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The Cpg Island of the Novel Tumor Suppressor Gene RASSF1A Is Intensely Methylated in Primary Small Cell Lung Carcinomas

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The Cpg Island of the Novel Tumor Suppressor Gene RASSF1A Is Intensely Methylated in Primary Small Cell Lung Carcinomas Oncogene (2001) 20, 3563 ± 3567 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc SHORT REPORTS The CpG island of the novel tumor suppressor gene RASSF1A is intensely methylated in primary small cell lung carcinomas Reinhard Dammann1, Takashi Takahashi2 and Gerd P Pfeifer*,1 1Department of Biology, Beckman Research Institute, City of Hope Cancer Center, Duarte, California 91010, USA; 2Division of Molecular Oncology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan Loss of heterozygosity at 3p21.3 occurs in more than genes include the RB gene (13q), the p53 gene (17p), 90% of small cell lung carcinomas (SCLCs). The Ras and the p16 CDK inhibitor gene (9p). association domain family 1 (RASSF1) gene cloned from Allelic loss at 3p21 is an early event in lung tumor the lung tumor suppressor locus 3p21.3 consists of two pathogenesis, occurs at the stage of hyperplasia/ major alternative transcripts, RASSF1A and RASSF1C. metaplasia (Sundaresan et al., 1992; Hung et al., Epigenetic inactivation of isoform A (RASSF1A) was 1995; Thiberville et al., 1995; Wistuba et al., 1999), observed in 40% of primary non-small cell lung and as such may be critically involved in tumor carcinomas and in several tumor cell lines. Transfection initiation. Cross-sectional examination of individual of RASSF1A suppressed the growth of lung cancer cells NSCLC tumors showed that all portions of the tumor in vitro and in nude mice. Here we have analysed the shared concordant LOH at 3p despite morphological methylation status of the CpG island promoters of diversity (Yatabe et al., 2000). RASSF1A and RASSF1C in primary SCLCs. In 22 of Homozygous deletions at 3p21.3 have been described 28 SCLCs (=79%) the promoter of RASSF1A was in several cancer cell lines and in primary lung tumors highly methylated at all CpG sites analysed. None of the (Killary et al., 1992; Yamakawa et al., 1993; Wei et al., SCLCs showed evidence for methylation of the CpG 1996; Kok et al., 1997; Todd et al., 1997). Recently, a island of RASSF1C. The results suggest that hyper- region of minimum homozygous deletion was nar- methylation of the CpG island promoter of the rowed to 120 kb using several lung cancer and a breast RASSF1A gene is associated with SCLC pathogenesis. cancer cell line (Sekido et al., 1998). Until now, several Oncogene (2001) 20, 3563 ± 3567. genes located in the 3p21.3-deleted chromosomal region have been isolated as candidates for tumor Keywords: RASSF1; methylation; CpG island; tumor suppressor genes. However, none of the genes located suppressor gene; small cell lung cancer; 3p21 within the 3p21.3 homozygous deletion area was found to be mutated in more than 5 ± 10% of lung tumors (Lerman and Minna, 2000). This supports the assump- Introduction tion that the putative 3p21.3 tumor suppressor gene is inactivated by mechanisms other than mutation of the Lung cancer currently is the leading cause of cancer coding sequence. death in the United States with more than 150 000 Transcriptional silencing by hypermethylation of deaths annually attributed to this disease (Wingo et al., CpG islands containing the promoter regions of tumor 1999). Lung cancers are broadly classi®ed into two suppressor genes is becoming recognized as a common groups: small cell lung cancer (SCLC) and non-small mechanism (Baylin et al., 1998; Jones and Laird, 1999; cell lung cancer (NSCLC), the latter being divided into Baylin and Herman, 2000). Recent studies have the major histological subtypes adenocarcinoma, demonstrated that the CpG islands in the RB, p16, squamous cell carcinoma, and large cell carcinoma. VHL, APC, MLH1,andBRCA1 genes are frequently Losses of heterozygosity (LOH) on chromosome arms methylated in a variety of human cancers, but are 3p, 9p, 13q and 17p are among the most frequent usually methylation-free in the corresponding normal alterations in lung tumors (Whang-Peng et al., 1982; tissues (Baylin et al., 1998; Jones and Laird, 1999; Yokota et al., 1987; Naylor et al., 1987; Hibi et al., Baylin and Herman, 2000). CpG islands that are 1992; Kohno and Yokota, 1999; Girard et al., 2000). hypermethylated in lung cancer include those of the However, only a limited number of tumor suppressor p16 gene (Shapiro et al., 1995; Otterson et al., 1995; genes have been identi®ed as potential targets for Merlo et al., 1995; Belinsky et al., 1998), the DNA chromosomal deletion events in these tumors. These repair gene MGMT (Palmisano et al., 2000), the death- associated protein kinase (Tang et al., 2000), and DMBT1, a candidate tumor suppressor gene located at 10q25.3 ± 26.1 (Wu et al., 1999). Inactivation of tumor *Correspondence: GP Pfeifer Received 5 January 2001; revised 27 February 2001; accepted 15 suppressor genes due to hypermethylation may play an