Oncogene (2010) 29, 2104–2117 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Identification of candidate tumour suppressor frequently methylated in renal cell carcinoma

MR Morris1,2, C Ricketts1,2, D Gentle1,2, M Abdulrahman2, N Clarke3, M Brown3, T Kishida4, M Yao4, F Latif1,2 and ER Maher1,2,5

1Cancer Research UK Renal Molecular Oncology Group, University of Birmingham, Birmingham, UK; 2Department of Medical and Molecular Genetics, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK; 3Genito-Urinary Cancer Research Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK; 4Department of Urology, Yokohama City University School of Medicine, Yokohama, Japan and 5West Midlands Region Genetics Service, Birmingham Women’s Hospital, Edgbaston, Birmingham, UK

Promoter region hyermethylation and transcriptional cancer are diagnosed in the world each year (Ferlay silencing is a frequent cause of tumour suppressor et al., 2007). The most common form of kidney cancer in (TSG) inactivation in many types of human cancers. adults is renal cell carcinoma (RCC). A majority of the Functional epigenetic studies, in which gene expression is RCC (B75%) are classified as clear cell (conventional) induced by treatment with demethylating agents, may and the next most frequent subtype is papillary RCC identify novel genes with tumour-specific methylation. We (B15% of all cases) (Mancini et al., 2008). The used high-density gene expression microarrays in a prognosis of advanced RCC is poor, although newer functional epigenetic study of 11 renal cell carcinoma treatments, based on knowledge of the molecular (RCC) cell lines. Twenty-eight genes were then selected pathology of RCC may provide a basis for developing for analysis of promoter methylation status in cell lines novel approaches to therapy. Thus, the most frequent and primary RCC. Eight genes (BNC1, PDLIM4, genetic event in the evolution of sporadic clear cell RCC RPRM, CST6, SFRP1, GREM1, COL14A1 and is inactivation of the von Hippel-Lindau (VHL) tumour COL15A1) showed frequent (430% of RCC tested) suppressor gene (TSG) (Latif et al., 1993; Foster et al., tumour-specific promoter region methylation. Hyper- 1994; Herman et al., 1994; Clifford et al., 1998). VHL methylation was associated with transcriptional silencing. inactivation leads to stabilisation of hypoxia-inducible Re-expression of BNC1, CST6, RPRM and SFRP1 factor (HIF)-1 and HIF-2 transcription factors and suppressed the growth of RCC cell lines and RNA activation of a wide repertoire of hypoxic response genes interference knock-down of BNC1, SFRP1 and (Maxwell et al., 1999). HIF-mediated RCC growth may COL14A1 increased the growth of RCC cell lines. be antagonised by multi-tyrosine kinase inhibitors such Methylation of BNC1 or COL14A1 was associated with as sunitinib and sorafenib (Chowdhury et al., 2008). a poorer prognosis independent of tumour size, stage or Hence, identification of mechanisms of tumourigenesis grade. The identification of these epigenetically inacti- in RCC can provide a basis for therapeutic intervention. vated candidate RCC TSGs can provide insights into renal Although large-scale mutation analysis studies of RCC tumourigenesis and a basis for developing novel therapies are in progress (see http://www.sanger.ac.uk/genetics/ and biomarkers for prognosis and detection. CGP/cosmic/), with the exception of VHL, none of the Oncogene (2010) 29, 2104–2117; doi:10.1038/onc.2009.493; thousands of genes tested to date are mutated in 415% published online 15 February 2010 of tumours. Epigenetic inactivation of TSGs by methylation promoter region of CpG dinucleotides has Keywords: renal cell carcinoma; methylation; epige- also been implicated in the pathogenesis of RCC and netics some important TSGs are frequently inactivated by promoter hypermethylation but rarely mutated (for example, RASSF1A) (Morrissey et al., 2001; Dallol et al., 2002; Morris et al., 2003). Hence, strategies to Introduction identify epigenetically inactivated TSGs are an impor- tant approach at elucidating the molecular pathogenesis Kidney cancers account for approximately 2% of all of RCC. Previously, to analyse the utility of a functional cancers and more than 200 000 new cases of kidney epigenomic approach to identify candidate novel epigenetically inactivated RCC TSGs, we performed a Correspondence: Professor ER Maher, Department of Medical and pilot study using gene expression profiling of four RCC Molecular Genetics, University of Birmingham, Institute of Biomedi- cell lines treated with the demethylated agent 5-aza-20- cal Research West, Edgbaston, Birminmgham, West Midlands B15 deoxycytidine (Morris et al., 2005, 2008). This led us to 2TT, UK. E-mail: [email protected] identify HAI-2/SPINT2 as a novel epigenetically in- Received 3 June 2009; revised 6 November 2009; accepted 22 November activated RCC TSG (Morris et al., 2005). We now 2009; published online 15 February 2010 report the results of a large functional epigenetic screen Identification of candidate TSG frequently methylated in RCC MR Morris et al 2105 of RCC in which 11 RCC cell lines were analysed using moter inspector (www.genomatix.de), 201 genes were a high-density gene expression microarray platform. left after this filter step. Genes known to be imprinted and genes that are not expressed in renal tissue (array express (www.ebi.ac.uk/arrayexpress/) were also re- moved, leaving 155 genes. Of these, 19 genes have Results previously been assessed for promoter methylation in RCC including KRT19, COL1A1 and IGFBP1 that are Identification of candidate silenced genes involved in RCC frequently methylated in RCC (Ibanez de Caceres et al., Eleven RCC-derived cell lines were treated with the 2006; Morris et al., 2008) (Supplementary Table 2 lists demethylating agent 5-aza-20-deoxycytidine (5 mM) for the genes identified by this filtering process in full). 5 days to re-activate epigenetically silenced/downregulated To evaluate the microarray fold-change filtering genes and changes in gene expression were measured using selection criteria, we analysed the methylation status Affymetrix (Santa Clara, CA, USA) U133 plus-2 micro- of three genes (LRCH1, KLF2 and FZD8) that had arrays that contain probes for 447 000 unique transcripts. expression fold-changes of o10 (range two- to seven- To prioritise genes for methylation analysis, we initially fold). All three genes were unmethylated in the cell lines focussed on genes, which showed a 10-fold increase in (Supplementary Table 1). expression after de-methylation in three or more cell In total, 24 genes from our shortlist were analysed for lines. Reverse transcriptase–PCR (RT–PCR) analyses promoter methylation in RCC cell lines and primary showed a good correlation with microarray-based tumours. In addition, we analysed promoter methyla- estimates of changes in gene expression (see Supplemen- tion status of RPRM which, although not meeting the tary Figure 1, Figures 1a and 2a), transcripts that selection criteria had previously been shown to be represented hypothetical were also removed. In inactivated by methylation in a number of other tumour all, 405 unique genes were left after this filter. Next, we types (Takahashi et al., 2005; Sato et al., 2006; Bernal excluded genes that did not have a predicted promoter- et al., 2008). One of the 25 genes (NOG) did not show specific CpG island (as predicted by the detectable promoter region hypermethylation. Six genes browser (www.genome.ucsc.edu) and Genomatix pro- (DMRTB1, SCYA20, CLDN6, TPM2L1, LOXL and a

