In Vitro Analysis of Integrated Global High-Resolution DNA Methylation Profiling with Genomic Imbalance and Gene Expression in Osteosarcoma

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks

Effects of THBS3, SPARC and SPP1 expression on biological behavior and survival in patients with osteosarcoma

BACKGROUND: Osteosarcoma is a very aggressive tumor with a propensity to metastasize and invade surrounding tissue. Identification of the molecular determinants of invasion and metastatic potential may guide the development of a rational strategy for devising specific therapies that target the pathways leading to osteosarcoma. METHODS: In this study, we used pathway-focused low density expression cDNA arrays to screen for candidate genes related to tumor progression. Expression patterns of the selected genes were validated by real time PCR on osteosarcoma patient tumor samples and correlated with clinical and pathological data. RESULTS: THBS3, SPARC and SPP1 were identified as genes differentially expressed in osteosarcoma. In particular, THBS3 was expressed at significantly high levels (p = 0.0001) in biopsies from patients with metastasis at diagnosis, which is a predictor of worse overall survival, event-free survival and relapse free survival at diagnosis. After chemotherapy, patients with tumors over-expressing THBS3 have worse relapse free survival. High SPARC expression was found in 51/55 (96.3%) osteosarcoma samples derived from 43 patients, and correlated with the worst event-free survival (p = 0.03) and relapse free survival (p = 0.07). Overexpression of SPP1 was found in 47 of 53 (89%) osteosarcomas correlating with better overall survival, event-free survival and relapse free survival at diagnosis. CONCLUSION: In this study three genes were identified with pattern of differential gene expression associated with a phenotypic role in metastasis and invasion. Interestingly all encode for proteins involved in extracellular remodeling suggesting potential roles in osteosarcoma progression. This is the first report on the THBS3 gene working as a stimulator of tumor progression. Higher levels of THBS3 maintain the capacity of angiogenesis. High levels of SPARC are not required for tumor progression but are necessary for tumor growth and maintenance. SPP1 is not necessary for tumor progression in osteosarcoma and may be associated with inflammatory response and bone remodeling, functioning as a good biomarker

Neurolastoma: molecular cytogenetics approach

O neuroblastoma (NB), um tumor solido extracranial derivado de celulas neuroectodermicas da crista neural, e uma das neoplasias infantis mais fascinantes apresentando uma complexa heterogeneidade clinica e biologica. Entre os tumores solidos, o NB e considerado um modelo pois os estudos citogeneticos e moleculares permitem um controle potencial do paciente. Diversas anormalidades geneticas caracteristicas e presentes nesse tumor tem sido identificadas, incluindo a delecao do braco curto do cromossomo 1, a amplificacao do oncogene MYCN e o grau de ploidia. Essas e outras alteracoes geneticas descobertas mais recentemente contribuiram para o conhecimento sobre o processo de transformacao maligna, progressao do tumor, prognostico do paciente, bem como a compreensao da heterogenidade presente em NB. Do ponto de vista citogenetico, a delecao do braco curto do cromossomo l (lp) constitui a aberracao cromossomica mais caracteristica de NB e sugere a perda ou inativacao de um ou mais genes supressores de tumor, criticos para o desenvolvimento ou progressao tumoral. A maioria dos NBs e diploide mas ha um numero representativo de tumores hiperdiploides ou quase triploides, com ganhos de cromossomos inteiros e ausencia ou presenca de poucos rearranjos cromossomicos, sendo estes pacientes considerados de prognostico favoravel. Entretanto, tumores quase diploides e hiperdiploides contendo rearranjos cromossamicos estruturais, apresentam prognostico desfavoravel. O estudo de ploidia em NB tem sido realizado atraves de analise de cariotipo das celulas tumorais ou mais frequentemente, por citometria de fluxo onde se obtem uma medida da quantidade de DNA celular sem a identificacao das aberracoes cromossomicas. A primeira evidencia associada ao prognostico em NB foi a amplificacao do oncogene MYCN sendo esta, um mecanismo particular da ativacao de proto-oncogenes, amplamente estudado em NB. Aproximadamente 20-25 por cento dos NBs primarios e a maioria (>90 por cento ) das linhagens celulares apresentam amplificacao do oncogene MYCN que se localiza em 2p23-24. Este proto-oncogene faz parte da familia de genes MYC e codifica uma proteina nuclear com caracteristicas de fator de transcricao, atuando no controle da proliferacao e da diferenciacao celulares. Acredita-se que seu papel na oncogenese envolva corpusculos cromatidicos acentricos denominados minusculos duplos extracromossomicos (DMs), que podem ser...(au)BV UNIFESP: Teses e dissertaçõe

