The authors have declared that no competing interests exist.
Conceived and designed the experiments: AF MET VCB GG. Performed the experiments: SB AK KA JJG. Analyzed the data: AK MS. Contributed reagents/materials/analysis tools: AC VKG. Wrote the paper: SB DAF.
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.
Malignant pleural mesothelioma (MPM) is an aggressive cancer caused by exposure to asbestos. It is increasing in incidence worldwide however there is a paucity of effective therapy
The proteasome inhibitor bortezomib has shown promising activity in preclinical models both in vitro and in vivo
In contrast to hematopoietic malignancies, the poor response of solid tumours to bortezomib treatment appears to be due to the existence of both primary and acquired resistance
Here, we show that specific components of the mitochondrial signalling pathway are lost or dysregulated in MPM, and can directly cause bortezomib resistance.
Bortezomib was courtesy of Millenium; MG132 was purchased from Sigma-Aldrich, (St. Louis, MO). Antibodies against c-Myc, BAX and BAK were from Cell Signaling (Danvers, MA, USA), anti-PARP from Alexis (Nottingham, UK), anti-NOXA from Calbiochem (Gibbstown, NY), GAPDH and β-Tubulin from Abcam (Cambridge, UK). Secondary antibodies were: goat anti-rabbit HRP (DAKO, Glostrup, Denmark), donkey anti-mouse HRP (GE Healthcare).
REN
Cell viability was assessed by a Vialight Plus kit (Lonza, Basel, Switzerland). For the caspase-3 luminescence assay, cells were analysed by using a Caspase-Glo 3/7 Assay (Promega, Southampton, Hampshire).
Cells were lysed in RIPA buffer containing protease inhibitors (Roche, Burgess Hill, UK). Cell lysates were separated on SDS-PAGE denaturing gels, transferred to nitrocellulose membranes, and blocked in 5% milk-PBS-0.1% tween. Membranes were probed with primary antibodies diluted in 5% milk-PBS-0.1% tween at 4°C overnight. Signal detection was performed with the ECL-plus chemiluminescent system (GE Healthcare, Amersham, UK).
Non-silencing control (NT), BAX, BAK, and NOXA targeting siRNAs were obtained from Qiagen. siRNA (50 nM for BAX and BAK, 20 nM for NOXA) transfections were performed using the RNAiMAX transfection reagent (Invitrogen) according to manufacturer’s instructions.
BAX/BAK DKO cells were transiently transfected with GST-tagged BAX and BAK (pEGFP-C3 vector), using X-tremeGENE transfection reagent (Roche) according to manufacturer’s instructions.
BAX and BAK protein expression was assessed by immunohistochemistry (IHC) on two sets of samples. 16 tissues were from ICORG-05 Study
Immunohistochemistry for the samples from the ICORG-05 study was performed within the Tissue Core Technology Unit at the Centre for Cancer Research and Cell Biology and sections were then scanned in the Queen’s University of Belfast Bioimaging Unit. BAK primary antibody was used at a 1∶800 dilution; BAX antibody was used at a 1∶50 dilution. IHC scoring of tissue slides from the Phase II trial was carried out through the PathXL™ TMA Toolbox (i-Path Diagnostics Ltd, Belfast, UK). The TMA was stained and scored in the Pathology Core Facility, Department of pathology, Bart’s and the London NHS Trust.
The staining results were semi-quantitatively assessed by two individual examiners. Tumours were graded for expression of BAX and BAK as follows: 0 = no cells stained, 1 = <25% cells positive (light staining), 2 = 25–75% cells positive (moderate staining) 3 = >75% cells positive (strong staining). In case of discrepancy between the two examiners, a result was obtained by consensus while reviewing the slides using a double-headed microscope. Survival data were available for 30 out of 70 patients; therefore the analysis has been carried out on this unselected population only. The survival analysis was performed by using Kaplan-Meier estimation and significance was measured by the log-rank test.
The adopted statistics software was SPSS17.0 (Chicago, IL, USA).
Cells were washed in Mitochondrial Isolation Buffer (200 mM Mannitol, 70 mM Sucrose, 1 mM EGTA, 10 mM HEPES, 0.5 mg/ml BSA, pH7.4). Mitochondria were then isolated by dounce homogenization followed by centrifugation at 800×g for 10 minutes at 4°C to remove debris and heavy membranes, then by centrifugation at 10,000×g for 10 minutes at 4°C. The mitochondrial-free cytosolic fraction was used for Western blot analysis
Total RNA was extracted using an RNeasy Plus mini kit (Qiagen Valencia, CA, USA) according to manufacturer’s instructions. Quality control was performed by Phalanx Biotech Group (Palo Alto, CA, USA). Reverse transcription was performed with M-MLV Reverse transcriptase (Invitrogen). Real-Time PCR was carried out using Power SYBR® Green PCR Master Mix (Applied Biosystem).
c-Myc reporter assay was performed using Cignal Reporter Assay Kits (SABiosciences, Frederick, MD). Transfections were carried out by using the Lipofectamine2000 transfection reagent (Invitrogen). Luciferase activity was measured using the dual-luciferase reporter assay system (Promega).
