Profiling and functional annotation of mRNA gene expression in pediatric rhabdomyosarcoma and Ewing's sarcoma

Using Affymetrix oligonucleotide microarrays, we analyzed mRNA gene expression patterns of 12 primary pediatric rhabdomyosarcomas (RMS) and 11 Ewing's sarcomas (EWS), which belong to the small round blue cell tumors (SRBCTs). Diagnostic classification of these cancers is frequently complicated by the highly similar appearance in routine histology, and additional molecular markers could significantly improve tumor classification. A combination of three independent statistical approaches (t‐test, SAM, k‐nearest neighborhood analysis) resulted in 101 highly significant probe sets that clearly discriminate between EWS and RMS. We identified novel marker transcripts that have not been previously associated with either RMS or EWS yet, including CITED2, glypican 3 (GPC3), and cyclin D1 (CCND1). Expression levels for selected candidate genes were validated by quantitative real‐time reverse‐transcription PCR. Furthermore, to identify biologically meaningful trends, functional annotations were assigned to 946 genes differentially expressed between EWS and RMS (t‐test). Genes involved in protein biosynthesis (n = 28) and complex assembly (n = 9), lipid metabolism (n = 23), energy generation (n = 22), and mRNA processing (n = 11) were expressed significantly higher in EWS. Thus, functional annotation of tumor‐specific genes reveals detailed insights into tumor biology and differentiation‐specific expression patterns and gives important clues related to the possible cellular origin of these pediatric tumors. Supplementary material for this article is available at the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020‐7136/suppmat/index.html. © 2004 Wiley‐Liss, Inc.

[1]  Helen C. Hurst,et al.  Physical and Functional Interactions among AP-2 Transcription Factors, p300/CREB-binding Protein, and CITED2* , 2003, The Journal of Biological Chemistry.

[2]  P. Khatri,et al.  Global functional profiling of gene expression ? ? This work was funded in part by a Sun Microsystem , 2003 .

[3]  Akihiro Umezawa,et al.  Upregulation of Id2, an oncogenic helix-loop-helix protein, is mediated by the chimeric EWS/ets protein in Ewing sarcoma , 2003, Oncogene.

[4]  Partha S. Vasisht.  Computational Analysis of Microarray Data , 2003 .

[5]  P. Khatri,et al.  Global functional profiling of gene expression. , 2003, Genomics.

[6]  D. Slonim.  From patterns to pathways: gene expression data analysis comes of age , 2002, Nature Genetics.

[7]  G. Grosveld,et al.  Pax3-FKHR Knock-In Mice Show Developmental Aberrations but Do Not Develop Tumors , 2002, Molecular and Cellular Biology.

[8]  K. Herrup,et al.  The essential role of Cited2, a negative regulator for HIF-1α, in heart development and neurulation , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[9]  T. Golub,et al.  Supplemental Information for , 2002 .

[10]  T. Shioda,et al.  Human CREB-binding Protein/p300-interacting Transactivator with ED-rich Tail (CITED) 4, a New Member of the CITED Family, Functions as a Co-activator for Transcription Factor AP-2* , 2002, The Journal of Biological Chemistry.

[11]  Toshifumi Ozaki,et al.  Expression analysis of pediatric solid tumor cell lines using oligonucleotide microarrays. , 2002, International journal of oncology.

[12]  J. Downing,et al.  Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. , 2002, Cancer cell.

[13]  A. Simeone,et al.  Folic acid prevents exencephaly in Cited2 deficient mice. , 2002, Human molecular genetics.

[14]  P. Khatri,et al.  Profiling gene expression using onto-express. , 2002, Genomics.

[15]  A. Donfrancesco,et al.  Id2 is critical for cellular proliferation and is the oncogenic effector of N-myc in human neuroblastoma. , 2002, Cancer research.

[16]  A. Iavarone,et al.  Id proteins at the cross-road of development and cancer , 2001, Oncogene.

[17]  Jennifer N. Murdoch,et al.  Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator , 2001, Nature Genetics.

[18]  Christopher T Denny,et al.  Biology of EWS/ETS fusions in Ewing's family tumors , 2001, Oncogene.

[19]  K. Pritchard-Jones,et al.  PAX3-FKHR induces morphological change and enhances cellular proliferation and invasion in rhabdomyosarcoma. , 2001, The American journal of pathology.

[20]  M. Ringnér,et al.  Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks , 2001, Nature Medicine.

