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[Cancer Research 66, 4617-4626, May 1, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Frequent Silencing of the Candidate Tumor Suppressor PCDH20 by Epigenetic Mechanism in Non–Small-Cell Lung Cancers

Issei Imoto1,4, Hiroyuki Izumi1,2, Sana Yokoi1,4, Hiroshi Hosoda5, Tatsuhiro Shibata6,7, Fumie Hosoda4,7, Misao Ohki7, Setsuo Hirohashi6 and Johji Inazawa1,3,4

1 Department of Molecular Cytogenetics, Medical Research Institute and Graduate School of Biomedical Science, 2 Thoracic and Cardiovascular Surgery, and 3 Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstitution of Tooth and Bone, Tokyo Medical and Dental University; 4 Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation; 5 Tokyo Kyosai Hospital; and 6 Pathology Division and 7 Cancer Genomics Project, National Cancer Center Research Institute, Tokyo, Japan

Requests for reprints: Johji Inazawa, Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Phone: 81-3-5803-5820; Fax: 81-3-5803-0244; E-mail: johinaz.cgen{at}mri.tmd.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protocadherins are a major subfamily of the cadherin superfamily, but little is known about their functions and intracellular signal transduction. We identified a homozygous loss of protocadherin 20 (PCDH20, 13q21.2) in the course of a program to screen a panel of non–small-cell lung cancer (NSCLC) cell lines (1 of 20 lines) for genomic copy number aberrations using an in-house array-based comparative genomic hybridization. PCDH20 mRNA was expressed in normal lung tissue but was not expressed in the majority of NSCLC cell lines without a homozygous deletion of this gene (10 of 19 lines, 52.6%). Expression of PCDH20 mRNA was restored in gene-silenced NSCLC cells after treatment with 5-aza 2'-deoxycytidine. The DNA methylation status of the PCDH20 CpG-rich region correlated inversely with the expression of the gene and a putative target region for methylation showed clear promoter activity in vitro. Methylation of this PCDH20 promoter was frequently observed in primary NSCLC tissues (32 of 59 tumors, 54.2%). Among our primary NSCLC cases, the methylated PCDH20 seemed to be associated with a shorter overall survival (P = 0.0140 and 0.0211 in all and stage I tumors, respectively; log-rank test), and a multivariate analysis showed that the PCDH20 methylation status was an independent prognosticator. Moreover, restoration of PCDH20 expression in NSCLC cells reduced cell numbers in colony formation and anchorage-independent assays. These results suggest that epigenetic silencing by hypermethylation of the CpG-rich promoter region of PCDH20 leads to loss of PCDH20 function, which may be a factor in the carcinogenesis of NSCLC. (Cancer Res 2006; 66(9): 4617-26)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cadherins play an important role in the communication between adjacent cells. There are at least 80 members of the cadherin superfamily in mammals including classic cadherins (1–3). They are characterized by a unique domain called the cadherin motif or extracellular domain, containing Ca2+-dependent homophilic-binding domains (4). The extracellular domain is tandemly repeated in the extracellular segment of all members of the cadherin superfamily, with the number of extracellular domains varying considerably among members (1), whereas the amino acid sequences of the other domains, in particular the cytoplasmic domain, vary significantly among members (2). More than 60 protocadherins have been identified and are currently a major subfamily of the cadherin superfamily (3, 5). Protocadherins have up to seven extracellular domains, a single transmembrane region, and divergent and distinct cytoplasmic portions, which also exhibit cell-to-cell adhesion activity, but the adhesion mechanism is thought to be distinct from that of classic cadherins (6–8). Protocadherins are thought to have other important activities as well, although the major functions of each have not been well elucidated. A few members of the protocadherin family have been suspected of involvement in the carcinogenesis of several tumors, such as colon cancer (9), hepatocellular carcinoma (9), renal cancer (9, 10), and prostate cancer (11), through their overexpression or inactivation. However, the association between protocadherin proteins and the pathogenesis of many other cancers remains unclear.

Genomic amplifications and homozygous deletions are believed to be useful for identifying oncogenes and tumor-suppressor genes, respectively, critical to tumorigenesis. For example, several typical tumor-suppressor genes were originally pinpointed by mapping regions of biallelic loss in cancer cells (12–15) although the homozygous deletion of these genes is a rare event and other genetic and epigenetic mechanisms, such as mutation and promoter methylation, mainly contribute to their functional inactivation. Therefore, the search for remarkable changes in copy number through the entire genome with high resolution will allow precise and rapid identification of tumor-suppressor genes as well as oncogenes in cancer genomes. For this approach, we have applied several types of in-house bacterial artificial chromosome (BAC)–based array for array-based comparative genomic hybridization (array-CGH) analysis (16–19).

