1. ¹Cancer Research UK Renal Molecular Oncology Group, University of Birmingham, Birmingham B15 2TT, UK
2. ²Department of Medical and Molecular Genetics, Department of Paediatrics and Child Health, University of Birmingham, Birmingham B15 2TT, UK
3. ³Paterson Institute for Cancer Research, University of Manchester, Manchester, M20 4BX, UK
4. ⁴Yokohama City University School of Medicine, Yokohama, Japan
5. ⁵Laboratory of Cancer Genetics, Van Andel Research Institute, Grand Rapids, MI, USA

Correspondence: Professor ER Maher, Department of Medical and Molecular Genetics, University of Birmingham, Institute of Biomedical Research, Edgbaston, Birmingham B15 2TT, UK. E-mail: E.R.Maher@bham.ac.uk

Received 6 August 2007; Revised 3 December 2007; Accepted 5 December 2007; Published online 15 January 2008.

Abstract

Promoter region hypermethylation and transcriptional silencing is a frequent cause of tumour suppressor gene (TSG) inactivation in many human cancers. Previously, to identify candidate epigenetically inactivated TSGs in renal cell carcinoma (RCC), we monitored changes in gene expression in four RCC cell lines after treatment with the demethylating agent 5-azacytidine. This enabled us to identify HAI-2/SPINT2 as a novel epigenetically inactivated candidate RCC TSG. To identify further candidate TSGs, we undertook bioinformatic and molecular genetic evaluation of a further 60 genes differentially expressed after demethylation. In addition to HAI-2/SPINT2, four genes (PLAU, CDH1, IGFB3 and MT1G) had previously been shown to undergo promoter methylation in RCC. After bioinformatic prioritisation, expression and/or methylation analysis of RCC cell linesprimary tumours was performed for 34 genes. KRT19 and CXCL16 were methylated in RCC cell lines and primary RCC; however, 22 genes were differentially expressed after demethylation but did not show primary tumour-specific methylation (methylated in normal tissue (n=1); methylated only in RCC cell lines (n=9) and not methylated in RCC cell lines (n=12)). Re-expression of CXCL16 reduced growth of an RCC cell line in vitro. In a summary, a functional epigenomic analysis of four RCC cell lines using microarrays representing 11 000 human genes yielded both known and novel candidate TSGs epigenetically inactivated in RCC, suggesting that this is valid strategy for the identification of novel TSGs and biomarkers.