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WT1 and WT1-AS genes are inactivated by promoter methylation in ovarian clear cell adenocarcinoma
Article first published online: 30 AUG 2005
Copyright © 2005 American Cancer Society
Volume 104, Issue 9, pages 1924–1930, 1 November 2005
How to Cite
Kaneuchi, M., Sasaki, M., Tanaka, Y., Shiina, H., Yamada, H., Yamamoto, R., Sakuragi, N., Enokida, H., Verma, M. and Dahiya, R. (2005), WT1 and WT1-AS genes are inactivated by promoter methylation in ovarian clear cell adenocarcinoma. Cancer, 104: 1924–1930. doi: 10.1002/cncr.21397
- Issue published online: 17 OCT 2005
- Article first published online: 30 AUG 2005
- Manuscript Accepted: 3 DEC 2004
- Manuscript Revised: 17 NOV 2004
- Manuscript Received: 13 AUG 2004
- National Institutes of Health and Veterans' Administration Research Enhancement Award Program (REAP) award and Merit Review grants. Grant Numbers: RO1AG21418, RO1CA101844, T32DK07790
- Wilms tumor suppressor 1 gene;
- WT1 antisense;
- ovarian carcinoma;
- cancers of the ovary
Ovarian clear cell adenocarcinoma is associated with one of the poorest prognoses among human epithelial ovarian cancers. The authors hypothesized that Wilms tumor suppressor 1 gene (WT1) sense and antisense (WT1-AS) expression and their promoter methylation status could characterize ovarian clear cell adenocarcinoma from ovarian serous adenocarcinoma.
To test this hypothesis, ovarian cancer cell lines and 42 cancer tissues (17 clear cell and 25 serous adenocarcinoma) were analyzed for expression and methylation of WT1 and WT1-AS genes.
These experiments demonstrated that all serous adenocarcinoma tissues expressed both WT1 and WT1-AS genes, although expression of these genes was lacking in clear cell adenocarcinoma. The WT1 and WT1-AS promoter were significantly methylated in clear cell adenocarcinoma (88.2% and 88.2%, respectively) compared with serous adenocarcinoma (24.0% and 20.0%, respectively). Significant correlation between methylation and mRNA expression status was observed for each gene. Also in agreement with these data, WT1 and WT1-AS negative ovarian cancer cell lines reexpressed these genes after treatment with the demethylating agent, 5-aza-2′-deoxycytidine.
The current study shows that CpG hypermethylation is an important mechanism of WT1 and WT1-AS gene inactivation in ovarian clear cell adenocarcinoma. This is the first report that has demonstrated differential expression and methylation of WT1-AS in ovarian clear cell and serous adenocarcinomas. This study presents new molecular characterizations between these two types of adenocarcinoma and may provide insight as to why clear cell adenocarcinoma has a poorer prognosis than serous adenocarcinoma of the ovary. Cancer 2005. © 2005 American Cancer Society.
Epithelial ovarian cancer is a significant cause of cancer death among women. The pathologic subtype of ovarian cancer is a key factor for determining patient prognosis. Clear cell adenocarcinoma typically imparts a poorer prognosis compared with other histologic types because of extreme chemoresistance despite being often identified at an early clinical stage.1 Therefore, efforts to biochemically characterize clear cell adenocarcinoma are very important, informative, and essential for treatment of this cancer. Differential expression of Wilms tumor suppressor 1 gene (WT1) has been reported in different pathologic types of ovarian cancer2–4 and promoter hypermethylation is considered to be an important factor for loss of gene function. Previous reports have shown that the WT1 promoter is often methylated in primary breast cancers5–7 and human colorectal cancers,8 interestingly, in both types of cancer, these tumors express WT1 despite methylation of the gene promoter. However, the mechanisms that regulate expression of the WT1 gene have not been extensively studied in ovarian cancer.
Factors involved in the regulation of expression of WT1 include several antisense transcripts of the WT1 gene9, 10 and the large antisense transcript known as WT1-antisense (WT1-AS).11 WT1-AS transcript includes both the first exon of the WT1 gene with the promoter positioned in intron 1 of the WT1 gene.11–13 Hypermethylation of the intron 1 region was observed in mesothelioma, renal cell carcinoma,14 and breast cancer;5, 7 however, these studies did not evaluate WT1-AS expression. Moreover, evaluation of WT1-AS expression and promoter methylation has not been studied in ovarian cancer.
