Nintedanib and Dasatinib Treatments Induce Protective Autophagy as a Potential Resistance Mechanism in MPM Cells

Our plain-English summary of Nintedanib and Dasatinib Treatments Induce Protective Autophagy as a Potential Resistance Mechanism in MPM Cells, by L. Hegedüs, K.D. Szücs, M. Kudla, et al (Frontiers in Cell and Developmental Biology, 2022).

The study

• Authors: L. Hegedüs, K.D. Szücs, M. Kudla, J. Heidenreich, V. Jendrossek, S. Peña-Llopis, T. Garay, A. Czirok, C. Aigner, T. Plönes, S. Vega-Rubin-de-Celis, B. Hegedüs
• Journal: Frontiers in Cell and Developmental Biology
• Published: 2022;10:852812
• Cited by: 9 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Malignant pleural mesothelioma (MPM) is a rare type of cancer with a grim prognosis. So far, no targetable oncogenic mutation was identified in MPM and biomarkers with predictive value toward drug sensitivity or resistance are also lacking. Nintedanib (BIBF1120) is a small-molecule tyrosine kinase inhibitor that showed promising efficacy preclinically and in phase II trial in MPM as an angiogenesis inhibitor combined with chemotherapy. However, the extended phase III trial failed. In this study, we investigated the effect of nintedanib on one of its targets, the SRC kinase, in two commercial and six novel MPM cell lines. Surprisingly, nintedanib treatment did not inhibit SRC activation in MPM cells and even increased phosphorylation of SRC in several cell lines. Combination treatment with the SRC inhibitor dasatinib could reverse this effect in all cell lines, however, the cellular response was dependent on the drug sensitivity of the cells. In 2 cell lines, with high sensitivity to both nintedanib and dasatinib, the drug combination had no synergistic effect but cell death was initiated. In 2 cell lines insensitive to nintedanib combination treatment reduced cell viability synergisticaly without cell death. In contrast, in these cells both treatments increased the autophagic flux assessed by degradation of the autophagy substrate p62 and increased presence of LC3B-II, increased number of GFP-LC3 puncta and decreased readings of the HiBiT-LC3 reporter. Additionaly, autophagy was synergistically promoted by the combined treatment. At the transcriptional level, analysis of lysosomal biogenesis regulator Transcription Factor EB (TFEB) showed that in all cell lines treated with nintedanib and to a lesser extent, with dasatinib, it became dephosphorylated and accumulated in the nucleus. Interestingly, the expression of certain known TFEB target genes implicated in autophagy or lysosomal biogenesis were significantly modified only in 1 cell line. Finally, we showed that autophagy induction in our MPM cell lines panel by nintedanib and dasatinib is independent of the AKT/mTOR and the ERK pathways. Our study reveals that autophagy can serve as a cytoprotective mechanism following nintedanib or dasatinib treatments in MPM cells.

Copyright © 2022 Hegedüs, Szücs, Kudla, Heidenreich, Jendrossek, Peña-Llopis, Garay, Czirok, Aigner, Plönes, Vega-Rubin-de-Celis and Hegedüs.

In plain English

This was a laboratory study of malignant pleural mesothelioma (MPM) cells. The drugs were tested on cells, not patients, and asbestos was not tested, though the six patient-derived lines' donors were recorded as asbestos-exposed. The authors first treated eight MPM cell lines with the drug nintedanib. Two were established lines, one bought and one donated; six were grown by the authors from patients' pleural fluid. Nintedanib did not reduce activation of one of its targets, SRC, in any line, and it increased it in five. Dasatinib, tested alone and with nintedanib in those five lines, reversed the increase in all five. In PF531, both drugs caused cell death even alone; in PF434, dasatinib increased cell death. In SPC111 and PF626, both insensitive to nintedanib, the drugs did not kill cells but increased autophagy, a process in which cells break down their own parts. In SPC111 cells, adding the autophagy blocker 3-MA to nintedanib had a strong synergistic effect in reducing cell viability. The authors concluded that some MPM cell lines induce autophagy as a protective mechanism of resistance against these drugs.

