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

Oncogene volume 36, pages 71–83 (2017)Cite this article

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Abstract

An integrative functional genomics study of multiple forms of data are 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 because of 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. Small interfering RNA 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 (ΔFMOD), 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. Chromatin immunoprecipitation–PCR experiments revealed that transforming growth factor beta-1 (TGF-β1) 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 Sma- and Mad-related protein-2 (SMAD2) and CBP. FMOD silencing inhibited the TGF-β1-mediated glioma cell migration significantly. In univariate and multivariate Cox regression analysis, both FMOD promoter methylation and transcript levels predicted prognosis in GBM. Thus, this 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-β1 pathway and forms a potential basis for therapeutic intervention in GBM.

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Abbreviations

ChIP:

chromatin immunoprecipitation

FAK:

focal adhesion kinase

FMOD:

fibromodulin

GBM:

glioblastoma

ROCK:

Rho-associated coiled-coil containing protein Kinase 1

SMADs:

Sma- and Mad-related proteins

TGF-β1:

transforming growth factor beta-1.

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Acknowledgements

The results published here are in whole or part based upon data generated by The Cancer Genome Atlas (TCGA) pilot project established by the NCI and NHGRI. Information about TCGA and the investigators and institutions, which constitute the TCGA research network, can be found at http://cancergenome.nih.gov/. The use of data sets from Institute NC and GSE22866 is acknowledged. IISc NGS facility is acknowledged for methylation and microarray experiments. KS thanks DBT, Government of India for financial support. KS is a JC Bose Fellow of the Department of Science and Technology. BM acknowledges IISc for the fellowship.

Author contributions

KS, VS, AA and ASH coordinated the study; BM and KS conceived and wrote the paper; BM designed, performed and analyzed all the experiments, but with help from MK for experiments shown in Figures 2j, 6f and g, VP for experiments shown in Figures 1 and 7; SDS and KSr performed the experiments shown in Figure 2f. All authors reviewed the results and approved the final version of the manuscript.

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Authors and Affiliations

  1. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India

    B Mondal, V Patil & K Somasundaram

  2. Department of Neuropathology, National Institute of Mental Health and Neuro Sciences, Bangalore, India

    S D Shwetha, K Sravani & V Santosh

  3. Department of Neurosurgery, Sri Satya Sai Institute of Higher Medical Sciences, Bangalore, India

    A S Hegde

  4. Department of Neurosurgery, National Institute of Mental Health and Neuro Sciences, Bangalore, India

    A Arivazhagan

  5. Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska University Hospital, Göteborg, Sweden

    M Kanduri

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Correspondence to K Somasundaram.

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Mondal, B., Patil, V., Shwetha, S. et al. Integrative functional genomic analysis identifies epigenetically regulated fibromodulin as an essential gene for glioma cell migration. Oncogene 36, 71–83 (2017). https://doi.org/10.1038/onc.2016.176

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