Purpose: Human DOC-2/DAB2 interactive protein (hDAB2IP) gene is a novel member of the Ras GTPase-activating family and has been demonstrated to be a tumor suppressor gene inactivated by methylation in prostate cancer. We analyzed methylation and expression status of hDAB2IP in breast cancer.
Experimental Design: The promoter region of hDAB2IP was divided into two regions (m2a and m2b) following our previous report on prostate cancer, and methylation status was determined in breast cancer cell lines with bisulfited DNA sequencing. Expression was semiquantified with real-time reverse transcription-PCR to find that aberrant methylation showed the inverse relationship with expression. On the basis of sequence data, we developed methylation-specific PCR for m2a and m2b regions and applied to samples.
Results: Aberrant methylation was detected in 11 of 25 breast cancer cell lines (44%) and 15 of 39 primary tumors (38%) at the m2a region and in 12 of 25 cell lines (48%) and 13 of 39 tumors (33%) at the m2b region. In addition, gene expression was restored in methylated cell lines with 5-aza-2′-deoxycytidine, confirming that methylation caused gene down-regulation. We also examined the relationship between hDAB2IP methylation and clinicopathologic features in primary tumors and found that methylation in the m2b region was associated with progressive nodal status of tumors.
Conclusions: We developed methylation-specific PCR for hDAB2IP and examined its methylation status in breast cancer. Our results demonstrate that hDAB2IP methylation frequently is present in breast cancer and plays a key role in hDAB2IP inactivation, suggesting the relationship between hDAB2IP methylation and lymph node metastasis of breast cancer.
Inactivation of tumor suppressor genes is an important event for carcinogenesis of malignant tumors. In addition to the classical genetic alteration (i.e., deletion or inactivating point mutations), tumor suppressor genes can be functionally inactivated by aberrant methylation of cytosine residues in the promoter region without alteration of the sequence in various kinds of cancers, including breast cancer (1, 2, 3) .
Human DOC-2/DAB2 interactive protein gene (hDAB2IP), located on chromosome 9q33.1-q33.3, is a novel member of the Ras GTPase-activating family (4, 5, 6) . It interacts directly with disabled 2 protein (DAB2; also known as DOC-2, differentially expressed in ovarian carcinoma 2), which appears to be a tumor suppressor in malignant cells, including mammary, prostate, and ovarian cancers (7 , 8) . hDAB2IP and DOC-2/DAB2 form a unique protein complex and have a negative regulatory activity to the Ras-mediated signal pathway (5) . It was reported recently that the P2 region (−598 to +44) in the hDAB2IP gene showed promoter activity and that the transcriptional silencing by the aberrant methylation of the P2 region was a critical event during the tumorigenesis of prostate cancer (9) .
In this study, we examined methylation and expression status of hDAB2IP in a series of breast cancer cell lines and developed methylation-specific PCR (MSP) assay for hDAB2IP to apply to samples. We also analyzed the relationship between methylation status and clinicopathologic features of surgically resected breast cancer specimens to investigate the clinical importance of hDAB2IP methylation in breast cancer.
MATERIALS AND METHODS
Breast Cancer Cell Lines.
The 21 human breast cancer cell lines (HCC38, HCC70, HCC202, HCC712, HCC1007, HCC1143, HCC1187, HCC1395, HCC1419, HCC1428, HCC1500, HCC1569, HCC1599, HCC1739, HCC1806, HCC1937, HCC1954, HCC2185, HCC2218, HCC2688, and HCC2713) were established by one of the authors (A. F. G.; Ref. 10 ), and the four human breast cancer cell lines (MDA-MB-231, MDA-MB-361, MCF7, and ZR-75–1) were obtained from the American Type Culture Collection (Manassas, VA). The characteristics of the cell lines are summarized in Table 1A⇓ . Most cells were grown in RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 5% or 10% fetal bovine serum and incubated in 5% CO2.
