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.
Highlights
Testicular germ cell tumors (TGCT) are the most frequent solid tumors in young adult men aged 18–45 years
Among the acquired variants found in NCCIT_CDDP cells we identified genes involved in cellular pathways crucial for survival, proliferation, metastatic dissemination as well as metabolic set up of cancer cells – e.g., genes involved in DNA damage repair (BCOR), chromatin remodeling (BRWD3, ATRX), major signaling pathways (MAP3K4, COMP), metabolic pathways (SLC22A2, HPSE) or maintenance of epithelial-mesenchymal balance (KDR)
Our study suggests that the development of CDDP resistance in TGCT is associated with increased number of genetic aberrations on the level of SNVs/tumor mutation burden (TMB) as well as CNVs
Summary
Testicular germ cell tumors (TGCT) are the most frequent solid tumors in young adult men aged 18–45 years. The genetic hallmark of invasive TGCT is the amplification of the short arm of chromosome 12, usually in the form of isochromosome i(12p), which may lead to the activation of genes located here, most importantly the core stem cell gene NANOG or the oncogene KRAS Another typical alteration is the KIT/KITL pathway activation, characteristic for early stages and pre-invasive tumors [11,12,13,14,15]. The interactions of germ cells with an altered interstitial microenvironment, including Sertoli and Leydig cells that influence germ cell growth and differentiation, represent another important aspect of TGCT development All these factors may combine in their effect, leading to the critical event in TGCT formation—the abnormal cell division leading to aneuploidy and copy number variations, regularly found in this type of tumor [16,17]
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