HOP/OB1/NECC1 Promoter DNA Is Frequently Hypermethylated and Involved in Tumorigenic Ability in Esophageal Squamous Cell Carcinoma Keishi Yamashita, 1 Myoung Sook Kim, 1 Hannah Lui Park, 1 Yutaka Tokumaru, 1 Motonobu Osada, 1 Hiroshi Inoue, 2 Masaki Mori, 2 and David Sidransky 1 1 Department of Otolaryngology, Head and Neck Cancer Research Institute, Johns Hopkins University, Baltimore, Maryland and 2 Department of Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, Tsurumibaru, Beppu, Japan Abstract Promoter DNA hypermethylation with gene silencing is a common feature of human cancer, and cancer-prone methylation is believed to be a landmark of tumor suppressor genes (TSG). Identification of novel methylated genes would not only aid in the development of tumor markers but also elucidate the biological behavior of human cancers. We identified several epigenetically silenced candidate TSGs by pharmacologic unmasking of esophageal squamous cell carcinoma (ESCC) cell lines by demethylating agents (5-aza-2¶-deoxycitidine and trichostatin A) combined with ESCC expression profiles using expression microarray. HOP/OB1/NECC1 was identified as an epigenetically silenced candidate TSG and further examined for (a ) expression status, (b ) methylation status, and (c ) functional involvement in cancer cell lines. (a ) The HOP gene encodes two putative promoters (promoters A and B) associated with two open reading frames (HOPA and HOPB, respectively), and HOPA and HOPB were both down-regulated in ESCC independently. (b ) Promoter B harbors dense CpG islands, in which we found dense methylation in a cancer-prone manner (55% in tumor tissues by TaqMan methylation-specific PCR), whereas promoter A does not harbor CpG islands. HOPB silencing was associated with DNA methylation of promoter B in nine ESCC cell lines tested, and reactivated by optimal conditions of demethylating agents, whereas HOPA silencing was not reactivated by such treatments. Forced expression of HOP suppressed tumorigenesis in soft agar in four different squamous cell carcinoma cell lines. More convincingly, RNA interference knockdown of HOP in TE2 cells showed drastic restoration of the oncogenic phenotype. In conclusion, HOP is a putative TSG that harbors tumor inhibitory activity, and we for the first time showed that the final shutdown process of HOP expression is linked to promoter DNA hypermethylation under the double control of the discrete promoter regions in cancer. (Mol Cancer Res 2008;6(1):31–41) Introduction Gene silencing by DNA methylation synergistic with its associated histone deacetylation is recognized at the promoter region of tumor suppressor genes (TSG), resulting in shutdown of the gene expression in human cancer (1, 2). As a result, the oncogenic signal is thought to be more efficiently transduced to the downstream molecules involved in aspects of cancer progression such as invasion, angiogenesis, and metastasis. This hypothesis is supported by studies that showed that pharmacologic treatment of cell lines with demethylating- associated agents such as 5-aza-2¶-deoxycitidine (5-aza-dC) and/or trichostatin A results in marked inhibition of tumori- genesis or angiogenesis, suggesting an important role for epigenetic silencing of TSGs in cancer progression (3, 4). We previously described a novel method of pharmacologic unmasking followed by microarray, which enabled us to identify unknown methylated TSG candidates in esophageal squamous cell carcinoma (ESCC; ref. 5). Subsequently, we improved the method of gene screening to detect more frequent and cancer-prone methylation, resulting in the identification of genes that have potent tumor-suppressive activity or display a clinically aggressive phenotype when methylated (6-8). For example, NMDAR2B is hypermethylated in more than 90% of primary ESCCs, and its expression induced drastic apoptosis in ESCC cells (8). In the current study, we further advanced our search for novel methylated TSGs showing a critical role in tumorigenesis through a combination of analyzing our previous database with expression profiles of primary ESCC tissues (5), and for the first time identified the promoter hypermethylation of homeo- box only protein (HOP)/OB1/NECC1 in human cancer and its potential role as a TSG. Received 5/10/07; revised 8/9/07; accepted 8/15/07. 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. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Conflict of Interest: Under a licensing agreement between OncoMethylome Sciences, SA and the Johns Hopkins University, Dr. Sidransky is entitled to a share of royalty received by the University on sales of products described in this article. Dr. Sidransky owns OncoMethylome Sciences, SA stock, which is subject to certain restrictions under University policy. Dr. Sidransky is a paid consultant to OncoMethylome Sciences, SA and is a paid member of the company’s Scientific Advisory Board. The term of this arrangement is being managed by the Johns Hopkins University in accordance with its conflict of interest policies. Requests for reprints: David Sidransky, Department of Otolaryngology, Head and Neck Cancer Research Division, Johns Hopkins University, Baltimore, MD 21231. Phone: 410-502-5152; Fax: 410-614-1411. E-mail: dsidrans@jhmi.edu Copyright D 2008 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-07-0213 Mol Cancer Res 2008;6(1). January 2008 31 Research. on December 2, 2015. © 2008 American Association for Cancer mcr.aacrjournals.org Downloaded from