Figure 1 (a) RT–PCR analysis of PDLIM4, CST6 and COL14A expression. PDLIM4 expression was restored (SKRC18, SKRC54, CAKI-1)/upregulated (RCC4, KTCL140, 786-0) in 6 of 11 cell lines after treatment with the demethylating agent, 5-aza-20- deoxycytidine. CST6 expression was restored (KTCL26, SKRC18, SKRC54, 786-0, CAKI-1)/upregulated (RCC4, KTCL140, SKRC45) in 8 of 11 cell lines. Global demethylation restored expression of COL14A in 5 of 11 cell lines (KTCL140, SKRC18, SKRC45, SKRC54, 786-0). (b–d) Bisulphite sequencing analysis of PDLIM4, CST6 and COL14A1 50 CpG island regions. Primers were designed to amplify the predicted promoter region of the 50 CpG island of PDLIM4, CST6 and COL14A. The schematic represents the region sequenced; vertical lines indicate individual CpGs. Sequencing indicated methylation in RCC cell lines correlated with gene silencing/downregulation: black circle; complete methylation; grey circle; partial methylation; empty circle; no methylation. Bisulfite sequencing of RCC tumours and adjacent normal tissue showed frequent methylation in tumours and infrequent methylation in normal tissue (see Results for details). Analysis of normal tissue derived from non-malignant kidneys indicated no CpG island methylation, with the exception of PDLIM4 in which three CpGs were partially and one CpG was fully methylated in one non- malignant sample (NMN2). T, tumour; N, normal tissue adjacent to tumour; NMN, non-malignant normal tissue. Representative sequencing traces are shown in Supplementary Figure 1.

Oncogene Identification of candidate TSG frequently methylated in RCC MR Morris et al 2106 Transcript Start Predicted Promoter

Col 14A1 CpG island Exon1 Cell Lines Non-malignant normal Adjacent Normal Tumour Figure 1 Continued.

ZFP42) were methylated in RCC cell lines and tumours frequent (methylation frequency 430%, range 35–53%) but also in non-malignant renal tissue and so were not methylation in primary RCC, no methylation in normal analysed further. A further 10 genes showed both kidney from non-RCC patients and upregulation of gene promoter region methylation in RCC cell lines but no expression after demethylation of methylated RCC cell (FOXG1B, RARRES1, DKK1 and UQCRH) or infre- lines (Table 1, Figures 1a and 2a). Five of eight genes quent (RGPD5 (7%), ITGBL (9%), KLF4 (10%), TLL1 were methylated in renal tumours but not (or rarely) in (10%), PTGIS (13%) and EMX2 (18%)) promoter matched normal renal tissue (BNC1, COL14A1, CST6, methylation in primary RCC tumours. PDLIM4 and SFRP1). Eight of 28 (29%) genes (BNC1, COL14A1, COL15A1 The promoter region of the 50 CpG island of BNC1 CST6, SFRP1, GREM1, RPRM and PDLIM4) showed was methylated in 46% (27/59) of primary RCC

Oncogene Identification of candidate TSG frequently methylated in RCC MR Morris et al 2107 Transcript Start Predicted promoter

PDLIM4 CpG Island Exon 1 Cell Lines Tumours Adjacent Normal Non-malignant normal

Figure 1 Continued.

(Figure 2b). One of 20 (5%) adjacent normal samples detected in 43% (13/30) primary RCC but not in any (from a patient with a methylated tumour) also showed normal renal tissue samples (n ¼ 22) (Figure 1c). SFRP1 promoter methylation. Methylation of the COL14A1 promoter methylation was present in 34% (20/58) promoter was detected in 44% (18/41) primary RCC sporadic primary RCC but not in adjacent normal tumours analysed (1/20 adjacent normal kidney samples tissue samples (Figure 2b). The CST6 predicted promo- (from a patient with a methylated tumour) and 0/6 ter was methylated in 46% (28/61) primary RCC. A samples from patients without kidney cancer) small number of normal kidney samples adjacent to (Figure 1b). Methylation of PDLIM4 promoter was RCC (11%, 4/35) showed a low level of promoter

Oncogene Identification of candidate TSG frequently methylated in RCC MR Morris et al 2108 Transcript start