In the course of a program to screen a panel of non–small-cell lung cancer (NSCLC) cell lines for copy number aberrations in a genome-wide manner using an in-house BAC array (16), we have identified a homozygous loss of protocadherin 20 (PCDH20, 13q21.2), of which the expression was absent in the majority of NSCLC cell lines without homozygous loss, although it was present in normal lung tissue. Further analysis using cell lines and primary tumors of NSCLC showed that the silencing of the PCDH20 gene predominantly by epigenetic mechanisms may be involved in the pathogenesis of NSCLC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and primary tumors. Of the 20 NSCLC cell lines employed, seven were derived from squamous-cell carcinomas (EBC-1, LK-2, PC10, VMRC-LCP, LC-1 sq, ACC-LC-73, and KNS-62), seven from adenocarcinomas (11-18, A549, ABC-1, RERF-LC-OK, VMRC-LCD, SK-LC-3, and RERF-LC-KJ), and six from large-cell carcinomas (86-2, LU65, PC-13, ACC-LC-33, NCI-H460, and LU99A). All cell lines were maintained as previously described (13).

Primary tumor samples were obtained during surgery from 112 patients being treated at the National Cancer Center Hospital in Tokyo or the Hokushin General Hospital in Nagano, Japan, with written consent from each patient in the formal style and after approval by the local ethics committees. Samples from 53 patients with adenocarcinoma were embedded in paraffin for laser-captured microdissection after fixation in methanol for 24 hours, as described elsewhere (20). Tumors from the other 59 patients (Table 1 ), along with adjacent non-cancerous lung tissues from 12 of them, were frozen immediately in liquid nitrogen and stored at –80°C until required. None of the patients had received preoperative radiation, chemotherapy, or immunotherapy.


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  		Table 1. Loci of high-level amplification (log 2 ratio > 2.5) detected in NSCLC cell lines by array-CGH analysis using MCG Whole Genome Array-4500

 
Array-CGH analysis. MCG Whole Genome Array-4500 (16), which contains 4523BAC/PAC clones covering the entire genome at intervals of ~0.7 Mb, was used for analysis. Hybridizations were carried out as described elsewhere (19). Hybridized slides were scanned with a GenePix 4000B (Axon Instruments, Foster City, CA) and acquired images were analyzed with GenePix Pro 4.1 imaging software (Axon Instruments). Fluorescence ratios were normalized so that the mean of the middle third of log 2 ratios across the array was zero. Average ratios that deviated significantly (>2 SD) from zero were considered abnormal.

Screening for homozygous deletions by genomic PCR using cell lines and LCM samples. Methanol-fixed, paraffin-embedded tissues were prepared for LCM with a PixCell II LCM system (Arcturus Engineering, Mountain View, CA). Genomic DNA was isolated in lysis buffer (10 mmol/L Tris-HCl at pH 7.5, 1 mmol/L EDTA, and 0.5% SDS) and amplified by adaptor-ligation–mediated PCR after end-filling as described by Tanabe et al. (21).

We screened DNAs from 20 cell lines and 53 primary NSCLCs (adenocarcinomas) for homozygous losses by genomic PCR. All primer sequences are available on request.

Reverse transcription-PCR. Single-stranded cDNAs were generated from total RNAs (17) and amplified with primers specific for each gene. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was amplified at the same time to allow estimation of the efficiency of cDNA synthesis.

Drug treatment. Cells were treated with various concentrations of 5-aza 2'-deoxycytidine (5-aza-dCyd) for 5 days and/or 100 ng/mL of trichostatin A (TSA) for various periods. For the synergistic study, 5 µmol/L 5-aza-dCyd was present in the cultures for 5 days and/or 100 ng/mL TSA was added for the last 12 hours.

Combined bisulfite restriction analysis and bisulfite sequencing. To investigate the methylation of DNA, combined bisulfite restriction analysis (COBRA) was done as described earlier (22). Genomic DNAs from frozen samples were treated with sodium bisulfite and subjected to PCR using primer sets designed to amplify the region of interest. For COBRA, PCR products were digested with Taq1 or Hha1 and electrophoresed. For bisulfite sequencing, PCR products were subcloned and then sequenced.

Promoter reporter assay. We obtained by PCR several DNA fragments around the CpG-rich region of PCDH20 predicted by the CpGPLOT program.8 Each fragment was ligated into the vector pGL3-Basic (Promega, Madison, WI) and an equal amount of each construct was introduced into cells along with an internal control vector (pRL-hTK, Promega) using FuGENE 6 (Roche Diagnostics, Tokyo, Japan). A pGL3-Basic vector without an insert served as a negative control. Firefly luciferase and Renilla luciferase activities were each measured 36 hours after transfection using the Dual-Luciferase Reporter Assay System (Promega); relative luciferase activities were calculated and normalized versus Renilla luciferase activity.

