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5' CpG Island Methylation of the FHIT Gene Is Correlated with Loss of Gene Expression in Lung and Breast Cancer¹

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

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Allele loss and loss of expression of fragile histidine triad (FHIT), a putative tumor suppressor gene located in chromosome region 3p14.2, are frequent in several types of cancers. Tumor-acquired methylation of promoter region CpG islands is one method for silencing tumor suppressor genes. We investigated 5' CpG island methylation of the FHIT gene in 107 primary non-small cell lung cancer (NSCLC) samples and corresponding nonmalignant lung tissues, 39 primary breast carcinomas, as well as in 49 lung and 22 breast cancer cell lines by a methylation-specific PCR assay. In addition, we analyzed brushes from the bronchial epithelium of 35 heavy smokers without cancer. FHIT methylation was detected in 37% of primary NSCLCs, 31% of primary breast cancers, and 65% of lung and 86% of breast cancer cell lines. The frequency of methylation in small cell and NSCLC cell lines were identical. Methylation was found in 9% of the corresponding nonmalignant lung tissues and in 17% of bronchial brushes from heavy cigarette smokers. FHIT methylation was significantly correlated with loss of FHIT mRNA expression by Northern blot analysis in lung cancer cell lines and with loss of Fhit expression in NSCLC and breast tumors by immunostaining. We conclude that methylation of FHIT is a frequent event in NSCLC and breast cancers and is an important mechanism for loss of expression of this gene. Methylation of FHIT commences during lung cancer pathogenesis and may represent a marker for risk assessment.

	Introduction

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The FHIT³ gene, located in chromosome region 3p14.2, undergoes frequent allele loss (LOH) and occasional homozygous deletions in various cancer types, including lung and breast cancer (1, 2, 3, 4, 5) . FHIT transcripts of abnormal size, including deletions of exons and insertions, are found in a high percentage of lung and breast cancers, whereas point mutations are rare (1 , 2 , 4 , 5) . Loss of Fhit protein expression also is frequent in these cancer types (6, 7, 8, 9, 10) . Although these findings suggest that FHIT is a TSG, which plays an important role in the pathogenesis of lung and breast cancer, there remains considerable controversy as to the role of the gene in tumorigenesis (reviewed in Ref. 11 ).

Aberrant methylation (referred to as methylation) of normally unmethylated CpG islands, located in the 5' promoter region of genes, has been associated with transcriptional inactivation of several genes in human cancer and can serve as an alternative to mutational inactivation (12 , 13) . Several genes, including p16, RARß, TIMP-3, DAPK, H-cadherin, and RASSF1A frequently undergo such methylation in lung and breast cancers (12 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) . In esophageal squamous cell carcinomas, methylation of the FHIT gene was found in three of four structurally unaltered but transcriptionally repressed tumor cell lines as well as in 5 of 35 primary tumors (24) .

Because of the potential role of FHIT in the pathogenesis of lung and breast cancers, we investigated the methylation status of FHIT in these tumors and correlated our findings with gene expression at the RNA and protein levels and with clinical features.

	Materials and Methods

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Tumor and Tissue Samples and Tumor Cell Lines.
Tissues were collected after obtaining appropriate Institutional Review Board permission and informed consent from the subjects. Primary NSCLC tumors (n = 107) and corresponding nonmalignant lung tissues (n = 104) were obtained from patients who had received surgical resections with curative intent at The Prince Charles Hospital. Areas of viable tumor tissue were macrodissected and stored at -70°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 females, ages 28–81 years (mean, 61 years) at diagnosis. Sixty-one patients had stage I disease, 21 patients had stage II disease, 24 patients had stage IIIA disease, and 1 patient had stage IIIB disease. Histological subtypes included 45 adenocarcinomas, 43 squamous cell carcinomas, 11 adenosquamous carcinomas, 4 large cell carcinomas, 3 atypical carcinoids, and 1 typical carcinoid. Ninety-eight patients were ever smokers (consisting of current and former smokers) with mean exposure of 31 pack-years, and 9 were never smokers. Survival data of 5 or more years were available for most patients. Primary breast tumors (n = 39) were obtained from women undergoing breast cancer resections at The University of Texas Southwestern Medical Center. The age of the patients ranged between 31 and 84 years (mean, 57 years) at diagnosis. 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 and 6 lobular carcinomas. Smokers (n = 35) with at least 30 pack-years of smoking history and with morphometric evidence of sputum dysplasia were examined under fluorescence bronchoscopy at the British Cancer Agency (27) . Bronchial brushes were obtained from a predetermined site (apical segment of right upper lobe). Bronchial biopsies were taken from multiple areas that demonstrated fluorescence abnormalities. Pathological examination revealed dysplasia in at least 1 biopsy in 33 of 35 cases (mild dysplasia in 26 cases and moderate dysplasia in 7 cases). Twenty-five NSCLCs, 22 SCLCs, and 2 mesothelioma cell lines (referred to as lung cancer cell lines) and 22 breast cancer cell lines generated by us have been described previously (28, 29, 30) . Corresponding B lymphoblastoid cell lines were available for 52 lines.

