BAP1 loss induces mitotic defects in mesothelioma cells through BRCA1-dependent and independent mechanisms

Our plain-English summary of BAP1 loss induces mitotic defects in mesothelioma cells through BRCA1-dependent and independent mechanisms, by A. Singh, S. Busacca, A. Gaba, et al (Oncogene, 2023).

The study

• Authors: A. Singh, S. Busacca, A. Gaba, M. Sheaff, C. Poile, A. Nakas, J. Dzialo, A. Bzura, A.G. Dawson, D.A. Fennell, A.M. Fry
• Journal: Oncogene
• Published: 2023;42(8):572-585
• Cited by: 14 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The tumour suppressor BRCA1-associated protein 1 (BAP1) is the most frequently mutated cancer gene in mesothelioma. Here we report novel functions for BAP1 in mitotic progression highlighting the relationship between BAP1 and control of genome stability in mesothelioma cells with therapeutic implications. Depletion of BAP1 protein induced proteasome-mediated degradation of BRCA1 in mesothelioma cells while loss of BAP1 correlated with BRCA1 loss in mesothelioma patient tumour samples. BAP1 loss also led to mitotic defects that phenocopied the loss of BRCA1 including spindle assembly checkpoint failure, centrosome amplification and chromosome segregation errors. However, loss of BAP1 also led to additional mitotic changes that were not observed upon BRCA1 loss, including an increase in spindle length and enhanced growth of astral microtubules. Intriguingly, these consequences could be explained by loss of expression of the KIF18A and KIF18B kinesin motors that occurred upon depletion of BAP1 but not BRCA1, as spindle and astral microtubule defects were rescued by re-expression of KIF18A and KIF18B, respectively. We therefore propose that BAP1 inactivation causes mitotic defects through BRCA1-dependent and independent mechanisms revealing novel routes by which mesothelioma cells lacking BAP1 may acquire genome instability and exhibit altered responses to microtubule-targeted agents.

© 2022. The Author(s).

In plain English

This was a laboratory study in malignant pleural mesothelioma cell lines, combined with immunohistochemistry on tumour samples from 26 chemonaive mesothelioma patients. BAP1 and BRCA1 were depleted with siRNAs in MSTO-211H and NCI-H2452 cells, and BRCA1 also by an inducible shBRCA1 construct in MSTO-211H cells. BAP1 depletion reduced BRCA1 protein, while BRCA1 depletion did not change BAP1; the proteasome inhibitor MG132 substantially rescued BRCA1, indicating BAP1 blocks proteasome-mediated BRCA1 degradation. In patient tumours, BAP1 and BRCA1 protein expression were positively and significantly correlated (chi-square test, p less than 0.05). Depletion of either protein increased misaligned chromosomes, multinucleated and micronucleated cells, multipolar spindles and amplified centrosomes, and reduced MAD2L1 protein; BUBR1 at kinetochores was reduced in cells arrested in prometaphase with vinorelbine. Loss of BAP1, but not BRCA1, reduced centrosome size, lengthened spindles (about 20 to 50 percent in MSTO-211H, 50 percent in NCI-H2452), increased astral microtubules, and lowered KIF18A and KIF18B; re-expressing these kinesins restored normal lengths. The authors proposed that BAP1 inactivation causes mitotic defects through BRCA1-dependent and independent mechanisms.