Methylation-specific PCR. Genomic DNA treated with sodium bisulfite was amplified using primers specific to the methylated and unmethylated forms of DNA sequences of interest. DNAs from cell lines and peripheral blood lymphocytes of a healthy male, recognized as unmethylated by bisulfite sequencing, were used as negative controls for the methylation-specific PCR (MSP) assay, whereas those from cell lines recognized as methylated were used as positive controls for methylated alleles. PCR products were visualized on 3% agarose gels stained with ethidium bromide.

Transient transfection, Western blotting, colony formation assay, and anchorage-independent growth assay. A plasmid expressing COOH-terminally Myc-tagged PCDH20 (pcDNA3.1-PCDH20-Myc) was obtained by cloning the reverse transcription-PCR (RT-PCR) product of the full coding sequence of PCDH20 in-frame along with the Myc-epitope into the eukaryotic expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). pcDNA3.1-PCDH20-Myc or the empty vector (pcDNA3.1-mock) control was transfected into cells for colony formation assays or anchorage-independent growth assays. The expression of PCDH20 protein in transiently transfected cells was confirmed 48 hours after transfection by Western-blotting as described elsewhere (14). For colony formation assays, cells were fixed with 70% ethanol and stained with crystal violet after 2 weeks of incubation in six-well plates with appropriate concentrations of G418 (6, 16). For anchorage-independent growth assays, 1 week after transfection and subsequent selection of G418-resistant cells, the same number (1 x 104) of living cells was plated onto a poly(2-hydroxyethyl methacrylate)-poly(HEMA)–coated 96-well plate (23) and cultured for an additional 1 week under the drug selection. The numbers of viable cells were assessed by a colorimetric water-soluble tetrazolium salt assay (cell counting kit-8, Dojindo Laboratories, Kumamoto, Japan; ref. 24).

Statistical analysis. The {chi}2 or Fisher's exact test was used to examine categorical data. To examine the relationship of multiple covariates to the methylation status of the PCDH20 promoter, unconditional logistic regression was used to adjust for the simultaneous effect of multiple variables on the prevalence of PCDH20 hypermethylation. For the analysis of survival, Kaplan-Meier survival curves were constructed for groups based on univariate predictors and differences between the groups were tested with the log-rank test. To examine the simultaneous effects of several variables on patient outcome, the Cox proportional hazards model was used. All tests of significance were two-sided and considered significant at a P < 0.05 level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Array-CGH analysis of NSCLC cell lines. Copy-number gains and losses were seen to some degree in all of the 20 NSCLC cell lines examined. Because the most common genetic aberrations had already been identified in NSCLC cell lines and primary tumors, we paid attention to more remarkable patterns of chromosomal abnormalities, such as high-level amplifications and homozygous deletions, which are likely to be landmarks of oncogenes and tumor-suppressor genes, respectively. Tables 1 and 2 summarize the clones showing high-level amplifications (log 2 ratio > 2.5) or homozygous deletions (log 2 ratio < -2.0), respectively. High-level amplifications were detected in 4 of the 20 NSCLC cell lines and four loci were represented (Table 1).


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  		Table 2. Loci of homozygous deletion (log 2 ratio < –2.0) detected in NSCLC cell lines by array-CGH analysis using MCG Whole Genome Array-4500

 
In all, nine loci were identified as candidate regions for homozygous deletions among eight of the 20 NSCLC cell lines (Table 2; Fig. 1A and B ; and data not shown). The region containing CDKN2A/p16 (four lines) was most frequently deleted. Genomic PCR analysis (Fig. 1B) confirmed complete losses of candidate genes located within or around BACs that had been detected as homozygous deletions by the analysis with array-CGH. Among them, the homozygous loss at 13q21.2, the location of TDRD3 and PCDH20 (Figs. 1A and B and 2A-C ), detected in VMRC-LCD cells, had never been documented in NSCLC before, prompting us to examine whether a tumor-suppressor gene(s) involved in tumorigenesis in the lung might lie within this region.


Figure 1
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  		Figure 1. Detection of candidate loci for homozygous deletions in NSCLC cell lines using the MCG Whole Genome Array-4500 and verification by genomic PCR. A, left, a representative copy number profile of chromosome 13 in VMRC-LCD cells determined by array-CGH. Arrowhead and arrow, candidate spots showing patterns of homozygous deletion (log 2 ratio < –2) at 13q21.2 and 13q32.2-q32.3, respectively. Right, a representative array-CGH image of the VMRC-LCD cell line. A remarkable decrease in the copy number ratio of RP11-307D17 at 13q21.2 was detected as a clear red signal (log 2 ratio = –2.084). B, images from genomic PCR experiments showing GAPDH, the functional control, and representative genes that were absent in at least one cell line for all cases examined. Results of three homozygously deleted loci, which harbor no candidate targets (13q31.1-q31.3 and 20p12.1 in Table 2) or PCDH20, were not included. PLC, normal peripheral lymphocytes.