Chromatin immunoprecipitation assays were performed as previously described
Dose-response curves were fitted using non-linear regression (GraphPad Prism version 4.0, GraphPad Software, Inc. LaJolla, CA, USA).
One or two-way analysis of variance was used to evaluate statistical significance and a Bonferroni post-test was performed. A p value less than 0.05 was considered significant.
Bortezomib induced apoptosis in wild type mouse embryonic fibroblasts (WT MEF), but this effect was dramatically reduced in cells with homozygous deletion of BAX and BAK (BAX/BAK DKO MEF), as evidenced by PARP cleavage (
A) WT MEF and BAX/BAK DKO MEF cells were treated with bortezomib 10 nM for 24 h. PARP cleavage was measured by western blot. B) Caspase3 activity was assessed by luminescence assay. Data were normalized to untreated control (WT: p<0.0001; DKO n.s.). C) BAX and BAK were transiently overexpressed in DKO cells and 24 h post transfection cells were treated with bortezomib 10 nM for a further 24 hours. BAX and BAK expression were then analysed by western blot. D) Caspase 3 activation after bortezomib treatment was also analysed by luminescence assay. Data were normalized to untreated control (EV: n.s.; GSTBAX: p = <0.0001 GSTBAK: p = <0.0001).
We then tested for the individual and combined contribution of BAX and BAK by using RNA interference to silence BAX or BAK or both in MPM cell lines (REN and JU77). Here in contrast to our findings using MEFs, we found that silencing of BAX alone did not reduced sensitivity to bortezomib. However, BAK proved to be important; BAK and BAX/BAK silencing significantly reduced bortezomib-induced caspase 3 activity in both REN (siNT: 3.25, siBAX: 3.14, siBAK: 0.83 and siBAX/BAK: 1.01 fold increase) (
A) REN and B) JU77 cells were transfected with siNT, siBAX, siBAK and the combination of siBAX and siBAK. 24 h following transfection, cells were treated with a concentration of bortezomib equal to the IC50 calculated for each cell line and caspase3 activity measured. Data were normalized to NT untreated control (REN: siNT p = 0.0003 siBAX p = 0.0159; siBAK n.s. siBAXsiBAK n.s.; JU77: siNT p = 0.0002 siBAX p = 0.0002; siBAK n.s.; siBAXsiBAK n.s.). C) BAX and BAK expression and PARP cleavage were confirmed by western blot analysis in REN and D) JU77 cells.
We used an immunohistochemistry-based approach to measure the expression of BAX and BAK in two different cohorts of MPM patients (
A) Representative image of normal tissue control and positive tissues stained for BAX and BAK by immunohistochemisty. B) Pie-charts representing the frequency of BAX, BAK and BAX-BAK negativity in previously treated 30 mesothelioma patients. C) Kaplan Meier curves correlating BAX, BAK, and double BAX/BAK expression respectively with survival in the total of 30 previously-treated patients.
A second cohort consisted of 16 specimens obtained from MPM patients treated with bortezomib in a phase II trial
Staining intensity | Staining intensity | ||||
Sample | BAX | BAK | Sample | BAX | BAK |
1 | 1 | 2 | 2 | ||
0 | 1 | 0 | 2 | ||
1 | 2 | 0 | 1 | ||
0 | 1 | 1 | 1 | ||
0 | 1 | 0 | 2 | ||
1 | 1 | 1 | 1 | ||
0 | 1 | 2 | 3 | ||
0 | 3 | 1 | 1 |
0 = No cells stained 1 = <25% cells stained 2 = 26–75% cells stained 3 = >75% cells stained.
