Key Players of Cisplatin Resistance: Towards a Systems Pharmacology Approach

Abstract

The major obstacle in the clinical use of the antitumor drug cisplatin is inherent and acquired resistance. Typically, cisplatin resistance is not restricted to a single mechanism demanding for a systems pharmacology approach to understand a whole cell's reaction to the drug. In this study, the cellular transcriptome of untreated and cisplatin-treated A549 non-small cell lung cancer cells and their cisplatin-resistant sub-line A549^rCDDP²⁰⁰⁰ was screened with a whole genome array for relevant gene candidates. By combining statistical methods with available gene annotations and without a previously defined hypothesis HRas, MAPK14 (p38), CCL2, DOK1 and PTK2B were identified as genes possibly relevant for cisplatin resistance. These and related genes were further validated on transcriptome (qRT-PCR) and proteome (Western blot) level to select candidates contributing to resistance. HRas, p38, CCL2, DOK1, PTK2B and JNK3 were integrated into a model of resistance-associated signalling alterations describing differential gene and protein expression between cisplatin-sensitive and -resistant cells in reaction to cisplatin exposure.

Keywords: CCL2; DOK1; HRas; JNK3; PTK2B; cellular signalling; cisplatin resistance; p38.

Figure 1

Flow chart of the array data processing (FDR: false discovery rate; GO: Gene Ontology; WB: Western blot).

Figure 2

Heat map of the whole transcriptome, regulated genes with fold change cut-off at 2.0 and a false discovery rate of 5% of all replicates in sensitive and resistant cells. Numbers above lanes indicate: 1, 2, 3, 4: A549, untreated; 5, 6, 7: A549, treated with 11 µM cisplatin; 8, 9, 14, 15, 16: A549^rCDDP²⁰⁰⁰, untreated; 10, 11, 17, 18, 19: A549^rCDDP²⁰⁰⁰, treated with 11 µM cisplatin; 12, 13, 20, 21, 22: A549^rCDDP²⁰⁰⁰, treated with 34 µM cisplatin. The p-value (2.6 × 10⁻²³) corresponds to the result of a global test [21], which assesses the statistical significance of the entire signature that discriminates A549 and A549^rCDDP²⁰⁰⁰ cells.

Figure 3

Venn diagram showing differentially expressed genes annotated with respective GO terms: The yellow sections indicate those genes, which were chosen for validation.

Figure 4

mRNA expression (all n = 6) of HRas, MAPK14 (p38), CCL2, DOK1 and PTK2B related to GAPDH mRNA expression; protein expression of HRas (n = 6), p38 (n = 6), CCL2 (n = 4), DOK1 (n = 7–8) and PTK2B (n = 3) related to GAPDH expression in A549 () and A549^rCDDP²⁰⁰⁰ () before (ctrl) and after treatment with 11 µM cisplatin (11) or 34 µM cisplatin (34) presented as mean ± SEM; as well as representative Western blots. * p < 0.05; ** p < 0.01; *** p < 0.01.

Figure 5

Expression of activated ERK (pERK1 and pERK2, both n = 7) related to GAPDH expression; mRNA expression of JNK3 (n = 6) related to GAPDH mRNA expression; protein expression of JNK3 (n = 9) related to GAPDH expression; expression of activated p38 (p-p38, n = 4) related to GAPDH expression in A549 () and A549^rCDDP²⁰⁰⁰ () after treatment with 11 µM cisplatin (11) or 34 µM cisplatin (34) expressed as mean ± SEM, as well as representative Western blots.* p < 0.05; ** p < 0.01; *** p < 0.01.

Figure 6

Model of resistance-associated signalling alterations indicating significant changes of mRNA expression (red arrows) or protein expression (green arrows) after cisplatin treatment in A549 and A549^rCDDP²⁰⁰⁰ cells, as well as an increase in basal mRNA levels (red circles), protein levels (green circles) or basal kinase activation (blue circles) in A549^rCDDP²⁰⁰⁰ (b) compared to A549 (a) cells. The model is based on the data presented and described here and the data previously published [6].