 

Figure 2
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  		Figure 2. Homozygous deletions and expression levels of PCDH20 in NSCLC. A, map of 13q21.2-21.31 covering the region homozygously deleted in the VMRC-LCD cell line. Horizontal white and black columns, BACs with log 2 ratio <–2 and >–2 in VMRC-LCD cells, respectively. Horizontal white closed arrow, the homozygously deleted region in VMRC-LCD cells as determined by array-CGH and genomic PCR analyses. Black or gray arrows, three genes located around this region, which are homozygously deleted or retained, respectively, that show positions and directions of transcription. B, homozygous deletions of PCDH20 and TDRD3, but not DIAPH3 (data not shown), in one NSCLC cell line (arrowhead), detected by genomic PCR analysis. C, expression of PCDH20 and TDRD3 in NSCLC cell lines and normal lung, detected by RT-PCR analysis. Arrowhead, cell line with the homozygous deletion indicated in (B). Expression of TDRD3 mRNAs was observed to some degree in most NSCLC cell lines whereas 10 of the 19 (52.6%) cell lines without a homozygous deletion of PCDH20 showed almost complete silencing of this gene and 4 (21.5%) showed decreased expression. D, representative results of RT-PCR to reveal PCDH20 expression in ABC-1 and Lu65 cells with or without treatment with 5-aza-dCyd (5 or 10 µmol/L) for 5 days and/or TSA (100 ng/mL) for 24 hours.

 
Identification of target genes involved in homozygous deletion at 13q21.2-21.31. Based on the result of the array-CGH analysis of VMRC-LCD cells (Figs. 1A and 2A and B), the estimated extent of the homozygous deletion was <2 Mb between RP11-218B22 and 79D3 at 13q21.2-21.31 (Fig. 2A, closed gray arrow), and information archived by genome databases9 indicated that only two genes, TDRD3 and PCDH20 (black arrows in Fig. 2A), are located within the candidate region (Fig. 2A). To confirm the homozygous loss of these potential target genes in VMRC-LCD cells and to determine whether this segment might be homozygously lost in other NSCLC lines, we did genomic PCR experiments. We detected a homozygous loss of TDRD3 and PCDH20 only in VMRC-LCD (1 of 27, 3.7%; Fig. 2B, arrows). DIAPH3 (Fig. 2A, gray arrow) was retained in all NSCLC cell lines examined (data not shown).

To determine whether the homozygous deletion of TDRD3 and PCDH20 genes might be a frequent event in primary NSCLC, we did genomic PCR for these genes using LCM-treated primary NSCLC tumors. However, no homozygous deletion of these genes was detected in 53 NSCLC tumors examined (data not shown).

Loss of PCDH20 expression in NSCLC cell lines. Next, we determined expression levels of TDRD3 and PCDH20 by means of RT-PCR in all 20 NSCLC cell lines and normal lung. The VMRC-LCD cells as well as 10 lines without homozygous loss within 13q21.2-21.31 (10 of 19, 52.6%) lacked expression of PCDH20 mRNA (Fig. 2C). The other nine NSCLC cell lines and normal lung did express PCDH20 mRNA, suggesting that loss of expression in some tumors might result from mechanisms other than genomic deletion, including epigenetic events. Six of the 12 lines that had shown a hemizygous loss around PCDH20 on our array-CGH analysis failed to express this gene (data not shown). On the other hand, the TDRD3 gene was expressed in all NSCLC cell lines, except VMRC-LCD, as well as normal lung tissue, suggesting that this gene is not a target for inactivation in NSCLC cells.

Effect of demethylation on PCDH20 expression. Aberrant methylation of DNA in 5' regulatory regions harboring a greater than expected number of CpG dinucleotides is a key mechanism by which genes relevant to cancer initiation and progression can be silenced (22). To investigate whether DNA demethylation could restore the expression of PCDH20 mRNA, we treated NSCLC cells lacking PCDH20 expression with 5-aza-dCyd, a methyltransferase inhibitor, for 5 days. Induction of PCDH20 mRNA expression occurred after treatment with 10 µmol/L 5-aza-dCyd both in ABC-1 and in LU65 cells (Fig. 2D). In addition, we observed an enhancement of PCDH20 mRNA expression by 5-aza-dCyd given along with TSA, a histone deacetylase inhibitor, in both lines although treatment with TSA alone had no effect on its expression, suggesting that histone deacetylation does play some role in the transcriptional silencing of PCDH20 among methylated NSCLC cells (Fig. 2D).

