Upregulation of Thymidylate Synthase Induces Pemetrexed Resistance in Malignant Pleural Mesothelioma

Our plain-English summary of Upregulation of Thymidylate Synthase Induces Pemetrexed Resistance in Malignant Pleural Mesothelioma, by Y. Sato, M. Tomita, T. Soga, et al (Frontiers in Pharmacology, 2021).

The study

• Authors: Y. Sato, M. Tomita, T. Soga, A. Ochiai, H. Makinoshima
• Journal: Frontiers in Pharmacology
• Published: 2021;12:718675
• Cited by: 15 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Malignant pleural mesothelioma (MPM) is an invasive malignancy that develops in the pleural cavity, and antifolates are used as chemotherapeutics for treating. The majority of antifolates, including pemetrexed (PMX), inhibit enzymes involved in purine and pyrimidine synthesis. MPM patients frequently develop drug resistance in clinical practice, however the associated drug-resistance mechanism is not well understood. This study was aimed to elucidate the mechanism underlying resistance to PMX in MPM cell lines. We found that among the differentially expressed genes associated with drug resistance (determined by RNA sequencing), TYMS expression was higher in the established resistant cell lines than in the parental cell lines. Knocking down TYMS expression significantly reduced drug resistance in the resistant cell lines. Conversely, TYMS overexpression significantly increased drug resistance in the parental cells. Metabolomics analysis revealed that the levels of dTMP were higher in the resistant cell lines than in the parental cell lines; however, resistant cells showed no changes in dTTP levels after PMX treatment. We found that the nucleic acid-biosynthetic pathway is important for predicting the efficacy of PMX in MPM cells. The results of chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays suggested that H3K27 acetylation in the 5'-UTR of TYMS may promote its expression in drug-resistant cells. Our findings indicate that the intracellular levels of dTMP are potential biomarkers for the effective treatment of patients with MPM and suggest the importance of regulatory mechanisms of TYMS expression in the disease.

Copyright © 2021 Sato, Tomita, Soga, Ochiai and Makinoshima.