MSP.
DNA was prepared from tissue samples and cell lines by standard methods, and bisulfite modification of genomic DNA was performed as reported by Herman et al. (31) . Briefly, 1 µg of genomic DNA was denatured with NaOH (final concentration, 0.2 M) and 10 mM hydroquinone (Sigma Chemical Co., St. Louis, MO), and 3 M sodium bisulfite (Sigma Chemical Co.) were added and incubated at 50°C for 16 h. Afterward, modified DNA was purified using Wizard DNA purification resin (Promega Corp., Madison, WI), followed by ethanol precipitation. Treatment of genomic DNA with sodium bisulfite converts unmethylated but not methylated cytosines to uracil, which are then converted to thymidine during the subsequent PCR step, producing sequence differences between methylated and unmethylated DNA. Primer sequences for the methylated FHIT reaction were 5'-TTG GGG CGC GGG TTT GGG TTT TTA CGC-3' (forward) and 5'-CGT AAA CGA CGC CGA CCC CAC TA-3' (reverse), and primer sequences for the unmethylated FHIT reaction were 5'-TTG GGG TGT GGG TTT GGG TTT TTA TG-3' (forward) and 5'-CAT AAA CAA CAC CAA CCC CAC TA-3' (reverse). Primer sequences were determined on the basis of the sequence data of the 5' CpG island of the gene as described in "Results." The PCR mixture contained 10x PCR buffer (Qiagen, Inc., Valencia, CA), deoxynucleotide triphosphates (1.25 mM), primers (final concentration, 0.6 µM each per reaction), 1 unit of HotStarTaq (Qiagen, Inc.) and bisulfite-modified DNA (100 ng). A touchdown PCR with an annealing temperature between 71°C and 64°C was performed. Amplification was carried out in a 9700 Perkin-Elmer Thermal Cycler. DNA from peripheral blood lymphocytes and buccal smears of healthy individuals were used as positive controls for the unmethylated form; DNA from peripheral blood lymphocytes treated with SssI methyltransferase (New England BioLabs, Inc., Beverly, MA) was used as a positive control for methylated alleles. Negative control samples without DNA were included for each set of PCR. PCR products were analyzed on 3% agarose gels and visualized under UV illumination. The PCR reactions for all samples demonstrating methylation were repeated to confirm these results.

5-Aza-2'-deoxycytidine Treatment and RT-PCR.
The NCI-H1299 NSCLC cell line was incubated in culture medium with and without 1 µM 5-aza-2'-deoxycytidine (Sigma Chemical Co.) for 6 days (16) . RNA was prepared, and RT-PCR was performed to detect FHIT expression using FHIT primers 5RT-F and 3D2, as described previously (24) . Primers for glyceraldehyde-3-phosphate dehydrogenase were used to confirm RNA integrity (16) . PCR products were analyzed on 2% agarose gels.

Other Molecular Markers.
Data on immunostaining of Fhit have been reported (in the case of NSCLCs) or were performed (in the case of breast cancer samples) as described previously (8) . LOH analysis at 3p14.2 using polymorphic markers (D3S1300, D3S4103, and D3S1234) has been reported previously for lung cancer (2 , 3) and was performed for the breast cancer samples as described (3) . Other available molecular markers from previous studies included K-ras codon 12, p53 exons 5–8 mutations, and the methylation status of the genes RARß, RASSF1A, TIMP-3, p16, O⁶-methylguanine-DNA-methyltransferase (MGMT), death-associated protein kinase (DAPK), E-cadherin (ECAD), p14^ARF (p14), and glutathione S-transferase P1 (GSTP1) in the 107 primary NSCLC samples (18 , 23 , 25) .