[21]  R. Tibshirani,et al.  Significance analysis of microarrays applied to the ionizing radiation response , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[22]  J. Filmus,et al.  Glypicans in growth control and cancer. , 2001, Glycobiology.

[23]  T. Golub,et al.  Genome-wide views of cancer. , 2001, The New England journal of medicine.

[24]  A. Iavarone,et al.  Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins , 2000, Nature.

[25]  D. Sinnett,et al.  Expression of glypican 3 (GPC3) in embryonal tumors , 2000, International journal of cancer.

[26]  K. Pritchard-Jones,et al.  Genes, chromosomes, and rhabdomyosarcoma , 1999, Genes, chromosomes & cancer.

[27]  S. Bhattacharya,et al.  Molecular cloning and chromosomal localization of the human CITED2 gene encoding p35srj/Mrg1. , 1999, Genomics.

[28]  Michael L. Bittner,et al.  cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene , 1999, Nature Genetics.

[29]  J. Mesirov,et al.  Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. , 1999, Science.

[30]  E. Lander.  Array of hope , 1999, Nature Genetics.

[31]  M. Bittner,et al.  Gene expression profiling of alveolar rhabdomyosarcoma with cDNA microarrays. , 1998, Cancer research.

[32]  W. Molenaar,et al.  Expression of neural cell adhesion molecules and neurofilament protein isoforms in skeletal muscle tumors. , 1998, Human pathology.

[33]  J. Moore,et al.  Insulin-like growth factor II and PAX3-FKHR cooperate in the oncogenesis of rhabdomyosarcoma. , 1998, Cancer Research.

[34]  J. Epstein,et al.  Tumor-Specific PAX3-FKHR Transcription Factor, but Not PAX3, Activates the Platelet-Derived Growth Factor Alpha Receptor , 1998, Molecular and Cellular Biology.

[35]  Marcienne M Wright,et al.  EWS/FLI1-induced manic fringe renders NIH 3T3 cells tumorigenic , 1997, Nature Genetics.

[36]  W. Winkelmann,et al.  Assessment of molecular genetic detection of chromosome translocations in the differential diagnosis of pediatric sarcomas , 1997, Klinische Padiatrie.

[37]  C. Denny,et al.  EAT-2 is a novel SH2 domain containing protein that is up regulated by Ewing's sarcoma EWS/FLI1 fusion gene. , 1996, Oncogene.

[38]  G. Thomas,et al.  An EWS/ERG fusion with a truncated N‐terminal domain of EWS in a Ewing's tumor , 1996, International journal of cancer.

[39]  A. Iavarone,et al.  Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins , 1996, Molecular and cellular biology.

[40]  D. Schlessinger,et al.  Mutations in GPC3, a glypican gene, cause the Simpson-Golabi-Behmel overgrowth syndrome , 1996, Nature Genetics.

[41]  J. Biegel,et al.  In vivo amplification of the PAX3-FKHR and PAX7-FKHR fusion genes in alveolar rhabdomyosarcoma. , 1996, Human molecular genetics.

[42]  J. Epstein,et al.  Pax3 Inhibits Myogenic Differentiation of Cultured Myoblast Cells (*) , 1995, The Journal of Biological Chemistry.

[43]  C. Denny,et al.  A variant Ewing's sarcoma translocation (7;22) fuses the EWS gene to the ETS gene ETV1. , 1995, Oncogene.

[44]  C. Denny,et al.  Multiple domains mediate transformation by the Ewing's sarcoma EWS/FLI-1 fusion gene. , 1995, Oncogene.

[45]  A. Iavarone,et al.  The helix-loop-helix protein Id-2 enhances cell proliferation and binds to the retinoblastoma protein. , 1994, Genes & development.

[46]  J. Biegel,et al.  Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma. , 1994, Cancer research.

[47]  P. Sorensen,et al.  A second Ewing's sarcoma translocation, t(21;22), fuses the EWS gene to another ETS–family transcription factor, ERG , 1994, Nature Genetics.

[48]  B. Emanuel,et al.  Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma , 1993, Nature Genetics.

[49]  H Kovar,et al.  MIC2 is a specific marker for ewing's sarcoma and peripheral primitive neuroectodermal tumors. Evidence for a common histogenesis of ewing's sarcoma and peripheral primitive neuroectodermal tumors from MIC2 expression and specific chromosome aberration , 1991, Cancer.

[50]  H. Kovar,et al.  Overexpression of the pseudoautosomal gene MIC2 in Ewing's sarcoma and peripheral primitive neuroectodermal tumor. , 1990, Oncogene.