessel growth. VEGF is a driving force that induces tip cell formation through activation of VEGFR2 signaling. Angiogenic endothelial cells produce PDGF-BB to recruit pericytes onto newly formed vessels to prevent excessive sprouting. The Dll4-Notch1 signaling also prevent excessive vascular sprouting from "stalk cells".

Thus, angiogenic factors whether within the same (Nissen et al., 2007). Therefore, assessment of family or in different families have distinctive roles angiogenic profiles in a given tumor tissue should in modulation of vessel growth and remodeling. consider potential interaction relations between During tumor angiogenesis, these signaling various angiogenic factors and signaling pathways. pathways may become uncoordinated, leading to the formation of disorganized and primitive Targeting angiogenic pathways vascular networks. in tumors Interplay between angiogenic The original idea of blocking tumor-derived angiogenic factors for cancer therapy was raised by factors Dr. Judah Folkman. In his conceptual paper, Co-existence of various angiogenic factors, Folkman wrote "One approach to the initiation of cytokines, signaling receptors and intracellular 'anti-angiogenesis' would be the production of an signaling components often results in crosstalk antibody against tumor angiogenic between various signaling pathways. Thus, in tumor factor" (Folkman, 1971). His prediction and vision tissues various angiogenic factors and cytokines not were validated in human cancer patients 33 years only transduce their signals vertically, but also later with approval of bevacizumab, an anti-VEGF interact each other horizontally. At the ligand level, neutralizing antibody, by US FDA in 2004 for various angiogenic factors with the same family can treatment of human colorectal cancer (Hurwitz et interact each other. For example, VEGF-A and al., 2004). In fact, bevacizumab remains as the most PlGF or VEGF-B can form heterodimers in commonly used antiangiogenic drugs for treatment addition to their respective homodimers and of various human cancers either in combination heterodimers may different biological functions with chemotherapy or monotherapy settings. (Cao et al., 1996; Olofsson et al., 1996). Similarly, Antiangiogenic drugs that block signaling pathways PDGF-A and PDGF-B can also form heterodimers can be divided into several categories according to that display overlapping but yet different functions their targets and specificity: 1) Inhibition of from their corresponding homodimers (Heldin and angiogenic factor production from various cell Westermark, 1989b). The same heterodimerization types of tumors. These may include inhibition of mechanism also exists in various VEGF receptor transcription and translation of a specific molecules and PDGF receptor molecules (Heldin angiogenic factor; 2) Functional neutralization of and Westermark, 1989a; Mac Gabhann and Popel, angiogenic factors. Bevacizumab targeting VEGF is 2007). Similarly, interactions between cell surface one of such neutralizing antibodies (Hurwitz et al., receptors beyond the same family also exist, 2004); 3) Anti-receptor neutralizing antibodies. demonstrating the complex signaling transduction Similar to angiogenic ligands, binding of antibodies of these receptors. Activation of a particular to specific regions of extracellular domains of a signaling pathway often induces and amplifies receptor could also effectively block their ligand- signaling pathways. For example, stimulation of triggered angiogenic signaling. Ramucizumab, an endothelial cells and angiogenesis by FGF-2 anti-VEGFR2 neutralizing antibody is an example induces expression levels of PDGFR expression, of such drugs that are under clinical development and thus triggering a synergistic angiogenic (Fuchs et al., 2014); 4) Tyrosine kinase inhibitors response (Cao et al., 2003). Such a synergistic (TKIs) that block angiogenic receptor functions. angiogenic activity in the tumor microenvironment There are 7 currently US-FDA-approved promotes tumor growth, invasion and metastasis antiangiogenic TKIs, including: axitinib,

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 449 Angiogenic factors and cancer therapy Cao Y