In this study, we hypothesize that the WT1 and WT1-AS genes may be differentially expressed and methylated in ovarian clear cell and serous adenocarcinomas. We tested this hypothesis by analysis of mRNA expression and methylation-specific polymerase chain reaction (MSP) of both WT1 and WT1-AS genes in ovarian clear cell and serous adenocarcinomas. To our knowledge, this is the first report on the expression and methylation status of these genes in different subtypes of human epithelial ovarian cancers.
MATERIALS AND METHODS
Primary Human Epithelial Ovarian Carcinoma Tissues and Nucleic Acid Isolation
Forty-two primary human epithelial ovarian carcinoma samples, comprising 25 serous adenocarcinoma and 17 clear cell adenocarcinoma, were obtained from the Department of Gynecology at the Hospital of Hokkaido University, Japan. Among them, 20 samples were fresh frozen tissues taken only from cancerous regions at the time of surgery. These tissues were confirmed pathologically to have a majority of cancer cells in the sample. The other 22 cancer tissues were microdissected from paraffin-embedded sections. Both DNA and RNA were extracted from fresh frozen tissues using TRI Reagent (Molecular Research Center, Cincinnati, OH). Extracted RNA samples were treated with DNase (DNA-free, Ambion, Austin, TX) before reverse transcription (RT). Isolation of genomic DNA from paraffin-embedded tissues was performed using a DNeasy tissue kit (Qiagen, Valencia, CA). Appropriate informed consent was obtained from patients in accordance with local ethical committee guidelines of the hospital.
Ovarian Cancer Cell Lines and Nucleic Acid Isolation
Human ovarian cancer cell lines were obtained from the Cell Culture Facility at the University of California, San Francisco. OVCAR-3 and SKOV-3 were used for these studies and cultured in media according to the recommendations of the American Type Culture Collection (Rockville, MD), in a humidified 5% CO2 atmosphere at 37 °C. The cells were treated with a freshly prepared solution of the demethylating agent, 5-azaC (Sigma, Santa Cruz, CA) and harvested as previously described.15 Messenger RNA from each cell line was extracted using Oligotex Direct mRNA Mini Kit (Qiagen, Valencia, CA). Genomic DNA was isolated using DNeasy tissue kit (Qiagen, Valencia, CA).
Reverse Transcription Polymerase Chain Reaction
A total of 1–5 μg of RNA was reverse-transcribed with random hexamers in a 40 μL reaction using a Reverse Transcription System kit (Promega Corp., Madison, WI). Complementary DNA was amplified by polymerase chain reaction (PCR) using primers and conditions shown in Table 1. Products for WT1 show two isoforms (198 base pairs [bp] and 147 bp) because of the splicing of exon 5. The primer pair used to detect WT1-AS mRNA product was designed to amplify a 343 bp fragment spanning the splicing sequence reported by Gessler and Bruns,10 and products were confirmed by sequencing. Schematic representation of the WT1 and WT1-AS gene structure is shown in Figure 1. PCR reactions (35 cycles for G3PDH, 40 cycles for WT1 and 45 cycles for WT1-AS) were performed in a PTC-200 thermal cycler (MJ Research, Watertown, Massachusetts) and PCR products were resolved by electrophoresis using 3% agarose gels.
|Gene||Primer namea||Sequences||Location||Accession number||PCR condition|
|WT1||WT1-RT-F||5′-CAAATGACATCCCAGCTTGA-3′||1072–1091||NM_024424||94°C, 1 min; 55°C, 1 min; 72°C, 1 min|
|WT1-Univ-F||5′-GGGGGAGGGTTGTGTTATAT-3′||1186–1205||X74840||94°C, 1 min; 55°C, 1 min; 72°C, 1 min|
|WT1-M-F||5′-GTTTTTAAGGAGTAGCGCGC-3′||1295–1316||X74840||94°C, 30 sec; 60°C, 45 sec; 72°C, 1 min|
|WT1-U-F||5′-GAGGGTGTTTTTAAGGAGTAGTGT-3′||1291–1314||X74840||94°C, 30 sec; 58°C, 45 sec; 72°C, 1 min|
|WT1-AS||WT1AS-RT-F||5′-GAGGACAGAGAGGCATGGAG-3′||51306–51325||AL049692||94°C, 1 min; 55°C, 1 min; 72°C, 1 min|
|WT1AS-Univ-F||5′-GAAGAGGGTAAATTATAGGGGTTT-3′||1440–1463||AF233371||94°C, 1 min; 55°C, 1 min; 72°C, 1 min|
|WT1AS-M-F||5′-TAAATTATAGGGGTTTCGTAGGTTC-3′||1448–1472||AF233371||94°C, 30 sec; 60°C, 45 sec; 72°C, 1 min|
|WT1AS-U-F||5′-AATTATAGGGGTTTTGTAGGTTTGG-3′||1450–1474||AF233371||94°C, 30 sec; 58°C, 45 sec; 72°C, 1 min|
|G3PDH||G3PDH-RT-F||5′-TCCCATCACCATCTTCCA-3′||291–308||NM_002046||94°C, 1 min; 55°C, 1 min; 72°C, 1 min|
Bisulfite Modification and Methylation-Specific PCR (MSP)
Bisulfite modification of genomic DNA was carried out using CpGenome DNA Modification Kit (Intergen, Purchase, NY). Sequences, references, PCR conditions, and orientation of each primer used for MSP analysis are summarized in Table 1 and Figure 1A. Two-step PCR procedure was used. For the first PCR, modified DNA was amplified for 40 cycles using primer sets indicated “-Uni-” in Table 1. These primer sets were prepared to amplify larger products than the targeted methylation-specific PCR products and designed not to contain cytosine guanine (CG) sequences. First PCR products were subjected to MSP analysis using primer sets indicated “-M-” or “-U-” in Table 1. The amplification cycles performed were 35 cycles for frozen tissue-extracted DNA and 40 cycles for paraffin-extracted DNA.
