Oncogene (2006) 25, 4890–4903 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Oncogenic HRAS suppresses clusterin expression through promoter hypermethylation P Lund1, K Weihaupt1, T Mikeska2, D Jammas2, X Chen1, R-J Kuban3, U Ungethu¨ m3, U Krapfenbauer1, H-P Herzel4, R Scha¨ fer1,3, J Walter2 and C Sers1 1Laboratory of Molecular Tumor Pathology, Institute of Pathology, Charite´, Berlin, Germany; 2Department of Natural Sciences – Technical Faculty III FR 8.3, Biological Sciences, Institute of Genetics/Epigenetics, University of Saarland, Saarbru¨cken, Germany; 3Laboratory of Functional Genome Research, Charite´, Berlin, Germany and 4Institute for Theoretical Biology, Humboldt University Berlin, Berlin, Germany Silencing of gene expression by methylation of CpG Introduction islands in regulatory elements is frequently observed in cancer. However, an influence of the most common Activation of RAS proteins by point mutations is oncogenic signalling pathways onto DNA methylation frequently observed in cancers of the colon, pancreas, has not yet been investigated thoroughly. To address this thyroid, lung and the myeloid system (Bos, 1989). RAS issue, we identified genes suppressed in HRAS-trans- genes encode small GTP-binding proteins that act as formed rat fibroblasts but upregulated after treatment major molecular switches in signal transduction pro- with the demethylating agent 5-Aza-2-deoxycytidine and cesses, transmitting extracellular signals to cytoplasmic with the MEK1,2 inhibitor U0126. Analysis of gene signalling cascades (Campbell et al., 1998). The onco- expression by microarray and Northern blot analysis genic forms of RAS proteins are locked in their active revealed the MEK/ERK target genes clusterin, matrix state and contribute to transformation, angiogenesis, metalloproteinase 2 (Mmp2), peptidylpropyl isomerase invasion and metastasis by constitutive activation of C-associated protein, syndecan 4, Timp2 and Thbs1 to be downstream pathways. Among these are the RAF/ repressed in the HRAS-transformed FE-8 cells in a MEK/ MEK/ERK (MAPK) cascade of cytoplasmic kinases ERK- and methylation-dependent manner. Hypermethy- (Mansour et al., 1994), the phosphoinositide 3-phos- lation of putative regulatory elements in HRAS-trans- phate lipid kinases (Downward, 1998), the small GTP- formed cells as compared to immortalized fibroblasts binding proteins RAC and RHO (Zohn et al., 1998) was detected within a CpG island 14.5 kb upstream of and the family of guanine nucleotide exchange factors clusterin, within the clusterin promoter and within a CpG (RalGDS, RGL and Rlf/RGL2) that serve as activators island of the Mmp2 promoter by bisulphite sequencing. of the small GTPases RalA and RalB (Wolthuis and Furthermore, hypermethylation of the clusterin promoter Bos, 1999). was observed 10 days after induction of HRAS in RAS signalling is linked to the activation of immortalized rat fibroblasts and a clear correlation transcription factors of the AP-1 and Ets families, and between reduced clusterin expression and hypermethlya- also to NF-kB, SRF and c-Myc, all of which induce tion could also be observed in distinct rat tissues. These a complex set of transcriptional targets. Meanwhile, a results suggest that silencing of individual genes by DNA number of RAS-dependent gene expression profiles have methylation is controlled by oncogenic signalling path- confirmed the quantitative and qualitative impact of ways, yet the mechanisms responsible for initial target RAS signalling on gene expression control (Zuber et al., gene suppression are variable. 2000; Brem et al., 2001; Gadal et al., 2003; Fensterer Oncogene (2006) 25, 4890–4903. doi:10.1038/sj.onc.1209502; et al., 2004; Tchernitsa et al., 2004). These studies published online 27 March 2006 revealed that similar numbers of genes are activated and repressed by RAS oncogenes and suggested that RAS Keywords: HRAS(G12V); MEK/ERK signalling; gene can influence the expression of defined groups of genes expression; CpG methylation; clusterin; Mmp2 in a signalling pathway-dependent manner (Tchernitsa et al., 2004). Transcriptional activation of critical RAS target genes involved in cell cycle control, mitogenic signalling or metastasis has been studied in much detail. In contrast, the mechanisms of RAS-dependent gene repression have been unfolded only for a few indivi- Correspondence: Dr C Sers, Laboratory of Molecular Tumorpathol- dual genes. A recent example of RAS-dependent gene ogy, Institute of Pathology, Charite´ , Schumannstr. 