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Loss of Expression and Aberrant Methylation of the CDH13 (H-Cadherin) Gene in Breast and Lung Carcinomas¹

Hamon Center for Therapeutic Oncology Research [K. O. T., S. T., A. K. V., V. G. S., A. F. G.] and Departments of Pathology [A. V. K., A. F. G.], Surgery [D. M. E.], Internal Medicine [J. D. M.], and Pharmacology [J. D. M.], University of Texas Southwestern Medical Center, Dallas, Texas 75390, and Department of Pathology, M. D. Anderson Medical Center, Houston, Texas 77030 [M. G.]

	ABSTRACT

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Expression of some members of the cadherin family is reduced in several human tumors, and CDH13 (H-cadherin), located on chromosome 16q24.2–3, may function as a tumor suppressor gene. In human tumors, loss of expression of many tumor suppressor genes occurs by aberrant promoter region methylation. We examined the methylation status of the CDH13 promoter in breast and lung cancers and correlated it with mRNA expression using methylation-specific PCR and reverse transcription-PCR. Methylation was frequent in primary breast tumors (18 of 55, 33%) and cell lines (7 of 20, 35%). In lung cancers, methylation was present more frequently in non-small cell lung cancer tumors (18 of 42, 43%) and cell lines (15 of 30, 50%) than in small cell lung cancer cell lines (6 of 30, 20%; P = 0.03). Only the methylated or unmethylated forms of the gene were present in most (73 of 80, 91%) tumor cell lines. CDH13expression was present in 24 of 30 (80%) of nonmethylated tumor lines. All 18 methylated lines tested lacked expression irrespective of whether the unmethylated form was present, confirming biallelic inactivation in methylated lines. Gene expression was restored in all five methylated cell lines tested after treatment with the demethylating agent 5'-aza-2-deoxycytidine. Our results demonstrate frequent aberrant methylation of CDH13in breast and lung cancers accompanied by loss of gene expression, although expression may occasionally be lost by other mechanisms.

	INTRODUCTION

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The cadherins are a family of cell surface glycoproteins responsible for selective cell recognition and adhesion (1) . Several family members, including CDH1 (E-cadherin) and CDH13 (H-cadherin) are located on the long arm of chromosome 16 (16q) (2) , while another gene cluster resides on the short arm of chromosome 5 (3) . The chromosomal locations of several of the cadherins are sites of frequent LOH³ in many tumor types (2) . Deletions of 16q are frequent in breast, lung, and other carcinomas (4, 5, 6, 7, 8) . Loss of expression of cadherins has been described in many epithelial cancers, and it may play a role in tumor cell invasion and metastasis (1 , 9, 10, 11) .

Although allelic loss is frequent in these cancers, Knudson’s hypothesis (12) states that both alleles of a tumor suppressor gene have to be inactivated for tumorigenesis. Inactivation of the second allele may occur via point mutations, homozygous deletions, or by aberrant methylation. Aberrant methylation of 5' gene promoter regions associated with gene silencing is an epigenetic phenomenon observed in many cancer types (13) , and the number of methylated genes in individual cancers is estimated to be very high (14) . CDH1 is the prototype cadherin family member, and its role in tumorigenesis has been studied extensively (11) . CDH1 may be inactivated by multiple mechanisms (15) . In breast, prostate, and thyroid cancers, 5' CpG island promoter methylation is a frequent method of CDH1 inactivation (16 , 17) . However, the methylation patterns of the CDH1 in breast cancers are unstable and reflect a dynamic, heterogeneous loss of gene expression during metastatic progression (18) .

CDH13 expression is diminished in breast and lung cancers (7 , 19) . In ovarian tumors, the combination of deletion and aberrant methylation has been reported to inactivate CDH13 (20) . Aberrant methylation of CDH13 has also been reported in lung cancers (7) . The purpose of this study was to determine the frequency of CDH13 methylation in human breast and lung cancers and to investigate the relationship between aberrant methylation and expression of CDH13 in tumor cell lines.

	MATERIALS AND METHODS

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Clinical Samples.
Surgically resected specimens from 55 primary breast tumors and 17 corresponding nonmalignant breast tissues from these patients were obtained from the Tumor and Tissue Repository at the Hamon Center. Tumor samples from 42 primary NSCLC (11 squamous cell and 31 adenocarcinomas) and 25 corresponding nonmalignant lung tissues were obtained from surgical resections performed at the M. D. Anderson Cancer Center. For gene expression studies, six nonmalignant tissue samples (two breast, two peripheral lung tissues, and one bronchial mucosa) were obtained as far from the tumor tissue as possible. Epithelial cells from buccal swabs of 8 healthy nonsmoking volunteers and peripheral blood lymphocytes from 12 healthy volunteers also were obtained. Appropriate Institutional Review Board permission was obtained from both participating centers, and written informed consent was obtained from all subjects. Tissues were stored at -80°C for up to 3 years prior to testing.