In plain English

This laboratory study used two human malignant pleural mesothelioma (MPM) cell lines, MSTO-211H and TCC-MESO-2, to study resistance to the chemotherapy drug pemetrexed. It did not study patients or asbestos exposure. The authors grew the cells in rising pemetrexed doses, up to 10 μM, to make resistant versions. The pemetrexed concentration that halved cell viability rose from 31.8 nM and 32.3 nM in the original lines to 413.6 nM and 869.2 nM in the resistant lines. Resistant cells had more of the enzyme thymidylate synthase (TYMS). Lowering TYMS in resistant cells reduced their resistance, and adding extra TYMS to original cells increased it. After pemetrexed, levels of dTTP, a DNA building block, fell in the original cells but were unchanged in resistant cells. Resistant and original cells differed significantly in a chemical tag on histone proteins (H3K27 acetylation) near the TYMS gene start. The authors proposed that this tag raises TYMS expression. The authors concluded that higher TYMS was linked to acquired resistance and that dTMP levels inside cells may be potential biomarkers. They stated that how the H3K27 acetylation arises was not determined.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) study of human mesothelioma cell lines, using gene expression, gene knockdown and overexpression, metabolite measurement, and chromatin immunoprecipitation experiments
• Who was studied: Two human malignant pleural mesothelioma cell lines: MSTO-211H (biphasic, purchased from the American Type Culture Collection) and TCC-MESO-2 (epithelial, established from Japanese patients with MPM). The study also used pemetrexed-resistant versions made from them (MSTO-211H_R and TCC-MESO-2_R). No patients were studied.
• Exposure: The chemotherapy drug pemetrexed, increased stepwise up to 10 μM to create resistant cells. 5-fluorouracil was also tested. Asbestos exposure was not studied.
• Main results:
  • The pemetrexed concentration that halved viability after 72 hours (IC50) was 31.8 nM for MSTO-211H and 413.6 nM for resistant MSTO-211H_R. It was 32.3 nM for TCC-MESO-2 and 869.2 nM for resistant TCC-MESO-2_R. The authors reported these as 13.0-fold and 28.0-fold higher.
  • Sensitivity to 5-fluorouracil did not differ significantly between parental and resistant cells. The authors concluded the resistance was specific to pemetrexed.
  • By RNA sequencing, TYMS expression was 51.23-fold higher in MSTO-211H_R and 15.90-fold higher in TCC-MESO-2_R than in their parental lines.
  • Genes previously reported as linked to drug resistance (ABCC1 to 5 and RFC) were not differentially expressed.
  • By RT-qPCR, TYMS mRNA was 35.8-fold higher in MSTO-211H_R and 16.4-fold higher in TCC-MESO-2_R than in their parental lines.
  • Knocking down TYMS in both resistant cell lines decreased their pemetrexed resistance compared with control-treated cells.
  • Parental cells engineered to overexpress TYMS were more resistant to pemetrexed than cells given the control vector.
  • After pemetrexed treatment, dUMP accumulated in both parental cell lines but was not detected in either resistant line.
  • dTMP was detected in both resistant lines treated with PBS (control), and dTMP increased after pemetrexed treatment.
  • After pemetrexed treatment, dTTP was reduced in both parental lines but did not change in the resistant lines.
  • Levels of the proteins FOXM1, c-MYC and E2F1 did not change with acquired resistance or with TYMS knockdown.
  • ChIP-qPCR found a significant difference in H3K27ac levels near the TYMS gene start between parental and resistant cell lines. H3K4me3 levels differed significantly only in MSTO-211H cells.
  • The authors proposed that long-term pemetrexed exposure causes H3K27 acetylation near the TYMS gene start, resulting in increased TYMS expression.
• The authors' conclusion: The authors concluded that their results link acquired antifolate resistance to increased TYMS expression, and that intracellular dTMP levels may be potential biomarkers for pemetrexed treatment.
• Limitations the authors state:
  • The authors stated that how H3K27 acetylation is induced, and which transcription factors are involved at the TYMS promoter, were not determined.
  • Cells made to overexpress TYMS were less resistant than the established resistant cells. The authors surmised that other genes may act together with TYMS.
  • RNA sequencing did not identify changes in genes for histone modification and transcription. The authors said single-cell analyses and ChIP-sequencing would be needed.
  • The authors considered that inborn and acquired drug resistance may work differently in MPM cells. They cited an earlier cell line that was resistant but had low TYMS.

Topics

Medical terms in this study, explained in our Lexicon: Pemetrexed (Alimta), Pleural Mesothelioma, Pleural Cavity, Antifolate, Biomarker.

Related studies

• Thymidylate synthase expression determines pemetrexed targets and resistance development in tumour cells — A. Buqué, U. Aresti, et al. (2013)
• Thymidylate synthase and folyl-polyglutamate synthase are not clinically useful markers of response to pemetrexed in patients with malignant pleural mesothelioma — D.E. Lustgarten, C. Deshpande, et al. (2013)
• Reduced folate carrier and folylpolyglutamate synthetase, but not thymidylate synthase predict survival in pemetrexed-treated patients suffering from malignant pleural mesothelioma — F. Mairinger, C. Vollbrecht, et al. (2013)
• Downregulation of thymidylate synthase by RNAi molecules enhances the antitumor effect of pemetrexed in an orthotopic malignant mesothelioma xenograft mouse model — A.S. Abu Lila, C. Kato, et al. (2016)
• New tricks for old biomarkers: thymidylate synthase expression as a predictor of pemetrexed activity in malignant mesothelioma — P.A. Zucali, E. Giovannetti, et al. (2010)
• Thymidylate synthase: a critical target for cancer chemotherapy — M.G. Rose, M.P. Farrell, et al. (2002)

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