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5 Cpg Island Methylation of the FHIT Gene Is Correlated with Loss Of

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5 Cpg Island Methylation of the FHIT Gene Is Correlated with Loss Of [CANCER RESEARCH 61, 3581–3585, May 1, 2001] Advances in Brief 5؅ CpG Island Methylation of the FHIT Gene Is Correlated with Loss of Gene Expression in Lung and Breast Cancer1 Sabine Zo¨chbauer-Mu¨ller, Kwun M. Fong, Anirban Maitra, Stephen Lam, Joseph Geradts, Raheela Ashfaq, Arvind K. Virmani, Sarah Milchgrub, Adi F. Gazdar, and John D. Minna2 Hamon Center for Therapeutic Oncology Research [S. Z-M., A. M., A. K. V., A. F. G., J. D. M.] and Departments of Pathology [A. M., R. A., S. M., A. F. G.], Internal Medicine [J. D. M.], and Pharmacology [J. D. M.], The University of Texas Southwestern Medical Center, Dallas, Texas 75390; Department of Thoracic Medicine, The Prince Charles Hospital, Brisbane 4032, Australia [K. M. F.]; British Columbia Cancer Agency, Vancouver V5Z 355, Canada [S. L.]; and Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom [J. G.] Abstract Aberrant methylation (referred to as methylation) of normally un- methylated CpG islands, located in the 5Ј promoter region of genes, Allele loss and loss of expression of fragile histidine triad (FHIT), a has been associated with transcriptional inactivation of several genes putative tumor suppressor gene located in chromosome region 3p14.2, are in human cancer and can serve as an alternative to mutational inac- frequent in several types of cancers. Tumor-acquired methylation of ␤ promoter region CpG islands is one method for silencing tumor suppres- tivation (12, 13). Several genes, including p16, RAR , TIMP-3, -sor genes. We investigated 5؅ CpG island methylation of the FHIT gene in DAPK, H-cadherin, and RASSF1A frequently undergo such methyl 107 primary non-small cell lung cancer (NSCLC) samples and corre- ation in lung and breast cancers (12, 14–23). In esophageal squamous sponding nonmalignant lung tissues, 39 primary breast carcinomas, as cell carcinomas, methylation of the FHIT gene was found in three of well as in 49 lung and 22 breast cancer cell lines by a methylation-specific four structurally unaltered but transcriptionally repressed tumor cell PCR assay. In addition, we analyzed brushes from the bronchial epithe- lines as well as in 5 of 35 primary tumors (24). lium of 35 heavy smokers without cancer. FHIT methylation was detected Because of the potential role of FHIT in the pathogenesis of lung in 37% of primary NSCLCs, 31% of primary breast cancers, and 65% of and breast cancers, we investigated the methylation status of FHIT in lung and 86% of breast cancer cell lines. The frequency of methylation in these tumors and correlated our findings with gene expression at the small cell and NSCLC cell lines were identical. Methylation was found in RNA and protein levels and with clinical features. 9% of the corresponding nonmalignant lung tissues and in 17% of bron- chial brushes from heavy cigarette smokers. FHIT methylation was sig- nificantly correlated with loss of FHIT mRNA expression by Northern Materials and Methods blot analysis in lung cancer cell lines and with loss of Fhit expression in NSCLC and breast tumors by immunostaining. We conclude that meth- Tumor and Tissue Samples and Tumor Cell Lines. Tissues were col- ylation of FHIT is a frequent event in NSCLC and breast cancers and is lected after obtaining appropriate Institutional Review Board permission and ϭ an important mechanism for loss of expression of this gene. Methylation informed consent from the subjects. Primary NSCLC tumors (n 107) and ϭ of FHIT commences during lung cancer pathogenesis and may represent corresponding nonmalignant lung tissues (n 104) were obtained from a marker for risk assessment. patients who had received surgical resections with curative intent at The Prince Charles Hospital. Areas of viable tumor tissue were macrodissected and stored Introduction at Ϫ70°C. This cohort of patients had been investigated previously for various genetic abnormalities (2, 8, 18, 23, 25, 26). There were 76 males and 31 The FHIT3 gene, located in chromosome region 3p14.2, undergoes females, ages 28–81 years (mean, 61 years) at diagnosis. Sixty-one patients frequent allele loss (LOH) and occasional homozygous deletions in had stage I disease, 21 patients had stage II disease, 24 patients had stage IIIA various cancer types, including lung and breast cancer (1–5). FHIT disease, and 1 patient had stage IIIB disease. Histological subtypes included 45 transcripts of abnormal size, including deletions of exons and inser- adenocarcinomas, 43 squamous cell carcinomas, 11 adenosquamous carcino- tions, are found in a high percentage of lung and breast cancers, mas, 4 large cell carcinomas, 3 atypical carcinoids, and 1 typical carcinoid. whereas point mutations are rare (1, 2, 4, 5). Loss of Fhit protein Ninety-eight patients were ever smokers (consisting of current and former smokers) with mean exposure of 31 pack-years, and 9 were