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

This dataset includes the text, figures and supplementary material associated with the Oncogene submission MS#ONC-2022-0172

Vorinostat increases the apoptotic response of mesothelioma cells within tumor fragment spheroids to treatment.

<p>(<b>A</b>) Tumor fragment spheroids were grown from six treatment-naïve mesothelioma samples: three were treated with bortezomib (100 nM) and three with cisplatin (250 µM) plus pemetrexed (10 µM) (C+P) with or without vorinostat (5 µM) for 24 h. Apoptosis was measured by the detection of merged cytokeratin and cleaved caspase 3 immunofluorescence. In all samples, vorinostat increased the apoptotic response of mesothelioma cells to treatment. (* p<0.05 compared to chemotherapy or vorinostat alone, n = 3). (<b>B</b>) Representative confocal fluorescent merged images of tumor fragment spheroids after treatment. Addition of vorinostat either to bortezomib or to cisplatin plus pemetrexed increased the number of apoptotic mesothelioma cells (positive for both pan-cytokeratin and for cleaved caspase-3 staining). White arrowheads highlight some of the apoptotic cells. Images are representative of all tumor fragment spheroids used in the experiment. Immunohistochemistry for Noxa (<b>C</b>) and Bim (<b>D</b>) showed that the addition of vorinostat to treatment increases both Noxa and Bim in tumor fragment spheroids (images representative of the 3 tumor fragment spheroids treated with C+P in panel A).</p

Vorinostat abolishes multicellular resistance of spheroids.

<p>(<b>A</b>) Four mesothelioma cell lines (M28, REN, SARC and VAMT) were grown as monolayers and spheroids and treated with bortezomib (25 nM), vorinostat (5 µM) or their combination for 24 h. Apoptosis was measured by analysis of nuclear condensation in Hoechst-stained cells. Spheroids grown from all the cell lines acquired multicellular resistance to bortezomib, a resistance that was effectively eliminated by the addition of vorinostat. Both vorinostat and bortezomib induced little apoptosis in spheroids when given alone. (* p<0.05 as compared to bortezomib alone, n = 3) (<b>B</b>) Apoptosis was confirmed by CaspaseGlo 3/7 assay in M28 and REN spheroids, treated with either bortezomib (25 nM), vorinostat (5 µM) or their combination for 24 h. Vorinostat had no effect on spheroids but increased their apoptotic response to bortezomib (* p<0.05 as compared to bortezomib or vorinostat alone, n = 3) (<b>C–D</b>) Bright field 10× images of the edge of M28 and REN spheroids treated with vorinostat, bortezomib or the combination for 24 h. Whereas vorinostat or bortezomib alone induced little or no morphologic change, the combination potently increased the number of cells detaching from the spheroids and floating in the medium. (Focus was adjusted in the lower panels to visualize some of these floating cells).</p

Vorinostat restores Noxa mRNA of spheroids to the level of monolayers.

<p>M28 and REN monolayers and spheroids were treated with bortezomib (100 nM), vorinostat (5 µM) or their combination for 4 h and Noxa mRNA was measured by qRT-PCR. After treatment with bortezomib, Noxa mRNA increased significantly in monolayers but was significantly lower in spheroids. The addition of vorinostat to bortezomib led to an increase in Noxa mRNA levels in spheroids to the same level as in monolayers. (* p<0.05, significant increase compared to bortezomib alone; † p<0.05, significantly less than in the monolayer exposed to same treatment; n = 3).</p

Noxa and Bim are each required for vorinostat to increase the apoptotic response of spheroids.

<p>REN cells were treated with siRNA to ablate Noxa or Bim, allowed to form spheroids over 24 h and then treated with bortezomib, vorinostat or the combination. Monolayers were treated similarly for 4 h and collected and lysed to verify the efficacy of Noxa siRNA (see immunoblots). After 24 h, Hoechst-stained cells were counted for the presence of apoptosis. Noxa and Bim siRNA significantly inhibited the ability of vorinostat to increase apoptosis in spheroids. (* p<0.05, significant difference compared to control siRNA; n = 3).</p

Vorinostat restores the upregulation of Noxa induced by bortezomib in spheroids.

<p>M28 and REN monolayers and spheroids were treated with bortezomib (100 nM), vorinostat (5 µM) or the combination for 6 h. Cells were then lysed and Noxa and Bim levels were analyzed by immunoblot. In both M28 and REN cells grown as monolayers, bortezomib increased levels of Noxa protein; however, in the same cells grown as spheroids, the bortezomib-induced increase in Noxa was blunted or absent. With the addition of vorinostat, bortezomib now increased Noxa protein in the spheroids to levels comparable to or greater than those in bortezomib-treated monolayers. Of note, vorinostat alone had little or no effect on Noxa protein levels. Vorinostat alone and particularly in combination with bortezomib increased the levels of Bim protein. (Densitometry of band intensities relative to tubulin is shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052753#pone.0052753.s002" target="_blank">Figure S2A and S2B</a></b>).</p

In MEF cells, BAX and BAK expression regulates bortezomib activity.

<p>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).</p

The BH3-only protein NOXA is essential for bortezomib-induced apoptosis.

<p>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.</p

Immunohistochemical analysis of BAX and BAK expression in malignant mesothelioma patients.

<p>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.</p

Scoring of samples from ICORG-05 Phase II Trial [7].

<p>0 = No cells stained 1 = <25% cells stained 2 = 26–75% cells stained 3 = >75% cells stained.</p