Integrative Analysis Reveals Relationships of Genetic and Epigenetic Alterations in Osteosarcoma

Background Osteosarcomas are the most common non-haematological primary malignant tumours of bone, and all conventional osteosarcomas are high-grade tumours showing complex genomic aberrations. We have integrated genome-wide genetic and epigenetic profiles from the EuroBoNeT panel of 19 human osteosarcoma cell lines based on microarray technologies. Principal Findings The cell lines showed complex patterns of DNA copy number changes, where genomic copy number gains were significantly associated with gene-rich regions and losses with gene-poor regions. By integrating the datasets, 350 genes were identified as having two types of aberrations (gain/over-expression, hypo-methylation/over-expression, loss/under-expression or hyper-methylation/under-expression) using a recurrence threshold of 6/19 (>30%) cell lines. The genes showed in general alterations in either DNA copy number or DNA methylation, both within individual samples and across the sample panel. These 350 genes are involved in embryonic skeletal system development and morphogenesis, as well as remodelling of extracellular matrix. The aberrations of three selected genes, CXCL5, DLX5 and RUNX2, were validated in five cell lines and five tumour samples using PCR techniques. Several genes were hyper-methylated and under-expressed compared to normal osteoblasts, and expression could be reactivated by demethylation using 5-Aza-2′-deoxycytidine treatment for four genes tested; AKAP12, CXCL5, EFEMP1 and IL11RA. Globally, there was as expected a significant positive association between gain and over-expression, loss and under-expression as well as hyper-methylation and under-expression, but gain was also associated with hyper-methylation and under-expression, suggesting that hyper-methylation may oppose the effects of increased copy number for detrimental genes. Conclusions Integrative analysis of genome-wide genetic and epigenetic alterations identified dependencies and relationships between DNA copy number, DNA methylation and mRNA expression in osteosarcomas, contributing to better understanding of osteosarcoma biology.

[1]  David M. Thomas,et al.  Cyclin E1 is amplified and overexpressed in osteosarcoma. , 2011, The Journal of molecular diagnostics : JMD.

[2]  M. van Glabbeke,et al.  Improvement in histologic response but not survival in osteosarcoma patients treated with intensified chemotherapy: a randomized phase III trial of the European Osteosarcoma Intergroup. , 2007, Journal of the National Cancer Institute.

[3]  G. K. Sandve,et al.  The Genomic HyperBrowser: inferential genomics at the sequence level , 2010, Genome Biology.

[4]  M. Ringnér,et al.  DNA methylation analyses of urothelial carcinoma reveal distinct epigenetic subtypes and an association between gene copy number and methylation status , 2012, Epigenetics.

[5]  Anne-Marie Cleton-Jansen,et al.  Tumor-Infiltrating Macrophages Are Associated with Metastasis Suppression in High-Grade Osteosarcoma: A Rationale for Treatment with Macrophage Activating Agents , 2011, Clinical Cancer Research.

[6]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[7]  Shishir Shah,et al.  Genome-wide array comparative genomic hybridization analysis reveals distinct amplifications in osteosarcoma , 2004, BMC Cancer.

[8]  N. Carter,et al.  Massive Genomic Rearrangement Acquired in a Single Catastrophic Event during Cancer Development , 2011, Cell.

[9]  川村 幹雄.  CXCL5, a promoter of cell proliferation, migration and invasion, is a novel serum prognostic marker in patients with colorectal cancer , 2013 .

[10]  宁北芳,et al.  疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A , 2005 .

[11]  Qinghua Wu,et al.  DNA methylation profiling of ovarian carcinomas and their in vitro models identifies HOXA9, HOXB5, SCGB3A1, and CRABP1 as novel targets , 2007, Molecular Cancer.

[12]  Peter Simpson,et al.  DNA methylome of familial breast cancer identifies distinct profiles defined by mutation status. , 2010, American journal of human genetics.

[13]  H. Saluz,et al.  Hypomethylation of PRAME is responsible for its aberrant overexpression in human malignancies , 2007, Genes, chromosomes & cancer.

[14]  Brad T. Sherman,et al.  DAVID: Database for Annotation, Visualization, and Integrated Discovery , 2003, Genome Biology.

[15]  S. Ferrari,et al.  Neoadjuvant chemotherapy for osteosarcoma of the extremities with metastases at presentation: recent experience at the Rizzoli Institute in 57 patients treated with cisplatin, doxorubicin, and a high dose of methotrexate and ifosfamide. , 2003, Annals of oncology : official journal of the European Society for Medical Oncology.

[16]  W. Winkelmann,et al.  Genetic imbalances revealed by comparative genomic hybridization in osteosarcomas , 2002, International journal of cancer.

[17]  K. Dudley,et al.  Loss of neuronatin expression is associated with promoter hypermethylation in pituitary adenoma. , 2009, Endocrine-related cancer.

[18]  T. Yoneda,et al.  Regulation of bone and cartilage development by network between BMP signalling and transcription factors. , 2012, Journal of biochemistry.

[19]  Anne-Marie Cleton-Jansen,et al.  mRNA expression profiles of primary high-grade central osteosarcoma are preserved in cell lines and xenografts , 2011, BMC Medical Genomics.

[20]  B. V. van Royen,et al.  Molecular alterations as target for therapy in metastatic osteosarcoma: a review of literature , 2011, Clinical & Experimental Metastasis.

[21]  Derek Y. Chiang,et al.  The landscape of somatic copy-number alteration across human cancers , 2010, Nature.

[22]  Anne-Marie Cleton-Jansen,et al.  Evaluation of high-resolution microarray platforms for genomic profiling of bone tumours , 2010, BMC Research Notes.

