Bone morphogenetic protein 3B silencing in non-small-cell lung cancer Zunyan Dai 1,2 , Anthony P Popkie 1,3 , Wei-Guo Zhu 4 , Cynthia D Timmers 1 , Aparna Raval 1 , Sarah Tannehill-Gregg 5 , Carl D Morrison 1,2 , Herbert Auer 1 , Robert A Kratzke 6 , Gloria Niehans 6 , Stefan Amatschek 7 , Wolfgang Sommergruber 8 , Gustavo W Leone 1,3 , Thomas Rosol 5 , Gregory A Otterson 4 and Christoph Plass* ,1 1 Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210, USA; 2 Department of Pathology, The Ohio State University, Columbus, OH 43210, USA; 3 Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA; 4 Division of Hematology/Oncology, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA; 5 Department of Veterinary Biosciences and the Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA; 6 Minneapolis VA Medical Center, 1 Veterans Drive, 111E, Minneapolis, MN 55417, USA; 7 Department of Dermatology, University of Vienna, Vienna 1090, Austria; 8 Boehringer Ingelheim Vienna 1120, Austria Bone morphogenetic protein 3B (BMP3B) is a member of the TGF-b superfamily. The BMP3B promoter sequence was previously identified as a target for aberrant DNA methylation in non-small-cell lung cancer (NSCLC). Aberrant DNA hypermethylation in the BMP3B promo- ter is associated with downregulation of BMP3B tran- scription in both primary human lung cancers as well as lung cancer cell lines. In order to understand the mechanisms of BMP3B silencing in lung cancer, a sample set of 91 primary NSCLCs was used to detect aberrant BMP3B promoter methylation, mutations in the coding sequence of BMP3B, and loss of heterozygosity (LOH). Our results showed that 45 of 91 (or 49.5%) tested primary NSCLCs exhibited increased promoter methyla- tion, and 40% demonstrated LOH in at least one of the flanking microsatellite markers sJRH and D10S196 (63 kb upstream or 3.338 Mbp downstream of BMP3B). The lung cancer cell line A549, a type II alveolar epithelial human lung cancer cell line, is characterized by aberrant DNA promoter methylation. We used retroviral vector constructs containing the BMP3B cDNA to re-express the gene in A549 cells and to investigate the effects on cell growth. No change in the cell growth rate was observed after BMP3B re-expression, as compared to the vector controls. Although the number of colonies formed in anchorage-dependent assays was only slightly decreased, the colony-forming ability of A549 cells after BMP3B expression in anchorage-independent assays in soft agar was significantly reduced to 10% (Po0.005, t-test). Moreover, the in vivo tumorigenicity assay in nude mice indicated that cells re-expressing BMP3B grew signifi- cantly slower than cells not expressing BMP3B (Po0.05, t-test). In conclusion, this study provides evidence that BMP3B expression is repressed by different mechanisms in lung cancer, and that the silencing of BMP3B promotes lung tumor development. Oncogene (2004) 23, 3521–3529; doi:10.1038/sj.onc.1207441 Keywords: bone morphogenetic protein 3B (BMP3B); DNA methylation; non-small-cell lung cancer Introduction DNA methylation of CpG islands in the promoter regions of genes has been correlated with silencing of tumor suppressor genes (Baylin et al., 1998; Plass and Soloway, 2002; Smiraglia and Plass, 2002). Genome scans for aberrant DNA methylation have shown that up to 10% of CpG islands can be methylated in human malignancies (Costello et al., 2000). Silencing of genes by DNA hypermethylation can be the sole mechanism of gene inactivation, or can cooperate with genetic mechanisms (including large deletions or other intra- genic mutations) to inactivate putative tumor suppressor genes in tumors. Aberrant promoter methylation con- stitutes an equal mechanism to gene silencing in tumor development. However, since DNA methylation is a reversible event, whereas genetic alterations are not, there might be fundamental differences in those genes that are silenced primarily or exclusively by genetic events compared with those that are silenced principally by DNA methylation. Only a small number of genes that are predominantly inactivated by promoter methy- lation have been identified thus far. These genes were either identified in a candidate cancer gene approach, or in genome scans using AP-PCR (Liang et al., 2002), MDA (Toyota and Issa, 2002) or restriction landmark genomic scanning (RLGS) (Costello et al., 2000). Examples include GSTP1 in prostate cancer (Lee et al., 1994), PAX6 (Liang et al., 2002) and Versican (Toyota et al., 1999). RASSF1A is a lung tumor suppressor gene that was identified based on aberrant promoter DNA Received 11 September 2003; revised 5 December 2003; accepted 10 December 2003 *Correspondence: C Plass, Division of Human Cancer Genetics, Medical Research Facility 494A, The Ohio State University, 420 West 12th Ave., Columbus, OH 43210, USA; E-mail: plass-1@medctr.osu.edu Oncogene (2004) 23, 3521–3529 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc