Multigene methylation analysis of Wilms' tumour and adult renal cell carcinoma

Abstract

To investigate the role of epigenetic gene silencing in the pathogenesis of Wilms' tumour and renal cell carcinoma (RCC), we determined their methylation profile using a candidate gene approach. Thus, 40 Wilms' tumours and up to 49 adult RCC were analysed by methylation-specific PCR for promoter methylation at CASP8, CDH1, CDH13, DAPK, MGMT, NORE1A, p14ARF and RARB2 in primary Wilms' tumours and CASP8, CDH1, CDH13, CRBP1, DAPK, MGMT, MT1G, NORE1A, p16INK4a, SDHB and RARB2 in primary RCC. Both tumour sample sets had previously been analysed for RASSF1A promoter methylation, and p16INK4a methylation results were also available for the Wilms' tumour samples. Wilms' tumours demonstrated a high incidence of methylation at CASP8 (43%) and MGMT (30%), intermediate frequencies at NORE1A (15%), p14ARF (15%), p16INK4a (10%), DAPK (11%) and CRBP1 (9%), but promoter methylation was rare or absent at RARB2 (0%), CDH13 (0%) and CDH1 (3%). No association was detected between methylation of RASSF1A, CASP8 or MGMT in individual tumours. The frequency of MGMT methylation was higher in stage 1 and 2 tumours (50%) than in stage 3 and 4 tumours (17%) but this did not reach statistical significance (P=0.06). RCC were most frequently methylated at DAPK (24%), MT1G (20%), NORE1A (19%), CDH1 (16%) and MGMT (9%) and not or rarely at SDHB (4%), RARB2 (0%), p16INK4a (0%) and CDH13 (3%). There were no associations between methylation of RASSF1A, DAPK and CDH1 in individual tumours. Papillary RCC demonstrated a higher frequency of DAPK methylation (43%) than clear cell tumours (19%) (P=0.14). We have demonstrated that de novo promoter methylation is frequent in Wilms' tumour and RCC, and these data enable methylation profiles to be constructed for each tumour type. Thus, combining our results with data published previously, it appears that promoter methylation occurs frequently (≥20% of primary tumours) at CASP8, SLIT2 and RASSF1A in Wilms' tumour and at RASSF1A, TIMP3, DAPK, SLIT2, MT1G and GSTP1 in RCC.

Introduction

Methylation of CpG dinucleotides in the promoter regions of tumour suppressor genes (TSGs) producing transcriptional silencing is a major mechanism of TSG inactivation in many human cancers (Baylin et al., 1998; Jones, 1999; Tycko, 2000; Costello and Plass, 2001). The frequency of TSG inactivation by de novo methylation varies between TSGs and between cancers. Thus, whereas the predominant causes of inactivation in the RB1 and VHL TSGs are mutation and allele loss, the 3p21 TSG RASSF1A is usually inactivated by epigenetic silencing (and allele loss) and only rarely by somatic mutations (Dammann et al., 2000, 2001b; Agathanggelou et al., 2001; Astuti et al., 2001a; Burbee et al., 2001; Dreijerink et al., 2001; Lo et al., 2001; Morrissey et al., 2001). The spectrum of genes frequently inactivated by methylation differs between tumour types such that certain tumours may exhibit specific patterns of methylation (Esteller et al., 2001). Characterization of tumour-specific TSG methylation profiles may provide insights into the molecular pathology of tumours and the definition of tumour-type-specific methylation profiles will provide opportunities to develop novel diagnostic tests. In addition, for certain cancers, notably colorectal cancer, some authors have suggested that a subset of tumours may demonstrate a ‘methylator phenotype’ with promoter methylation of a wide range of TSGs (Toyota et al., 1999).

Tumours of the kidney account for 2% of all human cancers. Renal cancers comprise a wide variety of histological subtypes and tumour types differ markedly in children and adults. Wilms' tumour is the most common childhood kidney tumour and is thought to be derived from primitive nephroblasts. Wilms' tumour is usually diagnosed between 1 and 6 years and is rare in adults. Renal cell carcinoma (RCC) is the most common adult kidney tumour and most cases are diagnosed after the age of 50 years. The majority (75%) of RCC are classified as clear cell (conventional) and the most frequent nonclear cell histopathological subtype is papillary (15% of all cases) (Storkel and Vandenberg, 1995). The investigation of inherited forms of renal tumours has led to the identification of susceptibility genes (e.g. WT1 and VHL TSGs and the MET proto-oncogene) that are implicated in both inherited and sporadic forms of renal cancer (Zbar, 2000; Lee and Haber, 2001). However, germline and somatic mutations in these genes are associated with specific tumour types (e.g. WT1 in Wilms' tumour, VHL in clear cell RCC and MET in papillary RCC) and so do not identify common pathways of tumorigenesis in different renal tumour types. Compared to many other human cancers, there is relatively little information on the frequency and pattern of de novo promoter methylation in renal tumours, particularly Wilms' tumour. Frequent hypermethylation of H19 is well described in Wilms' tumour and p16INK4a (CDKN2a) promoter methylation has been reported in advanced stage tumours (Steenman et al., 1994; Taniguchi et al., 1995; Arcellana-Panlilio et al., 2000). Recently we reported frequent RASSF1A methylation in Wilms' tumour and in clear cell and papillary RCC (Astuti et al., 2001a; Wagner et al., 2002). These findings suggested that disruption of specific signalling pathways might be implicated in different types of renal tumour. To investigate further the role of TSG promoter methylation in the pathogenesis of Wilms' tumour and RCC, we have determined the methylation status of a series of genes that have been reported to undergo frequent promoter methylation in other cancer types.

