Human Cancer Biology Methylation Induced Gene Silencing of HtrA3 in Smoking-Related Lung Cancer Daniah Beleford 1 , Zhixue Liu 3 , Ramandeep Rattan 1 , Lucio Quagliuolo 4 , Mariarosaria Boccellino 4 , Alfonso Baldi 4 , Jacie Maguire 1 , Julie Staub 1 , Julian Molina 2 , and Viji Shridhar 1 Abstract Purpose: Some 85% of lung cancers are smoking related. Here, we investigate the role of serine pro- tease HtrA3 in smoking-related lung cancer. Experimental Design: We assess HtrA3 methylation and its corresponding expression in the human bronchial cell line BEAS-2B following cigarette smoke carcinogen treatment, in lung cancer cell lines and in primary lung tumors from light, moderate, and heavy smokers. We also show the effects of HtrA3 downregulation on MTT reduction and clonogenic survival with etoposide and cisplatin treatment and the corresponding effects of HtrA3 re-expression during treatment. Results: We show for the first time that HtrA3 expression is reduced or completely lost in over 50% of lung cancer cell lines and primary lung tumors from heavy smokers. Treatment of HtrA3-deficient cell lines with 5-aza-2′-deoxycytidine resulted in a dose-dependent increase in HtrA3 transcription. Further, sequence analysis of bisulfite-modified DNA from lung cancer cell lines and from primary lung tumors showed an increased frequency of methylation within the first exon of HtrA3 with a corresponding loss of HtrA3 expression, particularly in tumors from smokers. In BEAS-2B, treatment with the cigarette smoke carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone resulted in HtrA3 downregulation with a corresponding increase in methylation. Additional studies indicate resistance to etoposide and cisplatin cytotoxicity as a functional consequence of HtrA3 loss. Finally, immunohistochemical analysis of primary lung tumors revealed a strong correlation between low HtrA3 expression and heavy smoking history. Conclusions: Collectively, these results suggest that cigarette smoke–induced methylation of HtrA3 could contribute to the etiology of chemoresistant disease in smoking-related lung cancer. Clin Cancer Res; 16(2); 398–409. ©2010 AACR. Lung cancer is the leading cause of cancer-related deaths in the United States and worldwide (1, 2). Standard first line treatment for small-cell lung cancer (SCLC) includes platinum-based chemotherapy, generally with the DNA and protein cross-linking agent cis-platinum-(II)-diamine dichloride (cisplatin) or carboplatin plus etoposide, a to- poisomerase II inhibitor, with or without radiation (3–6). Treatment of non–small cell lung carcinoma may include surgical resection, radiotherapy, and/or platinum-based chemotherapy (7–10). Despite a promising initial response to therapy, however, some 86% of lung cancer patients still die within 5 years of diagnosis, many from chemoresistant disease (2, 11–14). This grim prognosis underscores the need to characterize not only those factors involved in lung cancer etiology and progression but also those that compro- mise treatment by promoting chemoresistance. Smoking increases the risk of all histologic types of lung cancer but is most strongly linked with small-cell lung can- cer and squamous cell carcinoma (15, 16). Among carci- nogens that have been isolated from cigarette smoke, the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK) is widely accepted as a major causative agent for lung cancer in smokers (17–19). NNK metabolites can interact directly with DNA, forming meth- yl adducts primarily in target tissues most affected by NNK carcinogenicity (17, 18). Further, of adenocarcinomas in- duced by NNK in rats, 94% showed gene silencing through promoter hypermethylation within p16 (20, 21). In humans, p16 silencing is associated with promoter hy- permethylation in non–small cell lung cancers (22). HtrA3 was first characterized as an upregulated factor during mouse uterus placentation (23). More recently, HtrA3 has been identified as a possible target for ciga- rette smoke–induced changes in normal human bronchi- al epithelial cells, showing dramatic transcriptional downregulation within 2 hours of condensed cigarette smoke exposure in contrast to the steady HtrA1 and Authors' Affiliations: Departments of 1 Experimental Pathology and 2 Medical Oncology, Mayo Clinic College of Medicine, Rochester, Minnesota; 3 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia; and 4 Department of Biochemistry, Second University of Naples, Naples, Italy Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Viji Shridhar, Department of Experimental Pa- thology, Stabile 2-47, 200 First Street Southwest, Rochester, MN 55905. Phone: 507-266-2775; Fax: 507-266-5193; E-mail: shridhar. vijayalakshmi@mayo.edu. doi: 10.1158/1078-0432.CCR-09-1677 ©2010 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 16(2) January 15, 2010 398 Research. on March 18, 2016. © 2010 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Published OnlineFirst January 12, 2010; DOI: 10.1158/1078-0432.CCR-09-1677