title: OTUB1 inhibits the ubiquitination and degradation of FOXM1 in breast cancer and epirubicin resistance.
abstract: The forkhead transcription factor FOXM1 has a key role in DNA damage response, and its deregulated overexpression is associated with genotoxic drug resistance in breast cancer. However, little is known about the posttranslational mechanisms by which FOXM1 expression is regulated by genotoxic agents and how they are deregulated in resistant cells. Initial co-immunoprecipitation studies verified previous proteomic analysis finding that the OTUB1 is a novel FOXM1-interacting protein. Western blot analysis showed that both OTUB1 and FOXM1 expression reduced upon genotoxic agent treatment in MCF-7 cells, but remained relatively constant in resistant cells. FOXM1 expression reduced upon OTUB1 depletion by siRNA and increased with OTUB1 overexpression in MCF-7 cells, arguing that OTUB1 positively regulates FOXM1 expression. In agreement, co-immunoprecipitation experiments demonstrated that FOXM1 expression is associated with OTUB1 binding but inversely correlates with conjugation to the protein degradation-associated Lys-48-linked ubiquitin-chains. Overexpression of wild-type (WT) OTUB1, but not the OTUB1(C91S) mutant, disrupted the formation of Lys48-linked ubiquitin-conjugates on FOXM1. Importantly, knockdown of OTUB1 by siRNA resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide, whereas overexpression of WT OTUB1, but not the OTUB1(C91S) mutant, significantly enhances the half-life of FOXM1. In addition, proliferative and clonogenic assays also show that OTUB1 can enhance the proliferative rate and epirubicin resistance through targeting FOXM1, as OTUB1 has little effect on FOXM1-deficient cells. The physiological relevance of the regulation of FOXM1 by OTUB1 is further underscored by the significant correlations between FOXM1 and OTUB1 expression in breast cancer patient samples. Cox-regression survival analysis indicates that OTUB1 overexpression is linked to poorer outcome in particular in patients treated with chemotherapy. Collectively, these data suggest that OTUB1 limits the ubiquitination and degradation of FOXM1 in breast cancer and has a key role in genotoxic agent resistance.
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.
authors.forename: U
authors.initials: U
authors.lastname: Karunarathna
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.
authors.forename: M
authors.initials: M
authors.lastname: Kongsema
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.
authors.forename: S
authors.initials: S
authors.lastname: Zona
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.|Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
authors.forename: C
authors.initials: C
authors.lastname: Gong
authors.affiliation: Unidad de Investigación, Hospital Universitario de Canarias, Instituto de Tecnologías Biomédicas, Ofra s/n, La Laguna, Tenerife, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Cabrera
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.
authors.forename: A R
authors.initials: AR
authors.lastname: Gomes
authors.affiliation: Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
authors.forename: E P S
authors.initials: EP
authors.lastname: Man
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.
authors.forename: P
authors.initials: P
authors.lastname: Khongkow
authors.affiliation: Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
authors.forename: J W-H
authors.initials: JW
authors.lastname: Tsang
authors.affiliation: Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
authors.forename: U-S
authors.initials: US
authors.lastname: Khoo
authors.affiliation: Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: R H
authors.initials: RH
authors.lastname: Medema
authors.affiliation: Unidad de Investigación, Hospital Universitario de Canarias, Instituto de Tecnologías Biomédicas, Ofra s/n, La Laguna, Tenerife, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Freire
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.
authors.forename: E W-F
authors.initials: EW
authors.lastname: Lam
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D000072017:Deubiquitinating Enzymes; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D015251:Epirubicin / Q000494:pharmacology*; D005260:Female; D000072278:Forkhead Box Protein M1; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D061986:MCF-7 Cells; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D054875:Ubiquitination / Q000235:genetics
chemical_list: D000903:Antibiotics, Antineoplastic; C107585:FOXM1 protein, human; D000072278:Forkhead Box Protein M1; D051858:Forkhead Transcription Factors; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; D015251:Epirubicin; D003513:Cycloheximide; D000072017:Deubiquitinating Enzymes; C538826:OTUB1 protein, human; D003546:Cysteine Endopeptidases