Methylation of the PCDH20 CpG island in NSCLC cell lines. Inspection of the 5' of the PCDH20 genomic sequence revealed that this region is not representative of a CpG island but relatively CpG rich; 25 CpG sites were found in a 446-bp fragment (from –290 to +156, +1 was the translational start site) around exon 1 (Fig. 3A ). To explore the potential role of hypermethylation of this CpG-rich region in silencing the transcription of PCDH20, we first assessed the methylation status in NSCLC-derived cell lines by bisulfite sequencing using 494-bp PCR fragments from –314 to +180 encompassing 25 CpG sites (Fig. 3A). As shown in Fig. 3B, cells lacking PCDH20 expression without its homozygous loss (Lu65, ABC-1, 11-18, EBC1) were aberrantly hypermethylated whereas hypomethylation was seen in PCDH20-expressing cells (Lu99A). Although the degree of methylation varied among CpG sites in PCDH20-nonexpressing cells, CpG sites targeted for methylation were mapped between CpG-10 and CpG-15 in 25 CpG sites (Fig. 3B).


Figure 3
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  		Figure 3. Methylation status of the PCDH20 CpG-rich region in NSCLC cell lines. A, schematic map of the CpG-rich region around exon 1 of PCDH20. Vertical ticks, CpG sites on the expanded axis. Open box, exons; the transcription start site is marked at +1. Heavy black lines, the fragments examined in a promoter assay (fragments 1-4). Horizontal black and gray columns, the regions examined in the COBRA and bisulfite sequencing, respectively. Black and gray upward arrowheads, restriction sites for Taq1 and Hha1, respectively, for the COBRA. Arrows, positions of primers for MSP. B, representative results of bisulfite sequencing of the PCDH20 CpG-rich region examined in PCDH20-expressing NSCLC cell lines (+) and PCDH20-nonexpressing NSCLC cell lines (–). Each square indicates a CpG site: open squares, unmethylated; solid squares, methylated. Black and gray downward arrowheads, restriction sites for Taq1 and Hha1, respectively, for the COBRA. Horizontal white closed arrow, the possible most critical methylated region for silencing of PCDH20 expression. Arrows, positions of primers for MSP. C, representative results of the COBRA of the PCDH20 CpG-rich region in NSCLC cell lines after digestion with Hha1 and Taq1. Arrows, fragments specifically restricted at the sites recognized as methylated CpGs; arrowheads, undigested fragments indicating unmethylated CpGs. D, promoter activity of the PCDH20 CpG-rich region. pGL3 basic empty vectors (mock) and constructs containing one of four different sequences around exon 1 of PCDH20 (Fragments 1-4 in A) were transfected into ABC-1 and Lu65 cells. Luciferase activities were normalized versus an internal control. Columns, mean of three separate experiments, each done in triplicate; bars, SE.

 
Based on the results of bisulfite sequencing, we compared the methylation status of frequently methylated CpG-sites among more cell lines with or without expression of PCDH20 by means of COBRA (Fig. 3C). Six cell lines lacking PCDH20 expression without its homozygous loss showed a hypermethylation pattern whereas three lines with PCDH20 expression showed a hypomethylation pattern, suggesting that the methylation status in specific CpG-sites was likely to be inversely correlated with the expression of PCDH20.

Promoter activity of the PCDH20 CpG-rich region around exon 1. Because part of the PCDH20 CpG-rich region seems to be a target for methylation and closely related to transcriptional silencing of the gene, we tested the CpG-rich region for promoter activity using four fragments encompassing or within this region (519, 279, 58, and 182 bp; fragments 1-4, respectively, in Fig. 3A) that were linked to the luciferase reporter in cells lacking PCDH20 expression. As shown in Fig. 3D, a remarkable increase in transcriptional activity was a feature of constructs containing CpG-10 to CpG-15 targeted for methylation (fragments 1 and 3) whereas constructs not containing those CpG sites (fragments 2 and 4) showed lower transcriptional activity. This result with the correlation between expression and methylation pattern in NSCLC cells (Fig. 3B and C) suggests that the region around specific CpG sites of PCDH20 exhibiting promoter activity is a target for DNA methylation for silencing of this gene.

Methylation of the PCDH20 promoter region in primary NSCLC tumors. To determine whether the aberrant methylation of PCDH20 also takes place in primary tumors of NSCLC, we did MSP with primer sets targeting the sequence around the most frequently methylated sites (Fig. 2A) in a panel of 59 primary NSCLC tumors; corresponding normal lung tissues were available for 12 of them.