Methods

Patients and samples

In total, 40 primary Wilms' tumour (38 sporadic cases, one with Beckwith–Wiedemann syndrome and one familial) DNA samples were analysed for TSG promoter methylation. In 14 cases, normal tissue DNA (blood or kidney) was also available for study. A total of 63 primary RCCs were analysed for at least one gene (up to 49 for any one gene), subdivided into 39 clear cell, 20 papillary RCCs and four oncocytomas.

Sodium bisulphite modification

Sodium bisulphite modification was carried out using an adapted method (Esteller et al., 2000). Genomic DNA, (1 μg) was denatured at 37°C for 10 min in 0.3 M NaOH. Unmethylated cytosines were sulphonated by incubation in 3.12 M sodium bisulphite/1 M hydroquinone (pH 5) at (99°C (30 s) 50°C (15 min)) × 20 cycles. The resulting sulphonated DNA was purified using the Wizard DNA clean-up system (Promega), according to the manufacturer's instructions, except that DNA was eluted with distilled water (50 μl) at room temperature. Following elution, DNA was desulphonated in 0.3 M NaOH for 5 min at room temperature, then the DNA was precipitated with NaOAc (5 μl of 3 M) and ethanol (125 μl of 100%) overnight at −20°C and resuspended in 50 μl distilled water.

Methylation-specific polymerase chain reaction

Methylation-specific PCR primers binding specifically to either unmethylated or methylated bisulphite modified DNA were used (Herman et al., 1996). Primer sequences and conditions for PCR reactions were derived from previously published articles: CASP8 (Teitz et al., 2000), RARβ, CDH1, MGMT, DAPK, p14ARF (Zochbauer-Muller et al., 2001), CDH13 (Toyooka et al., 2001), CRBP1 (Esteller et al., 2002) and NORE1A (Hesson et al., 2003). Methylation-specific PCR for SDHB was performed using specific primers designed to amplify methylated and unmethylated SDHB promoter sequences: unmethylated-specific, 5′-IndexTermTGTGTTGTTATTGTG TTATTGTGTAT-3′ (forward) and 5′-IndexTermCCACCAAAAATTATAACCAACAACCA-3′ (reverse) and methylated-specific, 5′-IndexTermTGCGTCGTTATTGCGTTATTGCGTAC-3′ (forward) and 5′-IndexTermCCGCCAAAAATTATAACCGACAACCG-3′ (reverse). Taq DNA polymerase (Gibco) was added after a ‘hot start’ at 95°C for 5 min. Amplification was performed for 35 cycles at an annealing temperature of 53°C for the unmethylated specific primers and 61°C for the methylated specific primers on Omn-E (Hybaid) DNA thermal cycler. The expected sizes of the PCR products for both unmethylated and methylation-specific amplifications was 269 bp. Primer details for MT1G were: unmethylated-specific, 5′-IndexTermGTGAGTTGGTGTGAAAG GGGTT-3′ (forward) and 5′-IndexTermCCACACCACCCACAATCCCA-3′ (reverse) and methylated-specific, 5′-IndexTermTGCGAAAGG GGTCGTTTTGC-3′ (forward) and 5′-IndexTermGCGATCCCGACCTAAACTATACG-3′ (reverse). Amplification was performed for 35 cycles at an annealing temperature of 59°C for both the unmethylated and methylated specific primers on Omn-E (Hybaid) DNA thermal cycler. The expected sizes of the PCR products for unmethylated and methylation-specific amplifications were 113 and 93 bp, respectively. Normal human genomic DNA that had been treated with SssI methylase to achieve full CpG island methylation was used as a positive control for methylated PCRs.

Sequencing of PCR products

MSP products were excised from agarose gels and extracted using the QIAquick Gel Extraction Kit (Qiagen), according to the manufacturer's instructions. Products were confirmed by direct sequencing from the forward PCR primer using ABI PRISM® BigDyeTM Terminators v 3.0 Cycle Sequencing Kit according to the manufacturer's instructions and run using ABI 377 automatic sequencers.

Statistical analysis

Fisher's exact test was used as appropriate. Statistical significance was taken at 5% level.

Results

Methylation analysis of CASP8, CDH1, CDH13, DAPK, MGMT, NORE1A, p14ARF and RARβ in primary Wilms' tumours

No or very low levels of promoter methylation were detected for RARβ (0%), CDH13 (0%) and CDH1 (3%). High levels of methylation were detected for CASP8 (43%) and MGMT (30%), and intermediate levels for NORE1A (15%), p14ARF (15%), p16INK4a (10%) and DAPK (11%) (see Figures 1 and 2). All tumours with CpG island methylation for CASP8, MGMT, NORE1A, p14ARF, p16INK4a, DAPK and CDH1 also contained unmethylated alleles (of the relevant CpG island), which might be attributable to the presence of contaminating normal tissue (tumour samples were not microdissected) or partial methylation. For tumours with CpG island hypermethylation, all matching normal tissue samples did not show methylation, except that 3/6 tumours with MGMT tumour methylation also demonstrated methylation in the matched normal kidney tissue.