Cell Lines.
Human tumor cell lines (20 breast lines, 30 SCLC lines, and 30 NSCLC lines) and corresponding B-lymphoblastoid lines (n = 47) were established by us (21 , 22) . Most breast and NSCLC lines were established from primary tumors, and most SCLC lines were established from metastases. Cells cultures were grown in RPMI 1640 (Life Technologies, Inc., Rockville, MD) supplemented with 5% fetal bovine serum and incubated in 5% CO₂ at 37°C.

DNA Extraction.
Genomic DNA was obtained from cell lines, primary tumors, and nonmalignant cells by digestion with proteinase K (Life Technologies, Inc.) for 1 day at 37°C, followed by two extractions with phenol:chloroform (1:1) (23) .

MSP.
Aberrant promoter methylation of CDH13 was determined by the method of MSP as reported by Herman et al. (24) using primers specific for CDH13-methylated and -unmethylated sequences (7) . Forward and reverse primers for the methylated sequence were 5'-TCGCGGGGTTCGTTTTTCGC-3' and 5'-GACGTTTTCATTCATACACGCG-3', respectively, and for the unmethylated sequence were 5'-TTGTGGGGTTGTTTTTTGT-3' and 5'-AACTTTTCATTCATACACACA-3', respectively. Briefly, 1 µg of genomic DNA was denatured by NaOH and modified by sodium bisulfite, which converts all unmethylated cytosines to uracil while methylated cytosines remain unchanged (25) . The modified DNA was purified using a Wizard DNA purification kit (Promega, Madison, WI), treated with NaOH to desulfonate, precipitated with ethanol, and resuspended in water. Two sets of primers were used to amplify each region of interest: PCR amplification was done with bisulfite-treated DNA as template using specific primer sequences for the methylated (i.e., unmodified by bisulfite treatment) and unmethylated (i.e., bisulfite modified to UpG) forms of the gene. DNA from peripheral blood lymphocytes (n = 12) and buccal mucosae (n = 8) from healthy nonsmoking subjects were used as negative controls for methylation-specific assays. DNA from lymphocytes of healthy volunteers treated with Sss1 methyltransferase (New England Biolabs, Beverly, MA) and then subjected to bisulfite treatment was used as a positive control for methylated alleles. Water blanks were included with each assay. PCR products were visualized on 2% agarose gels stained with ethidium bromide. Results were confirmed by repeating bisulfite treatment and MSP assays for all samples.

Expression of CDH13.
Expression of CDH13was analyzed by the RT-PCR technique. Total RNA was extracted from the cell lines (20 breast, 19 NSCLC, and 9 SCLC cell lines) with TRIzol (Life Technologies, Inc.) following the manufacturer’s instructions. RT reaction was performed on 2 µg of total RNA with the SuperScript II First-Strand Synthesis using an oligo(dT) primer system (Life Technologies, Inc.). Primer sequences and conditions for RT-PCR product were previously described (forward primer, 5'-TTCAGCAGAAAGTGTTCCATAT-3' in exon 2 and reverse primer, 5'-GTGCATGGACGAACAGAGT-3' in exon 3) (7) and confirmed that genomic DNA was not amplified with these primers. The housekeeping gene GAPDH was used as an internal control to confirm the success of the RT reaction (forward primer, 5'-ACAGTCCATGCCATCACTGCC-3' and reverse primer, 5'-GCCTGCTTCACCACCTTCTTG-3') (26) . PCR products were analyzed on 2% agarose gels.

Aza-CdR Treatment.
Five tumor cell lines with CDH13 promoter methylation and absent gene expression were incubated in culture medium with and without Aza-CdR at a concentration of 2 µg/ml for 6 days, with medium changes on days 1, 3, and 5 (27) . Cells were harvested at the end of the sixth day for extraction of total RNA and tested for gene expression.

Analysis of LOH.
Three polymorphic microsatellite markers (D16S422, D16S3098, and D16S511) located at chromosome region 16q24.2–3 were selected for LOH analysis. DNA from 13 breast cancers, 9 SCLC, and 14 NSCLC cell lines and their corresponding B-lymphoblastoid lines (as sources of constitutional DNA) were analyzed (28) . Briefly, 100 ng of genomic DNA was amplified by PCR in the presence of [³²P]CTP using the microsatellite marker. PCR products were separated by electrophoresis in 6% polyacrylamide gels containing 7 M urea and were visualized by autoradiography. Subjects who yielded two distinguishable bands of different sizes but similar intensity in the lane having normal DNA were termed informative (i.e., heterozygous) for the marker. Samples having only a single major band in normal DNA were termed noninformative. LOH was defined as complete loss of a band corresponding to an allele present in informative cases.

