Promoter Hypermethylation of RASSF1A in Esophageal Squamous Cell Carcinoma 1 Tamotsu Kuroki, Francesco Trapasso, Sai Yendamuri, Ayumi Matsuyama, Hansjuerg Alder, Masaki Mori, and Carlo M. Croce 2 Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, Pennsylvania 19107 [T. K., F. T., S. Y., A. M., H. A., C. M. C.], and Department of Surgery, Medical Institute of Bioregulation, Kyushu University, Beppu 874-0838, Japan [M. M.] ABSTRACT Purpose: The RAS association domain family 1A (RASSF1A) gene, a candidate tumor suppressor gene, is frequently inactivated by hypermethylation of its promoter region in several human cancers. The aim of this study was to evaluate the promoter methylation status of the RASSF1A in esophageal squamous cell carcinoma. Experimental Design: We analyzed the methylation sta- tus of RASSF1A promoter by methylation-specific PCR in 23 esophageal squamous cell carcinoma cell lines and 48 primary tumors. Results: Hypermethylation of RASSF1A was found in 74% of cell lines and 52% of primary tumors. The presence of hypermethylation was statistically associated with loss of RASSF1A mRNA expression in both cell lines (P  0.007) and primary tumors (P  0.003). There was a statistically significant correlation between the presence of hypermethy- lation and tumor stage (P  0.009). Conclusions: Our findings suggest that epigenetic si- lencing of RASSF1A gene expression by promoter hyper- methylation could play an important role in primary esoph- ageal squamous cell carcinogenesis. INTRODUCTION The genesis of human cancers, including those of the esophagus, is a multistep process involving cumulative genetic alterations that include activation of oncogenes or inactivation of tumor suppressor genes. Although multiple genetic and epi- genetic changes have been detected in esophageal squamous cell carcinoma (1–3), the precise molecular mechanisms of devel- opment and/or progression of esophageal squamous cell carci- noma still remain unknown. Examples of genetic alterations involving homozygous de- letions and LOH 3 of chromosome 3p have been reported in many different human tumors, including esophageal cancer (4, 5). The fragile histidine triad gene, located at 3p14.2, is a tumor suppressor gene that is predominantly altered by deletions, and it plays an important role in the development of esophageal cancers with exposure to environmental carcinogens (1, 6, 7). LOH analyses revealed that 3p21.3 is another region commonly deleted in several cancers (8, 9). Furthermore, we recently reported abnormalities in the WWOX gene, a putative tumor suppressor gene, involving LOH and point mutation (3). Re- cently, RASSF1A has been proposed as a candidate tumor suppressor gene, isolated within the minimal homozygous de- letion at 3p21.3 in lung cancer (10). Recent studies have shown that the hypermethylation of CpG island into regulatory se- quences is another mechanism that could account for silencing genes involved in cancer (11–13). The most recurrent mecha- nism of inactivation of RASSF1A is the hypermethylation of a CpG island in its promoter sequences as reported in lung, breast, prostate, and kidney cancers (14 –16). To investigate whether the promoter hypermethylation of RASSF1A could play a role in human esophageal tumorigenesis, we performed a MSP on esophageal primary tumors and cell lines. We investigated the frequency and potential clinical implications of hypermethyla- tion of RASSF1A promoter in human esophageal squamous cell carcinomas. MATERIALS AND METHODS Cell Lines and Tissues. Twenty-three human esopha- geal carcinoma cell lines and 13 TE series were provided by the Cell Resource Center for Biomedical Research Institute of De- velopment, Aging and Cancer at Tohoku University (Tohoku, Japan), and 10 KYSE series were gifts from Dr. Shimada of Kyoto University (Kyoto, Japan). The tumor samples and their corresponding noncancerous tissues were obtained from 48 Jap- anese patients who underwent surgery for esophageal squamous cell carcinoma (45 male and 3 female; median age, 60.7 years; range, 48 –74 years). Four of them were stage I, 5 were stage II, 37 were stage III, and 2 were stage IV, according to the Tumor- Node-Metastasis classification. DNA and RNA Extraction. The tissue samples were excised and immediately stored at -80°C. DNA and RNA were Received 9/12/02; revised 12/3/02; accepted 12/5/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by USPHS Grants CA39860, CA56036, CA77738, and CA83698 from the National Cancer Institute. 2 To whom requests for reprints should be addressed, at Kimmel Cancer Institute, Jefferson Medical College, Thomas Jefferson University, BLSB Room 1050, 233S 10th Street, Philadelphia, PA 19107-5799. Phone: (215) 503-4645; Fax: (215) 923-3528; E-mail:carlo.croce@mail. tju.edu. 3 The abbreviations used are: LOH, loss of heterozygosity; RASSF1A, RAS association domain family 1A; MSP, methylation-specific PCR; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phos- phate dehydrogenase. 1441 Vol. 9, 1441–1445, April 2003 Clinical Cancer Research Cancer Research. on December 11, 2021. © 2003 American Association for clincancerres.aacrjournals.org Downloaded from