Predicted promoter

CST6 CpG Island Exon 1

1 2 34 56 7 8 9 10 1112 13141516171819202122 23242526 27 2829

SKRC18

KTCL26

SKRC54

RCC54 Cell Lines KTCL140

CAKI-1

SKRC39

581T

248T

536T

583T

151T

581N

248N

536N

583T Adjacent Normal 151N

1NMN

2NMN

3NMN

4NMN

5NMN Non-malignant normal Tumours 6NMN Figure 1 Continued.

methylation (all of which showed methylation in the methylation in normal renal tissue and in tumour matched tumour tissue) (Figure 1d). material. Thus, 8/9 normal renal tissue with COL15A1 Three genes, COL15A1, RPRM and GREM1 showed methylation in normal tissue also showed methylation in frequent promoter methylation in primary RCC (53% the corresponding tumour and the corresponding figures (34/65), 44% (23/52) and 41% (11/27), respectively) for RPRM and GREM1 were 7/8 and 5/7, respectively. (Figure 2b), but methylation was also detected relatively frequently in macroscopically normal renal tissue from patients with RCC (30% (9/30), 18% (8/44), 24% (7/ Functional analysis of the tumour suppressor activity 29), respectively (P ¼ 0.034, P ¼ 0.009 and P ¼ 0.025, of epigenetically inactivated genes respectively compared with primary RCC)). However, The effect of re-expressing four of the epigenetically there was a strong correlation between the presence of silenced RCC genes (SFRP1, RPRM, CST6 and BNC1)

Oncogene Identification of candidate TSG frequently methylated in RCC MR Morris et al 2109 Table 1 A summary of genes identified in this study and their frequency of promoter methylation in RCC-derived cell lines, sporadic tumours, kidney tissue resected from an area adjacent to the tumour and kidney tissue from patients with no kidney cancer Gene Accession Gene name Loci Cell line Tumour Adjacent kidney Non-malignant symbol no. methylation methylation methylation kidney methylation frequency frequency frequency frequency

COL15A1 NM_001855 Collagen, type XV, alpha 1 9q22 7/9 35/65 (53%) 9/30 (30%) 0/6 GREM1 NM_204978 Gremlin-1 15q13 7/11 11/27 (41%) 7/29 (24%) 0/6 COL14A1 NM_021110 Collagen, type XIV, alpha 1 8q24 7/11 18/41 (44%) 1/20 (5%) 0/6 PDLIM4 NM_003687 PDZ and LIM domain 4 5q31 5/10 13/30 (43%) 0/22 (0%) 1/6 RPRM NM_019845 Reprimo, TP53-dependant G2 arrest 2q23 4/9 23/52 (44%) 8/44 (18%) 0/6 mediator SFRP1 NM_003012 Secreted frizzled-related 1 8p11 5/10 20/58 (34%) 0/20 (0%) 0/6 CST6 NM_001323 Cystatin E/M 11q13 8/11 28/61 (46%) 4/35 (11%) 0/6 BNC1 NM_001717 Basonuclin 1 15q25 5/11 27/59 (46%) 1/20 (5%) 0/6

Abbreviation: RCC, renal cell carcinoma. on in vitro growth characteristics of RCC cell lines was RPRM and those with RPRM silenced grew robustly assessed using colony formation and anchorage-inde- when suspended in soft agar. pendent growth assays. As the BNC1 silenced RCC4 cell line did not grow well in an anchorage-independent manner, to analyse Colony formation. The tumour suppressor activity of the effect of alterations of BNC1 expression on cell SFRP1, RPRM, CST6 and BNC1 was assessed by growth, we produced BNC1 overexpressing clones of in vitro colony formation assays. After transfection of HEK293s. Although HEK293 cells transfected with an wild-type BNC1, CST6, RPRM and SFRP1 expression empty vector grew robustly, BNC1 overexpressing plasmids into non-expressing RCC cell lines (RCC4, clones produced statistically significantly fewer colonies 786-0, SKRC39 and SKRC47, respectively) there was a X100 mm (mean (±s.d.) reduction 54% (±5%), t ¼ 5.92 significantly reduced number of G418 resistant colonies P ¼ 0.001) (Figure 3b). compared with cell lines transfected with an empty To analyse the possible tumourigenic advantage of vector control for three independent experiments loss of expression of BNC1, SFRP1 and COL14A,we (Figure 3a). used RNA interference (RNAi) methodology to knock- The number of BNC1 expressing RCC4 clones was down BNC1 and SFRP1 expression in HEK293s and reduced by 77% compared with those transfected with COL14A expression in the KTCL26 RCC cell line an empty plasmid (s.d. ¼ 10%, t ¼ 12.43, Po0.0001). (insufficient knock-down of COL14A expression was Re-expression of CST6 in 786-0 cells reduced their obtained in HEK293 cells). Twenty-four hours after colony-forming ability by 47% (s.d. ¼ 6%, t ¼ 6.629, RNAi transfection cells were seeded into 3% agar, P ¼ 0.003). RPRM expressing SKRC39 clone numbers colonies 4100 mm were counted 21 days later. In all, were reduced by 40% (s.d. ¼ 5%, t ¼ 8.328, P ¼ 0.001). 55% more colonies 4100 mm were produced by The number of SFRP1 expressing SKRC47 clones was HEK293 cells with reduced BNC1 (s.d. ¼ 11%, reduced by 76% compared with SKRC47 clones t ¼À6.9, Po0.0001) expression compared with cells containing the empty vector (s.d. ¼ 5%, t ¼ 16.644, transfected with a control RNAi oligo (Figure 4a). Po0.0001) (Figure 3a). Reduced expression of SFRP1 resulted in 74% more colonies 4100 mm (s.d. ¼ 6%, t ¼À14.3, Po0.0001) than control cells (Figure 4a). The growth suppressing Anchorage-independent growth. The effect of re-expres- activity of re-expression of SFRP1 in RCC cell lines has sion of, RPRM and CST6 on anchorage-independent been shown earlier (Gumz et al., 2007). However, to our growth in a soft agar colony formation assay was knowledge, this is the first time that an increase in assessed in stably transfected RCC cell line clones. growth potential has been associated with reduced Colonies were counted after initial seeding and incuba- SFRP1 expression in RCC. The colony-forming cap- tion in soft agar for 5 weeks. Each experiment was ability of the RCC cell line KTCL26 was less than that carried out in triplicate with three independent clones. of HEK293s. However, this was increased by 93% in Cells transfected with empty vector showed robust those cells in which COL14A expression was reduced by colony growth, whereas, colony growth was greatly RNAi (s.d. ¼ 9%, t ¼À10.44, Po0.0001) (Figure 4b). reduced when CST6 was re-expressed in 786-0 cells, both the number and size of colonies was statistically significantly reduced The number of colonies X100 mm Analysis of patient survival and gene methylation was reduced by 53% (s.d. ¼ 4%, t ¼ 16.15, Po0.0001) in We initially analysed whether the presence of tumour clones expressing CST6 when compared with the control methylation for BNC1, PDLIM4, RPRM, CST6, clones (Figure 3b). SFRP1, GREM1, COL14A1 and COL15A1 was asso- Re-expression of RPRM in SKRC39 cells did not ciated with changes in patient survival (time to cancer significantly alter the anchorage-independent growth death) or recurrence/cancer death. No significant capabilities of SKRC39 cells. Both clones re-expressing association was detected for CST6 (P ¼ 0.38 and