Figure 1
figure1

(a) Methylation-specific PCR analysis of MGMT, p14, CASP8 and p16 in Wilms' tumours. MGMT promoter methylation is positive for tumours 198, 207, 231 and 244, p14 promoter methylation was detected in 142 and 146, but not 126 and 130; CASP8 methylation was present in 11 and 44, but not 12 and 32; and p16 promoter methylation was detected in 536, but not 424, 425 and 427 (b) Methylation-specific PCR analysis of p16, DAPK and RARβ2 in RCC. For p16, DAPK and RARβ2 examples of PCR results using primers for methylated (M) and unmethylated (U) DNA are shown. Tumours 331 and 103 were negative for p16 methylation and tumours 71 and 103 were negative for RARβ2 promoter methylation. RCC 69 was positive for DAPK methylation and tumour 69 was negative

Figure 2
figure2

(a) Methylation status of MT1G in RCC primary tumours using MSP. M=methylated specific PCR; U=unmethylated specific PCR. (b) NORE1A methylation status of RCCs and Wilms' kidney tumours using MSP. (1), 178 T and 183 T are examples of NORE1A methylated RCC tumours. (2), Tumours 420 and 249 are examples of NORE1A methylated Wilms' tumours. SssI in vitro methylated normal blood DNA was used as a positive control and water was used as the negative control

Previously, we had demonstrated that 56% of the same sample set of primary Wilms' tumours demonstrated RASSF1A hypermethylation (Wagner et al., 2002). We investigated the possible relationships between methylation of CASP8, RASSF1A and MGMT. There was no apparent association between (a) CASP8 and RASSF1A methylation (P=0.78) RASSF1A and CASP8 both methylated in 9/39 (23%), RASSF1A only 13/39 (33%), CASP8 only 8/39 (21%) neither 9/39 (23%); (b) CASP8 and MGMT methylation (P=1.0) or MGMT and RASSF1A methylation (P=0.72) in Wilms' tumours.

Methylation profile and clinicopathological status

Clinical information on tumour stage was available for 32 WTs. The frequency of MGMT methylation was lower in more advanced stage 3 and 4 tumours (17%, 3/18) than in stage 1 and 2 tumours (50%, 7/14) although this did not quite reach statistical significance (P=0.06). There was no association between (a) CASP8 methylation frequency and tumour stage (9/14 in stage 1 and 2 tumours and 9/18 in stage 3 and 4, P=0.49) or (b) tumour stage and the number of genes with CpG promoter methylation. In all, six patients relapsed following treatment of the primary tumour; however, the frequency of MGMT, CASP8 and DAPK methylation in these primary tumours was similar to the overall frequencies of methylation in all Wilms' tumours.

Methylation analysis of CASP8, CDH1, CDH13, CRBP1, DAPK, MGMT, MT1G, NORE1A, pl6ink4a, SDHB and RARβ primary renal cell carcinomas

No or very low levels of promoter methylation were detected for SDHB (1/25), RARβ (0/30), P16INK4a (0/17) and CDH13 (1/40). Methylation at DAPK (24%, 12/49) was most common followed by MT1G (5/25, 20%), NORE1A (5/26, 19%), CASP8 (8/49, 16%), CDH1 (16%, 4/26), MGMT (9%, 2/22) and CRBP1 (9%, 2/22) (see Figures 1 and 2). All tumours with promoter methylation also contained unmethylated alleles (of the relevant CpG island) that might be attributable to the presence of contaminating normal tissue (tumour samples were not microdissected). The identity of MSP products was confirmed by sequencing. Previously, we had analysed RASSF1A methylation in RCC and we investigated whether there was any association between RASSF1A, DAPK and CDH1 promoter methylation. However, we did not find any association between RASSF1A and DAPK methylation, RASSF1A and CDH1 methylation or DAPK and CDH1 methylation (all P>0.4).

Methylation profile and clinicopathological status

RCC were classified as clear cell RCC, papillary RCC or oncocytoma. The frequency of CDH1 methylation was similar in papillary and clear cell RCC, but papillary RCC demonstrated a higher frequency of DAPK methylation (43%) than clear cell tumours (19%) (P=0.14). There was no association between DAPK methylation and papillary RCC subtype. There were no significant differences between DAPK methylated and unmethylated clear cell RCC for age (mean 60±7.4 vs 56±7.8 years), tumour size (5.96±1.39 vs 6.41±3.25 cm (P=0.76)), grade or TNM status (data not shown).

There was no association (P=0.62) between DAPK and RASSF1A methylation and only 1/23 clear cell RCC was methylated for both genes. There were no significant differences between RASSF1A methylated and unmethylated clear cell RCC for age (mean 61.0 vs 61.7 years), tumour size (5.98±2.37 vs 6.07±3.85 cm (P=0.76)) or TNM status (data not shown).

Discussion

Methylation profiling of a wide variety of human cancers (e.g. colon, stomach, pancreas, liver kidney, bladder, brain, leukaemia and lymphomas) has demonstrated that de novo CpG island methylation has been reported in every tumour type, but that the patterns of the methylation profile vary between tumour types (reviewed in Esteller et al., 2001). Some genes (e.g. p16INK4a) demonstrate epigenetic silencing in many tumour types, whereas methylation of other genes is associated with specific tumour types (e.g. VHL in RCC) (Clifford et al., 1998; Herman et al., 1994). In this study of 40 primary Wilms' tumours, we detected significant levels of methylation for CASP8 (43%), MGWT (30%), NORE1A (15%), p14ARF (15%) and DAPK (11%), and previously we had reported methylation frequencies for RASSF1A (54%) and p16NK4a (10%) in the same set of tumours. In contrast, RARβ (0%), CDH13 (0%) and CDH1 (3%) methylation was absent or rare.