Decitabine-Induced Demethylation of 5′ CpG Island in GADD45A Leads to Apoptosis in Osteosarcoma Cells1

GADD45 genes are epigenetically inactivated in various types of cancer and tumor cell lines. To date, defects of the GADD45 gene family have not been implicated in osteosarcoma (OS) oncogenesis, and the role of this pathway in regulating apoptosis in this tumor is unknown. The therapeutic potential of Gadd45 in OS emerged when our previous studies showed that GADD45A was reexpressed by treatment with the demethylation drug decitabine. In this study, we analyze the OS cell lines MG63 and U2OS and show that on treatment with decitabine, a significant loss of DNA methylation of GADD45A was associated with elevated expression and induction of apoptosis. In vivo affects of decitabine treatment in mice showed that untreated control xenografts exhibited low nuclear staining for Gadd45a protein, whereas the nuclei from xenografts in decitabine-treated mice exhibited increased amounts of protein and elevated apoptosis. To show the specificity of this gene for decitabine-induced apoptosis in OS, GADD45A mRNAs were disrupted using short interference RNA, and the ability of the drug to induce apoptosis was reduced. Understanding the role of demethylation of GADD45A in reexpression of this pathway and restoration of apoptotic control is important for understanding OS oncogenesis and for more targeted therapeutic approaches

Recurrent RECQL4 Imbalance and Increased Gene Expression Levels Are Associated with Structural Chromosomal Instability in Sporadic Osteosarcoma12

Osteosarcoma (OS) is an aggressive bone tumor with complex abnormal karyotypes and a highly unstable genome, exhibiting both numerical- and structural-chromosomal instability (N- and S-CIN). Chromosomal rearrangements and genomic imbalances affecting 8q24 are frequent in OS. RECQL4 gene maps to this cytoband and encodes a putative helicase involved in the fidelity of DNA replication and repair. This protective genomic function of the protein is relevant because often patients with Rothmund-Thomson syndrome have constitutional mutations of RECQL4 and carry a very high risk of developing OS. To determine the relative level of expression of RECQL4 in OS, 18 sporadic tumors were studied by reverse transcription-polymerase chain reaction. All tumors overexpressed RECQL4 in comparison to control osteoblasts, and fluorescence in situ hybridization analysis of tumor DNA showed that expression levels were strongly copy number-dependent. Relative N- and S-CIN levels were determined by classifying copy number transitions within array comparative genomic hybridization profiles and by enumerating the frequency of break-apart fluorescence in situ hybridization within 8q24 using region-specific and control probes. Although there was no evidence that disruption of 8q24 in OS led to an elevated expression of RECQL4, there was a marked association between increased overall levels of S-CIN, determined by copy number transition frequency and higher levels of RECQL4

Three-Color FISH Analysis of TMPRSS2/ERG Fusions in Prostate Cancer Indicates That Genomic Microdeletion of Chromosome 21 Is Associated with Rearrangement

The recent description of novel recurrent gene fusions in ~80% of prostate cancer (PCa) cases has generated increased interest in the search for new translocations in other epithelial cancers and emphasizes the importance of understanding the origins and biologic implications of these genomic rearrangements. Analysis of 15 PCa cases by reverse transcription-polymerase chain reaction was used to detect six ERG-related gene fusion transcripts with TMPRSS2. No TMPRSS2/ETV1 chimeric fusion was detected in this series. Three-color fluorescence in situ hybridization confirms that TMPRSS2/ERG fusion may be accompanied by a small hemizygous sequence deletion on chromosome 21 between ERG and TMPRSS2 genes. Analysis of genomic architecture in the region of genomic rearrangement suggests that tracts of microhomology could facilitate TMPRSS2/ERG fusion events