Statistics.
Statistical analysis was performed using ² test for differences between groups and t tests between means. Overall survival was calculated using Kaplan-Meier log rank testing.

	Results

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Genomic Sequencing of the FHIT 5' CpG Island after Bisulfite Treatment.
Genomic sequencing of the FHIT 5' CpG island (GenBank accession numbers U76262 and U76263) after bisulfite treatment of DNA from seven primary NSCLCs that lacked Fhit immunostaining was performed as described (8 , 24) . The methylation pattern of the 5' CpG island of the FHIT gene in these seven samples was very similar to the pattern described by Tanaka et al. (24) and indicated that the region between nucleotides 195 and 283 (from GenBank sequence number U76263) was highly methylated. Two sets of PCR primers that distinguish between methylated and unmethylated DNA sequences in the 5' region of the gene were designed based on these results. Using the original primer set (32) , the correlation between positive methylation and loss of immunostaining was poor (data not shown). A second set of primers was designed, as described in "Materials and Methods," and used for all experiments described herein.

Frequency of FHIT Methylation.
Using the primers we designed, we determined the frequency of FHIT methylation in primary NSCLC samples and corresponding nonmalignant lung tissues, primary breast carcinomas, and lung and breast cancer cell lines by MSP (Fig. 1) , and the results are summarized in Table 1 . FHIT methylation was also found in 6 of 35 bronchial brushes from heavy smokers without lung cancer. Three of the positive cases were from subjects with mild dysplasia, and three were from subjects with moderate dysplasia. The unmethylated form of FHIT was found in 100% of the primary tumors that had been grossly dissected and thus had at least some contamination with normal cells and also in all nonmalignant specimens. In contrast to the tumor samples, the cancer cell lines represent pure populations of tumor cells, and we determined their FHIT allele status. We found that the vast majority of these tumor lines contained either the methylated or the unmethylated form, and only occasional cell lines contained both forms (Table 2) .

View larger version (52K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Methylation analysis of FHIT in various specimens. TU, tumor; NL, nonmalignant lung tissue; Lane U, amplified product with primers recognizing unmethylated sequence (74-bp PCR product); Lane M, amplified product with primers recognizing methylated sequence (74-bp PCR product). Positive controls include peripheral blood lymphocytes from healthy individuals for the unmethylated form and in vitro methylated DNA for the methylated form of FHIT. Water blanks were used as negative controls. In the case of primary breast carcinomas, lung and breast cancer cell lines, and bronchial brushes, the amplified product represents the methylated form of FHIT. Lanes that do not show a band represent samples that are not methylated.

FHIT Methylation and Loss of Fhit Expression by Immunostaining.
Fhit immunostaining was performed in 98 of the 107 NSCLC samples and was absent in 53% as described previously by Geradts et al. (8) . In addition, immunostaining for Fhit expression was also performed in 36 primary breast cancers. Loss of Fhit expression was observed in 26 of 36 (72%) breast cancer samples.

Of the 98 NSCLC samples studied, 38 were methylated; of these, 27 lacked immunostaining, 4 were uniformly positive, and 7 showed a heterogeneous pattern of immunostaining, with focal areas of positivity and negativity. Of the 36 breast cancer samples studied, 20 were methylated, and of these, 16 lacked immunostaining, 1 was uniformly positive for staining, and 3 showed a heterogeneous pattern. Because of the difficulty of interpreting the tumors showing heterogeneous staining patterns, they were excluded from the analysis of concordance. Of the remaining 48 methylated lung and breast cancer samples, 43 lacked expression. Of the methylation-negative samples, 35 of 60 (58%) NSCLC samples and 6 of 16 (38%) breast cancer samples were positive for immunostaining. Thus, of a total of 76 methylation-negative lung and breast tumors, 41 (54%) lacked immunostaining, suggesting that methods of gene silencing other than methylation may exist. The correlation between FHIT methylation and lack of Fhit expression was highly significant (P < 0.0001).