CASP8 methylation has been reported in 50% of neuroblastomas by us and others (Teitz et al., 2000; Astuti et al., 2001a; Harada et al., 2002b). Loss of CASP8 expression is reported to be common in primitive neuroectodermal brain tumour (PNET)/medullobastoma (Zuzak et al., 2002), and CASP8 methylation is common in medulloblastoma. Although both we and Harada et al. (2002b) detected similar levels of CASP8 methylation in neuroblastoma tumours (Astuti et al., 2001a), we detected a higher incidence of CASP8 promoter methylation in Wilms' tumours (43 vs 19%). Loss of CASP8 expression in neuroblastoma and PNET cell lines is associated with resistance to TRAIL-induced apoptosis, suggesting that CASP8 methylation could be implicated in chemotherapy resistance (Grotzer et al., 2000; Eggert et al., 2001). Although we did not detect an association between CASP8 methylation and relapse in Wilms' tumour, the number of cases was small and further studies are indicated. The frequency of CASP8 methylation in RCC (16%) was less than that in Wilms' tumour (P=0.0001). However, it seems that high levels of CASP8 methylation are not restricted to paediatric tumours as Shivapurkar et al. (2002) reported CASP8 methylation in 35% of adult small cell lung cancers (but none of 44 non-small-cell lung cancers). Interestingly, the CASP8 region studied by us and others does not target the promoter region of the gene, but does appear to correlate with epigenetic silencing when methylated (Harada et al., 2002b). Thus, it has been suggested that while methylation of this region is unlikely to be the direct cause of gene silencing, methylation in this region may reflect promoter methylation status (Harada et al., 2002b).

The DNA repair gene, MGMT (O6-methylguanine-DNA methyltransferase), is methylated in many tumour types including colorectal (39%), brain (34%), head and neck (32%), lung (21%), lymphoma (25%), oesophagus (20%) and pancreas (11%) (Esteller et al., 2001). We detected MGMT methylation in 9% of RCC, which is close to the previously reported estimate of 8% (Esteller et al., 1999). In contrast to the other genes tested, in a significant number of cases in which MGMT was methylated in the tumour, MGMT methylation was also detected in the corresponding normal tissue. We cannot exclude the fact that this may be due to contamination with adjacent malignant cells, but another possibility is that preneoplastic/preinvasive and even histologically normal epithelium may have suffered epigenetic changes, as described in lung tissue (Zochbauer-Muller et al., 2001). In our analyses, MGMT methylation was detected more frequently in Wilms' tumour than in adult RCC, but Harada et al. (2002a) did not detect MGMT methylation in 31 Wilms' tumours. Previously, it was suggested that MGMT methylation might promote chemosensitivity and our results suggest that the relationship between MGMT methylation and response to chemotherapy would merit further detailed analysis.

DAPK (death associated protein kinase) is a positive mediator of IFN-γ induced apoptosis. Although DAPK promoter methylation has been studied in many human cancers (Esteller et al., 2001), it has not been reported previously in RCC and only recently in Wilms' tumour (Harada et al., 2002a). DAPK methylation is very frequent in lymphoma and low to moderate frequencies have been reported for head and neck (18%), lung (16%), colon (13%), bladder (9%), ovary (9%) and breast (7%) cancers (Esteller et al., 2001 and references within). The frequency of DAPK methylation in Wilms' tumour in our study (11%) and that (0%) by Harada et al. (2002a) was not high, but the frequency in RCC (19%) was higher than average. In particular, the frequency of DAPK promoter methylation in papillary RCC (43%) was second only to that reported for lymphoma (Esteller et al., 2001).

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16INK4a and p14ARF. p16INK4a plays a central role in G1 cell cycle control and p14ARF regulates MDM2-mediated degradation of p53. Although p16INK4a and p14ARF methylation occurred in similar proportions of Wilms' tumours, there was no significant correlation between methylation of the two genes, and only one of six tumours with p14ARF methylation also had p16INK4a methylation. Homozygous methylation of both p16INK4a and p14ARF is frequent in colorectal cancers (Gonzalez-Zulueta et al., 1995) but in both colorectal and other types (e.g. uveal melanoma and bile duct cancers), p16INK4a methylation is independent of p14ARF methylation (Esteller et al., 2000; van der Velden et al., 2001; Caca et al., 2002; Robertson and Jones, 1998).

The NORE1 gene located at 1q32.1 is a member of the RASSF1 gene family and exists in three isoforms (NORE1Aalpha, NORE1Abeta and NORE1B). Both NORE1A and NORE1B isoforms have separate CpG islands spanning their first exons and previously we demonstrated methylation of the NORE1A, but not NORE1B, CpG island in lung and breast cell lines and primary tumours (Hesson et al., 2003). In addition, the NORE1A CpG island was methylated in three of nine kidney tumour cell lines tested. We have now demonstrated that NORE1A methylation occurs in a subset of primary RCC and Wilms' tumours, although the frequency is less than that in RCC cell lines. Previously, we did not find a correlation between RASSF1A and NORE1A methylation in non-small-cell lung cancer (Hesson et al., 2003), and no association was apparent in RCC and Wilms' tumour also.