[23]  David Haussler,et al.  Integrated molecular profiles of invasive breast tumors and ductal carcinoma in situ (DCIS) reveal differential vascular and interleukin signaling , 2011, Proceedings of the National Academy of Sciences.

[24]  W. Huber,et al.  Model-based variance-stabilizing transformation for Illumina microarray data , 2008, Nucleic acids research.

[25]  A. Simpson,et al.  Insights on PRAME and osteosarcoma by means of gene expression profiling , 2011, Journal of orthopaedic science : official journal of the Japanese Orthopaedic Association.

[26]  M. Relling,et al.  Integrated genomic analysis of relapsed childhood acute lymphoblastic leukemia reveals therapeutic strategies. , 2011, Blood.

[27]  G. Stein,et al.  The Role of RUNX2 in Osteosarcoma Oncogenesis , 2010, Sarcoma.

[28]  Chi-Hung Lin,et al.  Identification of chromosomal aberrations associated with disease progression and a novel 3q13.31 deletion involving LSAMP gene in osteosarcoma. , 2009, International journal of oncology.

[29]  D. Lev,et al.  Cancer‐testis antigens expressed in osteosarcoma identified by gene microarray correlate with a poor patient prognosis , 2012, Cancer.

[30]  Matthew W. Wilson,et al.  A Novel Retinoblastoma Therapy from Genomic and Epigenetic Analyses , 2011, Nature.

[31]  Anne-Marie Cleton-Jansen,et al.  Identification of osteosarcoma driver genes by integrative analysis of copy number and gene expression data , 2012, Genes, chromosomes & cancer.

[32]  Yusuke Yamamoto,et al.  RPN2 gene confers docetaxel resistance in breast cancer , 2008, Nature Medicine.

[33]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[34]  A. Huvos,et al.  Frequent amplification and rearrangement of chromosomal bands 6p12‐p21 and 17p11.2 in osteosarcoma , 2004, Genes, chromosomes & cancer.

[35]  B. Bjerkehagen,et al.  Management of high-grade bone sarcomas over two decades: The Norwegian Radium Hospital experience , 2006, Acta oncologica.

[36]  S. Ferrari,et al.  Emerging drugs for high-grade osteosarcoma , 2010, Expert opinion on emerging drugs.

[37]  E. Kleinerman,et al.  Epigenetic Regulation of Apoptosis and Cell Cycle in Osteosarcoma , 2010, Sarcoma.

[38]  Bekim Sadikovic,et al.  Identification of interactive networks of gene expression associated with osteosarcoma oncogenesis by integrated molecular profiling. , 2009, Human molecular genetics.

[39]  R. Sciot,et al.  Somatic mosaic IDH1 or IDH2 mutations are associated with enchondroma and spindle cell hemangioma in Ollier disease and Maffucci syndrome , 2011, Nature Genetics.

[40]  Cornelis J H van de Velde,et al.  Disrupted Expression of CXCL5 in Colorectal Cancer Is Associated with Rapid Tumor Formation in Rats and Poor Prognosis in Patients , 2008, Clinical Cancer Research.

[41]  W. Winkelmann,et al.  Primary metastatic osteosarcoma: presentation and outcome of patients treated on neoadjuvant Cooperative Osteosarcoma Study Group protocols. , 2003, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[42]  M. Burdick,et al.  Overexpression of CXCL5 is associated with poor survival in patients with pancreatic cancer. , 2011, The American journal of pathology.

[43]  L. van Neste,et al.  Re-expression of CXCL14, a common target for epigenetic silencing in lung cancer, induces tumor necrosis , 2010, Oncogene.

[44]  A. Llombart‐Bosch,et al.  Functional characterization of osteosarcoma cell lines provides representative models to study the human disease , 2011, Laboratory Investigation.

[45]  Saraswati Sukumar,et al.  The Hox genes and their roles in oncogenesis , 2010, Nature Reviews Cancer.

[46]  O. Myklebost,et al.  Molecular characterization of commonly used cell lines for bone tumor research: A trans‐European EuroBoNet effort , 2010, Genes, chromosomes & cancer.

[47]  Stine H. Kresse,et al.  LSAMP, a novel candidate tumor suppressor gene in human osteosarcomas, identified by array comparative genomic hybridization , 2009, Genes, chromosomes & cancer.

[48]  R. Bernards,et al.  A causal role for the human tumor antigen preferentially expressed antigen of melanoma in cancer. , 2006, Cancer research.

[49]  M. Ladanyi,et al.  Cell Cycle Regulator Gene CDC5L, a Potential Target for 6p12-p21 Amplicon in Osteosarcoma , 2008, Molecular Cancer Research.

[50]  Gang Meng,et al.  Changes in genomic imprinting and gene expression associated with transformation in a model of human osteosarcoma. , 2008, Experimental and molecular pathology.

[51]  Peter A. Jones,et al.  A decade of exploring the cancer epigenome — biological and translational implications , 2011, Nature Reviews Cancer.

[52]  Wan-chun Wang,et al.  Epigenetic changes in osteosarcoma. , 2011, Bulletin du cancer.

[53]  Raj Chari,et al.  An integrative multi-dimensional genetic and epigenetic strategy to identify aberrant genes and pathways in cancer , 2010, BMC Systems Biology.

[54]  Y. Surh,et al.  eEF1A2 as a Putative Oncogene , 2009, Annals of the New York Academy of Sciences.

[55]  M. Rosemann,et al.  Genomic Alterations and Allelic Imbalances Are Strong Prognostic Predictors in Osteosarcoma , 2010, Clinical Cancer Research.

[56]  Bekim Sadikovic,et al.  In Vitro Analysis of Integrated Global High-Resolution DNA Methylation Profiling with Genomic Imbalance and Gene Expression in Osteosarcoma , 2008, PloS one.