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 experimental study (siRNA/shRNA depletion, Western blotting, immunofluorescence, flow cytometry) with immunohistochemistry on patient tumour tissue
• Who was studied: Malignant pleural mesothelioma cell lines MSTO-211H and NCI-H2452, primary patient-derived lines MEDCL85, MEDCL91 and MEDCL96, NCI-H226 cells, and tumour samples from 26 chemonaive mesothelioma patients in the MEDUSA cohort (Leicester, UK)
• Exposure: Not an asbestos-exposure study. The experimental manipulations were siRNA depletion of BAP1 or BRCA1, an inducible shBRCA1 construct in MSTO-211H cells, plasmid re-expression of KIF18A and KIF18B, and drug treatments (MG132, STLC, vinorelbine). Asbestos is named only as the cause of mesothelioma in the introduction
• Main results:
  • BAP1 depletion with two independent siRNAs significantly reduced BRCA1 protein in MSTO-211H and NCI-H2452 cells, whereas BRCA1 depletion did not change BAP1 protein
  • Proteasome inhibition with MG132 for 6 h substantially rescued BRCA1 expression after 72 h of BAP1 depletion in both cell lines
  • In the 26 patient tumours, BAP1 and BRCA1 protein expression were positively and significantly correlated
  • Depletion of either BAP1 or BRCA1 increased the frequency of multipolar spindles in MSTO-211H and NCI-H2452 cells
  • Depletion of either BAP1 or BRCA1 produced a similar, approximately ten-fold increase in the percentage of cells with amplified centrosomes (more than 2 centrosomes). The text continues after a figure legend to say this was in MSTO-211H cells
  • MAD2L1 protein fell after BAP1 or BRCA1 depletion in MSTO-211H cells, and after BAP1 depletion in NCI-H2452 cells
  • Total BUBR1 protein did not differ on Western blot after BAP1 or BRCA1 depletion in MSTO-211H cells
  • BUBR1 association with kinetochores was reduced after BAP1 or BRCA1 depletion. The sentence continues after a page break to say this was in MSTO-211H and NCI-H2542 cells arrested in prometaphase with vinorelbine
  • Spindles were longer after BAP1 depletion (about 20 to 50 percent in MSTO-211H, 50 percent in NCI-H2452), with no effect of BRCA1 depletion on spindle length
  • Microtubules in monopolar asters were about 20 percent longer (MSTO-211H) and about 80 percent longer (NCI-H2452) after BAP1 depletion, with no change after BRCA1 depletion
  • BAP1 depletion reduced KIF18A and KIF18B protein. Overexpressing GFP-KIF18A restored spindle length, and GFP-KIF18B reversed the roughly five-fold increase in astral microtubule volume
  • No significant difference in cell cycle distribution or percentage of cells in G2/M was seen by flow cytometry after BAP1 or BRCA1 depletion
• The authors' conclusion: The authors proposed that BAP1 inactivation causes mitotic defects through both BRCA1-dependent and BRCA1-independent mechanisms. They suggested these are routes by which BAP1-deficient mesothelioma cells may acquire genome instability and respond differently to microtubule-targeted drugs
• Limitations the authors state:
  • The authors noted that different responses in other cell types could not be excluded from their flow cytometry findings
  • Endogenous KIF18B could not be detected by immunofluorescence despite several commercial antibodies
  • The mechanisms by which BAP1 regulates BRCA1 stability and KIF18A/KIF18B expression remain undetermined, and further work, including restoring BRCA1 in BAP1-deficient cells, is needed
  • Whether the mitotic consequences also occur in other BAP1-deficient cancers was not examined

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

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

Related studies

• A Novel BRCA1-Associated Protein-1 Isoform Affects Response of Mesothelioma Cells to Drugs Impairing BRCA1-Mediated DNA Repair — R. Parrotta, A. Okonska, et al. (2017)
• BRCA1/MAD2L1 Deficiency Disrupts the Spindle Assembly Checkpoint to Confer Vinorelbine Resistance in Mesothelioma — S. Busacca, L. O'Regan, et al. (2021)
• Functional Genomic Screen in Mesothelioma Reveals that Loss of Function of BRCA1-Associated Protein 1 Induces Chemoresistance to Ribonucleotide Reductase Inhibition — A. Okonska, S. Bühler, et al. (2020)
• Loss of BRCA1-associated protein 1 (BAP1) expression is rare in non-small cell lung cancer — D. Owen, B.S. Sheffield, et al. (2017)
• Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells — K.K. Kolluri, C. Alifrangis, et al. (2018)
• Loss of BRCA1-associated protein 1 (BAP1) expression is useful in diagnostic cytopathology of malignant mesothelioma in effusions — I. Cozzi, F.A. Oprescu, et al. (2018)

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