Chromosome 16q allele loss occurs in up to 25% of Wilms' tumours and has been associated with a poorer prognosis (Austruy et al., 1995; Grundy et al., 1996, 1998; Klamt et al., 1998; Skotnicka-Klonowicz et al., 2000). Candidate 16q Wilms' tumour suppressor genes include CDH1 and CDH13 (H-cadherin, 16q24.2–3). Cadherin molecules such as E-cadherin and H-cadherin may enhance tumour progression and invasion by multiple mechanisms including reduced cell–cell adhesion. Somatic CDH1 inactivation by mutation or promoter methylation is frequent in many human cancers, and germline CDH1 mutations predispose to familial gastric and colorectal cancer (Keller et al., 1999; Richards et al., 1999; Esteller et al., 2001). Although mutation analysis of CDH1 in Wilms' tumours did not reveal somatic mutations, 50% of Wilms' tumours demonstrate abnormal E-cadherin protein staining (CDH1 methylation status has not been analysed previously in Wilms' tumour) (Schulz et al., 2000). CDH13 expression is reduced in lung and breast cancers and epigenetic inactivation of CDH13 is frequent in lung, breast, prostate and ovarian cancers (Lee 1996; Kawakami et al., 1999; Toyooka et al., 2001; Maruyama et al., 2002). However, we did not find any evidence for frequent CDH1 and CDH13 promoter methylation in Wilms' tumours and these findings are consistent with those of Harada et al. (2002a) Although these results do not exclude completely a role for CDH1 and CDH13 inactivation in Wilms' tumours with 16q allele loss (CDH13 mutations have not been sought and E-cadherin protein expression may be lost by other mechanisms) our data suggest that, if CDH13 mutation analysis is negative, other 16q candidate genes should be prioritized for investigation. Although the frequency of CDH13 methylation was also very low in RCC, the frequency of CDH1 methylation was nonsignificantly higher than in Wilms' tumour (1/40 vs 4/26, P=0.07). However, the frequency of CDH1 methylation in our series of RCC was less than that (15 vs 67%) reported from Japan (Nguyen et al., 2000).

The RARβ2 gene encodes nuclear receptors for retinoic acid, which mediate the antiproliferative effects of retinoids. It has long been appreciated that retinoids play an essential role in kidney organogenesis (Burrow, 2000). The RARβ2 and RARβ4 isoforms are frequently methylated in lung, breast and prostate cancers (Virmani et al., 2000; Maruyama et al., 2002), but neither we nor Harada et al. (2002a) detected de novo methylation of their promoter (P2) in Wilms' tumour. RARβ maps to 3p24. Although chromosome 3p allele loss is frequent in RCC and not Wilms' tumour, we have found frequent methylation of the 3p21 RASSF1A TSG in both tumour types demonstrating that the absence of allele loss does not exclude TSG inactivation (Astuti et al., 2001a; Morrissey et al., 2001). However, although multiple 3p TSGs (including VHL, RASSF1A and FHIT) have been implicated in the pathogenesis of RCC (Huebner, 2001), we did not find any evidence that RARβP2 methylation is implicated in RCC or Wilms' tumour. The cellular retinol-binding protein 1 (CRBP1) also plays a key role in retinol (vitamin A) transport and hypermethylation of CRBP1 has been reported in a variety of tumours and cancer cell lines (Esteller et al., 2002). The highest frequency of CRBP1 promoter methylation is observed in lymphoma and gastrointestinal cancers and CRBP1 methylation frequently occurs in association with RARβ2 methylation (Esteller et al., 2002). Hence our observation that RARβ2 and CRBP1 methylation is uncommon in RCC suggests that altered vitamin A metabolism may not have a major role in the pathogenesis of RCC.

Metallothioneins are a family of cysteine-rich, metal-binding intracellular proteins which have been linked with cell proliferation. Differential expression of metallothioneins has been reported in RCC tissue samples with significant upregulation of MT2A and downregulation of MT1A and MT1G transcripts (Nguyen et al., 2000). In addition, MT1G promoter region methylation has been reported in oesophageal and thyroid papillary carcinoma (Yamashita et al., 2002, Huang et al., 2003). Thus, our findings suggest a mechanism for MT1G transcript alterations in RCC and extend the tumour types in which MTIG methylation has been reported.

There is increasing evidence of the importance of germline mutations of genes involved in the Krebs cycle in tumour formation, and germline fumarate hydratase (FH) mutations have been associated with renal cancer susceptibility (Tomlinson et al., 2002). Germ line SDHB mutations are associated with inherited susceptibility to paraganglioma and phaeochromocytoma and, although somatic SDHB mutations have not been identified in these tumours, SDHB promoter methylation occurs in neuroblastoma and in phaeochromocytoma (Astuti et al., 2001b, Astuti et al submitted). Inactivation of the VHL TSG occurs in most clear cell RCC and results in upregulation of hypoxia-inducible gene expression via an HIF-dependent pathway (Foster et al., 1994; Clifford et al., 1998; Maxwell et al., 1999). However, it has been suggested that upregulation of HIF-1 in clear cell RCC might involve both VHL-dependent and VHL-independent mechanisms (Turner et al., 2002). SDHB inactivation in phaeochromocytoma has been suggested to result in the activation of hypoxic response pathways (Gimenez-Roqueplo et al., 2002), but we did not find any evidence that SDHB inactivation in clear cell RCC represents a frequent mechanism for a non-VHL pathway for HIF dysregulation (unpublished results demonstrated no somatic SDHB mutations in RCC samples).

To compare the methylation profile of Wilms' tumour and RCC to that of other common cancers, we combined our own results with those of other groups (see Figure 3). It is of interest to note that RASSF1A and SLIT2 are most consistently methylated in all tumour types, colorectal cancer probably demonstrates the widespread pattern of gene methylation, whereas Wilms' tumour and RCC (and neuroblastoma) demonstrate a more restricted pattern of gene methylation. Combining our results with data from previous studies, RCC is characterized by methylation of TIMP3, RASSF1A, DAPK, SLIT2, MT1G and GSTP1 in ≥20% of tumours (Esteller et al., 2001; Nojima et al., 2001; Astuti et al., submitted). Although somatic VHL inactivation is usually by somatic mutation rather than methylation, VHL methylation is specifically associated with clear cell RCC (Herman et al., 1994; Clifford et al., 1998). Similarly, methylation profiling of Wilms' tumours has revealed de novo promoter methylation of CASP8 and RASSF1A (and H19), in ≥20% of all cancers (p161NK4a in advanced tumours).