March 2001 important role in carcinogenesis. RASSF1A methylation in lung cancer R Dammann et al 3564 In previous work we have cloned and characterized the Ras association domain family 1A gene (RASS- F1A) located within the 120 kb 3p21.3 minimal homozygous deletion region and found that this gene is epigenetically inactivated in 40% of primary non- small cell lung cancers (Dammann et al., 2000). The RASSF1 protein is 55% homologous to the mamma- lian Ras eector Nore1 (Vavvas et al., 1998). Re- expression of RASSF1A in A549 lung cancer cells reduced colony formation, suppressed anchorage-in- dependent growth and inhibited tumor formation in nude mice (Dammann et al., 2000). In the present study we have investigated the methylation status of the RASSF1A gene in primary SCLC samples. Previously, we have observed that all 17 of the 17 analysed SCLC cell lines had lost the expression of RASSF1A and silencing of RASSF1A correlated with hypermethylation of the CpG island promoter sequence (Dammann et al., 2000). However, cell lines are known to have unusually high frequencies of CpG island methylation (Antequera et al., 1990; Jones et al., 1990), which makes an analysis of primary tumors a necessity. To analyse the RASSF1 gene in primary SCLCs, we determined the methylation status of the two CpG islands, in which the alternative RASSF1A and RASSF1C transcripts initiate. We used bisul®te sequencing of genomic DNA (Clark et al., 1994) to determine the methylation status of CpG dinucleo- tides. We analysed 16 CpGs in a 205 bp fragment containing three Sp1 consensus binding sites and the putative transcription and translation initiation sites of RASSF1A. All guanines present after bisul®te sequen- cing in Figure 1 are derived from methylated cytosines on the complementary strand. In Figure 1, one SCLC cell line (H345) shows complete methylation of the Figure 1 Methylation analysis of the RASSF1A gene in primary small cell lung carcinomas. Sequences of PCR products from sequence analysed. Further, we analysed the methyla- bisul®te-treated DNA for the CpG island spanning the RASSF1A tion status of the RASSF1A promoter in 28 primary promoter were obtained from dierent cancer and normal non-microdissected SCLC samples. Of the 28 samples samples. Methylated cytosines appear as a G signal in the analysed, 22 (=79%) were methylated in the complementary strand (bold Gs). The sequence is shown before and after bisul®te conversion at the top. The position of a TaqI promoter region of RASSF1A. Five matching normal restriction site is underlined. Two Sp1 consensus sites are boxed. tissue samples were available. Several examples, The data shown were obtained by direct sequencing of PCR including two matching tumor and normal tissues, products from bisul®te-treated DNA or from PCR products are shown in Figure 1. One of the normal tissue subcloned into plasmids (two primary tumors and matched samples was methylated, but to a lower degree than normal tissue controls). Small cell lung carcinomas and the corresponding normal lung tissues were obtained from the Aichi the corresponding tumor (25% versus 80%). The Cancer Center Research Institute and the City of Hope National other four normal matching tissue samples were Medical Center. DNA was isolated from tumors, and the unmethylated. In Figure 1, ®ve methylated primary methylation status of the RASSF1A promoter region was small cell lung carcinomas are shown. We estimated determined by a bisul®te genomic sequencing protocol (Clark et al., 1994; Dammann et al., 2000). DNA sequences were ampli®ed from the sequence scans of the uncloned PCR by mixing 100 ng of bisul®te-treated DNA with primers MU379 products that the tumor samples SC-1, SC-3, and (5'-GTTTTGGTAGTTTAATGAGTTTAGGTTTTTT) and SC-7 showed more than 80% bisul®te modi®cation- ML730 (5'-ACCCTCTTCCTCTAACACAATAAAACTAACC) resistant cytosines at all CpGs analysed. In total, more in 100 mMl reaction buer containing 200 mM of each dNTP and than 75% of the SCLC samples that were methylated Taq polymerase (Roche Diagnostics Corp.; Indianapolis, IN, USA) and incubating at 958C for 15 s, 558C for 15 s and 748C for contained more than 60% bisul®te-resistant cytosines 30 s, for 20 cycles. A semi-nested PCR was performed using 1/50 at all CpG sequences. This high level of methylation of the initially ampli®ed products and an internal primer ML561 indicates that a large fraction of the tumor cell (5'-CCCCACAATCCCTACACCCAAAT) and primer MU379 population has the promoter of RASSF1A methylated. with similar conditions as described for the preceding PCR ampli®cation, but for 30 cycles. PCR products were initially Since LOH at 3p21.3 occurs in almost 100% of all sequenced directly to obtain average methylation levels and were SCLC tumors (Whang-Peng et al., 1982; Naylor et al., then subcloned for con®rmation 1987; Hibi et al., 1992; Kok et al., 1997; Wistuba et Oncogene RASSF1A methylation in lung cancer R Dammann et al 3565 al., 2000; Girard et al., 2000; Lindblad-Toh et al., 2000), the remaining allele is silenced by methylation.
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