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

• Sung HY
• Ju W
• Ahn JH

• KMID: 2070216
• DOI: http://doi.org/10.3349/ymj.2014.55.6.1656

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.

Reference

1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014; 64:9–29. PMID: 24399786.

2. Lengyel E. Ovarian cancer development and metastasis. Am J Pathol. 2010; 177:1053–1064. PMID: 20651229.

3. Kipps E, Tan DS, Kaye SB. Meeting the challenge of ascites in ovarian cancer: new avenues for therapy and research. Nat Rev Cancer. 2013; 13:273–282. PMID: 23426401.

4. Feki A, Berardi P, Bellingan G, Major A, Krause KH, Petignat P, et al. Dissemination of intraperitoneal ovarian cancer: Discussion of mechanisms and demonstration of lymphatic spreading in ovarian cancer model. Crit Rev Oncol Hematol. 2009; 72:1–9. PMID: 19179094.

5. Tan DS, Agarwal R, Kaye SB. Mechanisms of transcoelomic metastasis in ovarian cancer. Lancet Oncol. 2006; 7:925–934. PMID: 17081918.

6. Jeon BH, Jang C, Han J, Kataru RP, Piao L, Jung K, et al. Profound but dysfunctional lymphangiogenesis via vascular endothelial growth factor ligands from CD11b+ macrophages in advanced ovarian cancer. Cancer Res. 2008; 68:1100–1109. PMID: 18281485.

7. Bussink J, Kaanders JH, van der Kogel AJ. Tumor hypoxia at the micro-regional level: clinical relevance and predictive value of exogenous and endogenous hypoxic cell markers. Radiother Oncol. 2003; 67:3–15. PMID: 12758235.

8. Ivanov S, Liao SY, Ivanova A, Danilkovitch-Miagkova A, Tarasova N, Weirich G, et al. Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer. Am J Pathol. 2001; 158:905–919. PMID: 11238039.

9. de Martino M, Klatte T, Seligson DB, LaRochelle J, Shuch B, Caliliw R, et al. CA9 gene: single nucleotide polymorphism predicts metastatic renal cell carcinoma prognosis. J Urol. 2009; 182:728–734. PMID: 19539328.

10. Bui MH, Seligson D, Han KR, Pantuck AJ, Dorey FJ, Huang Y, et al. Carbonic anhydrase IX is an independent predictor of survival in advanced renal clear cell carcinoma: implications for prognosis and therapy. Clin Cancer Res. 2003; 9:802–811. PMID: 12576453.

11. Giatromanolaki A, Koukourakis MI, Sivridis E, Pastorek J, Wykoff CC, Gatter KC, et al. Expression of hypoxia-inducible carbonic anhydrase-9 relates to angiogenic pathways and independently to poor outcome in non-small cell lung cancer. Cancer Res. 2001; 61:7992–7998. PMID: 11691824.

12. Liao SY, Ivanov S, Ivanova A, Ghosh S, Cote MA, Keefe K, et al. Expression of cell surface transmembrane carbonic anhydrase genes CA9 and CA12 in the human eye: overexpression of CA12 (CAXII) in glaucoma. J Med Genet. 2003; 40:257–261. PMID: 12676895.

13. Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004; 3:Article3. PMID: 16646809.

14. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Methodol. 1995; 57:289–300.

15. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009; 4:44–57. PMID: 19131956.

16. Wilson WR, Hay MP. Targeting hypoxia in cancer therapy. Nat Rev Cancer. 2011; 11:393–410. PMID: 21606941.

17. Brahimi-Horn MC, Bellot G, Pouysségur J. Hypoxia and energetic tumour metabolism. Curr Opin Genet Dev. 2011; 21:67–72. PMID: 21074987.

18. Supuran CT. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov. 2008; 7:168–181. PMID: 18167490.

19. De Simone G, Supuran CT. Carbonic anhydrase IX: Biochemical and crystallographic characterization of a novel antitumor target. Biochim Biophys Acta. 2010; 1804:404–409. PMID: 19679200.

20. Hilvo M, Baranauskiene L, Salzano AM, Scaloni A, Matulis D, Innocenti A, et al. Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes. J Biol Chem. 2008; 283:27799–27809. PMID: 18703501.

21. McDonald PC, Winum JY, Supuran CT, Dedhar S. Recent developments in targeting carbonic anhydrase IX for cancer therapeutics. Oncotarget. 2012; 3:84–97. PMID: 22289741.

22. Robertson N, Potter C, Harris AL. Role of carbonic anhydrase IX in human tumor cell growth, survival, and invasion. Cancer Res. 2004; 64:6160–6165. PMID: 15342400.

23. Williams E, Martin S, Moss R, Durrant L, Deen S. Co-expression of VEGF and CA9 in ovarian high-grade serous carcinoma and relationship to survival. Virchows Arch. 2012; 461:33–39. PMID: 22699808.

24. Svastová E, Zilka N, Zat'ovicová M, Gibadulinová A, Ciampor F, Pastorek J, et al. Carbonic anhydrase IX reduces E-cadherin-mediated adhesion of MDCK cells via interaction with beta-catenin. Exp Cell Res. 2003; 290:332–345. PMID: 14567991.

25. Wykoff CC, Beasley NJ, Watson PH, Turner KJ, Pastorek J, Sibtain A, et al. Hypoxia-inducible expression of tumor-associated carbonic anhydrases. Cancer Res. 2000; 60:7075–7083. PMID: 11156414.

26. Cho M, Uemura H, Kim SC, Kawada Y, Yoshida K, Hirao Y, et al. Hypomethylation of the MN/CA9 promoter and upregulated MN/CA9 expression in human renal cell carcinoma. Br J Cancer. 2001; 85:563–567. PMID: 11506497.

27. Cho M, Grabmaier K, Kitahori Y, Hiasa Y, Nakagawa Y, Uemura H, et al. Activation of the MN/CA9 gene is associated with hypomethylation in human renal cell carcinoma cell lines. Mol Carcinog. 2000; 27:184–189. PMID: 10708480.

28. Nakamura J, Kitajima Y, Kai K, Hashiguchi K, Hiraki M, Noshiro H, et al. Expression of hypoxic marker CA IX is regulated by site-specific DNA methylation and is associated with the histology of gastric cancer. Am J Pathol. 2011; 178:515–524. PMID: 21281785.