never smokers. expression also is frequent in these cancer types (6–10). Although Survival data of 5 or more years were available for most patients. Primary these findings suggest that FHIT is a TSG, which plays an important breast tumors (n ϭ 39) were obtained from women undergoing breast cancer role in the pathogenesis of lung and breast cancer, there remains resections at The University of Texas Southwestern Medical Center. The age considerable controversy as to the role of the gene in tumorigenesis of the patients ranged between 31 and 84 years (mean, 57 years) at diagnosis. (reviewed in Ref. 11). Three patients had stage I, 16 stage IIA, 3 stage IIB, 8 stage IIIA, 3 stage IIIB, and 6 stage IV disease. Histological subtypes included 33 ductal carcinomas ϭ Received 2/19/01; accepted 3/13/01. and 6 lobular carcinomas. Smokers (n 35) with at least 30 pack-years of The costs of publication of this article were defrayed in part by the payment of page smoking history and with morphometric evidence of sputum dysplasia were charges. This article must therefore be hereby marked advertisement in accordance with examined under fluorescence bronchoscopy at the British Cancer Agency (27). 18 U.S.C. Section 1734 solely to indicate this fact. Bronchial brushes were obtained from a predetermined site (apical segment of 1 Supported by Grants J1658-MED and J1860-MED from the Austrian Science Foun- dation, Lung Cancer Specialized Program of Research Excellence Grant P50 CA70907, right upper lobe). Bronchial biopsies were taken from multiple areas that and grants from The G. Harold and Leila Y. Mathers Charitable Foundation, The Susan demonstrated fluorescence abnormalities. Pathological examination revealed G. Komen Foundation and the Cancer Research Foundation of North Texas. dysplasia in at least 1 biopsy in 33 of 35 cases (mild dysplasia in 26 cases and 2 To whom requests for reprints should be addressed, at Hamon Center for Therapeutic moderate dysplasia in 7 cases). Twenty-five NSCLCs, 22 SCLCs, and 2 Oncology Research, The University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-8593. Phone: (214) 648-4900; Fax: (214) 648-4940; mesothelioma cell lines (referred to as lung cancer cell lines) and 22 breast E-mail: [email protected]. cancer cell lines generated by us have been described previously (28–30). 3 The abbreviations used are: FHIT, fragile histidine triad; TSG, tumor suppressor Corresponding B lymphoblastoid cell lines were available for 52 lines. gene; NSCLC, non-small lung cancer; SCLC, small cell lung cancer; MSP, methylation- MSP. DNA was prepared from tissue samples and cell lines by standard specific PCR; LOH, loss of heterozygosity; RT-PCR, reverse transcription-PCR; p16, p16INK4a; RAR, retinoic acid receptor; TIMP, tissue inhibitor of metalloproteinase; methods, and bisulfite modification of genomic DNA was performed as DAPK, death-associated protein kinase. reported by Herman et al. (31). Briefly, 1 ␮g of genomic DNA was denatured 3581 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2001 American Association for Cancer Research. FHIT METHYLATION with NaOH (final concentration, 0.2 M) and 10 mM hydroquinone (Sigma Frequency of FHIT Methylation. Using the primers we designed, Chemical Co., St. Louis, MO), and 3 M sodium bisulfite (Sigma Chemical Co.) we determined the frequency of FHIT methylation in primary NSCLC were added and incubated at 50°C for 16 h. Afterward, modified DNA was samples and corresponding nonmalignant lung tissues, primary breast purified using Wizard DNA purification resin (Promega Corp., Madison, WI), carcinomas, and lung and breast cancer cell lines by MSP (Fig. 1), and followed by ethanol precipitation. Treatment of genomic DNA with sodium the results are summarized in Table 1. FHIT methylation was also bisulfite converts unmethylated but not methylated cytosines to uracil, which found in 6 of 35 bronchial brushes from heavy smokers without lung are then converted to thymidine during the subsequent PCR step, producing sequence differences between methylated and unmethylated DNA. Primer cancer. Three of the positive cases were from subjects with mild sequences for the methylated FHIT reaction were 5Ј-TTG GGG CGC GGG dysplasia, and three were from subjects with moderate dysplasia. The TTT GGG TTT TTA CGC-3Ј (forward) and 5Ј-CGT AAA CGA CGC CGA unmethylated form of FHIT was found in 100% of the primary tumors CCC CAC TA-3Ј (reverse), and primer sequences for the unmethylated FHIT that had been grossly dissected and thus had at least some contami- reaction were 5Ј-TTG GGG TGT GGG TTT GGG TTT TTA TG-3Ј (forward) nation with normal cells and also in all nonmalignant specimens. In and 5Ј-CAT AAA CAA CAC CAA CCC CAC TA-3Ј (reverse). Primer contrast to the tumor samples, the cancer cell lines represent pure sequences were determined on the basis of the sequence data of the 5Ј CpG populations of tumor cells, and we determined their FHIT allele island of the gene as described in “Results.” The PCR mixture contained 10ϫ status.
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