Representative results are shown in Fig. 4A . Consistent with the results of the bisulfite sequencing and COBRA (Fig. 3B and C), representative cell lines without PCDH20 expression (EBC1 and LU65) were mostly methylated whereas the PCDH20-expressing cell line (LU99A) was unmethylated, as expected (first line in Fig. 4A). We detected PCDH20 hypermethylation in 32 of the 59 primary NSCLC tissues (54.2% second to sixth lines of Fig. 4A and data not shown) and in 7 of 12 paired samples (58.3%); 3 of those 7 samples were methylated in the corresponding noncancerous lung tissues. Other 5 of 12 paired samples were unmethylated in both tumors and corresponding noncancerous tissues (second line in Fig. 4A and data not shown), suggesting that the aberrant methylation of PCDH20 promoter region may occur early in the lung carcinogenesis (18).


Figure 4
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  		Figure 4. A, representative results of a MSP analysis of the PCDH20 promoter region in primary NSCLC tissues (T). Some of the tumors have corresponding normal lung tissue (N). Parallel amplification reactions were done using primers specific for unmethylated (U) or methylated (M) DNA. The positions of primers are shown in Fig. 3A and B. B, methylation status of the PCDH20 promoter region, determined by bisulfite sequencing in tumor samples and corresponding normal tissues. N, normal; T, tumor. See legend to Fig. 3B for interpretation. C, expression of PCDH20 in three primary cases of NSCLC and paired normal lung tissues. In cases 420 and 425, where tumors had shown aberrant hypermethylation of the PCDH20 promoter region, tumors showed reduced PCDH20 expression compared with normal; case 434, where both tumor and normal tissue were unmethylated in the PCDH20 promoter region, showed no difference in PCDH20 expression. D, Kaplan-Meier curve for overall survival rates of patients with NSCLC. The overall survival rate for NSCLC tumors with a methylated PCDH20 promoter region was significantly lower than that for tumors with an unmethylated PCDH20 promoter region in all 59 patients (P = 0.0140, log-rank test) as well as 39 patients with stage I tumors (P = 0.02110, log-rank test).

 
To confirm the results of the MSP analysis quantitatively and compare PCDH20 methylation status with its expression status, we did bisulfite sequencing (Fig. 4B) and RT-PCR (Fig. 4C), respectively, in representative primary tumor samples and their corresponding noncancerous tissues, although DNA and RNA suitable for assays were available in a few cases. Aberrant hypermethylation and decreased PCDH20 expression were observed in NSCLC tissues, which showed a methylation pattern in MSP analysis (Fig. 4A), compared with the corresponding noncancerous tissue. The presence of hypomethylated alleles was probably due to normal tissue components contaminating the tumors. On the other hand, tumors with an unmethylated pattern in the MSP analysis (Fig. 4A) showed a similar hypomethylation pattern and expression level to corresponding noncancerous tissues. These findings indicate that the PCDH20 promoter methylation was not an artifact arising during the passage of NSCLC cell lines in vitro, but rather may be a true cancer-related event during the pathogenesis of NSCLC.

Association between clinicopathologic characteristics and methylation status of the PCDH20 promoter region in primary cases. To clarify the significance of DNA methylation within the PCDH20 promoter region in the pathogenesis of NSCLC, we first analyzed the relationship between the PCDH20 methylation status determined by MSP and clinicopathologic characteristics of all 59 primary tumors. The methylation status of the PCDH20 promoter region in each sample was not associated with age, gender, smoking history, histologic subtype, or tumor staging (Table 3 ). A multivariate logistic regression analysis to control for the potential confounding effects of variables did not detect any association between the PCDH20 methylation status and the clinicopathologic characteristics described above (data not shown).


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  		Table 3. Relation between clinicopathologic data and PCDH20 promoter hypermethylation

 
Hypermethylation of the PCDH20 promoter region and patient survival. As shown in Fig. 4D, Kaplan-Meier survival estimates showed that hypermethylation of the PCDH20 promoter was significantly associated with a worse overall survival in all 59 NSCLC patients (P = 0.014). Even after stratification of the data by stage, hypermethylation of the PCDH20 promoter was still significantly associated with a poor prognosis in 39 patients with stage I tumor (P = 0.0211, Fig. 4D). In addition, when the data were stratified for the multivariate analysis using the Cox proportional hazard regression analysis, hypermethylation of the PCDH20 promoter remained significant at P = 0.0493 (hazard ratio, 3.21) for overall survival in all patients, suggesting that the methylation status of the PCDH20 promoter might be an independent predictor of overall survival (Table 4 ).


