RESUMO
Rationale: A number of guanine nucleotide exchange factors (GEFs) including epithelial cell transforming factor ECT2 are believed to drive carcinogenesis through activating distinct oncogenic GTPases. Yet, whether GEF-independent activity of ECT2 also plays a role in tumorigenesis remains unclear. Methods: Immunohistochemical (IHC) staining, colony formation and xenograft assays were used to examine the role of ECT2 in breast carcinogenesis. Co-immunoprecipitation, immunofluorescent stainings, in vivo deubiquitination and in vitro deubiquitination experiments were performed to examine the physical and functional interaction between ECT2 and ubiquitin-specific protease USP7. High-throughput RNA sequencing, quantitative reverse transcription-PCR and Western blotting were employed to investigate the biological significance of the interplay between ECT2 and USP7. Results: We report that ECT2 plays a tumor-promoting role in breast cancer, and GEF activity-deficient ECT2 is able to alleviate ECT2 depletion associated growth defects in breast cancer cells. Mechanistically, we demonstrated that ECT2 physically interacts with ubiquitin-specific protease USP7 and functionally facilitates USP7 intermolecular self-association, -deubiquitination and -stabilization in a GEF activity-independent manner. USP7 in turn, deubiquitinates and stabilizes ECT2, resulting in a feedforward regulatory circuit that ultimately sustains the expression of oncogenic protein MDM2. Conclusion: Our study uncovers a GEF-independent role of ECT2 in promoting survival of breast cancer cells, provides a molecular insight for the reciprocal regulation of ECT2 and USP7, and supports the pursuit of ECT2/USP7 as potential targets for breast cancer intervention.
Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Ensaios Enzimáticos , Retroalimentação Fisiológica , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Ligação Proteica/genética , Estabilidade Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/isolamento & purificação , RNA-Seq , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/isolamento & purificação , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal tail near the catalytic site. Here we delineate how the target protein promotes the activation of USP7. Using NMR analysis and biochemistry we describe the order of activation steps, showing that ubiquitin binding is an instrumental step in USP7 activation. Using chemically synthesised p53-peptides we also demonstrate how the correct ubiquitinated substrate increases catalytic activity. We then used transient reaction kinetic modelling to define how the USP7 multistep mechanism is driven by target recognition. Our data show how this pleiotropic DUB can gain specificity for its cellular targets.