Figure 3
figure3

Comparison of methylation profiles in different tumour types (Wilms' tumour, RCC, breast cancer, lung cancer, colorectal cancer and neuroblastoma). Data for p14ARF promoter methylation in neuroblastoma were not available. (Data were derived from the present study and reports from Merlo et al., 1995; Cote et al., 1998; Sato et al., 1998; Dammann et al., 2000, 2001a, Burbee et al., 2001; Agathanggelou et al., 2001; Dammann et al., 2001a, Dreijerink et al., 2001, Esteller et al., 2001; Morrissey et al., 2001; Toyooka et al., 2001, 2002a, 2002b; Wheeler et al., 2001; Yoon et al., 2001; Zochbauer-Muller et al., 2001; Dallol et al., 2002, 2003; Harada et al., 2002a, 2002b; van Engeland et al., 2002; Wagner et al., 2002)

For certain tumours such as colorectal cancer, a subset of tumours display frequent methylation of many genes (methylator phenotype) (Toyota et al., 1999). We found a higher frequency of p16INK4a methylation in tumours with RASSF1A methylation than those without RASSF1A methylation. However, this association was not statistically significant and there was no evidence for a ‘methylator phenotype’ in a subset of Wilms' tumours. Compared to many human cancers (e.g. colon, breast, lung, etc.), the role of epigenetic events in Wilms' tumour and RCC has not been investigated extensively. Although our analyses did not demonstrate significant associations between clinicopathological features and methylation of specific genes, this aspect requires further study in a larger series. The identification of associations between prognosis and response to chemotherapy and methylation status of Wilms' tumour and RCC would be advantageous because of the potential for rapid methylation profiling using tumour-type-specific microarrays.

References

  1. Agathanggelou A, Honorio S, Macartney DP, Martinez A, Dallol A, Rader J, Fullwood P, Chauhan A, Walker R, Shaw JA, Hosoe S, Lerman MI, Minna JD, Maher ER and Latif F . (2001). Oncogene, 20, 1509–1518.

  2. Arcellana-Panlilio MY, Egeler RM, Ujack E, Pinto A, Demetrick DJ, Robbins SM and Coppes MJ . (2000). Gene Chromosome Cancer, 29, 63–69.

  3. Astuti D, Agathanggelou A, Honorio S, Dallol A, Martinsson T, Kogner P, Cummins C, Neumann HPH, Voutilainen R, Dahia P, Eng C, Maher ER and Latif F . (2001a). Oncogene, 20, 7573–7577.

  4. Astuti D, Latif F, Dallol A, Dahia PL, Douglas F, George E, Skoldberg F, Husebye ES, Eng C and Maher ER . (2001b). Am. J. Hum. Genet., 69, 49–54.

  5. Austruy E, Candon S, Henry I, Gyapay G, Tournade MF, Mannens M, Callen D, Junien C and Jeanpierre C . (1995). Gene Chromosome Cancer, 14, 285–294.

  6. Baylin SB, Herman JG, Graff JR, Vertino PM and Issa JP . (1998). Adv. Cancer Res., 72, 141–196.

  7. Burbee DG, Forgacs E, Zochbauer-Muller S, Shivakumar L, Fong K, Gao B, Randle D, Kondo M, Virmani A, Bader S, Sekido Y, Latif F, Milchgrub S, Toyooka S, Gazdar AF, Lerman MI, Zabarovsky E, White M and Minna JD . (2001). J. Natl. Cancer Inst., 93, 691–699.

  8. Burrow CR . (2000). Exp. Nephrol., 8, 219–225.

  9. Caca K, Feisthammel J, Klee K, Tannapfel A, Witzigmann H, Wittekind C, Mossner J and Berr F . (2002). Int. J. Cancer, 97, 481–488.

  10. Clifford SC, Prowse AH, Affara NA, Buys CHCM and Maher ER . (1998). Gene Chromosome Cancer, 22, 200–209.

  11. Costello JF and Plass C . (2001). J. Med. Genet., 38, 285–303.

  12. Cote S, Sinnett D and Momparler RL . (1998). Anticancer Drugs, 9, 743–750.

  13. Dallol A, Da Silva NF, Viacava P, Minna JD, Bieche I, Maher ER and Latif F . (2002). Cancer Res., 62, 5874–5880.

  14. Dallol A, Morton D, Maher ER and Latif F . (2003). Cancer Res., 63, 1054–1058.

  15. Dammann R, Li C, Yoon JH, Chin PL, Bates S and Pfeifer GP . (2000). Nat. Genet., 25, 315–319.

  16. Dammann R, Takahashi T and Pfeifer GP . (2001a). Oncogene, 20, 3563–3567.

  17. Dammann R, Yang G and Pfeifer GP . (2001b). Cancer Res., 61, 3105–3109.

  18. Dreijerink K, Braga E, Kuzmin I, Geil L, Duh FM, Angeloni D, Zbar B, Lerman MI, Stanbridge EJ, Minna JD, Protopopov A, Li J, Kashuba V, Klein G and Zabarovsky ER. (2001). Proc. Natl. Acad. Sci., USA, 98, 7504–7509.

  19. Eggert A, Grotzer MA, Zuzak TJ, Wiewrodt BR, Ho R, Ikegaki N and Brodeur GM . (2001). Cancer Res., 61, 1314–1319.

  20. Esteller M, Cora PG, Baylin SB and Herman JG . (2001). Cancer Res., 61, 3225–3229.

  21. Esteller M, Guo M, Moreno V, Peinado MA, Capella G, Galm O, Baylin SB and Herman JG . (2002). Cancer Res., 62, 5902–5905.

