Elucidating BAP1-microRNA regulatory networks in mesothelioma

BRCA-1 Associated Protein 1 (BAP1) is a nuclear deubiquitylase (DUB) frequently inactivated by mutation in specific cancers including malignant pleural mesothelioma (MPM). Mutations cause loss of nuclear BAP1 expression or catalytic inactivation and, as BAP1 regulates both ubiquitylation and transcription, its loss in cancer may dysregulate expression of specific proteins, and potentially non-coding RNAs such as miRNAs. To begin to address these questions, the proteome response to BAP1 mutation was previously profiled in isogenic MeT5A mesothelial cells, and an unbiased DUB siRNA screen revealed that BAP1 regulates cellular abundance of the histone deacetylase HDAC2. In this thesis, I set out to (1) validate BAP1-dependent protein expression in isogenic MeT5A BAP1-mutant cells and patient-derived MPM cell lines, (2) address the mechanism by which BAP1 regulates HDAC2, and (3) to identify BAP1-miRNA regulatory networks in MPM. Profiling the effect of BAP1-deficiency on the MeT5A proteome had revealed alterations in cytoskeletal and metabolic pathways. I verified reduced expression of glycolytic enzymes but increased expression of TCA cycle and associated anaplerotic pathway proteins in MeT5A BAP1-mutant cells. Altered expression of certain cytoskeletal proteins correlated with BAP1-status in a panel of patient-derived MPM cell lines, confirming responses to BAP1 loss in isogenic cell models may be evident on more complex genetic backgrounds. Altered expression of HDAC2 is reported in cancer and our siRNA screen suggested this may be linked to BAP1 status. I found that BAP1 depletion regulates HDAC2 transcript abundance rather than protein stability, and that BAP1 status influences the response of MPM cell lines to HDAC inhibitors. As BAP1 can influence transcription, I next explored the idea that BAP1 may also regulate expression of miRNAs. I used a NanoString platform to screen for BAP1-dependent miRNAs in isogenic MeT5A mesothelial cells. In total, ~400 unique miRNAs were identified and, compared with parental cells, 113 miRNAs were significantly modulated (P≤0.05, >1.5-fold) in BAP1-deficient cells. Unsupervised hierarchical clustering generated five distinct clusters, including a large group from the chromosome 14q32.31 miRNA tumour-suppressor locus that was downregulated in BAP1-deficient cells. Gene ontology analysis suggested these miRNAs may affect the cytoskeleton and transcriptional regulation. I then used the Nanostring platform to profile miRNAs in a panel of 20 MPM cell lines. I observed differential miRNA expression in MPM cell lines grouped by BAP1 status, including downregulation of the ch14q32.31 locus. As loss of BAP1 protein may be more frequent than the reported mutation rate in MPM, post-transcriptional regulation of BAP1 may also be of importance in cancer. To explore this idea, I used computational algorithms to predict miRNA target sites in the BAP1 3’UTR. Several candidate miRNAs were identified, including members of the miR-125 and miR-200 families. We found significantly increased expression of miR-125b-5p in BAP1-deficient isogenic cells (P≤0.01) and an inverse correlation between miR-125b-5p and BAP1 protein expression (R2=0.85) in MPM cell lines. In addition, a miR-125b-5p mimic could reduce BAP1 expression, suggesting that miR-125b-5p may act as a post-transcriptional modulator of BAP1, operating in a double-negative feedback loop to control BAP1 expression. Together, the data in this thesis show that BAP1 deficiency leads to a significant deregulation of proteins involved in cytoskeletal and metabolic pathways, a reduction in HDAC2 transcript abundance, and profoundly altered miRNA expression profiles. My data suggest for the first time that BAP1-dependent miRNAs may in part account for the BAP1-deficient MPM phenotype and could provide new biomarkers for BAP1 loss.