Characterization of the functional roles and regulation of BAP1 in hepatocellular carcinoma

Abstract

BRCA1 associated protein 1 (BAP1) is a deubiquitinase and its germline mutations lead to the “BAP1 cancer syndrome” characterized by development of cancers, particularly uveal melanoma and mesothelioma. However, the role of BAP1 in hepatocellular carcinoma (HCC) is unclear. Here I investigated the role of BAP1 in HCC from multiple angles. Clinically, BAP1 was up-regulated at mRNA level in human HCCs as compared with the non-tumorous livers in both TCGA-LIHC and in-house RNA sequencing datasets, followed by validation in an independent HCC patient cohort. More than one-third of our HCC cases had up-regulation of BAP1 at mRNA (qPCR) and protein (immunohistochemistry on tissue microarrays) levels. Clinicopathological analysis showed that high expression of BAP1 mRNA was significantly correlated with aggressive tumor behavior with more tumor microsatellite formation and less tumor encapsulation, and shorter overall survival. However, high expression of BAP1 protein was found only in the cytoplasm and almost none in the nuclei of tumor cells of human HCC. Mutations of BAP1 in HCC were rare, with only 6% in TCGA-LIHC cohort and none in our in-house whole exome sequencing cohort. Functionally, with both in vitro (cell proliferation and migration assays) and in vivo assays (two liver orthotopic injection mouse models), I observed that silencing BAP1 expression had no significant effects on cell proliferation but fostered cell migratory ability in HepG2 hepatoblastoma cells. Using RNA-sequencing on HepG2 cells with BAP1 knockdown, I identified RHOJ amongst the down-regulated targets, and subsequently verified that overexpression of RHOJ suppressed cell migration in HCC cells. This finding suggested that BAP1 might up-regulate RHOJ resulting in reduced cell migratory ability of HCC cells. With regard to the upstream regulation of BAP1 transcription in HCC, I found a positive and significant association between copy number gain and the increased mRNA level and four transcription factors (CTCF, NRF1, ETS2, ELF1) involved in activating BAP1 transcription by binding to promoter region of BAP1 gene using public chromatin immunoprecipitation (ChIP)-sequencing data and in-silico search, which was confirmed by dual luciferase reporter and ChIP assays. At protein level, I noticed that nuclear BAP1 accumulated upon treatment with MG132, a proteasome inhibitor of the ubiquitin proteasome system (UPS). In addition, polyubiquitination of BAP1 was higher in the nucleus than cytoplasm in HCC cells. Two lysine sites on BAP1, K127A and K187A, were revealed to be possible polyubiquitinated sites responsible for nuclear BAP1 stability. Considering these, degradation by UPS was likely an alternative mechanism other than gene mutation contributing to absence of nuclear BAP1 protein in HCC. Moreover, I uncovered that O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) physically bound to BAP1 in the nucleus and diminished the nuclear BAP1 stability independent of its catalytic activity. To conclude, BAP1 was up-regulated at mRNA and cytoplasmic protein levels in HCC. Up-regulation of BAP1 mRNA level was associated with aggressive tumor behavior and shorter overall survival. The absence of nuclear BAP1 protein wa partly mediated by UPS and/or OGT, contributing to enhanced cell migration through suppression of RHOJ transcription in promoting aggressive tumor behavior of human HCCs.