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. 2022 Oct 4;23(19):11787.
doi: 10.3390/ijms231911787.

Pharmacogenomic Profiling of Cisplatin-Resistant and -Sensitive Human Osteosarcoma Cell Lines by Multimodal Targeted Next Generation Sequencing

Claudia Maria Hattinger  1 , Chiara Casotti  1   2 , Maria Pia Patrizio  3 , Silvia Luppi  3 , Leonardo Fantoni  1   2 , Katia Scotlandi  3 , Toni Ibrahim  1 , Massimo Serra  1
Affiliations

Pharmacogenomic Profiling of Cisplatin-Resistant and -Sensitive Human Osteosarcoma Cell Lines by Multimodal Targeted Next Generation Sequencing

Claudia Maria Hattinger et al. Int J Mol Sci. .

Abstract

Cisplatin (CDDP) is a drug for high-grade osteosarcoma (HGOS) treatment. Several germline pharmacogenetic studies have revealed associations between single nucleotide polymorphisms (SNPs) and CDDP-based therapy response or CDDP-related toxicity in patients with HGOS. Whether these variants could play a biological role in HGOS cells has not been studied so far. The aim of this study was to explore 28 SNPs of 14 genes in 6 CDDP-resistant and 12 drug-sensitive human HGOS cell lines. An innovative multimodal targeted next generation sequencing (mmNGS) approach with custom primers designed for the most commonly reported SNPs of genes belonging to DNA repair, CDDP transport or detoxification, or associated with CDPP-related toxicity was applied. The mmNGS approach was validated by TaqMan genotyping assays and emerged to be an innovative, reliable tool to detect genetic polymorphisms at both the DNA and RNA level. Allele changes in three SNPs (ERCC2 rs13181 and rs1799793, ERCC1 rs11615) were identified on both DNA and RNA derived libraries in association with CDDP resistance. A change of the GSTP1 rs1695 polymorphism from AA to AG genotype was observed in the RNA of all six CDDP-resistant variants. These SNPs emerged to be causally associated with CDDP resistance in HGOS cells.

Keywords: cisplatin resistance; next generation sequencing; osteosarcoma; pharmacogenomics; single nucleotide polymorphism.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
ERCC2 rs13181 genotypes identified by multimodal targeted next generation sequencing (mmNGS) in U-2OS cell line and U-2OS/CDDP4μg variant. Mismatched nucleotides were evidenced by the CLC Genomics Workbench (GWB) with a background color according to the Rasmol color scheme. The red square box indicates the polymorphism alleles.
Figure 2
Figure 2
ERCC2 rs1799793 genotypes identified by multimodal targeted next generation sequencing (mmNGS) in U-2OS cell line and U-2OS/CDDP4μg variant. Mismatched nucleotides were evidenced by the CLC Genomics Workbench (GWB) with a background color according to the Rasmol color scheme. The red square box indicates the polymorphism alleles.
Figure 3
Figure 3
Amino acid changes identified by multimodal targeted next generation sequencing (mmNGS) in the U-2OS cell line for the ERCC2 rs13181 (a) and ERCC2 rs1799793 (b). Amino acids are colored by the CLC Genomics Workbench (GWB) according to the Rasmol color scheme.
Figure 4
Figure 4
ERCC2 rs13181 genotypes identified by multimodal targeted next generation sequencing (mmNGS) in Saos-2 cell line and Saos-2/CDDP6μg variant. Mismatched nucleotides are evidenced by the CLC Genomics Workbench (GWB) with a background color according to the Rasmol color scheme. The red square box indicates the polymorphism alleles.
Figure 5
Figure 5
ERCC1 rs11615 genotypes identified by multimodal targeted next generation sequencing (mmNGS) in Saos-2 cell line and Saos-2/CDDP6μg variant. Mismatched nucleotides are evidenced by the CLC Genomics Workbench (GWB) with a background color according to the Rasmol color scheme. The red square box indicates the polymorphism alleles.
Figure 6
Figure 6
Amino acids indicated by multimodal targeted next generation sequencing (mmNGS) in the Saos-2/CDDP1μg variant for the non-synonymous ERCC2 rs13181 (a) and the synonymous ERCC2 rs11615 (b). Amino acids are colored by the CLC Genomics Workbench (GWB) according to the Rasmol color scheme.
Figure 7
Figure 7
Fold-changes of RNA expression in U-2OS cisplatin (CDDP)-resistant variants (a) and Saos-2 CDDP-resistant variants (b) compared to their parental cell lines. Thick lines indicate thresholds for overexpression (2-fold) and under-expression (0.5-fold). * indicates those genes that were significantly differentially expressed in the groups of U-2OS and Saos-2 CDDP-resistant variants compared to their respective parental cell line.
Figure 8
Figure 8
Heatmap plus hierarchical clustering analysis of 12 drug-sensitive and 6 cisplatin (CDDP)-resistant human osteosarcoma cell lines generated from RNAseq data using the CLC Genomics Workbench (GWB) tool.
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