cabozantinib, pazopanib, regorafenib, sorafenib, metastasis. Among these known lymphangiogenic sunitinib, and vandetanib (Choueiri et al., 2012; factors, the VEGF-C-VEGFR3 signaling system is Cohen et al., 2008; Escudier et al., 2007; George et probably the most well characterized signaling al., 2012; Houvras and Wirth, 2011; Motzer et al., pathway in regulation of physiological and 2013; Motzer et al., 2007). In general, TKIs lacks pathological lymphangiogenesis (Adams and specificity and each TKI blocks the activity of Alitalo, 2007; Stacker et al., 2002). VEGFR3 seems several tyrosine kinases. The antiangiogenic TKIs to be exclusively expressed on lymphatic share overlapping, but yet target different spectrums endothelial cells although it is also found in of angiogenic pathways. VEGFR2, as a key angiogenic endothelial cells (Tammela et al., 2008). functional receptor for VEGF-induced Several lymphangiogenic factors indirectly induce angiogenesis, is one of the common targets of these lymphangiogenesis through activation of the TKIs. TKIs have been clinically used for treatment VEGF-C-VEGFR3 signaling system. This indirect of various human cancers; 5) Inhibition of regulatory mechanism occurs at both VEGF-C downstream signaling components. For example, ligand and VEGFR3 receptor levels at which other mTOR (mammalian targets of rapamycin) factors often upregulate expression of these two inhibitors including temsirolimus and everolimus signaling molecules. Notably, the VEGFR3- potently suppress tumor angiogenesis (Fazio et al., mediated lymphatic endothelial tip cell formation is 2007; Yao et al., 2011); 6) Generic angiogenesis probably essential for other lymphangiogenic inhibitors. Thalidomide is an example of generic factor-induced lymphangiogenesis. For example, angiogenesis inhibitor that blocks a common FGF-2-induced lymphangiogenesis requires the pathway of angiogenesis that is currently used for VEGFR3 signaling system for sprouting and treatment of multiple myeloma (Singhal et al., inhibition VEGFR3 completely ablates FGF-2- 1999); 7) Endogenous angiogenesis inhibitors. induced lymphangiogenesis (Cao et al., 2012). These inhibitors such as angiostatin and endostatin Given the essential role of lymphangiogenesis in exhibit a broad-spectrum inhibitory activity against cancer metastasis, inhibition of lymphangiogenesis tumor angiogenesis induced by various stimuli would in principle be a valid approach for anti- (O'Reilly et al., 1997; O'Reilly et al., 1994). A metastatic cancer therapy. However, development variant version of endostatin has been approved by of drugs to block cancer metastasis for clinical use the Chinese FDA for treatment of lung cancer in remains a challenging issue and pharmaceutical human patients (Han et al., 2011). companies remain reluctant to pursue this avenue. Despite successful development of these drugs for Mechanistic challenges of treatment of human cancers, the survival beneficial effects, in general, are rather modest for most antiangiogenic cancer therapy cancer types. A majority of cancer patients show The initial antiangiogenic concept for treatment of intrinsic resistance toward these antiangiogenic cancer raised by Judah Folkman has led to drugs (Cao and Langer, 2008, 2010). For those successful development of antiangiogenic drugs for patients who initially respond to antiangiogenic treatment of various human cancers. Despite the drugs can also develop evasive refractoriness. fact that the growth of all solid tumors is dependent Additionally, antiangiogenic drugs also produce on angiogenesis, the response rate of antiangiogenic various side effects in cancer patients. Given the therapy in human cancer patients is rather modest essential roles of VEGF in regulation of human (Cao and Langer, 2010; Kerbel, 2008). In most physiology, it is perhaps not unexpected to observe types of cancers, antiangiogenic drugs are delivered broad side effects of anti-VEGF-based together with chemotherapeutics or in combination antiangiogenic drugs in human patients. with other therapeutic modalities and Lymphangiogenic factors and antiangiogenic monotherapy produce insignificant improvement of patient survivals. This clinical therapeutic targets finding is in marked contrast to the treatment Tumor-produced angiogenic factors not only regimen in preclinical tumor models in which most stimulate angiogenesis, but often induce antiangiogenic agents show potent antitumor effects lymphangiogenesis, which significantly contribute when delivered as a single agent. Why would to lymph node metastasis (Cao, 2005). Similar to human and mouse tumors respond so differently to blood vessel angiogenesis, tumor the same drug (Cao, 2011)? This important question lymphangiogenesis is also regulated by multiple remains unresolved at this time of writing. One of growth factors. Members in the VEGF, FGF, challenging issues of translating preclinical findings PDGF, Ang, HGF, and IGF families have been into clinical practice is the relevance of preclinical found to actively participate in regulation of mouse tumor models to human patients. In mouse lymphangiogenesis (Cao, 2005). Moreover, these tumor models, we often use genetically identical factors induce intra- and peri-tumoral mice to study the effect of a given antiangiogenic lymphangiogenesis that facilitate lymphatic agent whereas each human cancer patient carries

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 450 Angiogenic factors and cancer therapy Cao Y