Oncogene Identification of candidate TSG frequently methylated in RCC MR Morris et al 2110 P ¼ 0.76 respectively by Kaplan–Meier analysis), (CI) ¼ 1.27–10.93)) and P ¼ 0.018 (HR ¼ 3.07 (95% COL15A1 (P ¼ 0.25 and P ¼ 0.16), GREM1 (0.07 and CI ¼ 1.22–8.16)), COL14A1 (P ¼ 0.005 (HR ¼ 4.07 0.08), RPRM (P ¼ 0.83 and 0.94) or PDLIM4 (P ¼ 0.38 (95% CI ¼ 1.73–20.3)) and P ¼ 0.007 (HR ¼ 3.49 (95% and 0.32). However, methylation of BNC1 (P ¼ 0.017 CI ¼ 1.55–16.1)) or SFRP1 (P ¼ 0.002 (HR ¼ 4.88 (95% (hazard ratio (HR) ¼ 3.69 (95% confidence interval CI ¼ 2.76–87.95)) and P ¼ 0.003 (HR ¼ 4.44 (95%

a

b

Oncogene Identification of candidate TSG frequently methylated in RCC MR Morris et al 2111 CI ¼ 2.75–86.2)) was associated with a significantly mutated frequently. Thus, apart from VHL, there are poorer prognosis (see Figures 5a–c). We then analysed no reports of a TSG that is frequently mutated in the relationship between survival, tumour grade, size RCC (http://www.sanger.ac.uk/genetics/CGP/cosmic/). and stage and methylation status of BNC1, COL14A1 In contrast, at least 10 genes have been reported to be and SFRP1 using Cox proportional hazard analysis. hypermethylated 420% of RCC (13 and references Analysis for SFRP1 methylation, grade, size and stage within) and we have now reported a further eight genes gave a strong association with survival (P ¼ 0.0002) but that show promoter methylation in 430% of RCC. the effect of SFRP1 methylation status was not Functional epigenomic screens provide a useful statistically significant (P ¼ 0.4). However, for a similar strategy to identify novel TSGs and have been used to analysis for BNC1, tumour stage (P ¼ 0.019; identify epigenetically inactivated TSGs in a number of HR ¼ 2.4536 (95% CI ¼ 1.16–5.19)), BNC1 methylation different tumour types (Yamashita et al., 2002; Sato (P ¼ 0.033; HR ¼ 4.87 (95% CI ¼ 1.14–20.888)) and et al., 2003; Lodygin et al., 2005; Shames et al., 2006), tumour size (P ¼ 0.04; HR ¼ 1.23 (95% CI ¼ 1.009– including two studies of RCC (Morris et al., 2005, 2008; 1.49)) were each significantly associated with prognosis, Ibanez de Caceres et al., 2006). Previously, we identified and under a similar analysis for COL14A1, methylation SPINT2 as a novel RCC TSG (Morris et al., 2005), but of COL14A1 (P ¼ 0.0067) (HR ¼ 6.56 (95% CI ¼ 1.69– many candidate TSGs upregulated after treatment of 25.38)) and tumour stage (P ¼ 0.0009) (HR ¼ 3.42 (95% RCC cell lines with 5-Aza-20-deoxycytidine do not show CI ¼ 1.67–7.02)) were identified as significant prognostic tumour-specific promoter methylation (Morris et al., factors. A combined analysis was then performed for 2008). Therefore, to maximise the potential for identify- survival and BNC1 and COL14A1 methylation status, ing additional genes epigenetically silenced in RCC, we tumour grade, size and stage. Methylation of COL14A1 extended our previous study (Morris et al., 2005, 2008) (P ¼ 0.0067; HR ¼ 18.37 (95% CI ¼ 2.27–148.8)) was to include more cell lines (11 versus 4) and higher the most significant predictor of survival, followed by density microarrays (47 000 transcripts versus 11 000 stage (P ¼ 0.015; HR ¼ 3.5 (95% CI ¼ 1.28–9.57)) and genes). Seven of the eight genes we report in this study grade (P ¼ 0.08; HR ¼ 2.03 (95% CI ¼ 0.92–4.46)) (BNC1, RPRM, CST6, PDLIM4, GREM1, COL14A1 (BNC1 methylation status was not a significant prog- and COL15A1) have not previously been reported to be nostic factor (P ¼ 0.38)) (Figure 5d). methylated in RCC, and, to our knowledge, COL14A1 There were no significant differences in BNC1, and COL15A1 have not previously been reported to be PDLIM4, RPRM, CST6, SFRP1, GREM1, COL14A1 methylated in neoplasia. Although, while this study was and COL15A1 promoter methylation frequencies in progress SFRP1 wasreportedtobemethylatedinRCC bwtween clear cell RCC and non-clear cell RCC and (Awakura et al., 2007; Dahl et al., 2007; Gumz et al., no significant differences between methylation status 2007). of VHL mutated and VHL wild-type clear cell RCC. SFRP1 encodes a negative regulator the Wnt/beta- catenin pathway. Thus, SFRP1 molecules bind to and sequester Wnt molecules away from their cognate receptors, the Frizzled family. Activation of the Wnt/ Discussion beta-catenin pathway is associated with upregulation of a range of oncogenic targets (for example, Cyclin D1, Cancer-specific genetic and epigenetic alterations result- vascular endothelial growth factor, cMYC and cMET) ing in TSG inactivation are frequent events in RCC. implicated in the pathogenesis of cancer. We detected However, although the mutation spectrum causing SFRP1 promoter methylation in 34% of RCC com- TSG inactivation is usually diverse (so limiting the pared with 68% in the study of Dahl et al. (2007). Re- utility for tumour screening programmes of detecting an expression of SFRP1 in a null RCC-cell line has individual TSG mutation), TSG inactivation by pro- previously been shown to associate with reduced tumour moter hypermethylation provides a more homogeneous cell growth (Gumz et al., 2007). We confirmed this and target for molecular screening strategies. Furthermore, also have shown that reduced expression of endogenous large-scale gene sequencing studies of RCC and other SFRP1 increases the tumourogenic potential of an cancers have revealed that relatively few genes are RCC-cell line.

