Subcellular Localization of BRCA1-Associated Protein (BAP1) in Uveal Melanoma Cases and Assessment of Its Functions

Abstract

Background: BRCA1-associated protein 1 (BAP1) is a nuclear-localized deubiquitylase (DUB) that belongs to the ubiquitin carboxy terminal hydrolase family of DUBs. BAP1 acts as a tumor suppressor inactivated in various cancers. Several studies in cancer cells containing BAP1 mutations indicated that BAP1 nuclear localization and catalytic activity are required for its growth and suppressive properties. Methods: Subcellular localization of BAP1 was studied in B-16 cells (WT BAP1), as well as 15 fresh frozen samples of Uveal Melanoma (UM) cases using immunofluorescence staining and confocal microscopy imaging. Wound Healing Assay was used to evaluate the role of BAP1 in the regulation of cell migration in B-16 cells. Clonogenic Assay was used to assess cell proliferation and the effect of BAP1 on DNA repair after irradiation (IR). Results: WT BAP1 was mostly nuclear and small amount was cytoplasmic. Among 15 UM samples 2 showed nuclear localization, while 4 showed only extranuclear BAP1. In 3 samples BAP1 was both nuclear and extranuclear, while 6 specimens appeared with no BAP1. In wound healing assay, BAP1 stimulated cell migration, while BAP1 knock-out slowed it down. In clonogenic Assay, more proliferation rate was detected in B-16 cells colonies with WT BAP1 even after IR confirming the role of BAP1 in cell proliferation and DNA repair. Conclusion: Mutant BAP1 is localized in a different way in the same type of cells (nuclear or cytoplasmic or absent). BAP1 is important for cell proliferation, migration