RESUMO
Co-culture of intestinal organoids with a colibactin-producing pks+E. coli strain (EcC) revealed mutational signatures also found in colorectal cancer (CRC). E. coli Nissle 1917 (EcN) remains a commonly used probiotic, despite harboring the pks operon and inducing double strand DNA breaks. We determine the mutagenicity of EcN and three CRC-derived pks+E. coli strains with an analytical framework based on sequence characteristic of colibactin-induced mutations. All strains, including EcN, display varying levels of mutagenic activity. Furthermore, a machine learning approach attributing individual mutations to colibactin reveals that patients with colibactin-induced mutations are diagnosed at a younger age and that colibactin can induce a specific APC mutation. These approaches allow the sensitive detection of colibactin-induced mutations in â¼12% of CRC genomes and even in whole exome sequencing data, representing a crucial step toward pinpointing the mutagenic activity of distinct pks+E. coli strains.
Assuntos
Neoplasias Colorretais , Escherichia coli , Peptídeos , Policetídeos , Humanos , Escherichia coli/genética , Mutação , Dano ao DNA , Mutagênicos , OrganoidesRESUMO
Childhood cancer survivors are confronted with various chronic health conditions like therapy-related malignancies. However, it is unclear how exposure to chemotherapy contributes to the mutation burden and clonal composition of healthy tissues early in life. Here, we studied mutation accumulation in hematopoietic stem and progenitor cells (HSPC) before and after cancer treatment of 24 children. Of these children, 19 developed therapy-related myeloid neoplasms (t-MN). Posttreatment HSPCs had an average mutation burden increase comparable to what treatment-naïve cells accumulate during 16 years of life, with excesses up to 80 years. In most children, these additional mutations were induced by clock-like processes, which are also active during healthy aging. Other patients harbored mutations that could be directly attributed to treatments like platinum-based drugs and thiopurines. Using phylogenetic inference, we demonstrate that most t-MN in children originate after the start of treatment and that leukemic clones become dominant during or directly after chemotherapy exposure. SIGNIFICANCE: Our study shows that chemotherapy increases the mutation burden of normal blood cells in cancer survivors. Only few drugs damage the DNA directly, whereas in most patients, chemotherapy-induced mutations are caused by processes similar to those present during normal aging. This article is highlighted in the In This Issue feature, p. 1825.
Assuntos
Antineoplásicos , Segunda Neoplasia Primária , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Criança , Células-Tronco Hematopoéticas/patologia , Humanos , Mieloma Múltiplo/induzido quimicamente , Mieloma Múltiplo/genética , Mutação , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Neoplasias/genética , Segunda Neoplasia Primária/induzido quimicamente , Segunda Neoplasia Primária/genética , Segunda Neoplasia Primária/patologia , FilogeniaRESUMO
Mutational signatures have been identified in cancer genomes, providing information about the causes of cancer and treatment vulnerabilities. This protocol describes an assay to determine the genotoxic mechanisms underlying these signatures using cord-blood derived hematopoietic stem and progenitor cells (CB-HSPCs). CB-HSPCs have a low mutation background, enabling sensitive detection of mutations. First, CB-HSPCs are exposed in vitro, sorted, and clonally expanded. This expansion enables whole-genome sequencing to detect the mutation load and respective patterns induced during genotoxic exposure. For complete details on the use and execution of this protocol, please refer to de Kanter et al. (2021).
Assuntos
Sangue Fetal , Células-Tronco Hematopoéticas , Dano ao DNA , Genoma , Humanos , Sequenciamento Completo do GenomaRESUMO
Enterotoxigenic Bacteroides fragilis (ETBF) is consistently found at higher frequency in individuals with sporadic and hereditary colorectal cancer (CRC) and induces tumorigenesis in several mouse models of CRC. However, whether specific mutations induced by ETBF lead to colon tumor formation has not been investigated. To determine if ETBF-induced mutations impact the Apc gene, and other tumor suppressors or proto-oncogenes, we performed whole-exome sequencing and whole-genome sequencing on tumors isolated after ETBF and sham colonization of Apcmin/+ and Apcmin/+Msh2fl/flVC mice, as well as whole-genome sequencing of organoids cocultured with ETBF. Our results indicate that ETBF-induced tumor formation results from loss of heterozygosity (LOH) of Apc, unless the mismatch repair system is disrupted, in which case, tumor formation results from new acquisition of protein-truncating mutations in Apc. In contrast to polyketide synthase-positive Escherichia coli (pks+ E. coli), ETBF does not produce a unique mutational signature; instead, ETBF-induced tumors arise from errors in DNA mismatch repair and homologous recombination DNA damage repair, established pathways of tumor formation in the colon, and the same genetic mechanism accounting for sham tumors in these mouse models. Our analysis informs how this procarcinogenic bacterium may promote tumor formation in individuals with inherited predispositions to CRC, such as Lynch syndrome or familial adenomatous polyposis (FAP). IMPORTANCE Many studies have shown that microbiome composition in both the mucosa and the stool differs in individuals with sporadic and hereditary colorectal cancer (CRC). Both human and mouse models have established a strong association between particular microbes and colon tumor induction. However, the genetic mechanisms underlying putative microbe-induced colon tumor formation are not well established. In this paper, we applied whole-exome sequencing and whole-genome sequencing to investigate the impact of ETBF-induced genetic changes on tumor formation. Additionally, we performed whole-genome sequencing of human colon organoids exposed to ETBF to validate the mutational patterns seen in our mouse models and begin to understand their relevance in human colon epithelial cells. The results of this study highlight the importance of ETBF colonization in the development of sporadic CRC and in individuals with hereditary tumor conditions, such as Lynch syndrome and familial adenomatous polyposis (FAP).