DNA Sequencing.
The MSP products of five cell lines with promoter methylation were isolated from the gels and purified. After amplification with the same primers used for MSP, 20 ng of PCR products were sequenced by the Applied Biosystems PRISM dye terminator cycle sequencing method (Perkin-Elmer Corp., Foster City, CA). In addition, we amplified 411 nucleotides encompassing nucleotides 1315 and 1725 of the 5' region of the CDH13 gene (accession no. AB001090) in bisulfite treated genomic DNA by primers we designed (forward, 5'-TTGGAAAAGTGGAATTAGTTGG-3'; reverse, 5'-CCTCTTCCCTACCTAAAACA-3'). These primers were designed to exclude binding to any CpG site and to ensure amplification of both methylated and unmethylated sequences. PCR products were sequenced from both ends after purification. This region included the MSP primer sites and amplicon and encompassed 24 CpG sites.

Data Analysis.
Statistical differences between groups were examined using ² tests and Fisher’s exact test with continuity correction. Probability values of <0.05 were regarded as statistically significant.

	RESULTS

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Aberrant Promoter Methylation and Expression of CDH13.
Results of aberrant promoter methylation of CDH13 are detailed in Table 1 and representative examples are illustrated in Fig. 1A . Aberrant methylation was found in primary breast tumors (18 of 55, 33%) and cell lines (7 of 20, 35%). In lung cancers, aberrant methylation was present more frequently in NSCLC primary tumors (18 of 42, 43%; including 3 of 11 squamous cell carcinomas and 15 of 31 adenocarcinomas) and cell lines (15 of 30, 50%) than in SCLC cell lines (6 of 30, 20%; P = 0.03 between cell lines; Table 1 ). Differences in frequencies between breast and NSCLC tumors and their respective cell lines were not significant. Only the methylated or unmethylated forms of the gene were present in 73 of 80 (91%) cell lines and both forms were present in 7 of 80 (9%) cell lines (Table 2) . Aberrant methylation was absent in DNA from peripheral blood lymphocytes and buccal swabs from volunteers. In DNA from nonmalignant tissues from breast and NSCLC resections, aberrant methylation was present in 1 of 17 breast and 2 of 25 lung nonmalignant samples (Table 1) . The corresponding tumor samples were also methylated in the three cases where the nonmalignant tissues were methylated. In tumor samples, most of which consist of mixtures of tumor cells and nonmalignant cells, either the unmethylated band only or both methylated and unmethylated bands were present (data not shown). The presence of unmethylated CDH13 promoter sequences in all of the tissues analyzed confirmed the integrity of the DNA in these samples.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Methylation and expression of CDH13 in tumors and cell lines. A, presence of a methylated band (demonstrated by MSP assay) in 6 of 10 breast cancer specimens. N, blank control. B, CDH13 expression (by (RT-PCR) in 7 of 10 breast cancer cell lines. N, blank control. Expression of the housekeeping gene GAPDH was run as a control for RNA integrity. C, lack of CDH13 expression in five methylated cell lines in the untreated state and restoration of gene expression after Aza-CdR treatment. The cell lines were derived from NSCLC (HCC78 and NCI-H2887, SCLC (NCI-H1339) or breast (HCC1395 and HCC2218) cancers.

View larger version (41K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Representative examples of autoradiographs demonstrating LOH in breast and lung cancer cell lines and using three microsatellite makers for chromosome 16q24.2–3 (D16S422, D16S3098, and D16S511). N, normal sample (corresponding B-lymphoblastoid line); T, tumor cell line. Examples of breast (HCC1187 and HCC2713) and SCLC (NCI-H2141) cell lines are illustrated.

Using methylation-independent primers, we amplified and sequenced the 5' region of the CDH13 gene which included the MSP primer attachment sites and their amplicon. Two cell lines (NSCLC line NCI-H1770 and breast cancer line HCC38) lacking methylated bands after MSP assay were completely unmethylated at all 24 CpG sites in the methylation-independent amplicon. Of the three lines that yielded a product after MSP assay (NSCLC cell lines NCI-2087 and NCI-2887 and breast cancer line HCC2157), all nine CpG sites at forward (five sites) and reverse (four sites) primer attachments were fully methylated. However, the seven CpG sites that were encompassed in the MSP amplicon region showed heterogeneity of methylation.

