RESUMO
A remarkable observation emerging from recent cancer genome analyses is the identification of chromothripsis as a one-off genomic catastrophe, resulting in massive somatic DNA structural rearrangements (SRs). Largely due to lack of suitable model systems, the mechanistic basis of chromothripsis has remained elusive. We developed an integrative method termed "complex alterations after selection and transformation (CAST)," enabling efficient in vitro generation of complex DNA rearrangements including chromothripsis, using cell perturbations coupled with a strong selection barrier followed by massively parallel sequencing. We employed this methodology to characterize catastrophic SR formation processes, their temporal sequence, and their impact on gene expression and cell division. Our in vitro system uncovered a propensity of chromothripsis to occur in cells with damaged telomeres, and in particular in hyperploid cells. Analysis of primary medulloblastoma cancer genomes verified the link between hyperploidy and chromothripsis in vivo. CAST provides the foundation for mechanistic dissection of complex DNA rearrangement processes.
Assuntos
Cromossomos Humanos/genética , Rearranjo Gênico , Genoma Humano/genética , Instabilidade Genômica/genética , Neoplasias/genética , Aneuploidia , Divisão Celular , Linhagem Celular , Aberrações Cromossômicas , Variações do Número de Cópias de DNA/genética , Humanos , Meduloblastoma/genética , Poliploidia , Telômero/genética , Telômero/patologia , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismoRESUMO
TP53 deficiency is the most common alteration in cancer; however, this alone is typically insufficient to drive tumorigenesis. To identify genes promoting tumorigenesis in combination with TP53 deficiency, we perform genome-wide CRISPR-Cas9 knockout screens coupled with proliferation and transformation assays in isogenic cell lines. Loss of several known tumor suppressors enhances cellular proliferation and transformation. Loss of neddylation pathway genes promotes uncontrolled proliferation exclusively in TP53-deficient cells. Combined loss of CUL3 and TP53 activates an oncogenic transcriptional program governed by the nuclear factor κB (NF-κB), AP-1, and transforming growth factor ß (TGF-ß) pathways. This program maintains persistent cellular proliferation, induces partial epithelial to mesenchymal transition, and increases DNA damage, genomic instability, and chromosomal rearrangements. Our findings reveal CUL3 loss as a key event stimulating persistent proliferation in TP53-deficient cells. These findings may be clinically relevant, since TP53-CUL3-deficient cells are highly sensitive to ataxia telangiectasia mutated (ATM) inhibition, exposing a vulnerability that could be exploited for cancer treatment.
Assuntos
Proteínas Culina/genética , Proteína Supressora de Tumor p53/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carcinogênese/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Proteínas Culina/metabolismo , Transição Epitelial-Mesenquimal , Estudo de Associação Genômica Ampla , Instabilidade Genômica , Humanos , NF-kappa B/metabolismo , Epitélio Pigmentado da Retina/citologia , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismoRESUMO
Extensive prior research focused on somatic copy-number alterations (SCNAs) affecting cancer genes, yet the extent to which recurrent SCNAs exert their influence through rearrangement of cis-regulatory elements (CREs) remains unclear. Here we present a framework for inferring cancer-related gene overexpression resulting from CRE reorganization (e.g., enhancer hijacking) by integrating SCNAs, gene expression data and information on topologically associating domains (TADs). Analysis of 7,416 cancer genomes uncovered several pan-cancer candidate genes, including IRS4, SMARCA1 and TERT. We demonstrate that IRS4 overexpression in lung cancer is associated with recurrent deletions in cis, and we present evidence supporting a tumor-promoting role. We additionally pursued cancer-type-specific analyses and uncovered IGF2 as a target for enhancer hijacking in colorectal cancer. Recurrent tandem duplications intersecting with a TAD boundary mediate de novo formation of a 3D contact domain comprising IGF2 and a lineage-specific super-enhancer, resulting in high-level gene activation. Our framework enables systematic inference of CRE rearrangements mediating dysregulation in cancer.
Assuntos
Variações do Número de Cópias de DNA/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Substratos do Receptor de Insulina/genética , Fator de Crescimento Insulin-Like II/genética , Neoplasias/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Regiões Promotoras GenéticasRESUMO
The Acidobacteria phylum is of high ecological interest. Its members are ubiquitous and particularly abundant in soils but many are recalcitrant to cultivation in the laboratory. Thus, the ability of Acidobacteria to capture and maintain plasmids remains largely unexplored. In this work we tested the transfer and the stability of (i) the PromA plasmid pMOL98 and (ii) the IncQ plasmid pKT230 to the acidobacterial strain Edaphobacter aggregans DSM 19364. To this end quantitative conjugation assays were performed and transconjugants were scored for plasmid-borne antibiotic selection markers. The tested plasmids were transferred and maintained in the new host. Plasmid pMOL98 was more stable than pKT230 in Ed. aggregans in the absence of positive selection. Thus, from an ecological point of view, we have extended the host range of PromA and IncQ plasmids for the first time to an acidobacterial strain. Furthermore, we have uncovered the potential of Acidobacteria to capture as-yet-unknown plasmids and to foster the development of new cloning and expression systems for the exploitation of biotechnologically valuable soil resources.