Oncogene (2003) 22, 7862–7865
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Epigenetic inactivation of RASSF1A candidate tumor suppressor gene at 3p21.3 in brain tumors Keishi Horiguchi*,1, Yoshio Tomizawa2, Masahiko Tosaka1, Shogo Ishiuchi1, Hideyuki Kurihara1, Masatomo Mori2 and Nobuhito Saito1 1 2
Department of Neurosurgery, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan; First Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan
The human Ras association domain family 1A (RASSF1A) gene, recently isolated from the lung and breast tumor suppressor locus 3p21.3, is highly methylated in primary lung, breast, nasopharyngeal and other tumors, and re-expression of RASSF1A suppresses the growth of several types of cancer cells. Epigenetic inactivation of RASSF1A by promoter hypermethylation is also important in the development of several human cancers. The methylation status of the promoter region of RASSF1A was analysed in primary brain tumors and glioma cell lines by methylation-specific polymerase chain reaction. In primary brain tumors, 25 of 46 (54.3%) gliomas and five of five (100%) medulloblastomas showed RASSF1A methylation. In benign tumors, only one of 10 (10%) schwannomas and two of 12 (16.7%) meningiomas showed RASSF1A methylation. The RASSF1A promoter region was methylated in all four glioma cell lines. RASSF1A was re-expressed in all methylated cell lines after treatment with the demethylating agent 5-aza-20 -deoxycytidine. Methylation of the promoter CpG islands of the RASSF1A may play an important role in the pathogenesis of glioma and medulloblastoma. Oncogene (2003) 22, 7862–7865. doi:10.1038/sj.onc.1207082 Keywords: 3p21.3; methylation; glioma; medulloblastoma; meningioma; schwannoma
Allelic loss of chromosome 3p frequently occurs during the development of several types of human cancer, including brain tumors (Kanno et al., 1997; Kok et al., 1997; Maitra et al., 2001; Zabarovsky et al., 2002). According to Knudsen’s hypothesis, two hits are required to inactivate both alleles of a tumor suppressor gene and promote tumorigenesis. These insults can occur by deletion, mutation or methylation (Jones and Laird, 1999). There is high incidence of loss of heterozygosity of chromosome 3p in gliomas (Fults et al., 1990; Kanno et al., 1997), suggesting that tumor suppressor genes in 3p, such as the von Hippel–Lindau (VHL) tumor suppressor gene, may cause tumorigenesis *Correspondence: K Horiguchi; E-mail:
[email protected] Received 12 December 2002; revised 30 July 2003; accepted 5 August 2003
of gliomas. However, since somatic sense mutation of the VHL tumor suppressor gene was detected in only two of 15 gliomas, chromosome 3p may contain some other tumor suppressor genes (Kanno et al., 1997). The RASSF1 gene is a candidate tumor suppressor gene that was isolated from the 120-kb region of the minimal homozygous deletion region at 3p21.3 in lung and breast cancers (Dammann et al., 2000; Vos et al., 2000; Yoon et al., 2001). The RASSF1 locus encodes several major transcripts by alternative promoter selection and alternative mRNA splicing. RASSF1A, one of these transcripts, encodes a predicted Mr 39 000 peptide with a Ras association domain and a predicted NH2terminal diacylglycerol-binding domain. Transfection and expression of RASSF1A in lung and renal carcinoma cells result in suppression of colony formation, anchorage-independent soft agar growth and nude mouse tumorigenicity (Dammann et al., 2000; Yoon et al., 2001; Dreijerink et al., 2001). RASSF1A is frequently inactivated in various cancers, including lung cancers, breast cancers, ovarian cancers, bladder carcinomas, nasopharyngeal cancers, renal cell cancers, clear cell-type renal cell cancers, gastric adenocarcinomas, malignant mesotheliomas, pheochromocytomas and neuroblastomas, by promoter hypermethylation (Agathanggelou et al., 2001; Astuti et al., 2001; Byun et al., 2001; Dreijerink et al., 2001; Lee et al., 2001; Lo et al., 2001; Morrissey et al., 2001; Toyooka et al., 2001; Harada et al., 2002; Tomizawa et al., 2002). However, the epigenetic inactivation of RASSF1A gene in brain tumors remains unknown. The present study investigated the methylation status of the promoter region of RASSF1A in common types of brain tumors, including various grades of glioma (World Health Organization (WHO) grades II and III, and glioblastomas), pilocytic astrocytoma (WHO grade I), medulloblastoma, meningioma and schwannoma. Intracranial gliomas, including astrocytic tumors, oligodendroglial tumors, glioblastoma and mixed gliomas, are related to tumor development (WHO grades II, III and IV). However, pilocytic astrocytoma is essentially benign (WHO grade I), so is a special case of glioma, and rarely transforms via a different route of malignant transformation from other primary gliomas. Medulloblastoma is a malignant, invasive embryonal tumor of the cerebellum with a preference for pediatric onset.