	DISCUSSION

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Decreased expression of cadherin molecules in invasive carcinomas results in cell scattering and decreased mediated cell-cell adhesion (29, 30, 31) , which may enhance tumor progression and invasion. Although the role of CDH1 has been studied extensively, there is evidence that CDH13 also may function as a tumor suppressor gene. CDH13 is located at chromosome 16q24.2–3, a site of frequent LOH in several human cancers (5 , 32 , 33) , and expression of CHD13 is significantly reduced in some breast, lung, and ovarian cancers (7 , 19 , 20 , 34) .

A frequent method of suppression of tumor suppressor gene function is via aberrant methylation of the promoter region resulting in down-regulation of gene expression. In lung cancers, aberrant promoter methylation CDH13 has been reported without mutation (7) . However, it is important to demonstrate the biological relevance of gene methylation (28 , 35 , 36) . We and others have described the criteria required for the demonstration of biological significance (35 , 37) : (a) aberrant methylation is frequent in the tumor type studied; (b) methylation is a rare event in nonmalignant and control tissues; (c) loss of expression is a frequent event in tumors; (d) aberrant methylation and gene expression are correlated with each other; (e) gene expression is restored after exposure to a demethylating agent; and (f) there is a high frequency of 16q allelic loss in the tumors, suggesting a mechanism for biallelic loss. However, because the polymorphic regions examined for allelic loss are perigenic rather than intragenic, the loss may have targeted other genes in the region including CDH1 and certain other members of the CDH family.

With the assay conditions used, all control tissues from healthy volunteers were negative for CDH13 promoter methylation, and the gene was expressed in nonmalignant breast tissues, peripheral lung, and bronchial epithelium. Only occasional methylation was present in nonmalignant tissues adjacent to cancers, whereas a relatively high percentage of breast and NSCLC tumors and cell lines (33–50%) were positive. While heterogeneity of methylation was noted in the 5' region of the gene, the MSP primer attachment sites were consistently methylated in three MSP-positive cell lines and completely unmethylated in two MSP-negative cell lines. Thus, the MSP results would be expected to be consistent even if the resultant amplicon showed heterogeneity of methylation. The excellent concordance (88%) between the positive MSP assay result and loss of CDH13 expression provides powerful evidence that the MSP assay results correlate with gene silencing. Treatment with Aza-CdR restored transcript expression in methylated cell lines, confirming that methylation was responsible for loss of gene expression in these lines.

The methylation frequencies in both breast and NSCLC tumors were not significantly different from their respective cell lines, indicating that cell lines are suitable models for studying CDH13 promoter methylation. Although SCLC tumors were not available for study, the frequency of methylation in SCLC cell lines (20%) was significantly lower than the frequency in NSCLC cell lines (50%). Significant differences between SCLC and NSCLC have been reported for the methylation frequencies of other genes (38 , 39) , suggesting that the two major forms of lung cancer arise via different pathogenetic pathways.

Knudson’s hypothesis (12) states that both alleles of a tumor suppressor gene have to be inactivated for tumorigenesis. In ovarian tumors, inactivation of CDH13 has been reported to occur by the combination of allelic loss and aberrant methylation (20) . Although there was a relatively high frequency of allelic loss in breast and lung cell lines, the concordance between LOH at chromosome 16q24.2–3 and methylation status was moderate (72%). However, most methylated cell lines lacked an unmethylated allele, indicating biallelic inactivation. Because 20% of unmethylated cell lines lacked gene expression, alternative methods of gene silencing must exist in some tumors. Of interest, binding of the transcription factor Snail to the CDH1 promoter results in gene silencing (40 , 41) . It is not known whether Snail binds to CDH13.

Our results strongly suggest that silencing of CDH13 gene expression by methylation plays a role in the pathogenesis of breast and lung cancers. Future studies, including transfection of the gene, will be required to identify the biological effects of gene silencing and their relationship to the malignant phenotype.

	ACKNOWLEDGMENTS

 
We thank Dr. James Herman for assistance with the MSP methodology and Dr. Chun X. Huang and Thomas Cunningham for technical help.

	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 This work was supported by grants from the Early Detection Research Network (5U01CA8497102), the University of Texas Specialized Program of Research Excellence in Lung Cancer (P50CA70907), National Cancer Institute, Bethesda, MD, and the G. Harold and Leila Y. Mathers Charitable Foundation, The Susan G. Komen Foundation.

² To whom requests for reprints should be addressed, at Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390. Phone: (214) 648-4921; Fax: (214) 648-4940; E-mail: gazdar{at}simmons.swmed.edu

³ The abbreviations used are: LOH, loss of heterozygosity; MSP, methylation-specific PCR; SCLC, small cell lung cancer; NSCLC, non- small cell lung cancer; RT, reverse transcription; Aza-CdR, 5-aza-2'-deoxycytidine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Received 12/ 4/00. Accepted 3/27/01.

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