Surgically resected specimens of 39 primary breast cancers, 37 invasive ductal carcinoma, and 2 mucinous carcinoma and 10 corresponding nonmalignant breast tissues were obtained from Okayama University Hospital (Okayama, Japan) after acquiring informed consent from each patient between 1998 and 2002. Patient demographics are shown in Table 1B⇓ . The Tumor-Node-Metastasis classification was determined according to the American Joint Committee on Cancer Cancer Staging Manual, 6th edition. Peripheral blood lymphocytes were obtained from five healthy volunteers. The appropriate Institutional Review Board of our center approved the procedure.
DNA Extraction and Bisulfite Treatment.
Genomic DNA was isolated by digestion with proteinase K, followed by phenol:chloroform (1:1) extraction and ethanol precipitation from 25 breast cancer cell lines, 39 primary breast tumors, 10 nonmalignant breast tissues, and 5 peripheral blood lymphocytes (11) . For bisulfite treatment, 1 μg of genomic DNA was denatured by NaOH and modified by sodium bisulfite, which converts all of the unmethylated cytosines to uracils, whereas methylated cytosines remain unchanged (12) . The modified DNA was purified using a Wizard DNA cleanup system (Promega, Madison, WI).
Map of 5′-Flanking Region of hDAB2IP and Bisulfited DNA Sequencing Analysis.
Our previous report on prostate cancer identified the promoter region of hDAB2IP (P2), which was divided into m2a (237 bp) and m2b (401 bp) regions (9) . The location of the CpG dinucleotides, P2, m2a, and m2b regions in the 5′-flanking region of hDAB2IP (GenBank accession no. AL365274) are shown in Fig. 1A⇓ . The m2a and m2b regions were sequenced following our previous report (9) . The forward and reverse primers for m2a and m2b were as followed: hDAB2IPm2a forward, 5′-GGATTTTTTTAGGTGGGTGT-3′; hDAB2IPm2a reverse, 5′-CCCTAAACCRCTATTACCTTAAC-3′; hDAB2IPm2b forward, 5′-GTTAAGGTAATAGYGGTTTAGGG-3′; and hDAB2IPm2a reverse, 5′-ACRAACTCACCTCTCATTATCC-3′ (9) . A touchdown PCR with annealing temperature between 54°C and 50°C for m2a and between 51°C and 47°C for m2b was performed, and PCR amplicons were cloned into pCR2.1-TOPO vector using TOPO TA cloning kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. To determine the methylation status of hDAB2IP, individual eight clones from each sample were sequenced by the Applied Biosystems PRISM dye terminator cycle sequencing method (Foster City, CA) using forward and reverse M13 primers.
hDAB2IP mRNA Expression by Semiquantitative Real-Time Reverse Transcription-PCR.
Total cellular RNA was isolated from cell lines and three nonmalignant breast tissues with RNeasy minikit (Qiagen,Valencia, CA) according to the manufacturer’s instructions and then treated with 2 units/μl of DNase I (Ambion, Austin, TX) for 30 min at 37°C. Reverse transcription reaction was performed for 2 μg of total RNA with the SuperScript II first-strand synthesis using the oligo (dT) primer system (Invitrogen) in 20 μl reaction mixture. cDNAs were semiquantified by fluorescence-based real-time reverse transcription-PCR using TaqMan technology (Applied Biosystems) with the GeneAmp 5700 Sequence Detection System (Applied Biosystems). TATA box binding protein (TBP) was used to normalize the expression of hDAB2IP (13 , 14) . The sequences of the primers and probe for hDAB2IP expression were as follows: 5′-CGACAATGAGAGGTCCCATCTG-3′ (forward primer), 6FAM-5′-CGGTGGAGGCGCTGGACCTCA-3′-TAMRA (TaqMan probe), and 5′-TTTTCTCACGGCATTTCTTCACA-3′ (reverse primer). The 25-μl reaction mixtures contained the forward and reverse primers at 300 nm each, the probe at 100 nm, TaqMan Universal PCR Master Mix (Applied Biosystems), and 5 μl cDNA from 20-μl volume of RT reaction mixture. PCR was performed under the following conditions: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. We used serial dilutions of the positive control cDNA to create a standard curve. The expression ratio was defined as the hDAB2IP PCR products compared with those of the TBP, multiplied by 100. All of the experiments were performed in duplicate.
