Candidate DNA methylation drivers of acquired cisplatin resistance in ovarian cancer identified by methylome and expression profiling

Abstract

Multiple DNA methylation changes in the cancer methylome are associated with the acquisition of drug resistance; however it remains uncertain how many represent critical DNA methylation drivers of chemoresistance. Using isogenic, cisplatin-sensitive/resistant ovarian cancer cell lines and inducing resensitizaton with demethylating agents, we aimed to identify consistent methylation and expression changes associated with chemoresistance. Using genome-wide DNA methylation profiling across 27 578 CpG sites, we identified loci at 4092 genes becoming hypermethylated in chemoresistant A2780/cp70 compared with the parental-sensitive A2780 cell line. Hypermethylation at gene promoter regions is often associated with transcriptional silencing; however, expression of only 245 of these hypermethylated genes becomes downregulated in A2780/cp70 as measured by microarray expression profiling. Treatment of A2780/ cp70 with the demethylating agent 2-deoxy-5 0 -azacytidine induces resensitization to cisplatin and re-expression of 41 of the downregulated genes. A total of 13/41 genes were consistently hypermethylated in further independent cisplatin-resistant A2780 cell derivatives. CpG sites at 9 of the 13 genes (ARHGDIB, ARMCX2, COL1A, FLNA, FLNC, MEST, MLH1, NTS and PSMB9) acquired methylation in ovarian tumours at relapse following chemotherapy or chemoresistant cell lines derived at the time of patient relapse. Furthermore, 5/13 genes (ARMCX2, COL1A1, MDK, MEST and MLH1) acquired methylation in drug-resistant ovarian cancersustaining (side population) cells. MLH1 has a direct role in conferring cisplatin sensitivity when reintroduced into cells in vitro. This combined genomics approach has identified further potential key drivers of chemoresistance whose expression is silenced by DNA methylation that should be further evaluated as clinical biomarkers of drug resistance.

Characterization of regulatory sequences in alternative promoters of hypermethylated genes associated with tumor resistance to cisplatin

“…As the mechanism associated with cisplatin resistance is multifactorial (1-3), our results suggest that the genes encoding cisplatin resistance harbor more than one option to initiate their transcripts to enhance the efficiency of mRNA production. It has been well documented that CGIs are prone to DNA methylation and this epigenetic mechanism is associated with gene silencing, initiation and maintenance of malignancy and drug resistance (6)(7)(8)(9)(37)(38)(39). Hypermethylation in CGIs and subsequent gene inactivation contributes to cisplatin resistance (9), as described earlier in this study.…”

“…However, it remains puzzling that genomic expression patterns of platinum-sensitive̸-resistant determinants in several cell lines, including squamous cell carcinoma of the head and neck, bladder and ovarian cancers, do not always correlate with the results of biochemical analyses (4)(5)(6). Additionally, epigenetic changes in the DNA methylation profiles during cancer development are associated with the acquisition of cisplatin resistance (6)(7)(8)(9). However, the problem of platinum resistance at the genomic and epigenomic levels has not been clearly defined.…”

“…Cell and molecular biology research over several decades has provided insight into this problem and has demonstrated that alterations in drug influx and efflux, detoxification via glutathione and glutathione transferases and enhanced DNA repair (1)(2)(3) are all involved, either singly or in combination, in cisplatin resistance. However, it remains puzzling that genomic expression patterns of platinum-sensitive̸-resistant determinants in several cell lines, including squamous cell carcinoma of the head and neck, bladder and ovarian cancers, do not always correlate with the results of biochemical analyses (4)(5)(6). Additionally, epigenetic changes in the DNA methylation profiles during cancer development are associated with the acquisition of cisplatin resistance (6)(7)(8)(9).…”

“…The TFBS and the CGIs targeted for DNA methylation, significantly affect the functional activities of alternative promoters (6)(7)(8)(9)18,19). To date, the role and molecular characteristics of alternative promoters have not been investigated in platinum drug resistance.…”

“…Therefore, it is paramount to gain a better understanding of gene expression machinery components that control platinum resistance genes. A promising approach to , RUIWEN ZHANG 6 and KALKUNTE S. SRIVENUGOPAL these questions appears to be the characterization of regulatory elements, CpG islands (CGIs) and transcription factor-binding sites (TFBS) in alternative promoters of genes that have been validated and their association with cisplatin resistance proven. It has been well established that alternative promoters are involved in the transcription of nearly half of all eukaryotic genes and are considered one of the main molecular characteristics of eukaryotic genomes (10,11).…”