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Integrative functional genomic analysis identifies epigenetically regulated Fibromodulin as an essential gene for glioma cell migration

ABSTRACT: 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-ß1 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-ß1 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-ß1 pathway and forms a potential basis for therapeutic intervention in GBM.

ORGANISM(S): Homo sapiens

PROVIDER: GSE79122 | GEO | 2016/03/12

SECONDARY ACCESSION(S): PRJNA314994

REPOSITORIES: GEO

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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).

Project description:Background: Excess fibrotic remodeling leads to cardiac dysfunction in ischemic heart disease and is driven by MAP kinase-dependent transforming growth factor-ß1 (TGF-ß1) activation by coagulation signaling of myeloid cells. How coagulation-inflammatory circuits can be specifically targeted to achieve beneficial macrophage reprogramming after myocardial infarction (MI) is incompletely understood. Methods: Mice with permanent ligation of the proximal left anterior descending artery (LAD) were used to model ischemic heart disease and analyzed by single cell RNA sequencing, protein expression changes, confocal microscopy, and longitudinal monitoring of recovery. We probed the role of the tissue factor (TF)-factor 7 (F7)-integrin ß1-protease activated receptor (PAR) 2 signaling complex by utilizing genetic mouse models and pharmacological intervention. Results: Cleavage-insensitive PAR2R38E and myeloid cell integrin ß1-deficient mice had improved cardiac function after MI compared to controls. Proximity ligation assays of monocytic cells in the infarcted myocardium demonstrated that colocalization of F7 with integrin ß1 was diminished in monocyte/macrophage F7-deficient mice. F7fl/fl CX3CR1Cre relative to littermate control mice showed reduced TGF-ß1 and MAP kinase activation, as well as cardiac dysfunction after MI, despite unaltered overall recruitment of myeloid cells into the infarct zone. Single cell mRNA sequencing of CD45+ cells 3 and 7 days after MI uncovered a trajectory from ischemic myocardium-recruited monocytes to inflammatory TF+/F7+/TREM1+ macrophages. As early as 7 days after MI, macrophage F7-deletion led to an expansion of Olfml3+ macrophages with a reparative phenotype and, conversely, to a reduction of TF+/F7+/TREM1+ macrophages, which were also attenuated in activation-resistant PAR2R38E mice. To demonstrate the therapeutic potential of inhibiting the TF-F7-PAR2 signaling complex,, we injected a specific monoclonal anti-TF antibody that lacks anticoagulant activity, but interferes with macrophage migration in vitro. Short-term treatment from day 1-5 after non-reperfused MI improved cardiac dysfunction, decreased excess fibrosis, attenuated vascular endothelial dysfunction, and increased survival 28 days after MI. Conclusions: Extravascular TF-F7-PAR2 complex signaling drives inflammatory macrophage polarization in ischemic heart disease. Targeting this signaling complex for specific therapeutic macrophage reprogramming following MI attenuates cardiac fibrosis and improves cardiovascular function.

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Integrative functional genomic analysis identifies epigenetically regulated Fibromodulin as an essential gene for glioma cell migration

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