Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell lines
Introduction: Methotrexate (MTX) is one of the most important drugs included in the first-line protocols to treat high-grade osteosarcoma (HGOS). Although several polymorphisms have been reported to be associated with drug response or MTX-related toxicity in pharmacogenetic studies, their role in th...
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Frontiers Media S.A.
2023-11-01
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| Series: | Frontiers in Pharmacology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fphar.2023.1294873/full |
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| author | Chiara Casotti Chiara Casotti Claudia Maria Hattinger Maria Pia Patrizio Silvia Luppi Leonardo Fantoni Leonardo Fantoni Michela Pasello Katia Scotlandi Toni Ibrahim Massimo Serra |
| author_facet | Chiara Casotti Chiara Casotti Claudia Maria Hattinger Maria Pia Patrizio Silvia Luppi Leonardo Fantoni Leonardo Fantoni Michela Pasello Katia Scotlandi Toni Ibrahim Massimo Serra |
| author_sort | Chiara Casotti |
| collection | DOAJ |
| description | Introduction: Methotrexate (MTX) is one of the most important drugs included in the first-line protocols to treat high-grade osteosarcoma (HGOS). Although several polymorphisms have been reported to be associated with drug response or MTX-related toxicity in pharmacogenetic studies, their role in the development of MTX resistance in HGOS is still unclear.Methods: Therefore, in this study, 22 single nucleotide polymorphisms (SNPs) in 4 genes of the folate metabolism, 7 MTX transporter genes, and 2 SNPs of the tumor protein p53 (TP53) gene were investigated using a custom multimodal-targeted next-generation sequencing (mmNGS) approach in 8 MTX-resistant and 12 MTX-sensitive human HGOS cell lines. The panel was validated by TaqMan genotyping assays.Results: High instability of TP53 rs1642785 was observed in all U-2OS/MTX variants. Allele changes of the solute carrier family 19 member 1/replication factor C subunit 1 (SLC19A1, previously known as RFC1) and rs1051266 were identified in all Saos-2/MTX-resistant variants in both DNA- and RNA- derived libraries compared to the parental Saos-2 cell line. Allele changes of methylenetetrahydrofolate reductase (MTHFR) rs1801133 were identified only in the RNA-derived libraries of the two U2OS variants with the highest MTX resistance level. Significantly upregulated gene expression associated with the development of MTX resistance was revealed for dihydrofolate reductase (DHFR) whereas SLC19A1 was downregulated. In addition, a fusion transcript of DHFR (ex4) and MutS Homolog 3 (MSH3) (ex9) was identified in the RNA libraries derived from the two U-2OS variants with the highest MTX resistance level.Conclusion: This innovative mmNGS approach enabled the simultaneous exploration of SNPs at DNA and RNA levels in human HGOS cell lines, providing evidence of the functional involvement of allele changes associated with the development of MTX resistance. |
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| institution | Directory Open Access Journal |
| issn | 1663-9812 |
| language | English |
| last_indexed | 2024-03-10T08:21:35Z |
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| spelling | doaj.art-8139d9c37b0e48caa53e1ce404be48712023-11-22T09:53:22ZengFrontiers Media S.A.Frontiers in Pharmacology1663-98122023-11-011410.3389/fphar.2023.12948731294873Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell linesChiara Casotti0Chiara Casotti1Claudia Maria Hattinger2Maria Pia Patrizio3Silvia Luppi4Leonardo Fantoni5Leonardo Fantoni6Michela Pasello7Katia Scotlandi8Toni Ibrahim9Massimo Serra10Osteoncology, Bone and Soft Tissue Sarcomas and Innovative Therapies, IRCCS Istituto Ortopedico Rizzoli, Bologna, ItalyDepartment of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, ItalyOsteoncology, Bone and Soft Tissue Sarcomas and Innovative Therapies, IRCCS Istituto Ortopedico Rizzoli, Bologna, ItalyLaboratory of Experimental Oncology, IRCCS