Assuntos
Polipose Adenomatosa do Colo , Infecções Bacterianas , Neoplasias do Colo , Neoplasias Colorretais Hereditárias sem Polipose , Neoplasias Colorretais , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Animais , Infecções Bacterianas/patologia , Bacteroides fragilis/genética , Bacteroides fragilis/metabolismo , Colo/microbiologia , Neoplasias do Colo/genética , Neoplasias do Colo/microbiologia , Neoplasias do Colo/patologia , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Modelos Animais de Doenças , Escherichia coli/genética , Genes APC , Camundongos , MutaçãoRESUMO
During life, the DNA of our cells is continuously exposed to external damaging processes. Despite the activity of various repair mechanisms, DNA damage eventually results in the accumulation of mutations in the genomes of our cells. Oncogenic mutations are at the root of carcinogenesis, and carcinogenic agents are often highly mutagenic. Over the past decade, whole genome sequencing data of healthy and tumor tissues have revealed how cells in our body gradually accumulate mutations because of exposure to various mutagenic processes. Dissection of mutation profiles based on the type and context specificities of the altered bases has revealed a variety of signatures that reflect past exposure to environmental mutagens, ranging from chemotherapeutic drugs to genotoxic gut bacteria. In this review, we discuss the latest knowledge on somatic mutation accumulation in human cells, and how environmental mutagenic factors further shape the mutation landscapes of tissues. In addition, not all carcinogenic agents induce mutations, which may point to alternative tumor-promoting mechanisms, such as altered clonal selection dynamics. In short, we provide an overview of how environmental factors induce mutations in the DNA of our healthy cells and how this contributes to carcinogenesis. A better understanding of how environmental mutagens shape the genomes of our cells can help to identify potential preventable causes of cancer.
RESUMO
Genetic instability is a major concern for successful application of stem cells in regenerative medicine. However, the mutational consequences of the most applied stem cell therapy in humans, hematopoietic stem cell transplantation (HSCT), remain unknown. Here we characterized the mutation burden of hematopoietic stem and progenitor cells (HSPCs) of human HSCT recipients and their donors using whole-genome sequencing. We demonstrate that the majority of transplanted HSPCs did not display altered mutation accumulation. However, in some HSCT recipients, we identified multiple HSPCs with an increased mutation burden after transplantation. This increase could be attributed to a unique mutational signature caused by the antiviral drug ganciclovir. Using a machine learning approach, we detected this signature in cancer genomes of individuals who received HSCT or solid organ transplantation earlier in life. Antiviral treatment with nucleoside analogs can cause enhanced mutagenicity in transplant recipients, which may ultimately contribute to therapy-related carcinogenesis.
Assuntos
Antivirais/efeitos adversos , Infecções por Citomegalovirus , Transplante de Células-Tronco Hematopoéticas , Mutação , Neoplasias , Antivirais/uso terapêutico , Infecções por Citomegalovirus/tratamento farmacológico , Humanos , Neoplasias/genética , TransplantadosRESUMO
Changes in the microbiome are associated with the development of colorectal cancer, but causal explanations have been lacking. We recently demonstrated that pks+ Escherichia coli induce a specific mutational pattern using intestinal organoids and these mutations are present in the genomes of colorectal cancer. This finding warrants further studies on the microbial role in oncogenic mutation induction, cancer development and future preventive strategies.