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  		Table 4. Cox proportional hazard regression analysis for overall survival

 
Suppression of cell growth after restoration of PCDH20 expression. The frequent silencing of PCDH20 by promoter methylation in NSCLC cell lines and primary tumors suggests that PCDH20 is likely a functional tumor suppressor. To investigate whether restoration of PCDH20 expression would suppress growth of NSCLC cells in which the gene had been silenced, we did colony formation assays and anchorage-independent growth assays using an expression construct of the full coding sequence of PCDH20 (Fig. 5A ). As shown in Fig. 5B, 2 weeks after transfection and subsequent selection of drug-resistant colonies, the numbers of large colonies produced by PCDH20-transfected cells decreased compared with those of cells containing empty vector. In addition, ectopic expression of PCDH20 reduced the number of living A549 cells on poly(HEMA)-coated plates (Fig. 5C), suggesting that anchorage-independent growth was suppressed after restoration of PCDH20 in these cells. However, we have not been able to obtain cell lines stably expressing PCDH20 protein using constitutive or inducible strategies (data not shown).


Figure 5
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  		Figure 5. Effect of restoration of PCDH20 expression on growth of NSCLC cells. A COOH-terminally Myc-tagged construct containing PCDH20 (pcDNA3.1-PCDH20-Myc) or empty vector (pcDNA3.1-mock) as a control was transfected into ABC-1 or A549 cells, which lack expression of the PCDH20 gene because the CpG-rich region is methylated. A, Western blot analysis using 10 µg of protein extract, anti-Myc antibody, and the internal control antibody (anti-ß-actin), showing that cells transiently transfected with pcDNA3.1-PCDH20-Myc expressed Myc-tagged PCDH20 protein. B, 3 weeks after transfection and subsequent selection of drug-resistant colonies in six-well plates (16, 19), the colonies formed by PCDH20-transfected ABC-1 and A549 cells were less numerous than those formed by mock-transfected cells (top). Bottom, quantitative analysis of colony formation in ABC-1 and A549 cells. Colonies larger than 2 mm were counted. Columns, mean of three separate experiments, each done in triplicate; bars, SE. C, 1 week after transfection and subsequent selection of G418-resistant cells, the same number of living cells was plated onto poly(HEMA)-coated 96-well plates and cultured for an additional 5 days under drug selection. Poly(HEMA) was prepared by dissolving it in 95% ethanol to a concentration of 50 mg/mL and added to cell culture wells at a density of 5 mg/cm2, then allowed to dry overnight under sterile conditions in a laminar flow hood. Larger colonies had formed in mock-transfected A549 cells than PCDH20-transfected cells 5 days after plating onto poly(HEMA)-coated plates (top). Bottom, quantitative analysis of the number of living cells 2 and 5 days after plating onto poly(HEMA)-coated 96-well plates determined by water-soluble tetrazolium salt assay. Columns, mean of three separate experiments, each done in triplicate; bars, SE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The introduction of higher-density array-CGH and the vast amounts of mapping data that have recently become available through the human genome effort have greatly facilitated the analysis of tumor-related amplified or deleted chromosomal regions. In this study, we applied our in-house MCG Whole Genome Array-4500, covering the entire human genome with a total of 4523BACs (16), to scan for extreme aberrations, such as high-level amplifications and homozygous deletions in a panel of NSCLC cell lines. As observed in our previous array-CGH analyses of gastric cancer cell lines (19, 25), homozygous losses were more effectively identified with the MCG Whole Genome Array-4500 than MCG Cancer Array-800 (18), probably due to its greater density.

Among the nine homozygously deleted loci identified in our panel of NSCLC cell lines, we chose to focus on the complete loss at 13q21 observed in one of the cell lines because (a) relatively frequent loss at 13q was observed in our panel of cell lines (13 of 20, 65%) and has been previously reported in NSCLC (26–28), but (b) tumor-suppressor genes have not been known to be harbored within or around this region, except RB located in 13q14, in NSCLC (28). Among genes located within this homozygously deleted region, the expression of PCDH20 was detected in normal lung but frequently silenced in our panel of NSCLC cell lines, prompting us to focus on this gene as the possible target for inactivation in NSCLC. Homozygous deletion was not detected in primary tumors of NSCLC whereas hypermethylation of CpG sites was observed in the relatively CpG-rich region exhibiting clear promoter activity and likely to be inversely correlated with the expression of PCDH20 mRNA both in cell lines and in primary tumors of NSCLC. Those results suggest that the inactivation of PCDH20 occurs mainly through either deletion of one allele and methylation of the other or methylation of both alleles during the tumorigenesis of NSCLC.