Istituto Ortopedico Rizzoli, Bologna, ItalyLaboratory of Experimental Oncology, IRCCS Istituto Ortopedico Rizzoli, Bologna, ItalyOsteoncology, Bone and Soft Tissue Sarcomas and Innovative Therapies, IRCCS Istituto Ortopedico Rizzoli, Bologna, ItalyDepartment of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, ItalyLaboratory of Experimental Oncology, IRCCS Istituto Ortopedico Rizzoli, Bologna, ItalyLaboratory of Experimental Oncology, IRCCS Istituto Ortopedico Rizzoli, Bologna, ItalyOsteoncology, Bone and Soft Tissue Sarcomas and Innovative Therapies, IRCCS Istituto Ortopedico Rizzoli, Bologna, ItalyOsteoncology, Bone and Soft Tissue Sarcomas and Innovative Therapies, IRCCS Istituto Ortopedico Rizzoli, Bologna, ItalyIntroduction: Methotrexate (MTX) is one of the most important drugs included in the first-line protocols to treat high-grade osteosarcoma (HGOS). Although several polymorphisms have been reported to be associated with drug response or MTX-related toxicity in pharmacogenetic studies, their role in the development of MTX resistance in HGOS is still unclear.Methods: Therefore, in this study, 22 single nucleotide polymorphisms (SNPs) in 4 genes of the folate metabolism, 7 MTX transporter genes, and 2 SNPs of the tumor protein p53 (TP53) gene were investigated using a custom multimodal-targeted next-generation sequencing (mmNGS) approach in 8 MTX-resistant and 12 MTX-sensitive human HGOS cell lines. The panel was validated by TaqMan genotyping assays.Results: High instability of TP53 rs1642785 was observed in all U-2OS/MTX variants. Allele changes of the solute carrier family 19 member 1/replication factor C subunit 1 (SLC19A1, previously known as RFC1) and rs1051266 were identified in all Saos-2/MTX-resistant variants in both DNA- and RNA- derived libraries compared to the parental Saos-2 cell line. Allele changes of methylenetetrahydrofolate reductase (MTHFR) rs1801133 were identified only in the RNA-derived libraries of the two U2OS variants with the highest MTX resistance level. Significantly upregulated gene expression associated with the development of MTX resistance was revealed for dihydrofolate reductase (DHFR) whereas SLC19A1 was downregulated. In addition, a fusion transcript of DHFR (ex4) and MutS Homolog 3 (MSH3) (ex9) was identified in the RNA libraries derived from the two U-2OS variants with the highest MTX resistance level.Conclusion: This innovative mmNGS approach enabled the simultaneous exploration of SNPs at DNA and RNA levels in human HGOS cell lines, providing evidence of the functional involvement of allele changes associated with the development of MTX resistance.https://www.frontiersin.org/articles/10.3389/fphar.2023.1294873/fullosteosarcomamethotrexate resistancenext-generation sequencingsingle-nucleotide polymorphismpharmacogenomics |
| spellingShingle | Chiara Casotti Chiara Casotti Claudia Maria Hattinger Maria Pia Patrizio Silvia Luppi Leonardo Fantoni Leonardo Fantoni Michela Pasello Katia Scotlandi Toni Ibrahim Massimo Serra Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell lines Frontiers in Pharmacology osteosarcoma methotrexate resistance next-generation sequencing single-nucleotide polymorphism pharmacogenomics |
| title | Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell lines |
| title_full | Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell lines |
| title_fullStr | Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell lines |
| title_full_unstemmed | Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell lines |
| title_short | Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell lines |
| title_sort | single nucleotide polymorphism profiling by multimodal targeted next generation sequencing in methotrexate resistant and sensitive human osteosarcoma cell lines |
| topic | osteosarcoma methotrexate resistance next-generation sequencing single-nucleotide polymorphism pharmacogenomics |
| url | https://www.frontiersin.org/articles/10.3389/fphar.2023.1294873/full |
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