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) experiments on human malignant pleural mesothelioma cell lines
• Who was studied: Eight MPM cell lines. Two were established lines from biphasic MPM tumors: SPC111, purchased from Sigma-Aldrich, and SPC212, a gift from a researcher at the University of Zurich. Six were new lines (PF626, PF142, PF531, PF434, PF588, PF655) established by the authors from pleural effusion samples of MPM patients. The Ethics Committee at the University Hospital Essen, Germany, approved the study. PF588 and PF655 came from the same patient. The patient characteristics table records asbestos as 'yes' for each of the six new lines. No dates of sample collection are given.
• Exposure: Laboratory drug treatments, not asbestos. Nintedanib was given mainly at 1 μM, with 0.3 to 5 μM in viability tests. Dasatinib was given mainly at 50 nM and 300 nM, with 25 nM to 1 μM in viability tests. The drugs were used alone and in combination. Some experiments also used the autophagy inhibitors bafilomycin A1 and 3-MA.
• Main results:
  • In all eight cell lines, nintedanib did not reduce SRC activation (phosphorylation at Y416). In five lines, SRC activation increased after treatment.
  • Nintedanib substantially reduced cell viability in five lines (SPC212, PF531, PF434, PF142, PF655). Three lines (SPC111, PF626, PF588) were insensitive.
  • Dasatinib, alone and combined with nintedanib, was then studied in the five lines in which nintedanib increased SRC phosphorylation. These were SPC111, PF531, PF434, SPC212 and PF626.
  • In these lines, dasatinib reversed the nintedanib-induced SRC activation when the two drugs were combined.
  • The effect of the drug combination on viability differed by line. In SPC111 it was strongly synergistic at all concentrations. In PF434 and PF531 it was additive only at higher concentrations and antagonistic at lower ones.
  • In PF531 cells, both drugs strongly induced apoptosis (a form of cell death), even when given alone. In PF434 cells, dasatinib increased cell death in a concentration-dependent manner. In the other three lines tested, no treatment induced cell death.
  • In the nintedanib-insensitive lines SPC111 and PF626, the treatments significantly induced autophagy. In the more sensitive lines SPC212, PF434 and PF531, no significant autophagy induction was detected.
  • Nintedanib, and to a lesser extent dasatinib, caused the protein TFEB to become dephosphorylated and to accumulate in the cell nucleus. However, only SPC212 cells showed a solid induction of the TFEB target genes tested.
  • The authors reported that the movement of TFEB into the nucleus after treatment was independent of the AKT1-mTORC1 and ERK1/2 signalling pathways.
  • In SPC111 cells, combining nintedanib with the autophagy inhibitor 3-MA had a strong synergistic effect. The authors set up this test to see whether the combination would further decrease cell viability.
• The authors' conclusion: The authors concluded that some MPM cell lines induce autophagy as a protective mechanism of resistance against nintedanib and dasatinib. They also concluded that nintedanib-induced autophagy had a cytoprotective effect in SPC111 cells. They reported that the two sarcomatoid cell lines were highly sensitive to both drugs and that the treatments induced cell death in them.
• Limitations the authors state:
  • The authors state that the mechanism behind the different responses among their cell lines remains to be determined.
  • The authors state that the role of TFEB's movement into the nucleus remains to be studied, because its effects on target genes were mild and varied across cell lines.
  • The HiBiT-LC3 autophagy reporter could not be established in PF434 cells.

Topics

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, Prognosis, Biomarker, Tyrosine Kinase Inhibitor, Angiogenesis, Chemotherapy, Synergistic Effect.

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• Screening of Pleural Mesothelioma Cell Lines for Kinase Activity May Identify New Mechanisms of Therapy Resistance in Patients Receiving Platin-Based Chemotherapy — S. Borchert, P.M. Suckrau, et al. (2019)
• Functional inhibition of heat shock protein 70 by VER-155008 suppresses pleural mesothelioma cell proliferation via an autophagy mechanism — K. Sakai, M. Inoue, et al. (2021)
• Asbestos induces mesothelial cell transformation via HMGB1-driven autophagy — J. Xue, S. Patergnani, et al. (2020)
• Non-thermal plasma induces a stress response in mesothelioma cells resulting in increased endocytosis, lysosome biogenesis and autophagy — L. Shi, F. Ito, et al. (2017)
• Cisplatin and resveratrol induce apoptosis and autophagy following oxidative stress in malignant mesothelioma cells — Y.J. Lee, G.J. Lee, et al. (2016)

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