title: Methylation profiling and evaluation of demethylating therapy in renal cell carcinoma.
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.
authors.affiliation: 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.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Ricketts
authors.affiliation: 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.|School of Applied Sciences University of Wolverhampton, Wolverhampton WV1 1SV, UK.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Morris
authors.affiliation: 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.
authors.forename: Dean
authors.initials: D
authors.lastname: Gentle
authors.affiliation: 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.|Department of Pathology, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, 56000, Kuala Lumpur, Malaysia.
authors.forename: Salwati
authors.initials: S
authors.lastname: Shuib
authors.affiliation: Institute for Cancer Sciences, Cancer Research UK Paterson Institute for Cancer Research, Manchester Academic Health Science Centre, University of Manchester, Manchester M20 4BX, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Brown
authors.affiliation: Institute for Cancer Sciences, Cancer Research UK Paterson Institute for Cancer Research, Manchester Academic Health Science Centre, University of Manchester, Manchester M20 4BX, UK.|The Christie Hospital, Wilmslow Road, Manchester M20 4BX, UK.
authors.forename: Noel
authors.initials: N
authors.lastname: Clarke
authors.affiliation: School of Cancer Sciences, University of Birmingham, Birmingham, UK.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Wei
authors.affiliation: Mount Vernon Cancer Centre - Medical Oncology, Rickmansworth Road, Northwood, Middlesex HA6 2RN, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Nathan
authors.affiliation: 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.
authors.forename: Farida
authors.initials: F
authors.lastname: Latif
authors.affiliation: 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.|West Midlands Region Genetics Service, Birmingham Women's Hospital, Edgbaston, Birmingham B15 2TG, UK.|Department of Medical Genetics, University of Cambridge, Addenbrooke's Treatment Centre, Cambridge Biomedical Research Campus, Cambridge CB2 0QQ, UK.
authors.forename: Eamonn R
authors.initials: ER
authors.lastname: Maher
references.citation: Cancer Res. 2003 Dec 15;63(24):8695-9
references.pmid: 14695183
references.citation: Genes Chromosomes Cancer. 2011 Jul;50(7):479-88
references.pmid: 21456047
references.citation: Autophagy. 2009 Jan;5(1):52-60
references.pmid: 19029833
references.citation: PLoS One. 2011;6(6):e21120
references.pmid: 21698121
references.citation: Nat Genet. 2006 May;38(5):500-1
references.pmid: 16642009
references.citation: Cell Res. 2011 Mar;21(3):502-17
references.pmid: 21321605
references.citation: Cancer Res. 2010 Apr 1;70(7):2718-27
references.pmid: 20233874
references.citation: Nat Genet. 2009 May;41(5):521-3
references.pmid: 19330029
references.citation: Cancer Cell. 2012 Apr 17;21(4):532-46
references.pmid: 22516261
references.citation: Expert Rev Anticancer Ther. 2010 Jan;10(1):9-22
references.pmid: 20014881
references.citation: Ann Oncol. 2007 Mar;18(3):581-92
references.pmid: 17287242
references.citation: Cancer Res. 2011 Feb 15;71(4):1431-41
references.pmid: 21303982
references.citation: J Clin Endocrinol Metab. 2008 Sep;93(9):3568-76
references.pmid: 18583467
references.citation: Hum Mol Genet. 1994 Dec;3(12):2169-73
references.pmid: 7881415
references.citation: Genome Med. 2010 Sep 03;2(9):59
references.pmid: 20815920
references.citation: Cancer Res. 2008 Apr 1;68(7):2112-21
references.pmid: 18381416
references.citation: Nature. 2011 Jan 27;469(7331):539-42
references.pmid: 21248752
references.citation: Nature. 1999 May 20;399(6733):271-5
references.pmid: 10353251
references.citation: Hum Mol Genet. 2010 May 15;19(10):2028-38
references.pmid: 20190276
references.citation: Cancer Res. 2006 Feb 15;66(4):2000-11
references.pmid: 16488999
references.citation: Clin Cancer Res. 2004 Jun 15;10(12 Pt 1):3972-9
references.pmid: 15217927
references.citation: Science. 1993 May 28;260(5112):1317-20
references.pmid: 8493574
references.citation: Oncogene. 2007 Nov 15;26(52):7283-301
references.pmid: 17533367
references.citation: Epigenetics. 2012 Mar;7(3):278-90
references.pmid: 22430804
references.citation: Mol Cancer. 2009 Jun 03;8:31
references.pmid: 19493342
references.citation: Clin Cancer Res. 2004 Oct 15;10(20):7011-21
references.pmid: 15501981
references.citation: Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9700-4
references.pmid: 7937876
references.citation: Nat Biotechnol. 2010 Jan;28(1):47-55
references.pmid: 20037582
references.citation: Biochem Biophys Res Commun. 2008 May 2;369(2):622-9
references.pmid: 18294959
references.citation: Nat Rev Cancer. 2008 Nov;8(11):865-73
references.pmid: 18923434
references.citation: Bioinformatics. 2008 Aug 15;24(16):i139-45
references.pmid: 18689815
references.citation: Oncology (Williston Park). 2011 Mar;25(3):220-6, 228
references.pmid: 21548464
references.citation: Cancer Res. 2004 Aug 1;64(15):5511-7
references.pmid: 15289362
references.citation: Nature. 2011 Feb 3;470(7332):59-65
references.pmid: 21293372
references.citation: Cancer Res. 2001 Oct 1;61(19):7277-81
references.pmid: 11585766
references.citation: Nat Med. 1995 Aug;1(8):822-6
references.pmid: 7585187
references.citation: Hum Mutat. 2012 Jul;33(7):1059-62
references.pmid: 22461374
references.citation: Oncogene. 2004 Nov 11;23(53):8705-10
references.pmid: 15467763
references.citation: Cancer Res. 2009 Jun 1;69(11):4674-81
references.pmid: 19470766
references.citation: Nature. 2010 Jan 21;463(7279):360-3
references.pmid: 20054297
references.citation: Proc Natl Acad Sci U S A. 2007 Dec 11;104(50):20007-12
references.pmid: 18077431
references.citation: PLoS Genet. 2006 Feb;2(2):e20
references.pmid: 16482228
references.citation: Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):993-8
references.pmid: 9448273