Reexpression of FHIT after Treatment with 5-Aza-2'-deoxycytidine.
Loss of FHIT expression in the NSCLC cell line NCI-H1299 has been described previously by Northern blot analysis and RT-PCR (2) and confirmed in the present study. We found this cell line methylated for FHIT, making it a suitable candidate for treatment with the demethylating agent 5-aza-2'-deoxycytidine. Reexpression of FHIT was seen after treatment of NCI-H1299 cells with 5-aza-2'-deoxycytidine, confirming the role of the 5' region CpG methylation in regulating FHIT expression (Fig. 2) .

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Expression study of FHIT in the NCI-H1299 NSCLC cell line by RT-PCR. The expression of FHIT is lost in the cell line NCI-H1299 but can be restored after treatment with 5-aza-2'-deoxycytidine (5-aza-dC). -, NCI-H1299 without 5-aza-dC; +, NCI-H1299 with 5-aza-dC; Lane H2O, water blank; Lane Pos. Control, positive control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

LOH analysis on the FHIT locus was also performed on 20 breast cancer cell lines. Allele loss was seen in 10 (50%) cell lines, whereas 8 (40%) cell lines were heterozygous and 2 (10%) cell lines were classified as not informative. In addition, we analyzed 24 microdissected primary breast carcinomas and found FHIT locus LOH in 12 (50%) samples. Eleven (46%) samples were heterozygous, and 1 (4%) sample was scored as not informative. The concordance between LOH and methylation was 56% for breast cancer cell lines and 48% for primary breast carcinomas, respectively.

Clinicopathological Characteristics.
The methylation results from the primary NSCLCs and breast cancers were compared with clinicopathological characteristics from these patients including sex, age, histology, and Tumor-Node-Metastasis classification of the tumors. For the NSCLC patients, data about smoking history and overall survival also were available. No significant correlation between FHIT methylation and any of these parameters was observed for the NSCLC or breast cancer patients. However, there appeared to be a tendency for FHIT methylation to be associated with an increase in pack-years smoked (mean pack-years, 38 versus 49; P = 0.08) in the NSCLC patients. We also observed that none of the lobular breast carcinomas examined were methylated. Although the sample size (n = 6) is too small to reach statistical significance, this possible association needs to be investigated in a larger series.

FHIT Methylation and Other Markers.
No correlation between FHIT methylation and K-ras codon 12 mutations and p53 exons 5–8 mutations were observed in the primary NSCLC samples. FHIT methylation was also not correlated with the methylation status of RARß, RASSF1A, TIMP-3, p16, MGMT, DAPK, p14, and GSTP1 but was correlated with the methylation status of ECAD (P = 0.01) in the primary NSCLC samples.

	Discussion

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We studied the methylation status of the 5' CpG island of FHIT in lung and breast cancers and also their adjacent nonmalignant tissues and the bronchial epithelium of smokers without lung cancer. We first sequenced sodium bisulfite-treated DNA in the FHIT 5' CpG island region as reported by Tanaka et al. (24) . The methylation pattern we obtained by sequencing several primary NSCLCs with loss of Fhit expression by immunostaining was very similar to the pattern that had been described previously (24) . On the basis of the results, we designed two sets of primer pairs for use in MSP assays. The results presented in this report exclusively used the redesigned primer set as described in "Materials and Methods." Using the redesigned primer set, we found a high percentage of primary NSCLC samples, primary breast carcinomas, as well as lung and breast cancer cell lines methylated for FHIT. The higher percentage of methylation found in the lung and breast cancer cell lines compared with primary tumors might be explained by additional changes acquired in culture, or that the tumor cell lines were derived from more aggressive tumors and that these had acquired more changes.