  22. Esteller M, Hamilton SR, Burger PC, Baylin SB and Herman JG . (1999). Cancer Res., 59, 793–797.

  23. Esteller M, Tortola S, Toyota M, Capella G, Peinado MA, Baylin SB and Herman JG . (2000). Cancer Res., 60, 129–133.

  24. Foster K, Prowse A, van den Berg A, Fleming S, Hulsbeek MMF, Crossey PA, Richards FM, Cairns P, Affara NA, Ferguson-Smith MA, Buys CHCM and Maher ER . (1994). Hum. Mol. Genet., 3, 2169–2173.

  25. Gimenez-Roqueplo AP, Favier J, Rustin P, Rieubland C, Kerlan V, Plouin PF, Rotig A and Jeunemaitre X . (2002). J. Clin. Endocrinol., 87, 4771–4774.

  26. Gonzalez-Zulueta M, Bender CM, Yang AS, Nguyen T, Beart RW, Van Tornout JM and Jones PA . (1995). Cancer Res., 55, 4531–4535.

  27. Grotzer MA, Eggert A, Zuzak TJ, Janss AJ, Marwaha S, Wiewrodt BR, Ikegaki N, Brodeur GM and Phillips PC . (2000). Oncogene, 19, 4604–4610.

  28. Grundy P, Telzerow P, Moksness J and Breslow NE . (1996). Med. Pediatr. Oncol., 27, 429–433.

  29. Grundy RG, Pritchard J, Scambler P and Cowell JK . (1998). Br. J. Cancer, 78, 1181–1187.

  30. Harada K, Toyooka S, Maitra A, Maruyama R, Toyooka KO, Timmons CF, Tomlinson GE, Mastrangelo D, Hay RJ, Minna JD and Gazdar AF . (2002a). Oncogene, 21, 4345–4349.

  31. Harada K, Toyooka S, Shivapurkar N, Maitra A, Reddy JL, Malta H, Miyajima K, Timmons CF, Tomlinson GE, Mastrangelo D, Hay RJ, Chaudhary PM and Gazdar AF . (2002b). Cancer Res., 62, 5897–5901.

  32. Herman JG, Graff JR, Myohanen S, Nelkin BD and Baylin SB . (1996). Proc. Natl. Acad. Sci. USA, 93, 9821–9826.

  33. Herman JG, Latif F, Weng Y, Lerman MI, Zbar B, Liu S, Samid D, Duan DS, Gnarra JR and Linehan WM . (1994). Proc. Natl. Acad. Sci. USA, 91, 9700–9704.

  34. Herman JG, Merlo A, Mao L, Lapidus RG, Issa JP, Davidson NE, Sidransky D and Baylin SB . (1995). Cancer Res., 55, 4525–4530.

  35. Hesson L, Dallol A, Minna JD, Maher ER and Latif F . (2003). Oncogene, 22, 947–954.

  36. Huang Y, de la Chapelle A and Pellegata NS . (2003). Int. J. Cancer, 104, 735–744.

  37. Huebner K . (2001). Proc. Natl. Acad. Sci. USA, 98, 14763–14765.

  38. Jones PA . (1999). Trends Genet., 15, 34–37.

  39. Kawakami M, Staub J, Cliby W, Hartmann L, Smith DI and Shridhar V . (1999). Int. J. Oncol., 15, 715–720.

  40. Keller G, Vogelsang H, Becker I, Hutter J, Ott K, Candidus S, Grundei T, Becker KF, Mueller J, Siewert JR and Hofler H . (1999). Am. J. Pathol., 155, 337–342.

  41. Klamt B, Schulze M, Thate C, Mares J, Goetz P, Kodet R, Scheurlen W, Weirich A, Graf N and Gessler M . (1998). Gene Chromosome Cancer, 22, 287–294.

  42. Lee S and Haber DA . (2001). Exp. Cell Res., 264, 74–99.

  43. Lee SW . (1996). Nat. Med., 2, 776–782.

  44. Lo KW, Kwong J, Hui ABY, Chan SYY, To KF, Chan SC, Chow LSN, Teo PML, Johnson PJ and Huang DP . (2001). Cancer Res., 61, 3877–3881.

  45. Maruyama R, Toyooka S, Toyooka KO, Virmani AK, Zochbauer-Muller S, Farinas AJ, Minna JD, McConnell J, Frenkel EP and Gazdar AF . (2002). Clin. Cancer Res., 8, 514–519.

  46. Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, Wykoff CC, Pugh CW, Maher ER and Ratcliffe PJ . (1999). Nature, 399, 271–275.

  47. Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger, PC, Baylin SB and Sidransky D . (1995). Nat. Med., 1, 686–692.

  48. Morrissey C, Martinez A, Zatyka M, Agathanggelou A, Honorio S, Astuti D, Morgan NV, Moch H, Richards FM, Kishida T, Yao M, Schraml P, Latif F and Maher ER . (2001). Cancer Res., 61, 7277–7281.

  49. Nguyen A, Jing Z, Mahoney PS, Davis R, Sikka SC, Agrawal KC and Abdel-Mageed AB . (2000). Cancer Lett., 160, 133–140.

  50. Nojima D, Nakajima K, Li LC, Franks J, Ribeiro L, Ishii N and Dahiya R . (2001). Mol. Carcinogen., 32, 19–27.

  51. Richards FM, McKee SA, Rajpar MH, Cole TRP, Evans DGR, Jankowski JA, McKeown C, Sanders DSA and Maher ER . (1999). Hum. Mol. Genet., 8, 607–610.

