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Methylation profiling and evaluation of demethylating therapy in renal cell carcinoma

Christopher J Ricketts1, Mark R Morris12, Dean Gentle1, Salwati Shuib13, Michael Brown4, Noel Clarke45, Wenbin Wei6, Paul Nathan7, Farida Latif1 and Eamonn R Maher189*

Author Affiliations

1 Centre for Rare Diseases and Personalised Medicine, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK

2 School of Applied Sciences University of Wolverhampton, Wolverhampton WV1 1SV, UK

3 Department of Pathology, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, 56000, Kuala Lumpur, Malaysia

4 Institute for Cancer Sciences, Cancer Research UK Paterson Institute for Cancer Research, Manchester Academic Health Science Centre, University of Manchester, Manchester M20 4BX, UK

5 The Christie Hospital, Wilmslow Road, Manchester M20 4BX, UK

6 School of Cancer Sciences, University of Birmingham, Birmingham, UK

7 Mount Vernon Cancer Centre - Medical Oncology, Rickmansworth Road, Northwood, Middlesex HA6 2RN, UK

8 West Midlands Region Genetics Service, Birmingham Women’s Hospital, Edgbaston, Birmingham B15 2TG, UK

9 Department of Medical Genetics, University of Cambridge, Addenbrooke’s Treatment Centre, Cambridge Biomedical Research Campus, Cambridge CB2 0QQ, UK

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Clinical Epigenetics 2013, 5:16  doi:10.1186/1868-7083-5-16

Published: 13 September 2013

Abstract

Background

Despite therapeutic advances in targeted therapy, metastatic renal cell carcinoma (RCC) remains incurable for the vast majority of patients. Key molecular events in the pathogenesis of RCC include inactivation of the VHL tumour suppressor gene (TSG), inactivation of chromosome 3p TSGs implicated in chromatin modification and remodelling and de novo tumour-specific promoter methylation of renal TSGs. In the light of these observations it can be proposed that, as in some haematological malignancies, demethylating agents such as azacitidine might be beneficial for the treatment of advanced RCC.

Results

Here we report that the treatment of RCC cell lines with azacitidine suppressed cell proliferation in all 15 lines tested. A marked response to azacitidine therapy (>50% reduction in colony formation assay) was detected in the three cell lines with VHL promoter methylation but some RCC cell lines without VHL TSG methylation also demonstrated a similar response suggesting that multiple methylated TSGs might determine the response to demethylating therapies. To identify novel candidate methylated TSGs implicated in RCC we undertook a combined analysis of copy number and CpG methylation array data. Candidate novel epigenetically inactivated TSGs were further prioritised by expression analysis of RCC cell lines pre and post-azacitidine therapy and comparative expression analysis of tumour/normal pairs. Thus, with subsequent investigation two candidate genes were found to be methylated in more than 25% of our series and in the TCGA methylation dataset for 199 RCC samples: RGS7 (25.6% and 35.2% of tumours respectively) and NEFM in (25.6% and 30.2%). In addition three candidate genes were methylated in >10% of both datasets (TMEM74 (15.4% and 14.6%), GCM2 (41.0% and 14.6%) and AEBP1 (30.8% and 13.1%)). Methylation of GCM2 (P = 0.0324), NEFM (P = 0.0024) and RGS7 (P = 0.0067) was associated with prognosis.

Conclusions

These findings provide preclinical evidence that treatment with demethylating agents such as azacitidine might be useful for the treatment of advanced RCC and further insights into the role of epigenetic changes in the pathogenesis of RCC.

Keywords:
Renal cancer; Epigenetics; Methylation; Demethylation; VHL; Renal cell carcinoma; Methylation; Therapy