Figure 2 (a) RT–PCR analysis of BNC1, SFRP1 and COL15A1, GREM1, and RPRM expression. BNC1 expression was restored (RCC4, KTCL140, SKRC18)/upregulated (SKRC45, 786-0) in 5 of 11 cell lines after treatment with the demethylating agent, 5-aza-20- deoxycytidine. SFRP1 expression was restored (RCC4, SKRC47)/upregulated (KTCL140, SKRC18, SKRC45) in 5 of 11 cell lines. COL15A1 expression was restored (RCC4)/upregulated (KTCL140, SKRC18, SKRC47, SKRC54) in 5 of 11 cell lines. GREM1 expression was restored (RCC4, SKRC140, SKRC54, CAKI-1)/upregulated (SKRC39) in 5 of 11 cell lines after treatment with the demethylating agent, 5-aza-20-deoxycytidine. Global demethylation restored expression of RPRM in 3 of 11 cell lines (KTCL26, SKRC18, SKRC39). (b) Methylation-specific PCR analysis of BNC1, SFRP1 and COL15A, GREM1, and RPRM1. MSP analysis showed frequent (34–53%) promoter methylation in tumours, infrequent or absent methylation in adjacent tissue and no methylation in samples derived from non-malignant normal tissue (see Results for details). RCC tumour cell lines were used for each MSP assay as positive controls for methylated and unmethylated DNA. Representative samples are shown. M, product derived from methylation- specific primers; U, products derived from unmethylated-specific primers; T, tumour sample; N, non-malignant sample; *, methylated tumours. MSP and USP product are of a similar size. Smaller bands are primer dimmers.

Oncogene Identification of candidate TSG frequently methylated in RCC MR Morris et al 2112 CST6 Colony Formation SFRP1 Colony Formation RPRM Colony Formation BNC1 Colony Formation

200 250 200 160 786-O SKRC47 SKRC39 RCC4 180 180 140 160 200 160 120 140 140

120 150 120 100

100 100 80 Colonies Colonies Colonies 80 100 Colonies 80 60 60 60 40 40 50 40

20 20 20

0 0 0 0 EV CST6 EV SFRP1 EV RPRM EV BNC1

CST6 Soft Agar Assay BNC1 Soft Agar Assay

700 300 786-O HEK-293

600 250

500 m

m 200 µ µ 400 150 300 Colonies >100

Colonies >100 100 200

100 50

0 0 EV CST6 EV BNC1

Figure 3 (a) Re-expression of CST6, SFRP1, RPRM and BNC1 in RCC cells results in growth suppression. Exogenous re-expression of selected genes in RCC cell lines that do not express their respective genes resulted in a significant reduction of in vitro colony formation compared with RCC lines transfected with an empty vector (EV). Equal amounts of empty vector or pCDNA3.1-wtCST6, or pCDNA3.1-wtSFRP1, or pCDNA3.1-wtRPRM, or pCDNA3.1-wtBNC1 were transfected into 786-O (CST6), SKRC47 (SFRP1), RCC4 (BNC1) or SKRC39 (RPRM) cells. Each experiment was carried out in triplicate. There was a statistically significant reduction of colonies in each of the re-expression experiments (P ¼ 0.003, Po0.0001, Po0.0001 and P ¼ 0.001, respectively). Shown below each graph are representative plates showing reduction of colonies after gene re-expression. (b) Re-expression of CST6 or overexpression of BNC1 inhibits anchorage-independent growth. A, clones of 786-O-pCDNA3.1 and 786-0O-pCDNA3.1-wtCST6 or HEK293- pCDNA3.1 and HEK293-pCDNA3.1-wtBNC1were seeded at the same density into soft agar and incubated for 5 weeks. In all, 786-O clones not expressing CST6 (pCDNA3.1) produced many large (4100 mm) colonies. In contrast, 786-O clones expressing exogenous wtCST6 did not grow as robustly. Three independent experiments showed a 53% reduction of large colonies (P ¼ 0.0001) after 5 weeks of incubation. Similarly, exogenous overexpression of BNC1 resulted in 54% fewer large colonies (P ¼ 0.001). Below each graph is shown a representative image of clones after 5 weeks of incubation ( Â 100 magnification).

