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Oncogenic HRAS suppresses clusterin expression through promoter hypermethylation

Oncogene volume 25, pages 4890–4903 (2006)Cite this article

Abstract

Silencing of gene expression by methylation of CpG islands in regulatory elements is frequently observed in cancer. However, an influence of the most common oncogenic signalling pathways onto DNA methylation has not yet been investigated thoroughly. To address this issue, we identified genes suppressed in HRAS-transformed rat fibroblasts but upregulated after treatment with the demethylating agent 5-Aza-2-deoxycytidine and with the MEK1,2 inhibitor U0126. Analysis of gene expression by microarray and Northern blot analysis revealed the MEK/ERK target genes clusterin, matrix metalloproteinase 2 (Mmp2), peptidylpropyl isomerase C-associated protein, syndecan 4, Timp2 and Thbs1 to be repressed in the HRAS-transformed FE-8 cells in a MEK/ERK- and methylation-dependent manner. Hypermethylation of putative regulatory elements in HRAS-transformed cells as compared to immortalized fibroblasts was detected within a CpG island 14.5 kb upstream of clusterin, within the clusterin promoter and within a CpG island of the Mmp2 promoter by bisulphite sequencing. Furthermore, hypermethylation of the clusterin promoter was observed 10 days after induction of HRAS in immortalized rat fibroblasts and a clear correlation between reduced clusterin expression and hypermethlyation could also be observed in distinct rat tissues. These results suggest that silencing of individual genes by DNA methylation is controlled by oncogenic signalling pathways, yet the mechanisms responsible for initial target gene suppression are variable.

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Accession codes

Accessions

GenBank/EMBL/DDBJ

Abbreviations

5-Aza-CdR:

5-Aza-2-deoxycytidine

Thbs1:

thrombospondin 1

Mmp2:

matrix metalloproteinase 2

Ppicap:

peptidylpropyl isomerase C-associated protein

Thbs1:

thrombospondin 1

Timp2:

tissue inhibitor of metalloproteinase 2

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Acknowledgements

We thank Conny Giesseler, Katrin Kremp, Kerstin Lehmann, Kristin Lucht and Christina Daniel for technical assistance and Karsten Juerchott for helpful discussion. We are grateful to Pablo Steinberg (German Institute of Human Nutrition, Potsdam-Rehbrücke), who provided us with samples from rat colon and small intestine. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB618 grant to RS and HpH) and the Stifterverband für die Deutsche Wissenschaft (grant to CS, RS, JW and HpH).

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Authors and Affiliations

  1. Laboratory of Molecular Tumor Pathology, Institute of Pathology, Charité, Berlin, Germany

    P Lund, K Weißhaupt, X Chen, U Krapfenbauer, R Schäfer & C Sers

  2. Department of Natural Sciences – Technical Faculty III FR 8.3, Biological Sciences, Institute of Genetics/Epigenetics, University of Saarland, Saarbrücken, Germany

    T Mikeska, D Jammas & J Walter

  3. Laboratory of Functional Genome Research, Charité, Berlin, Germany

    R-J Kuban, U Ungethüm & R Schäfer

  4. Institute for Theoretical Biology, Humboldt University Berlin, Berlin, Germany

    H-P Herzel

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  1. P Lund
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Correspondence to C Sers.

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Supplementary Information accompanies the paper on Oncogene website (http://www.nature.com/onc).

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Lund, P., Weißhaupt, K., Mikeska, T. et al. Oncogenic HRAS suppresses clusterin expression through promoter hypermethylation. Oncogene 25, 4890–4903 (2006). https://doi.org/10.1038/sj.onc.1209502

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