Modulating BAP1 expression affects ROS homeostasis, cell motility and mitochondrial function

Our plain-English summary of Modulating BAP1 expression affects ROS homeostasis, cell motility and mitochondrial function, by L. Hebert, D. Bellanger, C. Guillas, et al (Oncotarget, 2017).

The study

• Authors: L. Hebert, D. Bellanger, C. Guillas, A. Campagne, F. Dingli, D. Loew, A. Fievet, V. Jacquemin, T. Popova, D. Jean, F. Mechta-Grigoriou, R. Margueron, M.H. Stern
• Journal: Oncotarget
• Published: 2017;8(42):72513-72527
• Cited by: 24 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The tumor suppressor BAP1 associates with ASXL1/2 to form the core Polycomb complex PR-DUB, which catalyzes the removal of mono-ubiquitin from several substrates including histone H2A. This complex also mediates the poly-deubiquitination of HCFC1, OGT and PCG1-α, preventing them from proteasomal degradation. Surprisingly, considering its role in a Polycomb complex, no transcriptional signature was consistently found among BAP1-inactivated tumor types. It was hypothesized that BAP1 tumor suppressor activity could reside, at least in part, in stabilizing proteins through its poly-deubiquitinase activity. Quantitative mass spectrometry and gene expression arrays were used to investigate the consequences of BAP1 expression modulation in the NCI-H226 mesothelioma cell line. Analysis of differentially expressed proteins revealed enrichment in cytoskeleton organization, mitochondrial activity and ROS management, while gene expression analysis revealed enrichment in the epithelial-to-mesenchymal transition pathway. Functional assessments in BAP1 inactivated, BAP1 wild-type and BAP1 catalytically dead-expressing NCI-H226 and QR mesothelioma cell lines confirmed alteration of these pathways and demonstrated that BAP1 deubiquitinase activity was mandatory to maintain these phenotypes. Interestingly, monitoring intracellular ROS levels partly restored the morphology and the mitochondrial activity. Finally, the study suggests new tumorigenic and cellular functions of BAP1 and shows for the first time the interest of studying the proteome as readout of BAP1 inactivation.

In plain English

This laboratory study used cultured human mesothelioma cell lines to examine what happens when BAP1 expression is changed. In the BAP1-deleted NCI-H226 line, the authors compared cells carrying an empty vector with cells re-expressing wild-type BAP1, using quantitative mass spectrometry (SILAC/MS) and gene expression arrays. They found 1098 proteins with differential accumulation (556 over-represented, 542 under-represented with BAP1wt) and 734 differentially expressed genes; only 55 of the 1098 proteins (5%) also changed at the mRNA level. Pathway analysis pointed to cell morphology and motility and to mitochondrial function. In functional tests, NCI-H226 and QR cells expressing wild-type BAP1 were compared with cells carrying an empty vector or a catalytically dead BAP1 (C91S). In both lines, wild-type BAP1 cells showed actin stress fibres, reduced N-cadherin, greater invasion, lower respiratory capacity and higher intracellular ROS. Greater migration in a wound-healing assay was reported for NCI-H226. The catalytically dead mutant did not reproduce most of these changes. In NCI-H226 cells, the antioxidant N-acetylcysteine partly reversed the morphological, invasive and respiratory changes. The authors concluded that BAP1 deubiquitinase activity regulates these phenotypes, largely at the protein level.

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 cell-line study (proteomic and transcriptomic profiling plus functional assays in isogenic mesothelioma cell lines)
• Who was studied: Two human mesothelioma cell lines: NCI-H226 (BAP1-deleted, from ATCC) and QR (also known as MPM_33); work performed at Institut Curie, Paris (published 2017)
• Exposure: Modulation of BAP1 expression (empty vector, wild-type BAP1, catalytically dead BAP1 C91S). Additional treatments were hydrogen peroxide (200 or 400 micromolar) and the antioxidant N-acetylcysteine (500 nM for 10 days). No asbestos fibers were used in the experiments.
• Main results:
  • In NCI-H226, 1098 proteins differed in accumulation with BAP1wt (556 over-represented, 542 under-represented), by SILAC/MS
  • 734 genes were differentially expressed, and only 55 of the 1098 proteins (5%) also changed at mRNA level
  • Cortical actin and stress fibres appeared in more than 40% of NCI-H226 cells and more than 25% of QR cells expressing BAP1wt
  • Cells expressing empty vector or BAP1C91S showed only a diffuse signal, with no stress fibres
  • In the wound-healing assay, reported for NCI-H226, motility was more than 2-fold higher in BAP1wt cells than in EV or BAP1C91S cells
  • Invasion in Boyden chambers was 10-fold higher (NCI-H226) and 2-fold higher (QR) for BAP1wt cells than for EV or BAP1C91S cells (p less than 0.05)
  • Mitochondrial respiratory capacity was reduced 1.3-fold (NCI-H226) and 1.5-fold (QR) in BAP1wt cells versus EV or BAP1C91S (p less than 0.001)
  • Intracellular ROS were 1.8-fold (NCI-H226) and 1.3-fold (QR) higher in BAP1wt than in EV cells (p less than 0.001 and p less than 0.05)
  • After 400 micromolar H2O2, BAP1wt NCI-H226 cells had 50% survival and EV cells 15%, a 3-fold difference (p less than 0.01)
  • N-acetylcysteine treatment of BAP1wt NCI-H226 cells gave a 2-fold decrease in the number of cells with cortical actin and stress fibres
  • N-acetylcysteine treatment gave a 2-fold decrease in invasion of BAP1wt NCI-H226 cells (p less than 0.01)
  • N-acetylcysteine treatment gave a 1.5-fold increase in respiratory capacity of BAP1wt NCI-H226 cells (p less than 0.01)
• The authors' conclusion: The authors concluded that BAP1 has a major role in regulating cell morphology, migration and mitochondrial respiration in mesothelioma, and that this role is mainly visible at the proteome level. They proposed that increased intracellular ROS is at least partly responsible for the morphological and respiratory changes.
• Limitations the authors state:
  • The reduced active mitochondrial content seen in NCI-H226 was not seen in QR, which the authors say may be cell line specific
  • Some protein expression changes seen by mass spectrometry could not be confirmed with an antibody cocktail
  • The authors state the phenotypes are most likely indirect consequences of BAP1 modulation, and that the BAP1 targets responsible remain to be identified
  • The catalytically dead mutant produced some phenotypic differences, so it may retain some biological activity

Topics

Medical terms in this study, explained in our Lexicon: BAP1, Mesothelioma.

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