Identification of hypermethylated genes associated with cisplatin resistance in human cancers

Abstract

Cisplatin is among the most widely used cytotoxic anticancer agents in solid tumors; however, the development of secondary resistance remains a major obstacle to clinical efficacy. Treatment-related DNA hypermethylation may play a role in creating drug-resistant phenotypes by inactivating genes that are required for cytotoxicity. We applied a pharmacologic unmasking approach to detect hypermethylated genes whose inactivation contributes to cisplatin resistance. Using three pairs of isogeneic, cisplatin-sensitive, and cisplatin-resistant cell lines derived from two parental cell lines (KB-3-1 and SCC25), we identified several hundred genes that were downregulated in each resistant cell line and reactivated by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. Among them, 30 genes were common to two or more cell lines and/or reported to be downregulated in previous studies. Bisulfite sequencing confirmed that 14 genes were hypermethylated in resistant cell lines but not in the sensitive parental cell lines. Six of 14 genes (SAT, C8orf4, LAMB3, TUBB, G0S2, and MCAM) were cisplatin inducible in sensitive but not in resistant cell lines. Small interfering RNA knockdown of two genes, SAT and S100P, increased cell viability with cisplatin treatment in sensitive parental cell lines. S100P knockdown significantly decreased the S-phase fraction of parental sensitive cell lines and slowed cell proliferation, which was associated with decreased sensitivity to cisplatin. Based on these findings, we conclude that DNA methylation is a frequent event in cells that are chronically exposed to cisplatin and that methylation-induced gene silencing may play a role in the development of resistance to cytotoxic chemotherapeutic agents.

Figure 1

RT-PCR and real-time PCR analysis of candidate genes. A, RT was performed on all 3 cell line pairs. For select candidate genes, conventional PCR showed downregulation in the cisplatin-resistant daughter cell lines and re-activation after 5-Aza-dC treatment. Representative real-time PCR data displaying amplification curves for the individual cell lines are shown. B, Real-time PCR was performed on all 30 candidate genes identified by our algorithm. Each sample was normalized to GAPDH. Data are plotted as relative quantity as compared to parental untreated control. Values are expressed as mean ± SD. Representative data from 8 genes are displayed.

Figure 2

Bisulfite sequencing and methylation-specific PCR (MSP) on candidate genes. A, Direct bisulfite sequencing of gene promoters (C8orf4, S100P, SAT) are presented. Representative sequencing data from either parental or resistant cell lines are shown. For S100P and C8orf4, all guanines present (arrows) after sequencing are derived from methylcytosines on the complementary strand. Sequence data for SAT are shown in the sense strand on which methylcytosines (arrows) remain cytosines after bisulfite treatment while unmethylated cytosines are converted to thymidine. B, Representative MSP are shown for C8orf4, S100P, and SAT in the cell lines indicated. U: PCR amplification with primers recognizing unmethylated DNA; M: PCR amplification with primers recognizing methylated DNA.

Figure 3

Cisplatin treatment induces expression of 6 genes in CP-sensitive but not CP-resistant cell lines. Cells were treated with cisplatin (at a dose equivalent to the sensitive cell line’s IC₅₀) or vehicle (DMSO) for 24 hrs. RNA was collected and real-time RT-PCR was performed. Values are expressed as mean ± SD. * indicates significant difference (P < 0.05).

Figure 4

siRNA knockdown of SAT and S100P increases parental cell line resistance to cisplatin. KB-3-1 (A) and SCC-25 (B) cell lines were transfected with control, SAT or S100P siRNA duplex nucleotides. Cells were treated with cisplatin for 48hrs, and cell viability was assayed. Representative data are shown. Efficiency of SAT and S100P knockdown was confirmed by real-time RT-PCR (right). None and control indicate either untransfected or control transfected cells. Representative data are shown. Values are expressed as mean ± SD. * indicates significant difference (P < 0.05).

Figure 5

S100P knockdown slows cell growth and decreases the S-phase fraction (SPF) of KB-3-1 cells. A, KB-3-1 cells were transfected with S100P siRNA and cell growth rate was assayed on indicated days (top). The growth rate of KB-CP cells (clones 1 and 2) was also assayed for comparison. Relative expression of S100P was examined by real-time PCR and amplification curves are shown (bottom). * indicates significant difference between control siRNA transfected and S100P siRNA transfected cells (P < 0.05). B, Cell cycle analysis was performed on control and S100P siRNA-transfected cells (72 hrs after transfection). Cell cycle distribution and percentage are shown. Knockdown of S100P expression decreased SPF from 13% to 5%. Values are expressed as mean ± SD of three independent experiments. * indicates significant difference (P < 0.05).