We compared tumor FHIT methylation and loss of expression of FHIT mRNA by Northern blot analysis and Fhit expression by immunostaining. A significant correlation between FHIT methylation and loss of expression by Northern blot was seen for lung cancer and by immunostaining for lung and breast cancers. Seven methylated NSCLCs and 3 breast cancer samples exhibited heterogeneity of Fhit immunostaining. A possible explanation for this finding is that there was tumor heterogeneity for methylation. Of 78 NSCLC and breast cancer specimens that completely lacked Fhit immunoreactivity, only 43 (55%) were methylated. In the methylated tumors, we presume that lack of expression occurred via biallelic inactivation, by a combination of methylation and allelic loss. However, because 35 (45%) of the tumors lacking protein expression were not methylated, we presume that mechanisms of inactivating the second allele other than methylation must exist. Another fact that supports the importance of FHIT methylation in inactivating this gene is the reexpression of the FHIT gene after treatment with the demethylating agent 5-aza-2'-deoxycytidine. This finding is in agreement with the results found by Tanaka et al. (24) in esophageal squamous cell carcinomas. Taken together, these findings demonstrate that methylation of FHIT is an important mechanism for silencing this gene in lung and breast cancers.

We also compared the results on FHIT methylation with data on LOH in lung and breast cancers. Although both events were frequent in the two tumor types, the correlation between them was relatively low.

We did not find any significant correlation between FHIT methylation status and clinicopathological characteristics of the NSCLC and breast cancer patients. However, we observed a tendency that the number of pack-years smoked by the NSCLC patients may be associated with FHIT methylation. This finding is particularly interesting because several authors reported an association between loss of Fhit expression and the smoking history of lung cancer patients (6 , 7 , 9) . Only nine never smokers were included in our study. Of these, five tumors were not methylated, and the other four were methylated. However, the number of nonsmokers in our study is relatively small; therefore, a study with a larger number of nonsmokers is necessary to answer the question of whether FHIT methylation is associated with smoking exposure. Interestingly, FHIT methylation was only detected in ductal breast cancers but not in lobular breast cancers. This finding needs to be confirmed by study of a larger number of these cancers.

Sozzi et al. (6) reported loss of Fhit immunostaining in 85% of bronchial dysplastic lesions and in 100% of carcinoma in situ lesions adjacent to the tumor or at the resection margin of the surgical samples. Our data published previously indicated that allele loss at the FHIT locus (3p14.2) occurred relatively late (at the dysplastic stage) during lung cancer pathogenesis (3) . Therefore, we investigated whether FHIT methylation can also be detected in the bronchial epithelium of heavy smokers. Although almost all of the subjects had dysplastic changes in at least one of several bronchial biopsies, the brushes were obtained from a predetermined site, and cytological examination of the cells was not performed. We found a relatively high percentage (17%) of FHIT methylation in these samples, suggesting that FHIT methylation is an early event in the pathogenesis of lung cancer. FHIT methylation in the nonmalignant lung tissues adjacent to resected NSCLC tumors may be explained by the fact that methylation of certain genes occurs during multistage pathogenesis. Whether FHIT methylation represents a risk factor for development of central or peripheral lung cancers needs to be studied.

In conclusion, we found methylation of FHIT frequently in primary NSCLC samples and primary breast carcinomas, and it was also detected in the bronchial epithelium from heavy smokers. Moreover, we were able to show that FHIT methylation is associated with both loss of FHIT mRNA expression and loss of Fhit expression, and that methylation of this gene is reversible with 5-aza-2'-deoxycytidine. Finally, our findings of a frequent acquired tumor-related epigenetic alteration favor the candidacy of FHIT as a TSG.

	FOOTNOTES

 
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.

¹ Supported by Grants J1658-MED and J1860-MED from the Austrian Science Foundation, Lung Cancer Specialized Program of Research Excellence Grant P50 CA70907, and grants from The G. Harold and Leila Y. Mathers Charitable Foundation, The Susan G. Komen Foundation and the Cancer Research Foundation of North Texas.

² To whom requests for reprints should be addressed, at Hamon Center for Therapeutic 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; E-mail: John.Minna{at}UTSouthwestern.edu

³ The abbreviations used are: FHIT, fragile histidine triad; TSG, tumor suppressor gene; NSCLC, non-small lung cancer; SCLC, small cell lung cancer; MSP, methylation-specific PCR; LOH, loss of heterozygosity; RT-PCR, reverse transcription-PCR; p16, p16^INK4a; RAR, retinoic acid receptor; TIMP, tissue inhibitor of metalloproteinase; DAPK, death-associated protein kinase.

Received 2/19/01. Accepted 3/13/01.

	REFERENCES

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