For confirmation of MSP, first PCR products were purified by QIAquick PCR Purification kit (Qiagen, Valencia, CA) and 20 ng of PCR products were used as a template for sequencing. Double-strand sequence analysis was performed using each primer set, an ABI 377 Sequencer, and a Dye Terminator Cycle sequencing kit (Applied Biosystems, Foster City, CA).
All statistical analyses were performed using StatView Statistical Analysis software (SAS Institute, Cary, NC). The methylation status of WT1 and WT1-AS promoters between clear cell and serous adenocarcinoma tissues was compared using Mann–Whitney U-test. Chi-square analysis was used to determine the relation between expression and methylation status, and Fisher exact P value was used for evaluation.
Table 2 summarizes the methylation status of WT1 and WT1-AS promoters, determined by MSP using methylated and unmethylated sequence-specific primers, in clear cell and serous adenocarcinomas. Representative MSP results for each histologic type of ovarian cancer are shown in Figure 2A. The WT1 promoter was methylated in 88.2% of clear cell and 24.0% of serous adenocarcinoma tissues (P < 0.05). The WT1-AS promoter was methylated in 88.2% clear cell compared with 20.0% of serous adenocarcinoma tissues (P < 0.05). Figure 2B shows representative results of direct sequencing from the first PCR product from bisulfite-modified DNA. CpG sites are underlined, and all cytosines were deaminized and converted to thymines in the sample from serous adenocarcinoma, whereas 5-methylcytosines remained unaltered in the sample from clear cell adenocarcinoma.
|Histology||WT1 Promoter||WT1-AS Promoter|
|Methylated||6/25 (24.0%)||5/25 (20.0%)|
|Unmethylated||19/25 (76.0%)||20/25 (80.0%)|
|Clear cell adenocarcinoma|
|Methylated||15/17 (88.2%)||15/17 (88.2%)|
|Unmethylated||2/17 (11.8%)||2/17 (11.8%)|
To determine the relations between methylation and expression status of these genes, we analyzed mRNA of WT1 and WT1-AS expression by RT-PCR in 8 clear cell and 12 serous adenocarcinoma tissues of which RNAs were available (Fig. 3). WT1 and WT1-AS mRNA was not detected in any of the clear cell adenocarcinoma tissues; conversely, both WT1 and WT1-AS mRNA were detected in all serous cell tissues. Noteworthy is that presence or absence of mRNA expression of WT1 is identical to that of WT1-AS for each tissue. The methylation status of the WT1 promoter and WT1-AS promoter region were found to have significant correlation (P < 0.05) with expression of WT1 and WT1-AS mRNA, respectively, in serous and clear cell adenocarcinoma (Table 3). These results suggest that methylation of the WT1 and WT1-AS promoter silences the expression of each gene in clear cell adenocarcinoma compared with serous adenocarcinoma.
|WT1 promoter||WT1-AS promoter|
|WT1 expression||WT1-AS expression|
To determine whether mRNA expression of WT1 and WT1-AS genes is inactivated by promoter methylation, we performed RT-PCR and MSP analysis using SKOV-3 and OVCAR-3 cell lines. Although the OVCAR-3 cell line expresses WT1 and WT1-AS mRNA, the SKOV-3 cell line lacks mRNA for both genes (Fig. 4A). In agreement with mRNA expression results, both WT1 and WT1-AS promoters are methylated in SKOV-3 cells, and unmethylated in OVCAR-3 cells (Fig. 4B). To investigate the mechanisms of inactivation of WT1 and WT1-AS genes, we treated these cell lines with the demethylating agent, 5-azaC. After treatment with 5-azaC, both WT1 and WT1-AS promoters were demethylated, and mRNA expression of both genes was restored in the SKOV-3 cell line (Fig. 4A–B), suggesting that methylation is involved in the inactivation of the WT1 and WT1-AS genes. No significant changes were observed in the OVCAR-3 cell line.