With >60 members, the protocadherins represent the largest subgroup of the cadherin superfamily (3, 5). Loss of expression or functional inactivation, caused by mutations or methylation of the promoter of members of the cadherin superfamily (e.g., CDH1, CDH13, CDH11, PCDH-LKC, PCDH-{gamma}-A11, and PCD10), has been shown in a number of different cancer entities including colorectal, breast, liver, lung, nasopharyngeal, and esophageal cancers and astrocytoma (9, 29–35), resulting in tumor cell invasion and metastasis (34–36). In addition, the expression of PCDH7, PCDH8, and PCDH9 was up-regulated after pharmacologic demethylation in tumor cells, indicating that they could also be candidate genes aberrantly silenced by DNA methylation in tumors (35, 37). Those results are complementary to ours and suggest that epigenetic inactivation of members of the cadherin superfamily, including protocadherins in neoplasms, could be widespread indeed and more genes in this family may be targets for inactivation by epigenetic mechanisms during tumorigenesis, although protein structure, expression patterns in various tissues, and physiologic function differ among members.

Among our panel of 59 primary NSCLC tumors, the methylation status of the PCDH20 promoter region determined by MSP in each sample was not associated with any clinicopathologic variables, including smoking history, although recent reports have suggested that smoking increases DNA methyltransferase activity and that DNA hypermethylation is associated with exposure to tobacco smoke (38, 39). The most striking finding in our study was that hypermethylation of the PCDH20 promoter was significantly associated with a worse clinical outcome without a correlation with tumor stage, suggesting that the inactivation of PCDH20 by promoter hypermethylation may occur in a stage-independent manner and be involved in the malignant progression of NSCLC. In some cases, PCDH20 hypermethylation was observed both in tumors and in the corresponding noncancerous tissues (Fig. 4A), suggesting that the aberrant CpG methylation of this gene seems to follow a chronological order during carcinogenesis and might be established during an early stage in the multistep process of lung carcinogenesis as shown in DBC1 (18). Because the result of our multivariate analysis showed that hypermethylation of the PCDH20 promoter seems to be an independent prognosticator, additional studies using a larger set of primary NSCLC need to be conducted that will help in assessing the clinical importance of the present findings. In particular, the importance of hypermethylated PCDH20 DNA as a diagnostic as well as a prognostic marker in the plasma of NSCLC patients has yet to be determined.

Colony-formation assays and anchorage-independent growth assays using transiently PCDH20-transfected NSCLC cell lines showed growth-suppressing and/or antiproliferative activity of the PCDH20 protein under both anchorage-dependent and anchorage-independent conditions, suggesting that PCDH20 may possess important functions, such as in cell-cell adhesion, signal transduction, and growth control. However, the exact function of PCDH20 is poorly defined, especially with respect to tumorigenesis (40). Different from classic cadherins, protocadherins are not just simple cell adhesion proteins involved in homophilic interactions (40, 41) and their heterophilic interactions with other molecules may be more important for their physiologic functions, such as the involvement in signal transduction. However, no motif associated with specific functions of molecules has been reported in PCDH20. Because no system we have tried was successful for obtaining stable cell lines expressing PCDH20 among our panel of NSCLC cell lines, it will be difficult to investigate the mechanisms of tumor-suppressing activity of PCDH20 any further. However, the tumor-suppressing activity of PCDH20 in vitro, together with its expression in normal lung and the inverse correlation between the methylation status of the PCDH20 promoter and expression level of PCDH20 mRNA in NSCLC cells, supports the candidacy of this gene as a tumor suppressor for NSCLC. Additional studies will be required to unravel the carcinogenic consequences of loss of PCDH20 function in human lung tissue.


   Acknowledgments
 
Grant support: Grants-in-Aid for Scientific Research on Priority Areas (C) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; Grant-in-Aid from Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation; Center of Excellence program for Frontier Research on Molecular Destruction and Reconstitution of Tooth and Bone; program for promotion of Fundamental Studies in Health Sciences of the Pharmaceuticals and Medical Devices Agency; and the Third Term Comprehensive Control Research for Cancer of the Ministry of Health, Labour and Welfare.

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.

We thank Professor Yusuke Nakamura (Human Genome Center, Institute of Medical Science, The University of Tokyo) for continuous encouragement throughout this work; Professor Takashi Takahashi (Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University Graduate School of Medicine), Professor Akira Horii (Department of Molecular Pathology, Tohoku University Graduate School of Medicine), and Professor Takehiko Fujisawa (Department of Thoracic Surgery, Chiba University Graduate School of Medicine) for providing NSCLC cell lines; and Ai Watanabe and Ayako Takahashi for technical assistance.


   Footnotes
 
8 http://www.ebi.ac.uk/emboss/cpgplot/. Back

9 http://www.ncbi.nlm.nih.gov/ and http://genome.ucsc.edu/. Back

Received 12/14/05. Revised 1/29/06. Accepted 3/ 7/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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