RPRM (Reprimo, TP53-dependent G2 arrest media- cell cycle arrest is thought to be mediated by an indirect tor candidate) is a downstream mediator of p53-induced inhibition of Cdc2-CyclinB1 complex translocation to G2 cell cycle arrest (Ohki et al., 2000). Reprimo-induced the nucleus (Ohki et al., 2000). Epigenetic silencing of

Oncogene Identification of candidate TSG frequently methylated in RCC MR Morris et al 2113 HEK293 proteases. Cathepsins are involved in multiple biological 450 processes such as apoptosis, intracellular protein cata- 400 bolism and pericellular matrix re-modelling (Bromme 350 m and Kaleta, 2002). It has been suggested that an µ 300 imbalance of cathepsins and their respective inhibitors 250 may promote tumour cell invasion (Bellail et al.,2004) 200 and so loss of CST6 expression by epigenetic silencing 150 might be predicted to contribute to the malignant

Colonies >100 100 phenotype. Consistent with this we found that re- 50 expression of CST6 reduced the ability of RCC cells to 0 form colonies in an anchorage-independent manner. Ct RNAi BNC1 RNAi SFRP1 RNAi CST6 has also been shown to be epigenetically silenced and reduced in vitro tumourigenicity in breast cancer (Shridhar et al.,2004;Aiet al., 2006) and gliomas (Qiu et al.,2008). PDLIM4 encodes a LIM domain candidate tumour gene mapping to 5q31.1. Although methylation of PDLIM4 has not been reported previously in RCC Ct RNAi BNC1 RNAi SFRP1 RNAi tumours, Boumber et al. (2007) reported frequent PDLIM4 promoter methylation in acute myelogenous leukaemia and colon cancer. Gremlin (GREM1)isan KTCL26 inhibitor of transforming growth factor-b signalling that 160 has previously been reported to be frequently methy- 140 lated in lung, breast and bladder cancers (Suzuki et al.,

m 2005). We found frequent methylation of GREM1

µ 120 100 suggesting that this may contribute to the perturbed 80 transforming growth factor-b signalling that is detected in many human cancers, including RCC. GREM1 60 methylation was detected in some samples of adjacent 40

Colonies >100 normal renal tissue from RCC patients with GREM1 20 tumour methylation. Similar findings were obtained for 0 RPRM and COL15A1, these results would be consistent Ct RNAi COL14A1 RNAi with the hypothesis that GREM1, RPRM and COL15A1 methylation might occur as part of a premalignant field effect, as has been described in bronchial epithelium (Wistuba, 2007). Basonuclin (BNC1) is a zinc-finger transcription factor that interacts with the promoters of both RNA polymerases I and II. In silico analysis suggests that Ct RNAi COL14A1 RNAi basonuclin target genes may be implicated in chromatin Figure 4 Knock-down of expression of BNC1, SFRP1 or structure, transcription/DNA-binding, adhesion/cell– COL14A1 increases anchorage-independent growth potential. (a) cell junction, signal transduction and intracellular RNAi-induced reduced expression of BNC1 or SFRP1 in HEK-293 cells resulted in the growth of significantly more colonies 4100 mm transport (Wang et al., 2006). Shames et al. (2006) in diameter compared with cells transfected with a control RNAi reported that BNC1 was methylated in breast, lung, oligo when seeded at the same density into soft agar (Po0.0001 prostate and colon cancers, suggesting that BNC1 in both cases). (b) RNAi-induced reduced expression of COL14A1 methylation might be of relevance to the diagnosis and in KTCL26 cells resulted in the growth of significantly more colonies 4100 mm in diameter compared with cells transfected with management of a range of human cancers. a control RNAi oligo when seeded at the same density into soft Ibanez de Caceres et al. (2006) have reported agar (Po0.0001). epigenetic silencing of COL1A1 in RCC. In this study, we identified frequent methylation of COL15A1 and COL14A1 in RCC. COL15A1 encodes a nonfibrillary proteoglycan, which is found in many tissue types RPRM has previously been reported in a variety of forming an integral unit of the collagenous network cancers including gastric, gall bladder, colorectal, subjacent to the basement membrane (Amenta et al., oesophageal, breast and prostate (Takahashi et al., 2005). The COOH-terminal end of COL15A1, which 2005; Sato et al., 2006; Bernal et al., 2008). We have now has homology to endostatin has been shown to have shown that it is also frequently methylated in RCC and anti-angiogenic properties and is capable of inhibiting that re-expression RPRM reduces the tumour-forming tumour growth in a xenograph renal model (Ramchan- properties of RCC-derived cell lines. dran et al., 1999). Moreover, recently COL15A1 was CST6 is a type 2 secreted cystatin. It inhibits cathepsin identified in a fibroblast-tumour cell hybrid screen as a B, L, H and V, members of a family of lysosomal potent suppressor of tumour growth, and re-expression

Oncogene Identification of candidate TSG frequently methylated in RCC MR Morris et al 2114 100 100 U 80 80 U BNC1 60 60 SFRP1 M 40 40 M Survival (%) Survival (%) 20 20

