BAK and NOXA are critical determinants of mitochondrial apoptosis induced by bortezomib in mesothelioma

Our plain-English summary of BAK and NOXA are critical determinants of mitochondrial apoptosis induced by bortezomib in mesothelioma, by S. Busacca, A.D. Chacko, A. Klabatsa, et al (PloS One, 2013).

The study

• Authors: S. Busacca, A.D. Chacko, A. Klabatsa, K. Arthur, M. Sheaff, D. Barbone, L. Mutti, V.K. Gunasekharan, J.J. Gorski, M. El-Tanani, V.C. Broaddus, G. Gaudino, D.A. Fennell
• Journal: PloS One
• Published: 2013;8(6):e65489
• Cited by: 14 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Based on promising preclinical efficacy associated with the 20S proteasome inhibitor bortezomib in malignant pleural mesothelioma (MPM), two phase II clinical trials have been initiated (EORTC 08052 and ICORG 05-10). However, the potential mechanisms underlying resistance to this targeted drug in MPM are still unknown. Functional genetic analyses were conducted to determine the key mitochondrial apoptotic regulators required for bortezomib sensitivity and to establish how their dysregulation may confer resistance. The multidomain proapoptotic protein BAK, but not its orthologue BAX, was found to be essential for bortezomib-induced apoptosis in MPM cell lines. Immunohistochemistry was performed on tissues from the ICORG-05 phase II trial and a TMA of archived mesotheliomas. Loss of BAK was found in 39% of specimens and loss of both BAX/BAK in 37% of samples. However, MPM tissues from patients who failed to respond to bortezomib and MPM cell lines selected for resistance to bortezomib conserved BAK expression. In contrast, c-Myc dependent transactivation of NOXA was abrogated in the resistant cell lines. In summary, the block of mitochondrial apoptosis is a limiting factor for achieving efficacy of bortezomib in MPM, and the observed loss of BAK expression or NOXA transactivation may be relevant mechanisms of resistance in the clinic.

In plain English

This was a laboratory study, with analysis of tumour tissue, of why malignant pleural mesothelioma may resist the drug bortezomib. It did not study asbestos exposure. In mouse cells lacking the proteins BAX and BAK, bortezomib-induced caspase 3 activation (a sign of cell death) was 1.13-fold, compared with 4.13-fold in normal mouse cells. In two human mesothelioma cell lines (REN and JU77), silencing BAK, but not BAX, reduced this response. In archived mesothelioma tissue, 26 of 69 cases (37.7%) lacked BAK and 26 of 70 (37.1%) lacked BAX. Among 30 previously treated patients with survival data, median survival was 18 months when BAK was present and 6 months when it was absent. All 16 tumours from patients in a bortezomib trial expressed BAK. Cell lines made resistant to bortezomib kept BAX and BAK. However, they failed to switch on the protein NOXA, a step that depended on the protein c-Myc. The authors concluded that bortezomib requires functional BAK and NOXA to kill mesothelioma cells, and that BAK loss may contribute to resistance. The authors noted that their patient sample numbers were small.

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 study of cell lines (gene silencing and drug-resistance experiments) combined with immunohistochemistry on patient tumour tissue and a survival analysis
• Who was studied: Human malignant pleural mesothelioma cell lines REN and JU77; normal mouse embryonic fibroblasts and mouse embryonic fibroblasts lacking both BAX and BAK; 16 tumour tissues from the ICORG-05 phase II bortezomib trial; a tissue microarray of 100 archived mesotheliomas (69 to 70 cases assessable), with survival data for 30 previously treated patients
• Exposure: The drug bortezomib (a proteasome inhibitor), at concentrations such as 5 nM and 10 nM. Asbestos exposure was not studied; it is mentioned only in the introduction as the cause of mesothelioma.
• Main results:
  • In mouse embryonic fibroblasts, bortezomib caused a 4.13-fold increase in caspase 3 activation in normal cells and a 1.13-fold increase in cells lacking both BAX and BAK
  • In the REN mesothelioma cell line, the fold increase in bortezomib-induced caspase 3 activity was 3.25 with control silencing, 3.14 with BAX silenced, 0.83 with BAK silenced and 1.01 with both silenced. In JU77 cells the corresponding values were 4.8, 5.13, 0.9 and 0.76.
  • In the archived tissue cohort, 43 of 69 cases (62.3%) were BAK positive and 26 (37.7%) were BAK negative
  • In the archived tissue cohort, 44 of 70 cases (62.9%) were BAX positive and 26 (37.1%) were BAX negative
  • Among the 30 previously treated patients with survival data, median survival was 18 months in BAK-positive patients and 6 months in BAK-negative patients (HR = 0.518, 95% CI 0.36 to 0.75)
  • Among the same 30 patients, median survival was 18 months in BAX/BAK-positive samples and 5 months in BAX/BAK-negative samples (HR = 0.197, 95% CI 0.08 to 0.49)
  • In the 16 tumours from bortezomib-treated trial patients, BAX was absent in 50% and BAK was present in all (100%)
  • Bortezomib-resistant cell lines showed 6-fold (REN) and 7-fold (JU77) resistance compared with the original cells. Their basal BAX and BAK levels were not significantly different from the original cells.
  • Silencing NOXA significantly inhibited bortezomib-induced caspase 3 activity in REN and JU77 cells
  • Bortezomib raised NOXA mRNA 4.42-fold in REN and 3.19-fold in JU77 cells, but only 0.82-fold and 1.24-fold in the resistant versions of these lines
  • c-Myc bound the NOXA promoter in the original REN cells but not in the resistant REN cells, even after bortezomib treatment
• The authors' conclusion: The authors concluded that bortezomib requires functional BAK and NOXA to induce cell death in mesothelioma cells. They proposed that BAK loss in a subset of patients may contribute to clinical resistance, and that disrupted NOXA activation may be an alternative mechanism.
• Limitations the authors state:
  • Adjusted (Cox regression) analysis was possible only for histology, age and sex, because too few cases had survival data; the prognostic value of BAX/BAK needs a larger cohort
  • The 16 trial samples were too few for a statistically reliable analysis
  • Some tissue microarray cores were partly or fully lost during staining
  • Other resistance mechanisms may be involved in patients

Topics

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

Medical terms in this study, explained in our Lexicon: Apoptosis, Mesothelioma, Pleural Mesothelioma, Clinical Trial, Immunohistochemistry.

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• Phase II clinical trial of first or second-line treatment with bortezomib in patients with malignant pleural mesothelioma — D.A. Fennell, C. McDowell, et al. (2012)
• Bortezomib inhibits nuclear factor-kappaB dependent survival and has potent in vivo activity in mesothelioma — A. Sartore-Bianchi, F. Gasparri, et al. (2007)
• Usp9x- and Noxa-mediated Mcl-1 downregulation contributes to pemetrexed-induced apoptosis in human non-small-cell lung cancer cells — J. Yan, N. Zhong, et al. (2014)
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