In the current study, we tested the hypothesis that WT1 and WT1-AS genes are differentially expressed and methylated in ovarian clear cell and serous adenocarcinoma. The results of these experiments demonstrate that the WT1 promoter was methylated in 88.2% of clear cell adenocarcinoma tissues, whereas it was unmethylated in 76.0% of serous adenocarcinoma tissues. Similarly, the WT1-AS promoter was methylated in 88.2% of clear cell adenocarcinoma tissues and unmethylated in 80.0% of serous adenocarcinoma tissues. These results suggest that methylation profiles of both WT1 and WT1-AS are different between clear cell and serous adenocarcinoma. In agreement with these methylation profiles, WT1 and WT1-AS mRNA were not detected in any clear cell adenocarcinoma tissues but were detected in all serous adenocarcinoma tissues. Previous reports have shown that the WT1 promoter is often methylated in primary breast tumors5–7 and human colorectal cancer,8 which nevertheless express WT1. Conversely, hypermethylation of the intron 1 region of WT1, known to include the promoter region of WT1-AS gene, is also reported in mesothelioma, renal cell carcinoma,14 and breast tumor.5, 7 As observed in other cancers,5–8 2 of 12 serous adenocarcinoma tissues also expressed WT1 mRNA, although they had methylation in the WT1 promoter region. Therefore, the degree and significance of methylation for inactivation of WT1 may vary, depending upon which CpG sites are methylated in the promoter region or upon the cancer type. However, our results clearly demonstrate that the expression of WT1 and WT1-AS correlates with the methylation status of each promoter region in the majority of ovarian serous and clear cell adenocarcinoma. Previous reports have demonstrated that WT1 or WT1-AS promoter activity depends on a very limited region approximately 450 bp immediately upstream of the WT1 or WT1-AS start site, where several transcription factor-binding sites are located.11, 16 Because the primers used for the WT1 or WT1-AS methylation study were designed to cover this important region, we believe our methylation study reflects the total promoter activity of WT1 or WT1-AS. Moreover, the restoration of both WT1 and WT1-AS expression after treatment of ovarian cancer cell line (SKOV-3) with a demethylating agent (5-azaC) also showed the significance of CpG methylation in silencing these genes in ovarian cancer.
The WT1 gene has been reported to be an important molecular marker to characterize the different pathologic subtypes of ovarian cancer.2–4 The current study also confirmed the different expression status of WT1 gene between clear cell and serous adenocarcinoma. Moreover, we showed that expression of WT1-AS mRNA was also different among them. Of note, the presence or absence of mRNA expression of WT1 is identical to that of WT1-AS for each tissue, which is in contrast to the usual regulatory system in the expression of sense mRNA by antisense mRNA. Moorwood et al. reported that expression of WT1-AS mRNA parallels expression of WT1 mRNA in fetal kidney, and introduction of WT1-AS transcripts causes the up-regulation of WT1 mRNA.17 Although our study can support their findings, further studies are necessary to confirm the function of WT1-AS in ovarian cancer.
Among epithelial ovarian cancers, clear cell adenocarcinoma is known to show much more clinical aggressiveness compared with serous adenocarcinoma, which is the major type of ovarian cancer.1 Considering that the WT1 gene may transcriptionally regulate the expression of various other genes,18 differential expression and methylation of both WT1 and WT1-AS may characterize a key biochemical feature of clear cell adenocarcinoma compared with serous adenocarcinoma. A recent study by Gius et al.20 tested the hypothesis that the effects of gene expression of altered DNA methylation by 5-aza-2-deoxycutidine and genetic manipulation of DNA are similar.
In conclusion, our study presents a possible regulatory mechanism of WT1 and WT1-AS expression through promoter hypermethylation in clear cell adenocarcinoma and serous adenocarcinoma. Moreover, the differential expression and methylation status of the WT1 and WT1-AS genes in clear cell adenocarcinoma and serous adenocarcinoma is a noteworthy biochemical difference between these two forms of ovarian cancer.