The methylation-specific primers were designed based on the bisulfited DNA sequencing data of cell lines of which expressions were remarkably down-regulated (see below). The location of MSP primers is shown in Fig. 1⇓ . The methylated alleles for m2a and m2b regions were amplified using hDAB2IPm2a-MSP forward, 5′-GAGGTGAGCGGGGCGGTC-3′; hDAB2IPm2a-MSP reverse, 5′-CGCTATTACCTTAACGACGCCGA-3′; hDAB2IPm2b-MSP forward, 5′-CGGGTTTCGGTTCGTCGTC-3′; and hDAB2IPm2b-MSP reverse, 5′-AACTCACCTCTCATTATCCGCGAC-3′. The methylation status of m2a and m2b regions in breast cancer was determined by MSP (15) . Bisulfite-modified DNAs were mixed with 10× PCR buffer, 150 μm of deoxynucleotide triphosphates, 0.4 μm of primers, and 1 unit of HotStarTaq (Qiagen). The PCR condition for methylated alleles consisted of 12 min at 94°C, 35 cycles of 20 s at 94°C, 30 s at 63°C for m2a region or 61°C for m2b region, and 30 s at 72°C, followed 7 min elongation. The unmethylated allele-specific primers also were designed in m2a and m2b regions, and sequences were as follows: hDAB2IPm2a-USP forward, 5′-GAGGTGAGTGGGGTGGTT-3′; hDAB2IPm2a-USP reverse, 5′-CACTATTACCTTAACAACACCAA-3′; hDAB2IPm2b-USP forward, 5′-TGTGTAGGGTTTTTTTAGTTGTTG-3′; and hDAB2IPm2b-USP reverse, 5′-CAAAACCCCAACAAAATATAACA-3′. The PCR condition for unmethylated alleles was similar to those for methylated alleles except a touchdown PCR was done with an annealing temperature between 52°C and 48°C for m2a amplification and between 57°C and 53°C for m2b amplification. The PCR products were resolved by electrophoresis in 2% agarose gels containing ethidium bromide. The normal lymphocyte DNA that was 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 in each assay.
5-Aza-2′-deoxycytidine (5-Aza-CdR) Treatment.
Cell lines were treated with 5-Aza-CdR (Sigma-Aldrich, St. Louis, MO) at a concentration of 1–2 μg/ml for 6 days with medium changes on days 1, 3, and 5 (16 , 17) . Treated or untreated cells from individual triplicate flasks were harvested to semiquantify the gene expression level using real-time reverse transcription-PCR as described previously.
The frequencies of hDAB2IP methylation between two groups were compared using the χ2 test. The quantitative ratios of different groups were compared using the Mann-Whitney nonparametric U test. P < 0.05 was defined as statistically significant. All of the data were analyzed with StatView for Windows (SAS Institute Inc., Cary, NC).
Bisulfited Genomic DNA Sequencing.
The m2a and m2b regions that covered most of the P2 region were amplified separately by PCR for 10 cell lines to examine the methylation status by direct bisulfited sequence. According to the degree of methylation by direct sequence, seven cell lines (MDA-MB-231, MCF7, ZR-75–1, HCC70, HCC1569, HCC1806, and HCC1954) were selected for detailed analysis. The PCR amplicons of these seven cell lines were cloned, and individual eight clones were sequenced (Fig. 1B)⇓ . Two cell lines (MDA-MB-231 and MCF7) showed rare methylation at CpG sites (MDA-MB-231, 3.6%; MCF7, 3.2%) and were regarded as unmethylated cell lines. In contrast, three cell lines (HCC1569, HCC1806, and HCC1954) showed heavily methylated pattern (HCC1569, 96.3%; HCC1806, 96.1%; and HCC1954, 88.1%). Two cell lines, ZR-75–1 and HCC70, revealed partial methylation (ZR-75–1, 45.8%; HCC70, 76.1%).
hDAB2IP Expression in Breast Samples.