Assuntos
Neoplasias Colorretais/genética , Microbioma Gastrointestinal/genética , Transcriptoma , Células Cultivadas , Colo/metabolismo , Colo/microbiologia , Neoplasias Colorretais/microbiologia , Dano ao DNA/genética , Escherichia coli/genética , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Mutação INDEL , Mutação , Organoides/metabolismo , Organoides/microbiologia , Organoides/patologia , Peptídeos/genética , Peptídeos/fisiologia , Policetídeos , Polimorfismo de Nucleotídeo Único , Transcriptoma/fisiologiaRESUMO
Various species of the intestinal microbiota have been associated with the development of colorectal cancer1,2, but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin3. This compound is believed to alkylate DNA on adenine residues4,5 and induces double-strand breaks in cultured cells3. Here we expose human intestinal organoids to genotoxic pks+ E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Ilhas Genômicas/genética , Mutagênese , Mutação , Técnicas de Cocultura , Estudos de Coortes , Sequência Consenso , Dano ao DNA , Microbioma Gastrointestinal , Humanos , Organoides/citologia , Organoides/metabolismo , Organoides/microbiologia , Peptídeos/genética , PolicetídeosRESUMO
Mutation accumulation during life can contribute to hematopoietic dysfunction; however, the underlying dynamics are unknown. Somatic mutations in blood progenitors can provide insight into the rate and processes underlying this accumulation, as well as the developmental lineage tree and stem cell division numbers. Here, we catalog mutations in the genomes of human-bone-marrow-derived and umbilical-cord-blood-derived hematopoietic stem and progenitor cells (HSPCs). We find that mutations accumulate gradually during life with approximately 14 base substitutions per year. The majority of mutations were acquired after birth and could be explained by the constant activity of various endogenous mutagenic processes, which also explains the mutation load in acute myeloid leukemia (AML). Using these mutations, we construct a developmental lineage tree of human hematopoiesis, revealing a polyclonal architecture and providing evidence that developmental clones exhibit multipotency. Our approach highlights features of human native hematopoiesis and its implications for leukemogenesis.
Assuntos
Linhagem da Célula/genética , Senescência Celular/genética , Hematopoese/genética , Mutagênese/genética , Mutação/genética , Adulto , Embrião de Mamíferos/citologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Especificidade de ÓrgãosRESUMO
In this Letter, the received date should have been 23 March 2017 instead of 13 April 2018. Authors R.M.K. and O.D.K. were incorrectly denoted as 'equally contributing' authors. The labels for 'control' and 'IFNγ' in Extended Data Fig. 4g were reversed. These have been corrected online.
RESUMO
Epithelial surfaces form critical barriers to the outside world and are continuously renewed by adult stem cells1. Whereas dynamics of epithelial stem cells during homeostasis are increasingly well understood, how stem cells are redirected from a tissue-maintenance program to initiate repair after injury remains unclear. Here we examined infection by Heligmosomoides polygyrus, a co-evolved pathosymbiont of mice, to assess the epithelial response to disruption of the mucosal barrier. H. polygyrus disrupts tissue integrity by penetrating the duodenal mucosa, where it develops while surrounded by a multicellular granulomatous infiltrate2. Crypts overlying larvae-associated granulomas did not express intestinal stem cell markers, including Lgr53, in spite of continued epithelial proliferation. Granuloma-associated Lgr5- crypt epithelium activated an interferon-gamma (IFN-γ)-dependent transcriptional program, highlighted by Sca-1 expression, and IFN-γ-producing immune cells were found in granulomas. A similar epithelial response accompanied systemic activation of immune cells, intestinal irradiation, or ablation of Lgr5+ intestinal stem cells. When cultured in vitro, granuloma-associated crypt cells formed spheroids similar to those formed by fetal epithelium, and a sub-population of H. polygyrus-induced cells activated a fetal-like transcriptional program, demonstrating that adult intestinal tissues can repurpose aspects of fetal development. Therefore, re-initiation of the developmental program represents a fundamental mechanism by which the intestinal crypt can remodel itself to sustain function after injury.
Assuntos
Feto/citologia , Helmintos/fisiologia , Intestinos/citologia , Parasitos/fisiologia , Nicho de Células-Tronco , Células-Tronco/citologia , Animais , Antígenos Ly/biossíntese , Células Epiteliais/citologia , Feminino , Feto/metabolismo , Interferon gama/imunologia , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Nematospiroides dubius/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Infecções por Strongylida/parasitologiaRESUMO
PTEN is a tumor suppressor that is frequently lost in epithelial malignancies. A part of the tumor-suppressive properties of PTEN is attributed to its function in cell polarization and consequently its role in maintaining epithelial tissue integrity. However, surprisingly little is known about the function and regulation of PTEN during epithelial cell polarization. We used clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene disruption to delete PTEN in intestinal epithelial Ls174T:W4 cells, which upon differentiation form a microvillus-covered apical membrane (brush border) on a part of the cell cortex, independent of cell-cell junctions. We show that loss of PTEN results in the formation of a larger brush border that, in a fraction of the cells, even spans the entire plasma membrane, revealing that PTEN functions in the regulation of apical membrane size. Depletion of the phosphatase PTPL1 resulted in a similar defect. PTPL1 interacts with PTEN, and this interaction is necessary for apical membrane enrichment of PTEN. Importantly, phosphatase activity of PTPL1 is not required, indicating that PTPL1 functions as an anchor protein in this process. Our work thus demonstrates a novel function for PTEN during cell polarization in controlling apical membrane size and identifies PTPL1 as a critical apical membrane anchor for PTEN in this process.