title: Deubiquitinase USP9x confers radioresistance through stabilization of Mcl-1.
abstract: Myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family, is often overexpressed in tumor cells limiting the therapeutic success. Mcl-1 differs from other Bcl-2 members by its high turnover rate. Its expression level is tightly regulated by ubiquitylating and deubiquitylating enzymes. Interaction of Mcl-1 with certain Bcl-2 homology domain 3 (BH3)-only members of the Bcl-2 family can limit the access to Mcl-1 ubiquitin ligase E3 and stabilizes the antiapoptotic protein. In addition, the overexpression of the deubiquitinase ubiquitin-specific protease 9x (USP9x) can result in the accumulation of Mcl-1 by removing poly-ubiquitin chains from Mcl-1 preventing its proteasomal degradation. Analyzing radiation-induced apoptosis in Jurkat cells, we found that Mcl-1 was downregulated more efficiently in sensitive parental cells than in a resistant subclone. The decline of Mcl-1 correlated with cell death induction and clonogenic survival. Knockdown of BH3-only proteins Bim, Puma, and Noxa did not affect Mcl-1 level or radiation-induced apoptosis. However, ionizing radiation resulted in activation of USP9x and enhanced deubiquitination of Mcl-1 in the radioresistant cells preventing fast Mcl-1 degradation. USP9x knockdown enhanced radiation-induced decrease of Mcl-1 and sensitized the radioresistant cells to apoptosis induction, whereas USP9x knockdown alone did not change Mcl-1 level in unirradiated cells. Together, our results indicate that radiation-induced activation of USP9x inhibits Mcl-1 degradation and apoptosis resulting in increased radioresistance.
authors.affiliation: Department of Radiation Oncology, University Hospital Tübingen, Tübingen, Germany.
authors.forename: Donatella
authors.initials: D
authors.lastname: Trivigno
authors.forename: Frank
authors.initials: F
authors.lastname: Essmann
authors.forename: Stephan M
authors.initials: SM
authors.lastname: Huber
authors.forename: Justine
authors.initials: J
authors.lastname: Rudner
mesh_terms: D017209:Apoptosis / Q000528:radiation effects*; D015153:Blotting, Western; D003374:Coumarins / Q000378:metabolism; D005434:Flow Cytometry; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D019169:Jurkat Cells; D053078:Membrane Potential, Mitochondrial / Q000528:radiation effects; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D059748:Proteolysis / Q000528:radiation effects*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D011836:Radiation Tolerance; D011839:Radiation, Ionizing; D014410:Tumor Stem Cell Assay; D025801:Ubiquitin / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D003374:Coumarins; C579095:MCL1 protein, human; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; C506234:USP9X protein, human; D025801:Ubiquitin; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D043222:Ubiquitin Thiolesterase; C028743:7-amino-4-methylcoumarin