RESUMO
Adaptive evolution to cellular stress is a process implicated in a wide range of biological and clinical phenomena. Two major routes of adaptation have been identified: non-genetic changes, which allow expression of different phenotypes in novel environments, and genetic variation achieved by selection of fitter phenotypes. While these processes are broadly accepted, their temporal and epistatic features in the context of cellular evolution and emerging drug resistance are contentious. In this manuscript, we generated hypomorphic alleles of the essential nuclear pore complex (NPC) gene NUP58. By dissecting early and long-term mechanisms of adaptation in independent clones, we observed that early physiological adaptation correlated with transcriptome rewiring and upregulation of genes known to interact with the NPC; long-term adaptation and fitness recovery instead occurred via focal amplification of NUP58 and restoration of mutant protein expression. These data support the concept that early phenotypic plasticity allows later acquisition of genetic adaptations to a specific impairment. We propose this approach as a genetic model to mimic targeted drug therapy in human cells and to dissect mechanisms of adaptation.
Assuntos
Adaptação Fisiológica/genética , Alelos , Receptor Quinase 1 Acoplada a Proteína G/genética , Aptidão Genética , N-Glicosil Hidrolases/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HCT116 , Células HEK293 , Haploidia , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Células Mieloides/metabolismo , Células Mieloides/patologia , N-Glicosil Hidrolases/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transdução de Sinais , Transcriptoma , Proteína Vermelha FluorescenteRESUMO
The Fanconi anemia (FA) pathway is a pivotal genome maintenance network that orchestrates the repair of DNA interstrand crosslinks (ICLs). The tumor suppressors RUNX1 and RUNX3 were shown to regulate the FA pathway independent of their canonical transcription activities, by controlling the DNA damage-dependent chromatin association of FANCD2. Here, in further biochemical characterization, we demonstrate that RUNX3 is modified by PARP-dependent poly(ADP-ribosyl)ation (PARylation), which in turn allows RUNX binding to DNA repair structures lacking transcription-related RUNX consensus motifs. SILAC-based mass spectrometric analysis revealed significant association of RUNX3 with core DNA repair complexes, including PARP1, even in unstressed cells. After DNA damage, the increased interaction between RUNX3 and BLM facilitates efficient FANCD2 chromatin localization. RUNX-Walker motif mutations from breast cancers are impaired for DNA damage-inducible PARylation, unveiling a potential mechanism for FA pathway inactivation in cancers. Our results reinforce the emerging paradigm that RUNX proteins are tumor suppressors with genome gatekeeper function.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Processamento de Proteína Pós-Traducional , RecQ Helicases/genética , Animais , Células COS , Chlorocebus aethiops , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Dano ao DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitomicina/farmacologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação , Ligação Proteica , RecQ Helicases/metabolismoRESUMO
Studies of genomic instability have historically focused on intrinsic mechanisms rather than extrinsic mechanisms based in the tumor microenvironment (TME). TGFß is the most abundantly secreted cytokine in the TME, where it imparts various aggressive characteristics including invasive migration, drug resistance, and epithelial-to-mesenchymal transition (EMT). Here we show that TGFß also promotes genomic instability in the form of DNA double strand breaks (DSB) in cancer cells that lack the tumor suppressor gene RUNX3 Loss of RUNX3 resulted in transcriptional downregulation of the redox regulator heme oxygenase-1 (HO-1 or HMOX1). Consequently, elevated oxidative DNA damage disrupted genomic integrity and triggered cellular senescence, which was accompanied by tumor-promoting inflammatory cytokine expression and acquisition of the senescence-associated secretory phenotype (SASP). Recapitulating the above findings, tumors harboring a TGFß gene expression signature and RUNX3 loss exhibited higher levels of genomic instability. In summary, RUNX3 creates an effective barrier against further TGFß-dependent tumor progression by preventing genomic instability. These data suggest a novel cooperation between cancer cell-extrinsic TGFß signaling and cancer cell-intrinsic RUNX3 inactivation as aggravating factors for genomic instability.Significance: RUNX3 inactivation in cancer removes an antioxidant barrier against DNA double strand breaks induced by TGFß expressed in the tumor microenvironment. Cancer Res; 78(1); 88-102. ©2017 AACR.