Changes in DNA methylation are associated with the development of drug resistance in cervical cancer cells.

BACKGROUND AND PROPOSE: Changes in DNA methylation are associated with changes in somatic cell fate without the alteration of coding sequences. In addition to its use as a traceable biomarker, reversible DNA methylation could also serve as a therapeutic target. In particular, if the development of drug resistance is associated with changes in DNA methylation, then demethylation might reverse the resistance phenotype. The reversion of the drug-resistance might then be feasible if the association between abnormal DNA methylation and the development of drug-resistance could be identified. METHODS: Methylation differences between the drug-resistance cervical cancer cell, SiHa, and its derived oxaliplatin-resistant S3 cells were detected by methylation specific microarray. The drug-resistance cells were treated with demethylation agent to see if the resistance phenotype were reversed. Targeted methylation of one of the identified locus in normal cell is expected to recapitulate the development of resistance and a two-component reporter system is adopted to monitor the increase of DNA methylation in live cells. RESULTS: In this report, we identified methylation changes, both genome-wide and within individual loci, in the oxaliplatin-resistant cervical cancer cell S3 compared with its parental cell line SiHa. Treatment of S3 with a demethylation agent reversed increases in methylation and allowed the expression of methylation-silenced genes. Treatment with the demethylation agent also restored the sensitivity of S3 to cisplatin, taxol, and oxaliplatin to the same level as that of SiHa. Finally, we found that methylation of the target gene Casp8AP2 is sufficient to increase drug resistance in different cells. CONCLUSIONS: These results suggest that global methylation is associated with the development of drug resistance and could serve as a biomarker and therapeutic target for drug resistance in cervical cancer.

Clustered DNA methylation changes in polycomb target genes in early-stage liver cancer.

Polycomb-group proteins mark specific chromatin conformations in embryonic and somatic stem cells that are critical for maintenance of their "stemness". These proteins also mark altered chromatin modifications identified in various cancers. In normal differentiated cells or advanced cancerous cells, these polycomb-associated loci are frequently associated with increased DNA methylation. It has thus been hypothesized that changes in DNA methylation status within polycomb-associated loci may dictate cell fate and that abnormal methylation within these loci may be associated with tumor development. To assess this, we examined the methylation states of four polycomb target loci -Trip10, Casp8AP2, ENSA, and ZNF484 - in liver cancer. These four targets were selected because their methylation levels are increased during mesenchymal stem cell-to-liver differentiation. We found that these four loci were hypomethylated in most early-stage liver cancer specimens. For comparison, two non-polycomb tumor suppressor genes, HIC1 and RassF1A, were also examined. Whereas the methylation level of HIC1 did not differ significantly between normal and tumor samples, RassF1A was significantly hypermethylated in liver tumor samples. Unsupervised clustering analysis classified the methylation changes within polycomb and non-polycomb targets to be independent, indicating independent epigenetic evolution. Thus, pre-deposited polycomb marks within somatic stem cells may contribute to the determination of methylation changes during hepatic tumorigenesis.

Targeted Casp8AP2 methylation increases drug resistance in mesenchymal stem cells and cancer cells.

Casp8AP2 contains a FLASH functional domain and is critical for the formation of death complex and the relay of death signal into the cells. Genetic defects in Casp8AP2 are associated with several diseases. A CpG island within the Casp8AP2 promoter is differentially regulated during somatic stem cell differentiation, and aberrant DNA methylation within the Casp8AP2 promoter has been reported in cancers. We hypothesized that abnormal DNA methylation of Casp8AP2 promoter might contribute to prolonged cellular survival or drug resistance in cancer. The epigenetic state within the Casp8AP2 promoter was then determined in different cancer cell lines and patient samples by methylation-specific PCR. Targeted Casp8AP2 methylation within normal and tumor cells was performed to see whether methylation promoted drug resistance. We found differential Casp8AP2 methylation among the normal and tumoral samples. Global demethylation in a platinum drug-resistant human gastric cancer cell line reversed Casp8AP2 methylation and diminished drug resistance. Targeted methylation of the Casp8AP2 promoter in somatic stem cells and cancer cells increased their resistance to drugs including platinum drugs. These data demonstrate that methylation within the Casp8AP2 promoter correlates with the development of drug resistance and might serve as a biomarker and treatment target for drug resistance in cancer cells.