Yonsei Med J.  2014 Nov;55(6):1656-1663. 10.3349/ymj.2014.55.6.1656.

DNA Hypomethylation-Mediated Overexpression of Carbonic Anhydrase 9 Induces an Aggressive Phenotype in Ovarian Cancer Cells

Affiliations
  • 1Department of Biochemistry, School of Medicine, Ewha Womans University, Seoul, Korea. ahnj@ewha.ac.kr
  • 2Department of Obstetrics and Gynecology, School of Medicine, Ewha Womans University, Seoul, Korea. goodmorning@ewha.ac.kr

Abstract

PURPOSE
Both genetic and epigenetic alterations can lead to abnormal expression of metastasis-regulating genes in tumor cells. Recent studies suggest that aberrant epigenetic alterations, followed by differential gene expression, leads to an aggressive cancer cell phenotype. We examined epigenetically regulated genes that are involved in ovarian cancer metastasis.
MATERIALS AND METHODS
We developed SK-OV-3 human ovarian carcinoma cell xenografts in mice. We compared the mRNA expression and DNA methylation profiles of metastatic tissues to those of the original SK-OV-3 cell line.
RESULTS
Metastatic implants showed increased mRNA expression of the carbonic anhydrase 9 (CA9) gene and hypomethylation at CpG sites in the CA9 promoter. Treatment of wild-type SK-OV-3 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reduced methylation of the CA9 promoter and increased CA9 mRNA expression. Eight CpGs, which were located at positions -197, -74, -19, -6, +4, +13, +40, and +86, relative to the transcription start site, were hypomethylated in metastatic tumor implants, compared to that of wild-type SK-OV-3. Overexpression of CA9 induced an aggressive phenotype, including increased invasiveness and migration, in SK-OV-3 cells.
CONCLUSION
Alterations in the DNA methylation profile of the CA9 promoter were correlated with a more aggressive phenotype in ovarian cancer cells.

Keyword

Ovarian cancer; metastasis; mouse xenograft; CA9; DNA methylation

MeSH Terms

Animals
Azacitidine/*analogs & derivatives/pharmacology
Carbonic Anhydrases/metabolism
*DNA Methylation
Female
Gene Expression Regulation, Neoplastic/*drug effects
Humans
Mice
Neoplasm Invasiveness/genetics
Neoplasm Metastasis/genetics/*pathology
Neoplasms, Experimental
Neoplasms, Glandular and Epithelial/genetics/*metabolism/pathology
Ovarian Neoplasms/genetics/*metabolism/pathology
Phenotype
Promoter Regions, Genetic
RNA, Messenger/metabolism
Azacitidine
Carbonic Anhydrases
RNA, Messenger

Figure

  • Fig. 1 CA9 expression is up-regulated in metastatic implants from mouse xenografts. CA9 mRNA expression was measured by gene expression microarray (A) and qRT-PCR (B). The error bars indicate means±standard deviations of triplicate experiments. The metastatic implants from the mouse xenografts are labeled 1C-8C (n=7). qRT-PCR, quantitative reverse-transcription polymerase chain reaction.

  • Fig. 2 DNA methylation is altered at CpG sites in the CA9 promoter in metastatic implants from mouse xenografts. The DNA methylation status at the -6 CpG site was analyzed with the Illumina HumanMethylation 450 BeadChip (A) and qMSP (B). The DNA methylation status was analyzed by bisulfite sequencing (C). The CA9 promoter is located between positions 35673651 and 35674055 in the human GRCh37/hg19 assembly, and contains eight CpG residues on chromosome 9. The eight CpGs are at positions -197, -74, -19, -6, +4, +13, +40, and +86 relative to the transcription start site. Each circle represents a CpG dinucleotide. The methylation status of each CpG site is indicated with a black (methylated) or white (unmethylated) circle. The percentage of methylation at each site is indicated in a pie graph on the bottom line. The black segment of the pie graph indicates the percentage of methylated CpGs, whereas the white segment represents the percentage of unmethylated CpGs (C). Triangles above the circles in C indicate the specific CpG site that was used for qMSP. Statistical analyses were performed by one-way ANOVA and subsequent Bonferroni tests (*p<0.05). M, the percentage of methylated CpGs; U, the percentage of unmethylated CpGs; qMSP, quantitative methylation-specific PCR; PCR, polymerase chain reaction; CA9, carbonic anhydrase 9; ANOVA, analysis of variance.

  • Fig. 3 Altered expression of CA9 following demethylation in SK-OV-3 cells. SK-OV-3 cells were treated for three days with 0, 5, or 10 µM 5-aza-2'-deoxycytidine, respectively. After treatment with 5-aza-2'-deoxycytidine, CA9 mRNA expression was measured by qRT-PCR (A). The DNA methylation status at the specific CpG site was analyzed by quantitative MSP (B). Data are shown as the means±SDs (n=3). Statistical analyses were performed with one-way ANOVA and subsequent Bonferroni tests (*p<0.05). M, the percentage of methylated CpGs; U, the percentage of unmethylated CpGs; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; CA9, carbonic anhydrase 9; MSP, methylation-specific PCR; ANOVA, analysis of variance.

  • Fig. 4 CA9 promotes migration of SK-OV-3 cells. Migration of serum-starved cells towards 15% serum-containing media was examined in a transwell assay. Cells that migrated through an 8-µm pore filter were fixed and stained with crystal violet. Representative images of migrated cells transfected with EGFP or CA9 are shown (A). Quantitative analysis of migrated cells was performed by measuring the absorbance of extracts from cell stains at 595 nm. Data are shown as means±SDs for triplicate measurements (B). Statistical analysis was performed with a t-test (*p<0.05). CA9, carbonic anhydrase 9; EGFP, enhanced green fluorescent protein.

  • Fig. 5 CA9 enhances the invasiveness of SK-OV-3 cells. Invasion of serum-starved cells towards 10% serum-containing media was examined in a Matrigel-coated invasion chamber. The cells that invaded through Matrigel were fixed and stained with crystal violet. Representative images of invading cells transfected with EGFP or CA9 are shown (A). Quantitative analysis of invading cells was performed by measuring the absorbance of extracts from cell stains at 595 nm. Data are shown as means±SDs for triplicate measurements (B). Statistical analysis was performed with a t-test (*p<0.05). CA9, carbonic anhydrase 9; EGFP, enhanced green fluorescent protein.


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