Fig 2: BAP1 knockdown increases death receptor expression and TRAIL sensitivity in cancer cells.A, immunoblots of proapoptotic proteins in parental, BAP1 shRNA (shBAP1—clone 1) or empty vector (EV) shRNA transduced BAP1-wt MPM cells (H2818) across multiple time points (0, 6, 12, 24, and 48 h) post rTRAIL treatment (100 ng/ml). Duplet bands of DR5 represent two isoforms, DR5-short (DR5-S) and DR5-long (DR5-L). The bands were quantified and normalized to an average of parental cells data. B, cell viability assay of parental, shBAP1-transduced, or EV-transduced H2818 cells following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h. C, immunoblot analysis in BAP1-wt-clear cell renal cell carcinoma (CCRCC) cells (BB65 and Caki-1) and MPM cells (H2818) transduced with BAP1 (shBAP1 1 or shBAP1 2) or EV shRNA. The bands were quantified and normalized to EV. D, cell viability assay of EV-transduced or shBAP1-transduced wt-BAP1 CCRCC cells following treatment with a dose range of rTRAIL (0–1000 ng/ml) for 72 h. E, relative expression of DR4 and DR5 mRNA in CCRCC cells transduced with EV or shBAP1 assessed by quantitative PCR. Relative mRNA expression was normalized to beta-2-microgloblin (B2M) expression. Data shown are the mean ± SD of two experiments performed in triplicates. t test; *p < 0.05, **p < 0.01. BAP1, BRCA1-associated protein 1; MPM, malignant pleural mesothelioma; rTRAIL, recombinant tumor necrosis factor–related apoptosis-inducing ligand; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.

Fig 3: YY1 recruits BAP1 to the promoter regions of DR4 and DR5 and represses their transcriptional activities.A, co-immunoprecipitation (co-IP) of endogenous YY1 and BAP1 in MPM (H2818 and MPP89) and CCRCC (Caki-1) cells. B, co-IP of YY1 and BAP1 in BAP1 null early passage MPM Meso-8T cells transduced with wt BAP1 (wt-BAP1) or a control vector. C, enrichment of BAP1 and YY1 in the promoter regions of DR4 and DR5. Meso-8T cells were overexpressed with wt-BAP1, catalytically inactive BAP1-mutant (C91A-BAP1) or a control vector (cont). Chromatin immunoprecipitation (ChIP) was performed against BAP1, YY1, or IgG control followed by quantitative PCR using primers specific for promoter regions of DR4 or DR5. Error bars represent the SD. p Values are calculated to compare against IgG control using Student's t test (n = 3); *p < 0.05, **p < 0.01. D, schematic model of the transcriptional regulation of TRAIL DRs by BAP1 and YY1. BAP1, BRCA1-associated protein 1; CCRCC, clear cell renal cell carcinoma; DR, death receptor; MPM, malignant pleural mesothelioma; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand; YY1, Ying Yang 1.

Fig 4: The reverse signaling of tmTNF-a promotes AICD through upregulating FasL/Fas and TRAIL/DR4(A, B, C, D) Jurkat cells were simultaneously stimulated with PHA-P (5 µg/ml) and TNF-a pAb (1:100) for 24 h. PHA-preactivated primary T cells were restimulated with aCD3 (10 µg/ml) and TNF-a pAb for 24 h. Expression of FasL/Fas (A, B) and TRAIL/DR4/DR5 (C, D) was detected by flow cytometry. (E, F) Jurkat cells transfected with 100 nM siRNA for FasL or TRAIL for 48 h were activated with PHA-P (5 µg/ml) in the absence or presence of TNF-a pAb for 24 h. The mRNA levels of FasL and TRAIL were determined by Real-time qPCR (E) and the apoptosis was detected by Annexin V/PI (F). All the quantitative data represent means ± S.D. of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001. ns: no significance.

Fig 5: Expression levels of DR4 and DR5 are inversely correlated with BAP1 expression in malignant pleural mesothelioma.A, representative images of immunohistochemistry of DR4 and DR5 in a core from an MPM tissue microarray (TMA) with or without nuclear BAP1 expression (from 88 cores of 32 patients). B, semiquantitative analysis of DR4 and DR5 expression in MPM TMA cores with (n = 42) and without (n = 46) nuclear BAP1 expression. Each dot represents an average score per patient (n = 32). t Test; p = 0.024 (DR4) and p = 0.0092 (DR5). See Experimental procedures section for details. C, immunoblots of DR4, DR5, and BAP1 protein expression in BAP1-mutant (n = 7) versus wt-BAP1 (n = 7) MPM cell lines. Duplet bands of DR5 represent two isoforms, DR5-short (DR5-S) and DR5-long (DR5-L). Sensitivity to rTRAIL treatment is indicated as font color: green sensitive (S); orange partially sensitive (PS); and red resistant (R). D, quantitative analysis of immunoblot intensity of DR4 and DR5 in wt BAP1 and BAP1-mutant MPM cell lines (DR4 t test, p = 0.046; DR5 t test, p = 0.009). Dot color indicates the sensitivity to rTRAIL treatment as shown in (C). E, quantitative analysis of immunoblot intensity of DR4 and DR5 in early passage MPM cells with (+) and without (-) nuclear BAP1 expression. (DR4 t test, p = 0.033; DR5 t test, p = 0.049). F, flow cytometry analysis of DR4 and DR5 cell surface expression in early passage MPM cells with (BAP1+) and without (BAP1-) nuclear BAP1 expression alongside an isotype control. BAP1, BRCA1-associated protein 1; DR, death receptor; MPM, malignant pleural mesothelioma; rTRAIL, recombinant tumor necrosis factor–related apoptosis-inducing ligand.