20/21, D-10117 repression is the observed transcriptional block onto the Berlin, Germany. E-mail: [email protected] thrombospondin 1 gene (THBS1), which is repressed Received 20 May 2005; revised 8 February 2006; accepted 10 February in a RAS/PI-3K/RHO- and ROCK-dependent manner 2006; published online 27 March 2006 through a phosphorylated c-myc protein (Watnick et al., RAS induces hypermethylation of MAPK target genes P Lund et al 4891 2003). The Ets-2 protein is a member of the ETS family cation and proliferation, excluding cytotoxic effects. of transcription factors directly activated by mito- Western blot analysis demonstrated histone H3 hyper- gen-activated protein kinases and functionally involved acetylation after TSA treatment, whereas the U0126- in the activation of RAS target genes. However, induced inhibition of MEK1,2 reduced the level of p44/ Ets-2 can inhibit BRCA-1 transcription through recruit- 42 ERK phosphorylation. Total DNMT1 protein levels ment of components of the SWI/SNF repressive were identical in the two cell lines and were also not chromatin-remodelling complex (Baker et al., 2003; affected by treatment with U0126 (Figure 1c). Wei et al., 2003). Furthermore, HDAC4, which is a member of the histone deacetylase family, was shown to translocate into the nucleus in cells transfected A distinct set of genes downregulated in FE-8 cells is with oncogenic RAS (Zhou et al., 2000). These obser- concordantly upregulated after treatment with 5-Aza-CdR vations indicate that RAS-dependent signalling path- as well as with U0126 ways alter DNA–protein interactions at regulatory Silencing of genes by methylation, particularly with elements by stimulating the recruitment of components regard to tumour suppressor genes, is frequently des- determining DNA structure. Also, increased expression cribed in cancer cells and tumour samples. However, the of DNMT1 has been detected in tumour cells (Kautiai- mechanisms and signalling pathways involved in gene- nen and Jones, 1986; Issa et al., 1993). Stable expression specific methylation are largely unknown. Therefore, we of HRAS(G12V) induces transcription of DNMT1 via tested the hypothesis that methylation is involved in the an AP-1 site in the promoter region (Rouleau et al., downregulation of genes suppressed in a MEK/ERK- 1995), and elevated DNMT1 levels were correlated with dependent manner in HRAS(G12V)-transformed FE-8 the 5-methylcytosine content and demonstrated to be a cells. prerequisite for fos-mediated transformation (Bakin and First we investigated the expression of 73 RAS target Curran, 1999). Recently, a set of genes was identified genes previously shown to be suppressed in FE-8 cells that was commonly downregulated in rat fibroblasts by Northern blot analysis (Zuber et al., 2000). Eighteen transformed by c-fos,v-fos, Hras(G12V) or Dnmt1 of these genes are regulated in a MEK/ERK-dependent (Ordway et al., 2004) suggesting a close connection and 55 genes are regulated in a MEK/ERK-independent between RAS transformation and epigenetic gene manner. Treatment with 0.1 mM 5-Aza-CdR revealed regulation. a 5-Aza-CdR-dependent upregulation of 10 of the In the current manuscript, we investigated the impact 18 MEK/ERK target genes (Table 1). In addition, 31 of constitutive RAS activation on DNA methylation of 55 MEK/ERK-independent genes downregulated of putative regulatory elements by correlating gene in FE-8 cells are upregulated after treatment with expression data with bisulphite sequencing data. We 5-Aza-CdR, suggesting that alternative RAS-dependent identified a set of MEK/ERK target genes down- pathways influence the methylation of genes (data not regulated in FE-8 cells and re-expressed after treatment shown). with 5-Aza-2-deoxycytidine (5-Aza-CdR) and after To gain further access to candidate genes silenced treatment with the MEK1,2 inhibitor U0126. The by methylation in response to oncogenic signalling clusterin gene revealed suppression due to hypermethy- pathways, we hybridized cDNA probes derived from lation of regulatory regions in cells transformed stably untreated and 5-Aza-CdR-treated 208F and FE-8 cells or inducibly by activated HRAS, suggesting that to an Affymetrix rat U34A oligonucleotide microarray. oncogenic signalling can directly influence DNA After treatment of 208F cells with 0.3 mM 5-Aza-CdR methylation. for 96 h, 108 genes (9%) were significantly upregulated and 169 genes (13%) were significantly downregulated. In FE-8 cells, 115 genes (8%) were significantly Results upregulated after treatment with 5-Aza-CdR and 216 genes (15%) were significantly downregulated. Thus, the 5-Aza-CdR and Trichostatin A cause growth inhibition overall number of genes deregulated after treatment and cell death in immortalized and HRAS-transformed with 5-Aza-CdR is not related to the RAS status of the fibroblasts cells. Further analysis identified 16 genes significantly We investigated the effects of the demethylating agent lower expressed in untreated FE-8 cells compared to 5-Aza-CdR and the HDAC1 inhibitor trichostatin A untreated 208F cells but upregulated after treatment (TSA) on growth and viability of immortalized 208F rat with 5-Aza-CdR, exclusively in FE-8 cells (Table 2). fibroblasts and the HRAS-transformed derivative FE-8. Within this set of genes silenced by a methylation- A dose-dependent inhibition of cell growth was ob- dependent mechanism, syndecan 4, clusterin and Timp2 served for both cell lines after treatment with 0.1–10 mM had earlier been demonstrated to be suppressed in 5-Aza-CdR for 96 h and with 5–50 ng/ml TSA for 72 h a MEK/ERK-dependent manner in FE-8 cells (Zuber (Figure 1a).