0 0 0 50 100 150 0 50 100 150 Months Months

100 100

80 80 U U

60 COL14A1 60 COL14A1

40 M 40 Survival (%) Survival (%) 20 20 M 0 0 0 50 100 150 0 50 100 150 200 Months Months Figure 5 (a) Kaplan–Meier survival analysis for BNC1 methylation status and Survival (M, methylated; U, unmethylated); (b) Kaplan–Meier survival analysis for COL14A1 methylation status and survival (M, methylated; U, unmethylated); (c) Kaplan– Meier survival analysis for BNC1 methylation status and survival (M, methylated; U, unmethylated). (d) Cox proportional hazard analysis for COL14A1 methylation status (analysis also incorporates tumour stage, Grade and size and BNC1 methylation status).

of COL15A1 in a HeLa derivative cell line completely RPRM, CST6 and SFRP1. Although RPRM and CST6 suppressed tumour formation in nude mice (Harris have not previously been studied in RCC, these findings et al., 2007). We have identified frequent COL15A1 are consistent with those reported in cervical and breast methylation in RCC (53%). Collagen XIVA1 cancer cell lines (Ohki et al., 2000; Shridhar et al., 2004). (COL14A1) is a large extracellular matrix glycoprotein We have also shown that reducing the expression of that associates with mature collagen fibrils (Schuppan BNC1, SFRP1 and COL14A1 in an embryonic kidney et al., 1990). Although methylation of COL14A1 has not cell line or an RCC-cell line increased tumourogenic previously been reported in human cancer, Schuppan potential as assessed by soft agar colony formation et al. (1990) noted frequent absence of COL14A1 in the assays. vicinity of invading tumours such as Kaposi sarcoma The methylated genes identified in this and previous and oral squamous cell carcinoma. In addition, we note studies of RCC (Morris et al., 2003, 2005, 2008; Ibanez data from the Sanger cancer genome re-sequencing de Caceres et al., 2006), represent a variety of functions. project (http://www.sanger.ac.uk/genetics/CGP/Studies/) However, we note that many of these genes can be that identifies COL14A1 mutations in B1% (n ¼ 4) of linked to pathways associated with the VHL TSG that RCC sequenced (n ¼ 412). COL14A1 interacts with has a gatekeeper function for RCC (analogous to that of Decorin (Ehnis et al., 1997), which regulates fibrillogen- APC in colorectal cancer). The VHL gene product has a esis and downregulates activity of a number of receptors key role in regulating the HIF-1 and HIF-2 and, in turn, (EGFR, IGF-IR, LRP) implicated in cell growth and these influence a wide repertoire of target genes. survival (Santra et al., 2002; Brandan et al., 2006; Although dysregulation of hypoxia-inducible genes Schaefer et al., 2007). (particularly those regulated by HIF-2) is strongly Loss of expression of epigenetically silenced TSGs can linked to risk of RCC with VHL inactivation, HIF- affect a wide variety of cellular processes, including dysregulation appears to be necessary but insufficient those which directly affect cell growth and proliferation. for VHL-related RCC tumourigenesis (Clifford et al., We analysed whether re-expressing SFRP1, RPRM, 2001; Raval et al., 2005). A number of HIF-independent CST6 and BNC1 in RCC cell lines would influence functions have been ascribed to pVHL. In the context of in vitro analyses of tumourigenesis. BNC1 has not COL14A1 and COL15A1 methylation, it is interesting previously been shown to affect tumour cell growth, but to note that analysis of Caenorhabditis elegans worms we found that re-expression of BNC1 suppressed mutant for VHL-1 showed a clear HIF-independent link tumour cell growth. Similar results were found for between pVHL and extracellular matrix function

Oncogene Identification of candidate TSG frequently methylated in RCC MR Morris et al 2115 (Bishop et al., 2004). Recently, pVHL has been reported later, cells were treated with 5 mM 5-aza-20-deoxycytidine. The to regulate the p53 pathway (relevant to RPRM) and medium was changed 24 h after treatment and then changed the Wnt/beta-catenin pathway (downstream of SFRP1) again after 72 h. RNA was prepared 5 days after treatment by targeting beta-catenin for proteasomal degradation using RNABee (AMS Biotechnology, Abingdon, UK). Total 0 (Chitalia et al., 2008). RNA from all 11 cell lines ±5-aza-2 -deoxycytidine was isolated The identification of frequently methylated RCC using RNA-Bee reagent following manufacturer’s instructions (AMS Biotechnology) followed by purification using RNeasy TSGs can highlight critical pathways that might be Mini-columns (Qiagen, Crawley, UK). Complementary RNA targeted for novel therapeutic interventions. In addition, probes were prepared using the Affymetrix protocol and the detection of methylated DNA in urine or serum hybridised to HG-U133 plus2 GeneChip oligonucleotide might be used as biomarkers for the diagnosis, staging arrays (Affymetrix). Array hybridisation and data production or risk stratification of RCC (Battagli et al., 2003; was carried out by the CRUK Paterson Institute Microarray Hoque et al., 2004; Urakami et al., 2006). VHL Service (http://bioinformatics.picr.man.ac.uk/mbcf/). inactivation occurs early in tumourigenesis but, to date, has not been shown to be a significant prognostic RT–PCR conditions indicator (Smits et al., 2008). Thus, given the infre- PCR cycling conditions consisted of 5 min at 95 1C followed by quency of mutations in other TSGs in RCC, detection of 30 cycles of 45 s of denaturation at 95 1C, 45 s of annealing at TSG methylation would appear to presents a promising 55–60 1C and 45 s of extension at 72 1C. Semi-quantitative strategy for prognostic biomarkers in RCC. However, analysis of expression was carried out using LabWorks only a few potential RCC methylation prognostic software (Ultraviolet Products, Upland, CA, USA). (RT–PCR biomarkers have been reported (Gonzalgo et al., 2004; primers and conditions on request). Breault et al., 2005; Christoph et al., 2006; Yamada et al., 2006; Costa et al., 2007; McRonald et al., 2009). Bisulfite modification and methylation analysis We found that methylation of BNC1, COL14A1 and Bisulfite DNA sequencing was performed as described previously SFRP1 was each associated with significantly poorer (Morris et al., 2005, 2008). Briefly, 0.5–1.0 mg of genomic DNA patient survival. However, for SFRP1 this association was denatured in 0.3 M NaOH for 15 min at 37 1C, and then was not independent of conventional prognostic factors unmethylated cytosine residues were sulfonated by incubation in 3.12 M sodium bisulfite (pH 5.0; Sigma)/5 mM hydroquinone (stage, size and grade). In multivariate analysis with (Sigma) in a thermocycler (Hybaid, Franklin, MA, USA) for conventional prognostic factors, methylation of 20 cycles of 30 s at 99 1Cand15minat501C. The sulfonated COL14A1 and BNC1 were each significant predictors DNA was recovered using the Wizard DNA cleanup system of poorer survival. Combining the COL14A1 and BNC1 (Promega, Southampton, UK) in accordance with the manufac- methylation data identified COL14A1 methylation as an turer’s instructions. The conversion reaction was completed by independent prognostic factor with higher statistical desulfonating in 0.3 M NaOH for 10 min at room temperature. significance than stage, size and grade. The DNA was ethanol-precipitated and resuspended in water.