  52. Robertson KD and Jones PA . (1998). Mol. Cell Biol., 18, 6457–6473.

  53. Sato M, Mori Y, Sakurada A, Fujimura S and Horii A . (1998). Hum. Genet., 103, 96–101.

  54. Schulz S, Becker KF, Braungart E, Reichmuth C, Klamt B, Becker I, Atkinson M, Gessler M and Hofler H . (2000). J. Pathol., 191, 162–169.

  55. Shivapurkar N, Toyooka S, Eby MT, Huang CX, Sathyanarayana UG, Cunningham HT, Reddy JL, Brambilla E, Takahashi T, Minna JD, Chaudhary PM and Gazdar AF . (2002). Cancer Biol. Ther., 1, 65–69.

  56. Skotnicka-Klonowicz G, Rieske P, Bartkowiak J, Szymik-Kantorowicz S, Daszkiewicz P and Debiec-Rychter M . (2000). Eur. J. Surg. Oncol., 26, 61–66.

  57. Steenman MJ, Rainier S, Dobry CJ, Grundy P, Horon IL and Feinberg AP . (1994). Nat. Genet., 7, 433–439.

  58. Storkel S and Vandenberg E . (1995). World J. Urol., 13, 153–158.

  59. Taniguchi T, Sullivan MJ, Ogawa O and Reeve AE . (1995). Proc. Natl. Acad. Sci. USA, 92, 2159–2163.

  60. Teitz T, Wei T, Valentine MB, Vanin EF, Grenet J, Valentine VA, Behm FG, Look AT, Lahti JM and Kidd VJ . (2000). Nat. Med., 6, 529–535.

  61. Tomlinson IP, Alam NA, Rowan AJ, Barclay E, Jaeger EE, Kelsell D, Leigh I, Gorman P, Lamlum H, Rahman S, Roylance RR, Olpin S, Bevan S, Barker K, Hearle N, Houlston RS, Kiuru M, Lehtonen R, Karhu A, Vilkki S, Laiho P, Eklund C, Vierimaa O, Aittomaki K, Hietala M, Sistonen P, Paetau A, Salovaara R, Herva R, Launonen V and Aaltonen LA . (2002). Nat. Genet., 30, 406–410.

  62. Toyooka S, Toyooka KO, Harada K, Miyajima K, Makarla P, Sathyanarayana UG, Yin J, Sato F, Shivapurkar N, Meltzer SJ and Gazdar AF . (2002b). Cancer Res., 62, 3382–3386.

  63. Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB and Issa JP . (1999). Proc. Natl. Acad. Sci. USA, 96, 8681–8686.

  64. Toyooka KO, Toyooka S, Maitra A, Feng Q, Kiviat NC, Smith A, Minna JD, Ashfaq R and Gazdar AF . (2002a). Am. J. Pathol., 161, 629–634.

  65. Toyooka KO, Toyooka S, Virmani AK, Sathyanarayana UG, Euhus DM, Gilcrease M, Minna JD and Gazdar AF . (2001). Cancer Res., 61, 4556–4560.

  66. Turner KJ, Moore JW, Jones A, Taylor CF, Cuthbert-Heavens D, Han C, Leek RD, Gatter KC, Maxwell PH, Ratcliffe PJ, Cranston D and Harris AL . (2002). Cancer Res., 62, 2957–2961.

  67. Tycko B . (2000). J. Clin. Invest., 105, 401–407.

  68. van der Velden PA, Metzelaar-Blok JAW, Bergman W, Hurks HMH, Frants RR, Gruis NA and Jager MJ . (2001). Cancer Res., 61, 5303–5306.

  69. van Engeland M, Roemen GM, Brink M, Pachen MM, Weijenberg MP, de Bruine AP, Arends JW, van den Brandt PA, de Goeij AF and Herman JG . (2002). Oncogene, 21, 3792–3795.

  70. Virmani AK, Rathi A, Zochbauer-Muller S, Sacchi N, Fukuyama Y, Bryant D, Maitra A, Heda S, Fong KM, Thunnissen F, Minna JD and Gazdar AF . (2000). J. Natl. Cancer Inst., 92, 1303–1307.

  71. Wagner KJ, Cooper WN, Grundy RG, Caldwell G, Jones C, Wadey RB, Morton D, Schofield PN, Reik W, Latif F and Maher ER . (2002). Oncogene, 21, 7277–7282.

  72. Wheeler JM, Kim HC, Efstathiou JA, Ilyas M, Mortensen NJ and Bodmer WF . (2001). Gut, 48, 367–371.

  73. Yamashita K, Upadhyay S, Osada M, Hoque MO, Xiao Y, Mori M, Sato F, Meltzer SJ and Sidransky D . (2002). Cancer Cell, 2, 485–495.

  74. Yoon JH, Dammann R and Pfeifer GP . (2001). Int. J. Cancer, 94, 212–217.

  75. Zbar B . (2000). Semin. Cancer Biol., 10, 313–318.

  76. Zochbauer-Muller S, Fong KM, Virmani AK, Geradts J, Gazdar AF and Minna JD . (2001). Cancer Res., 61, 249–255.

  77. Zuzak TJ, Steinhoff DF, Sutton LN, Phillips PC, Eggert A and Grotzer MA . (2002). Eur. J. Cancer, 38, 83–91.

Download references

Acknowledgements

We thank the Cancer Research UK, Association for International Cancer Research and West Midlands NHS Executive for financial support.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Eamonn R Maher.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Morris, M., Hesson, L., Wagner, K. et al. Multigene methylation analysis of Wilms' tumour and adult renal cell carcinoma. Oncogene 22, 6794–6801 (2003). https://doi.org/10.1038/sj.onc.1206914

Download citation

Keywords

  • Wilms' tumour
  • renal cell carcimona
  • methylation profile
  • epigenetics

Further reading

Search