Molecular Basis of Cisplatin Resistance in Testicular Germ Cell Tumors

Abstract

The emergence of cisplatin (CDDP) resistance is the main cause of treatment failure and death in patients with testicular germ cell tumors (TGCT), but its biologic background is poorly understood. To study the molecular basis of CDDP resistance in TGCT we prepared and sequenced CDDP-exposed TGCT cell lines as well as 31 primary patients' samples. Long-term exposure to CDDP increased the CDDP resistance 10 times in the NCCIT cell line, while no major resistance was achieved in Tera-2. Development of CDDP resistance was accompanied by changes in the cell cycle (increase in G1 and decrease in S-fraction), increased number of acquired mutations, of which 3 were present within ATRX gene, as well as changes in gene expression pattern. Copy number variation analysis showed, apart from obligatory gain of 12p, several other large-scale gains (chr 1, 17, 20, 21) and losses (chr X), with additional more CNVs found in CDDP-resistant cells (e.g., further losses on chr 1, 4, 18, and gain on chr 8). In the patients' samples, those who developed CDDP resistance and died of TGCT (2/31) showed high numbers of acquired aberrations, both SNPs and CNVs, and harbored mutations in genes potentially relevant to TGCT development (e.g., TRERF1, TFAP2C in one patient, MAP2K1 and NSD1 in another one). Among all primary tumor samples, the most commonly mutated gene was NSD1, affected in 9/31 patients. This gene encoding histone methyl transferase was also downregulated and identified among the 50 most differentially expressed genes in CDDP-resistant NCCIT cell line. Interestingly, 2/31 TGCT patients harbored mutations in the ATRX gene encoding a chromatin modifier that has been shown to have a critical function in sexual differentiation. Our research newly highlights its probable involvement also in testicular tumors. Both findings support the emerging role of altered epigenetic gene regulation in TGCT and CDDP resistance development.

Keywords: cell cycle; cisplatin resistance; molecular aberrations; next generation sequencing; testicular germ cell tumor.

Figure 1

Final sensitivity of TGCTs cell lines to CDDP measured by MTS assays after 72 h of CDDP treatment: (a) CDDP-naive (NCCIT) and CDDP-treated (NCCIT_CDDP) NCCIT cell lines; (b) CDDP-naive (Tera-2) and CDDP-treated (Tera-2_CDDP) Tera-2 cell lines (the means and SDs of 4 independent assays for each cell line are displayed, each assay analyzed in triplicates or 6-plicates).

Figure 2

Proliferation and cell cycle of TGCTs cell lines. (a) proliferation of the cells expressed in total numbers of cells during two week cultivation; (b) pyronin/hoechst cell cycle analysis; (c) EdU cell cycle analysis; (d) apoptosis analysis without CDDP treatment and after 72 h of CDDP treatment.

Figure 3

Hierarchical clustering of original and CDDP-treated cell lines based on gene expression profiles of the top 50 differentially expressed genes.

Figure 4

Scattered plots displaying copy number variation ratios inferred from WES data normalized to pooled control samples. Segmental changes with predicted copy number alteration are marked in orange, normal in gray. Chromosomes 12 and 17 as the most clinically relevant displayed here, all are in the supplementary material (Supplementary Figure S2).

Figure 5

Somatic variants detected by amplicon sequencing in primary TGCT tumors (in comparison to germ-line control patient samples)—variants present in at least 20% of sequencing reads.