We examined the expression status in seven cell lines (MDA-MB-231, MCF7, HCC70, ZR-75–1, MDA-MB-361, HCC1569, and HCC1806) by semiquantitative real-time reverse transcription-PCR. The semiquantitative values of mRNA expression in each cell line are shown in Fig. 2A⇓ . In two unmethylated cell lines, the mean hDAB2IP expression ratios for MDA-MB-231 and MCF7 were 79.0 and 30.6, respectively. Expression ratios of partially methylated cell lines were less than those of unmethylated cell lines (13.3 in HCC70, 10.7 in ZR-75–1, and 8.5 in MDA-MB-361). Furthermore, expression ratios of heavily methylated cell lines were much less than those of unmethylated cell lines (7.2 in HCC1569 and 1.1 in HCC1806). These results indicated that the expression of hDAB2IP tended to be reduced depending on the degree of methylation at the P2 region. In three nonmalignant breast tissues, the expression ratios were 93.1, 83.8, and 83.6 (mean ± SD, 86.8 ± 5.4), indicating that hDAB2IP was normally expressed in breast tissue.
MSP Assay and Aberrant Methylation of hDAB2IP in Breast Cancers.
We determined each region for MSP forward and reverse primers in the m2a and m2b regions (Fig. 1B)⇓ . The CpG sites in these regions were almost constantly methylated in expression-reduced cell lines (Fig. 1B)⇓ . Three and four cytosines were included in forward and reverse primers, respectively, in the m2a region producing 163-bp amplicon, and five and three cytosines were included in forward and reverse primers, respectively, in the m2b region producing 209-bp amplicon (Fig. 1B)⇓ . MSP assay using these primers was performed to elucidate methylation status in breast cancer cell lines and primary tumors, and results are summarized in Table 2⇓ . Representative examples were shown in Fig. 3⇓ . Aberrant methylation of m2a and m2b regions was found in 15 of 39 (38%) and 13 of 39 (33%) cases of primary breast cancers, respectively. In cell lines, methylation was detected in 11 of 25 cell lines (44%) for the m2a region and in 12 of 25 cell lines (48%) for the m2b region. The unmethylated form of hDAB2IP was always found in primary tumor samples because these had been grossly dissected and thus had some contamination with normal cells. In contrast, cancer cell lines were pure populations of tumor cells, and 20 of 25 cell lines (80%) had either methylated or unmethylated hDAB2IP alleles. Five of 25 cell lines (20%) showed methylated and unmethylated forms for either the m2a or m2b region (HCC70, MDA-MB-361, and ZR-75–1 in Fig. 3A⇓ ) and were regarded as partially methylated cell lines; detailed CpG methylation status is shown in Fig. 1B⇓ (HCC70 and ZR-75–1). Nine cases (23%) of primary breast tumors and 11 cases (44%) of cell lines showed aberrant methylation in the m2a and m2b regions. Conversely, 10 cases (26%) of primary breast cancers and 1 case (4%) of cell lines represented methylation of either m2a or m2b region (Table 1)⇓ . The concordances of methylation status between m2a and m2b were 95% in cell lines and 74% in primary tumors. However, some cell lines showed partial methylation in either region, suggesting the presence of heterogeneity of these cell lines (Table 1A)⇓ , which also was demonstrated in bisulfited sequence data (Fig. 1B)⇓ . There was no significant difference in methylation rate between breast cancer cell lines and primary tumors at the m2a and m2b regions. Aberrant methylation was not present at both regions in 5 peripheral blood lymphocytes and 10 nonmalignant breast tissues.
5-Aza-CdR Treatment for Breast Cancer Cell Lines.
To confirm the responsibility of DNA methylation for hDAB2IP silencing, we treated four methylated cell lines with 5-Aza-CdR. hDAB2IP expression was up-regulated significantly (>4.5-fold) by 5-Aza-CdR treatment in heavily methylated cell lines (5.8-fold for HCC1569, P = 0.005; 4.5-fold for HCC1806, P = 0.006). In partially methylated cell lines, hDAB2IP expression also was up-regulated 3.2-fold for MDA-MB-361 (P = 0.035) and 1.5-fold for ZR-75–1 (NS; Fig. 2, B and C⇓ ). The degree of up-regulation was higher in heavily methylated cell lines than in partially methylated cell lines. The expression was not up-regulated in unmethylated cell lines (MCF7 and MDA-MB-231) with 5-Aza-CdR treatment.
hDAB2IP Methylation and Clinicopathologic Correlation.