Promoter methylation analysis CpG islands were identified on the human genome browser Materials and methods and putative promoter regions were predicted by Promoter Inspector software (Genomatix (www.genomatix.de)). Details Patients and samples of bisulphite sequencing primers and COL15A1 MSP primers DNA from up to 61 primary RCCs (B80% clear cell and 20% are provided in Supplementary Table 1. Examples of direct non-clear cell) and matched adjacent macroscopically normal tumour-DNA sequencing traces are shown in Supplementary renal tissue and normal renal tissue (not required for surgical Figure 2. Methylation-specific PCR analysis for the following pathology) from six patients undergoing non-renal cancer genes was carried out using previously described MSP primers: surgery (mean age 57 years, range 23–79 years) were analysed. BNC1 (Shames et al., 2006), SFRP1 (Nomoto et al., 2007) and Local research ethics committees approved the collection of REPRIMO (Sato et al., 2006). samples and informed consent was obtained from each patient. This study was conducted according to the principles expressed in the Declaration of Helsinki. Plasmid constructs and colony formation assay The CST6, SFRP1 and REPRIMO expression constructs were made by cloning the full-length human coding regions amplified Cell lines, 5-aza-20-deoxycytidine treatment and microarray from kidney cell lines into the EcoR1–BamHII sites of analysis pCDNA3.1. (Invitrogen) or pFLAG–CMV4 (Sigma) vectors. RCC cell lines KTCL 26, RCC4, UMRC2, UMRC3, BNC1 was amplified from the IMAGE clone 40080551. SKRC18, SKRC39, SKRC45, SKRC47, SKRC54, 786-0 and Plasmid constructs were verified by sequencing. Six micro- Caki-1 were routinely maintained in Dulbecco’s modified grams of empty vector and an equal Molar amount of Eagle’s medium (DMEM) (Invitrogen, San Diego, CA, USA) expression vector were transfected, using Fugene (Roche, supplemented with 10% fetal calf serum (FCS) at 37 1C, 5% Burgess Hill, UK), following the manufacturer’s instructions, 0 5 CO2. The demethylating agent 5-aza-2 -deoxycytidine (Sigma, into 5 Â 10 target cells (SKRC39, RCC4, 786-0, SKRC47 or Poole, UK) was freshly prepared in ddH2O and the filter HEK-293). Forty-eight hours after transfection, cells were seeded sterilised. Cell lines were plated in 75-cm2 flasks in DMEM in a serial dilution and maintained in DMEM and 10% fetal supplemented with 10% FCS at differing densities, depending bovine serum supplemented with 1 mg/ml G418 (Life Techno- on their replication factor, to ensure that both control and logies, Paisley, UK). Surviving colonies were stained with 0.4% 5-aza-20-deoxycytidine treated lines reached approximately 75% crystal violet (Sigma) in 50% methanol, 21 days after initial confluency at the point of RNA extraction. Twenty-four hours seeding, and counted. Each transfection was carried out in

Oncogene Identification of candidate TSG frequently methylated in RCC MR Morris et al 2116 triplicate. In addition, replicate experiments were carried out 200 ml of DMEM 10% FCS weekly. After 3 weeks of growth, a to obtain further clones for expression analysis. Expression final count of colonies was performed. Cells not seeded into was confirmed by RT–PCR and western blot analysis. agar were incubated for a further 24 h before efficiency of knock-down was assessed by RT–PCR. Anchorage-independent growth assay Statistical analysis was performed as indicated with a RCC clones stably expressing BNC1, CST6, REPRIMO or the significance level of 5%. empty vector control were suspended in 2-ml DMEM 10% FCS, 3% agar. Cells were maintained by addition of 200 mlof DMEM 10% FCS, supplemented with 1 mg/ml G418, weekly. Conflict of interest After 5 weeks of growth, a final count of colonies was performed. RNAi ‘silencer select’ oligos against BNC1 (s2012), The authors declare no conflict of interest. COL14A1 (s14677) and SFRP1 (s12713) or ‘silencer select’ control oligo no.1 (Ambion, Warrington, UK) were trans- fected into HEK-293 or KTCL26 cells using Interferin reagent (Polyplus, Illkirch, France) following the manufacture’s instruc- Acknowledgements tions. After 24-h incubation, cells were seeded into 2-ml DMEM 10% FCS, 3% agar. Cells were maintained by addition of We thank Cancer Research UK for financial support.

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

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