Unknown,Transcriptomics,Genomics,Proteomics

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Global methylation analysis of development and progression of colorectal carcinoma

ABSTRACT: Colorectal tumorigenesis proceedes through well defined clinical stages assoicated with charateristic mutations. Besides genetic alterations, epi-driver genes that are aberrantly expressed in cancers in a fashion that confers a seletive growth advantage can also contribute to tumor evolution. To gain a global view of methylation patterns in normal and maliganant colorectal epithelia, we performed genome-wide DNA methylation analysis on DNAs from 48 fresh frozen CRC samples at different stages of CRC progression. We used IlluminaHumanMethylation450 Beadchip to get a broad view of genome-wide DNA methylationdata during CRC progression and identified significantly differentially methylated genes during CRC progression A total of 48 macro-dissected tissues including normal colon tissue, adenomas, carcinomas and metastases were collected by the Department of Pathology at the University of Virginia under the supervision of an experienced pathologist. DNA was extracted and run on IlluminaHumanMethylation450 Beadchip by Expression Analyisis.

ORGANISM(S): Homo sapiens

SUBMITTER: Omar Kabbarah

PROVIDER: E-GEOD-77954 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:An integrative functional genomics of multiple forms of data is vital for discovering molecular drivers of cancer development and progression. Here, we present an integrated genomic strategy utilizing DNA methylation and transcriptome profile data to discover epigenetically regulated genes implicated in cancer development and invasive progression. More specifically, this analysis identified Fibromodulin (FMOD) as a glioblastoma (GBM) upregulated gene due to the loss of promoter methylation. Secreted FMOD promotes glioma cell migration through its ability to induce filamentous actin stress fiber formation. Treatment with Cytochalasin D, an actin polymerization inhibitor, significantly reduced the FMOD induced glioma cell migration. siRNA and small molecule inhibitor-based studies identified that FMOD-induced glioma cell migration is dependent on Integrin-FAK-Src-Rho-ROCK signaling pathway. FMOD lacking C terminus LRR11 domain (FMOD), which does not bind collagen type I, failed to induce integrin and promote glioma cell migration. Further, FMOD-induced integrin activation and migration was abrogated by a 9-mer wild type peptide from the FMOD C-terminus. However, the same peptide with mutation in two residues essential for FMOD interaction with collagen type I failed to compete with FMOD, thus signifying the importance of collagen type I-FMOD interaction in integrin activation. ChIP-PCR experiments revealed that TGF-Ã1 regulates FMOD expression through epigenetic remodeling of FMOD promoter that involved demethylation and gain of active histone marks with a simultaneous loss of DNMT3A and EZH2 occupancy, but enrichment of SMAD2 and CBP. FMOD silencing inhibited the TGF-Ã1 mediated glioma cell migration significantly. In univariate and multivariate cox regression analysis, both FMOD promoter methylation and transcript levels predicted prognosis in GBM. Thus, the present study identified several epigenetically regulated alterations responsible for cancer development and progression. Specifically, we found that secreted FMOD as an important regulator of glioma cell migration downstream of TGF-Ã1 pathway and forms a potential basis for therapeutic intervention in GBM. We used DNA isolated from 17 diffuse astrocytoma, 16 anaplastic astrocytoma, 36 glioblastoma and 9 control brain tissue for the array. P53 status: All cases were subjected to histopathology and the diagnosis of GBM was confirmed. Paraffin sections (4 Î¼m) from the tissues were collected on silane-coated slides, and the protein expression of p53 was assessed by immunohistochemistry (IHC). Antigen retrieval was done by heat treatment at 850W in citrate buffer or at 700W. After the initial processing steps, sections were incubated overnight with primary antibody for p53 (Mouse Monoclonal DO-7; Biogenex, USA)at 4Â°C with 1: 200 dilution. MIB: Paraffin blocks of all the cases were retrieved, and histological features were reviewed on freshly cut and stained (hematoxylin and eosin) sections. After confirming the diagnosis of glioblastoma, 4 Âµm thick sections were collected on silane-coated slides, and IHC was performed using MIB-1 (anti Ki-67 monoclonal BGX 297, diluted to 1:30) EGFR status: Paraffin blocks of all the cases were retrieved, and histological features were reviewed on freshly cut and stained (hematoxylin and eosin) sections. After confirming the diagnosis of glioblastoma, 4 Âµm thick sections were collected on silane-coated slides, and IHC was performed using EGFR (monoclonal E-30, diluted to 1:50) GFAP status: Paraffin blocks of all the cases were retrieved, and histological features were reviewed on freshly cut and stained (hematoxylin and eosin) sections. After confirming the diagnosis of glioblastoma, 4 Âµm thick sections were collected on silane-coated slides, and IHC was performed using glial fibrillary acidic protein (GFAP, monoclonal GA-5, diluted to 1:100).

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Global methylation analysis of development and progression of colorectal carcinoma

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