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Schwannoma and meningioma are typical benign extraaxial brain tumors, corresponding to WHO grade I, that originate from the neoplastic Schwann cells of the peripheral nerve and neoplastic meningothelial cells of the dura mater, respectively (Kleihues et al., 2000). The methylation status of the promoter region of RASSF1A was analysed in 73 primary brain tumors and four glioma cell lines by methylation-specific polymerase chain reaction (PCR). Representative results are shown in Figure 1. RASSF1A promoter methylation was found in 33 of 73 (45.2%) tumors. Details of the methylation frequency and the histological diagnosis are summarized in Table 1. Methylation of the RASSF1A promoter region was more common in grade II (6/8) and III (9/14) gliomas and glioblastomas (9/19) than in pilocytic astrocytomas (1/5). Methylation was very frequent in medulloblastomas (5/5), but much less common in schwannomas (1/10) and meningiomas (2/12). Additionally, sequencing of all exons of RASSF1A was performed in a subset of samples not exhibiting RASSF1A methylation. The individual six exons of RASSF1A were PCR amplified by previously reported RASSF1A-specific oligonucleotide primers (Lo et al., 2001; Lusher et al., 2002), and then directly sequenced. Mutations of the RASSF1A gene were not detected in all samples. The correlation between RASSF1A transcriptional silencing and hypermethylation of the respective promoter was investigated by methylation-specific PCR analysis of the promoter region of RASSF1A in four glioma cell lines. All four human glioma cell lines (U251, A172, T98G and CGNH-89) had methylated alleles and lacked an unmethylated allele of RASSF1A. The expression status of RASSF1A was assessed in these four glioma cell lines. All four glioma cell lines showed loss of RASSF1A expression (Figure 2). The levels of the shorter RASSF1 isoform C and the glyceraldehyde-3phosphate-dehydrogenase (GAPDH) gene, which are widely expressed and not susceptible to methylation (Burbee et al., 2001; Yoon et al., 2001), were also assessed as controls. RASSF1C and GAPDH were detectable in all four cell lines. To confirm that promoter hypermethylation causes the lack of expression of RASSF1A in the glioma cell lines, the effect of demethylating agent 5-aza-20 -deoxycytidine on RASSF1A expression was investigated. RASSF1A expression was restored in all four cell lines after treatment with 5-aza-20 -deoxycytidine (Figure 2). In contrast, almost no detectable changes in the expression of GAPDH or RASSF1C were found. The present study shows that the candidate tumor suppressor gene, RASSF1A, is methylated in primary brain tumors, especially gliomas and medulloblastomas. Primary gliomas (grades II and III, and glioblastomas) have highly methylated alleles at the CpG island of RASSF1A (Table 1). Medulloblastomas also had highly methylated alleles in this region. In contrast, pilocytic astrocytomas, schwannomas and meningiomas only had methylated alleles in a few cases. Methylated RASSF1A alleles were also found in four of four glioma cell lines (Figure 1). RASSF1A expression was absent in the
Figure 1 Methylation-specific PCR of the RASSF1A CpG island in brain tumors. Representative samples are shown. For resected primary brain tumors, U ¼ results with primers specific for unmethylated sequences; M ¼ results with primers specific for methylated sequences. A total of 73 primary brain tumors were obtained by surgical resection in Gunma University Hospital (Maebashi, Japan) including 19 glioblastomas, five anaplastic astrocytomas, five astrocytomas, five pilocytic astrocytomas, four anaplastic oligoastrocytomas, five anaplastic oligodendrogliomas, two oligodendrogliomas, one oligoastrocytoma, five medulloblastomas, 12 meningiomas and 10 schwannomas. Four human glioma and glioblastoma cell lines, U251 (RCB 0270; Riken Cell Bank, Tsukuba, Japan), T98G (ATCC CRL 1690; American Type Culture Collection, Manassas, VA, USA), A172 (the Japanese Cancer Research Resources Bank, Tokyo, Japan) and CGNH-89 (Ishiuchi et al., 2002), were also analysed. Genomic DNA was extracted from frozen stored primary tumors and cell lines by QIAamp DNA Mini Kit (Qiagen, Tokyo, Japan). For primary brain tumors and cell lines, the methylation status of the RASSF1A was analysed by methylation-specific PCR, as described previously (Burbee et al., 2001; Tomizawa et al., 2002), using bisulfitemodified genomic DNA (Herman et al., 1996). Briefly, 1 mg of genomic DNA was denatured with 0.2 M NaOH, and 10 mM hydroquinone (Sigma Chemical Co, St Louis, MO, USA) and 3 M sodium-bisulfite (Sigma Chemical Co.) were added and incubated at 551C for 16 h. The modified DNA was purified using DNA purification resin (Promega, Madison, WI, USA) followed by ethanol precipitation. Treatment of genomic DNA with sodium bisulfite converts unmethylated cytosines (but not methylated cytosines) to uracil, which are then converted to thymidine during the subsequent PCR step, giving sequence differences for methylated and unmethylated DNA. PCR primers that distinguish between these methylated and unmethylated DNA sequence were described previously (Burbee et al., 2001). The PCR products were analysed on 2% agarose gels, visualized under UV illumination and photographed. The PCR reactions for all samples were repeated to confirm these results. Two human non-small-cell lung carcinoma cell lines, NCI-H 1299 and NCI-H2009, of which the RASSF1A methylation status was previously characterized, were simultaneously assayed as methylated and unmethylated controls, respectively (Burbee et al., 2001)