We examined the relationship between methylation status and clinicopathologic factors, including age, stage, tumor size, lymph nodal status, distant metastasis, histology, degree of invasion, hormonal receptor, HER-2/neu status, and prognosis. The m2b methylation was associated significantly with progressive status of histologic nodal involvement, which was in accordance with the Tumor-Node-Metastasis classification of the American Joint Committee on Cancer (P = 0.027; Fig. 4A⇓ ), and the number of metastasis-positive lymph nodes in the methylation-positive group was significantly higher than that in the methylation-negative group (P = 0.002; Fig. 4B⇓ ). There was no significant difference between methylation-positive and -negative groups in other factors. However, the number of examined samples may not be large enough to conclude the negative relationship between hDAB2IP methylation and other clinicopathologic factors.
MSP assay is one of the convenient and sensitive assays to examine the methylation status developed by Herman et al. (15) . The primer is one of the most essential elements for MSP assay and ideally should be designed based on bisulfited DNA sequence and expression in promoter region (18 , 19) . The promoter of hDAB2IP in prostate cancer was reported by one of the authors (H. C.). We designed MSP primers in promoter regions and examined the methylation status of hDAB2IP gene in breast cancers. We found frequent methylation of hDAB2IP gene in breast cancer cell lines and primary tumors, and there was no significant difference in rate of methylation between them. This fact indicates that cell lines are proper models to study methylation of hDAB2IP gene in breast cancer (14) . We reported previously that m2a seemed to be the key regulatory region for expression in prostatic cancer (9) . This study also showed that the down-regulation of hDAB2IP expression correlates with DNA methylation of reported promoter region in breast cancer cell lines. However, the methylation of m2a and m2b occurred simultaneously in breast cancer cell lines examined, and we could not distinguish which region might act as a regulatory promoter in breast cancer. We showed that the expression of hDAB2IP was highly reduced in five methylated cell lines compared with unmethylated cell lines and was restored after 5-Aza-CdR treatment. These results indicate that DNA methylation of the promoter region is critical for hDAB2IP gene transcription and that methylation plays an important role in hDAB2IP inactivation.
Regarding clinicopathologic features, we found a significant relationship only between nodal metastatic status and aberrant methylation of the m2b region even though the number of examined samples was limited (39 cases). Because the tumor size and nodal status are practical parameters to estimate prognosis in breast cancer, hDAB2IP methylation may be one of the indicators for prognosis in breast cancer patients (20) . This result suggests that hDAB2IP gene may be related to metastasis, and its methylation may occur in the late stage of the development of breast cancers. Of interest, there was no correlation between lymph nodal metastasis and aberrant methylation of the m2a region, which was shown to be the key regulatory sequence in our previous report. The reason for the difference between methylation of the m2a and m2b regions for metastatic nodal status was unclear; however, there is the possibility that the m2b region, which includes transcription starting site and upstream, may strongly regulate hDAB2IP expression and show biological significance in breast cancer.
In summary, we developed the MSP assay for the hDAB2IP gene based on expression and bisulfited DNA sequence data and applied it to breast cancer samples. Our results demonstrate that aberrant methylation is a major mechanism for down-regulation of hDAB2IP gene and support the fact that the methylation-mediated transcriptional silencing of hDAB2IP gene may be a critical event in tumorigenesis of breast cancer. Additional study with large numbers of cases will be necessary to investigate the relationship between hDAB2IP methylation and clinicopathologic features in breast cancer.
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Requests for reprints: Shinichi Toyooka, Department of Cancer and Thoracic Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Phone: 81-86-235-7265; Fax: 81-86-235-7269; E-mail:
- Received September 19, 2003.
- Revision received December 11, 2003.
- Accepted December 29, 2003.