Both REN and JU77 cell lines were exposed to increasing concentrations of bortezomib leading to selection of two isogenic resistant cell lines (RENBZR, JU77BZR). REN BZR and JU77 BZR cells exhibited 6-fold and 7-fold resistance compared to parental cells (IC50 12 nM and 50 nM respectively) (
A) REN and JU77 selected for resistance after exposure to increasing doses of bortezomib were tested for cell viability after 24 h treatment with bortezomib at concentrations ranging from 0.5 nM to 50 nM and compared to parental cells. REN/RENBZR and JU77/JU77BZR cells were treated for 24 h with bortezomib 5 nM and 10 nM, respectively. PARP cleavage induced by bortezomib was analysed by western blot and caspase3 activity was measured by luminescence assay. Data were normalized to untreated control (REN: <0.0001; RENBZR: n.s.; JU77: p = 0.0002; JU77BZR: n.s.). B) Expression of BAX and BAK was investigated in parental and resistant cells pre- and after 6 h treatment with bortezomib 5 nM and 10 nM in REN/RENBZR and JU77/JU77BZR respectively. C) Cytochrome C release was assessed after 24 h treatment with bortezomib (5 nM and 10 nM in REN/RENBZR and JU77/JU77BZR, respectively). Mitochondrial-free cytosolic fraction has been used for western blot analysis.
Upregulation of NOXA has been implicated as a regulator of bortezomib induced apoptosis specifically in tumour cells
A) REN and B) JU77 cells were transfected with siRNA sequences targeting the BH3-only protein NOXA. 24 h following transfection, cells were treated with a concentration of bortezomib equal to the IC50 calculated for each cell line and caspase3 activity measured. Data were normalized to NT untreated control. C) NOXA expression level and PARP cleavage were assessed by western blot analysis in REN and D) JU77 cells.
The upregulation of NOXA protein expression following bortezomib was significantly reduced in resistant cells compared to parental cells (
A) The expression of NOXA and c-Myc was evaluated in by western blotting in REN/RENBZR and JU77/JU77BZR cells. Cells were left untreated or exposed to bortezomib 5 nM or 10 nM respectively for 6 hours. B) NOXA mRNA expression was evaluated by qRT-PCR on RNA extracted from parental and resistant cells treated for 6 h with 5 nM (REN/RENBZR) or 10 nM (JU77/JU77BZR) bortezomib. Data were normalized to untreated control (REN: p = 0.00241; RENBZR n.s.; JU77: p = 0.0001; JU77BZR n.s.) C) RENshNT and RENshc-Myc cells were generated by RNAi and NOXA induction was analysed by western blot after 24 h treatment with 5 nM bortezomib. D) c-Myc activity was measured by a reporter assay in REN and RENBZR treated for 24 h with 5 nM bortezomib. Data were normalized to untreated control (REN: p = 0.0438; RENBZR n.s.). E) The binding of c-Myc to the Noxa promoter was evaluated by ChIP in REN and RENBZR treated for 24 h with 5 nM bortezomib.
C-Myc is a transcriptional activator of NOXA
In bortezomib resistant MPM cells, c-Myc protein expression was lower at baseline compared to parental cells and was unaffected by exposure to bortezomib (
Chromatin immunoprecipitation revealed the interaction of c-Myc with the promoter for NOXA in REN parental cells, which increased after treatment with bortezomib. However, no interaction was observed in RENBZR cells, even after treatment with bortezomib (
Bortezomib exhibits significant preclinical activity in several solid tumour cell lines and animal models including MPM
The expression of BAX and BAK has been previously investigated in mesothelioma samples and 24% loss of BAK and 42% loss of BAX expression were found, but no correlation with histology was reported
Apoptosis block is a hallmark of cancer and may contribute to aggressive tumour progression in this sub-population of patients with MPM, as well as potentially conferring drug resistance
Bortezomib upregulates the BH3-only protein and Mcl-1 inhibitor NOXA, at both the protein and mRNA level after 6 hours from exposure
Noxa is commonly described as a p53 target gene as it contains p53 response elements on its promoter and it has also been reported as a key mediator of p53-driven apoptosis
The induction of NOXA and the subsequent activation of the mitochondrial apoptosis pathway by bortezomib has been reported not to correlate linearly with c-Myc protein or mRNA expression
These events may occur in cooperation with proteasome function in regulating histone acetylases
Alternative mechanisms of resistance to bortezomib have been proposed, in fact, increasing evidence in haematological tumours support the importance of the expression levels of proteasome subunits and their composition. Mutation of PSMB5 has been shown to be a cause of bortezomib resistance
These findings suggest that disruption of c-Myc-dependent NOXA mediated death signalling and BAK could play a potential role in resistance to bortezomib in the clinical setting, highlighting the putative role of BAK and NOXA as valid prognostic markers for bortezomib. However, our data from 16 patients enrolled in the Phase II clinical trial showed that NOXA was expressed in the tissues from all the MPM patients examined, including the one showing stable disease
In summary, bortezomib requires functional BAK and NOXA to induce apoptosis in MPM cells. The loss of BAK expression occurring in a subset of patients with MPM may contribute to resistance to this drug in the clinical setting. However, dysregulation of NOXA transactivation may be an alternative mechanism as evidenced in MPM cells selected for resistance to bortezomib.