genetic information that is different from others. Folkman J, Merler E, Abernathy C, Williams G. Isolation of Tumors in human patients contain different a tumor factor responsible for angiogenesis. J Exp Med. 1971 Feb 1;133(2):275-88 mutations of crucial genes related to cell growth and oncogenesis whereas mouse tumors are often Gospodarowicz D. Humoral control of cell proliferation: the role of fibroblast growth factor in regeneration, identical from the same cell line. Human cancers angiogenesis, wound healing, and neoplastic growth. Prog are treated at different stages of malignant Clin Biol Res. 1976;9:1-19 progression and mouse tumors are often treated at Langer R, Brem H, Falterman K, Klein M, Folkman J. the same time and similar size during cancer Isolations of a cartilage factor that inhibits tumor development. Importantly, in mouse tumor models neovascularization. Science. 1976 Jul 2;193(4247):70-2 therapeutic efficacy of antiangiogenic drugs is Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, assessed by measuring tumor size and beneficial Dvorak HF. Tumor cells secrete a vascular permeability effects of antiangiogenic therapy are often factor that promotes accumulation of ascites fluid. Science. determined based on survival improvement. During 1983 Feb 25;219(4587):983-5 clinical practice, it is increasingly noticed that Shing Y, Folkman J, Sullivan R, Butterfield C, Murray J, tumor size cannot be used as a reliable surrogate Klagsbrun M. Heparin affinity: purification of a tumor- marker to predict survival benefits and derived capillary endothelial cell growth factor. Science. antiangiogenic therapy. Another important issue is 1984 Mar 23;223(4642):1296-9 the mechanism that underlies combination therapy, Heldin CH, Westermark B. Platelet-derived growth factor: which remains unknown. three isoforms and two receptor types. Trends Genet. 1989a Apr;5(4):108-11 A couple of hypotheses have been proposed to explain therapeutic benefits underlying the Heldin CH, Westermark B. Platelet-derived growth factors: a family of isoforms that bind to two distinct receptors. Br combination of antiangiogenic drugs with Med Bull. 1989b Apr;45(2):453-64 chemotherapeutics. Anti-antiangiogenic drug-induced vascular Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, Connolly DT. Vascular permeability factor, an endothelial normalization modulates chemotherapeutic delivery cell mitogen related to PDGF. Science. 1989 Dec in tumor tissues offers an attractive mechanism for 8;246(4935):1309-12 explaining the beneficial effects of combination Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara therapy (Jain, 2005). N. Vascular endothelial growth factor is a secreted Despite some interesting findings in mouse tumor angiogenic mitogen. Science. 1989 Dec 8;246(4935):1306- model, the vascular normalization concept needs to 9 be validated in human cancer patients (Van der O'Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Veldt et al., 2012). Another interesting concept is Moses M, Lane WS, Cao Y, Sage EH, Folkman J. that antiangiogenic drugs significantly reduce Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. chemotherapy-related toxicity in cancer hosts Cell. 1994 Oct 21;79(2):315-28 (Zhang et al., 2011). Again, this interesting concept warrants clinical Cao Y, Chen H, Zhou L, Chiang MK, Anand-Apte B, Weatherbee JA, Wang Y, Fang F, Flanagan JG, Tsang validation. Taken together, the mechanisms by ML. Heterodimers of placenta growth factor/vascular which antiangiogenic drugs improve survivals of endothelial growth factor. Endothelial activity, tumor cell cancer patients remains an enigma despite these expression, and high affinity binding to Flk-1/KDR. J Biol drugs suppress tumor angiogenesis. Future Chem. 1996 Feb 9;271(6):3154-62 preclinical and clinical studies should focus on Olofsson B, Pajusola K, Kaipainen A, von Euler G, Joukov mechanisms that underlie clinical benefits of these V, Saksela O, Orpana A, Pettersson RF, Alitalo K, Eriksson U. Vascular endothelial growth factor B, a novel drugs in cancer patients. growth factor for endothelial cells. Proc Natl Acad Sci U S Acknowledgements A. 1996 Mar 19;93(6):2576-81 Y.C.'s laboratory is supported by research grants Lindahl P, Johansson BR, Levéen P, Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient from the Swedish Research Council, the Swedish mice. Science. 1997 Jul 11;277(5323):242-5 Cancer Foundation, the Karolinska Institute Foundation, the Karolinska Institute distinguished Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClain J, professor award, the Torsten Söderbergs Aldrich TH, Papadopoulos N, Daly TJ, Davis S, Sato TN, foundation, Söderbergs stiftelse, the European Yancopoulos GD. Angiopoietin-2, a natural antagonist for Union Integrated Project of Metoxia (Project no. Tie2 that disrupts in vivo angiogenesis. Science. 1997 Jul 222741), and the European Research Council 4;277(5322):55-60 (ERC) advanced grant ANGIOFAT (Project no O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane 250021). WS, Flynn E, Birkhead JR, Olsen BR, Folkman J. Endostatin: an endogenous inhibitor of angiogenesis and References tumor growth. Cell. 1997 Jan 24;88(2):277-85 Singhal S, Mehta J, Desikan R, Ayers D, Roberson P, Folkman J. Tumor angiogenesis: therapeutic implications. 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refractory multiple myeloma. N Engl J Med. 1999 Nov thyroid cancer: results from a phase II study. J Clin Oncol. 18;341(21):1565-71 2008 Oct 10;26(29):4708-13 Stacker SA, Achen MG, Jussila L, Baldwin ME, Alitalo K. Kerbel RS. Tumor angiogenesis. N Engl J Med. 2008 May Lymphangiogenesis and cancer metastasis. Nat Rev 8;358(19):2039-49 Cancer. 2002 Aug;2(8):573-83 Tammela T, Zarkada G, Wallgard E et al.. Blocking Cao R, Bråkenhielm E, Pawliuk R, Wariaro D, Post MJ, VEGFR-3 suppresses angiogenic sprouting and vascular Wahlberg E, Leboulch P, Cao Y. Angiogenic synergism, network formation. Nature. 2008 Jul 31;454(7204):656-60 vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2. Nat Med. 2003 Cao Y, Zhong W, Sun Y. Improvement of antiangiogenic May;9(5):604-13 cancer therapy by understanding the mechanisms of angiogenic factor interplay and drug resistance. Semin Hurwitz H, Fehrenbacher L, Novotny W et al.. 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Atlas Genet Cytogenet Oncol Haematol. 2014; 18(6) 452 Atlas of Genetics and Cytogenetics in Oncology and Haematology

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