Aberrant methylation of multiple genes in the upper aerodigestive tract epithelium of heavy smokers

An important method for silencing tumor suppressor genes in cancers is by aberrant methylation (referred to as methylation) of CpG islands in gene promoter regions. In lung cancer, methylation of the genes retinoic acid receptor β‐2 (RARβ‐2), CDH13 (H‐cadherin), p16INK4a (p16), RASSF1A (RAS association domain family I) is frequent. Thus, we investigated methylation of these genes in 4 different types of specimens (oropharyngeal brushes, sputum samples, bronchial brushes and bronchioloalveolar lavage [BAL] samples) of the upper aerodigestive tract epithelium from heavy smokers without evidence of cancer but with morphometric evidence of sputum atypia and compared the frequencies of methylation in the different types of specimens. In addition, we also analyzed sputum samples from 30 never smokers for methylation of these genes. Our major findings are: (i) At least one gene was methylated in one or more specimens from 48% of the smokers. However, methylation was statistically significant less frequently in never smokers compared to smokers. (ii) In general, methylation occurred more frequently in samples from the central airways (sputum, bronchial brushes) compared to the peripheral airways (BAL) and only occasionally in the oropharynx. (iii) RARβ‐2 was the most frequently methylated gene, whereas the frequency of methylation for the other genes was lower. (iv) Data from sputum samples and bronchial brushes were comparable. Our findings suggest that detection of methylation should be investigated as an intermediate marker for lung cancer risk assessment and response to chemopreventive regimens. © 2003 Wiley‐Liss, Inc.

[1]  J S Lee,et al.  Clonal genetic alterations in the lungs of current and former smokers. , 1997, Journal of the National Cancer Institute.

[2]  Diane D. Liu,et al.  Aberrant promoter methylation of multiple genes in bronchial brush samples from former cigarette smokers. , 2002, Cancer research.

[3]  J. Herman,et al.  Predicting lung cancer by detecting aberrant promoter methylation in sputum. , 2000, Cancer research.

[4]  J. Samet,et al.  Molecular damage in the bronchial epithelium of current and former smokers. , 1997, Journal of the National Cancer Institute.

[5]  S. Lam,et al.  High resolution chromosome 3p allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele loss and three regions of frequent breakpoints. , 2000, Cancer research.

[6]  W. Travis,et al.  United States lung carcinoma incidence trends: Declining for most histologic types among males, increasing among females , 1996, Cancer.

[7]  J. Herman,et al.  Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. , 1999, Cancer research.

[8]  Peter A. Jones,et al.  The fundamental role of epigenetic events in cancer , 2002, Nature Reviews Genetics.

[9]  A. Kraus,et al.  Differential frequencies of p16(INK4a) promoter hypermethylation, p53 mutation, and K-ras mutation in exfoliative material mark the development of lung cancer in symptomatic chronic smokers. , 2000, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[10]  J. Minna,et al.  Molecular detection of early lung cancer. , 1999, Journal of the National Cancer Institute.

[11]  J. Minna,et al.  DNA methylation profiles of lung tumors. , 2001, Molecular cancer therapeutics.

[12]  J. Minna,et al.  Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. , 2001, Journal of the National Cancer Institute.

[13]  Y. Cormier,et al.  Placebo-controlled trial of 13-cis-retinoic acid activity on retinoic acid receptor-beta expression in a population at high risk: implications for chemoprevention of lung cancer. , 1999, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[14]  P. Rubin,et al.  Cancer of the head and neck. , 1972, JAMA.

[15]  R. Lotan,et al.  Suppression of retinoic acid receptor beta in non-small-cell lung cancer in vivo: implications for lung cancer development. , 1997, Journal of the National Cancer Institute.

[16]  J. Minna,et al.  5' CpG island methylation of the FHIT gene is correlated with loss of gene expression in lung and breast cancer. , 2001, Cancer research.

[17]  S. Lippman,et al.  Nuclear retinoid acid receptor beta in bronchial epithelium of smokers before and during chemoprevention. , 1999, Journal of the National Cancer Institute.

[18]  J. Herman,et al.  Alterations in DNA methylation: a fundamental aspect of neoplasia. , 1998, Advances in cancer research.

[19]  D. Slaughter,et al.  “Field cancerization” in oral stratified squamous epithelium. Clinical implications of multicentric origin , 1953, Cancer.

[20]  A Coldman,et al.  Localization of bronchial intraepithelial neoplastic lesions by fluorescence bronchoscopy. , 1998, Chest.

[21]  J. Minna,et al.  Aberrant promoter methylation of multiple genes in non-small cell lung cancers. , 2001, Cancer research.

[22]  G. Pfeifer,et al.  The CpG island of the novel tumor suppressor gene RASSF1A is intensely methylated in primary small cell lung carcinomas , 2001, Oncogene.

[23]  Chun Xing Li,et al.  Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3 , 2000, Nature Genetics.

[24]  J. Herman,et al.  Gene promoter hypermethylation in tumors and serum of head and neck cancer patients. , 2000, Cancer research.

[25]  B Palcic,et al.  Detection and localization of early lung cancer by fluorescence bronchoscopy , 2000, Cancer.

[26]  A. Gazdar,et al.  Aberrant methylation during cervical carcinogenesis. , 2001, Clinical cancer research : an official journal of the American Association for Cancer Research.

[27]  D. Hürzeler.  Fluorescence Bronchoscopy , 1982, Definitions.

[28]  J. Herman,et al.  A gene hypermethylation profile of human cancer. , 2001, Cancer research.

[29]  Richard Doll,et al.  Smoking, smoking cessation, and lung cancer in the UK since 1950: combination of national statistics with two case-control studies , 2000, BMJ : British Medical Journal.

[30]  J. Minna,et al.  Promoter methylation and silencing of the retinoic acid receptor-beta gene in lung carcinomas. , 2001, Journal of the National Cancer Institute.

[31]  J. Minna,et al.  Molecular pathogenesis of lung cancer. , 1994, Annual review of physiology.

[32]  R. Weichselbaum,et al.  Frequent 3p allele loss and epigenetic inactivation of the RASSF1A tumour suppressor gene from region 3p21.3 in head and neck squamous cell carcinoma. , 2002, European journal of cancer.

[33]  F. Gilliland,et al.  Aberrant promoter methylation in bronchial epithelium and sputum from current and former smokers. , 2002, Cancer research.

[34]  A. Gazdar,et al.  Surrogate anatomic/functional sites for evaluating cancer risk: an extension of the field effect. , 1999, Clinical cancer research : an official journal of the American Association for Cancer Research.

[35]  L. Thiberville,et al.  Re: Promoter methylation and silencing of the retinoic acid receptor-beta gene in lung carcinomas. , 2001, Journal of the National Cancer Institute.

[36]  E Gabrielson,et al.  Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[37]  J. Herman,et al.  Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[38]  M. Strong,et al.  Field cancerization in the aerodigestive tract--its etiology, manifestation, and significance. , 1984, The Journal of otolaryngology.

[39]  J. Minna,et al.  Loss of expression and aberrant methylation of the CDH13 (H-cadherin) gene in breast and lung carcinomas. , 2001, Cancer research.

[40]  E. Myers,et al.  Cancer of the Head and Neck , 1989 .

[41]  S. Lam,et al.  Sex-related differences in bronchial epithelial changes associated with tobacco smoking. , 1999, Journal of the National Cancer Institute.

[42]  G. Liang,et al.  Relationship between transcription and DNA methylation. , 2000, Current topics in microbiology and immunology.