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RASSF1A methylation in brain tumors K Horiguchi et al
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Histological gradea
Glioma
Medulloblastoma Schwannoma Meningioma
No. of samples
IV III
Glioblastoma Anaplastic astrocytoma Anaplastic oligoastrocytoma Anaplastic oligodendroglioma
II
Astrocytoma Fibrillary astrocytoma Oligoastrocytoma Oligodendroglioma
I IV I I
Pilocytic astrocytoma
19 5 4 5 Total 14 3 2 1 2 Total 8 5 5 10 12
No. of methylated alleles (%) 9 3 2 4 9 3 1 1 1 6 1 5 1 2
(47%) (60%) (50%) (80%) (64%) (100%) (50%) (100%) (50%) (75%) (20%) (100%) (10%) (17%)
a
Based on the WHO classification
Figure 2 Expression of mRNA of RASSF1A and RASSF1C before and after 5-aza-20 -deoxycytidine (5Aza-CdR) treatment in glioma cell lines (T98G, A172, U251 and CGNH-89). The four cell lines, which contained methylated alleles and lacked an unmethylated allele of RASSF1A, were grown in the presence ( þ lanes) and absence (lanes) of 10 mM 5Aza-CdR for 4 days. Total RNA was isolated, complementary DNA was prepared, and isoform-specific reverse transcription–PCR (RT–PCR) was performed for RASSF1A, with RASSF1C and GAPDH as controls. Total RNA was isolated from the four glioma cell lines with the RNeasy Extraction kit (Qiagen, Tokyo, Japan). RT–PCR assays were used for analysis of RASSF1A and RASSF1C expression. Primers for RASSF1A and RASSF1C were described previously (Burbee et al., 2001). PCR was performed using a touch down method. Two human cell lines, NCI-H2009 and H1299, of which the RASSF1 expression status was previously characterized, were simultaneously assayed as expression and nonexpression controls, respectively (Burbee et al., 2001). GAPDH, a housekeeping gene, was used as a control for RNA integrity. Primers for GAPDH were described previously (Mollerup et al., 1999). RT–PCR products (5 ml) were resolved on 2% agarose gels, stained with ethidium bromide and visualized under UV illumination
presence of a methylated allele in these four cell lines, and was restored by 5-aza-20 -deoxycytidine treatment (Figure 2). Tumor progression and transformation of low-grade astrocytoma into high-grade glioma is a well-known clinical observation (Maher et al., 2001). Chromosome 3p allele loss is considered the early event in glioma Oncogene
progression, because it is detected more frequently in low-grade than in high-grade gliomas (Kanno et al., 1997). In our study, RASSF1A methylation was more frequently detected in grade II and III gliomas than in glioblastomas. The relatively high methylation frequency of the RASSF1A promoter region in grade II and III gliomas may correspond to an early event in the progression of gliomas. Glioblastoma is thought to occur as primary or de novo glioblastoma and secondary glioblastoma (Maher et al., 2001). All our cases had a histological diagnosis of glioblastoma at first biopsy, with a relatively short clinical history and no evidence of a less malignant precursor lesion, suggesting that these cases were primary or de novo glioblastoma. Therefore, the loss of RASSF1A might be less involved in the formation of de novo glioblastoma than in the formation of secondary glioblastoma. Methylation of the promoter CpG islands of RASSF1A may be important in the formation and progression of gliomas. Medulloblastoma, a representative malignant brain tumor, showed frequent disruption at chromosome 3p by a deletion mapping study, suggesting a novel tumor suppressor gene in this region (Avet-Loiseau et al., 1999; Bayani et al., 2000). Recently, hypermethylation of the RASSF1A promoter region in medulloblastoma has been reported (Harada et al., 2002; Lusher et al., 2002). Medulloblastoma has the highest RASSF1A methylation frequencies (88%) among various systemic pediatric tumors. In the present study, all five medulloblastomas showed methylation in the promoter region of RASSF1A. These results indicate a strong relationship between the inactivation of RASSF1A by promoter methylation and the pathogenesis of medulloblastoma. Methylation of the RASSF1A promoter region in pilocytic astrocytoma, a special type of benign glioma, was clearly rare in our specimens. Methylated alleles of the RASSF1A gene were detected in only a few cases of schwannomas and meningiomas. These results suggest that methylated RASSF1A is not common in benign intracranial tumors, including pilocytic astrocytoma.
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Epigenetic inactivation of RASSF1A may play an important role in the pathogenesis of glioma and medulloblastoma. Additional investigations are required to elucidate the importance of RASSF1A methylation as a pathological diagnostic marker in the management of gliomas and medulloblastomas.
Acknowledgements We thank Ms Mitsue Maniwa and Ms Mayumi Umemura for technical assistance. This study was partly supported by grants from the Ministry of Education, Science, Sports and Culture, Japan.
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