title: Commensal bacteria drive endogenous transformation and tumour stem cell marker expression through a bystander effect.
abstract: OBJECTIVE
  Commensal bacteria and innate immunity play a major role in the development of colorectal cancer (CRC). We propose that selected commensals polarise colon macrophages to produce endogenous mutagens that initiate chromosomal instability (CIN), lead to expression of progenitor and tumour stem cell markers, and drive CRC through a bystander effect.
  
  
  METHODS
  Primary murine colon epithelial cells were repetitively exposed to Enterococcus faecalis-infected macrophages, or purified trans-4-hydroxy-2-nonenal (4-HNE)-an endogenous mutagen and spindle poison produced by macrophages. CIN, gene expression, growth as allografts in immunodeficient mice were examined for clones and expression of markers confirmed using interleukin (IL) 10 knockout mice colonised by E. faecalis.
  
  
  RESULTS
  Primary colon epithelial cells exposed to polarised macrophages or 4-hydroxy-2-nonenal developed CIN and were transformed after 10 weekly treatments. In immunodeficient mice, 8 of 25 transformed clones grew as poorly differentiated carcinomas with 3 tumours invading skin and/or muscle. All tumours stained for cytokeratins confirming their epithelial cell origin. Gene expression profiling of clones showed alterations in 3 to 7 cancer driver genes per clone. Clones also strongly expressed stem/progenitor cell markers Ly6A and Ly6E. Although not differentially expressed in clones, murine allografts positively stained for the tumour stem cell marker doublecortin-like kinase 1. Doublecortin-like kinase 1 and Ly6A/E were expressed by epithelial cells in colon biopsies for areas of inflamed and dysplastic tissue from E. faecalis-colonised IL-10 knockout mice.
  
  
  CONCLUSIONS
  These results validate a novel mechanism for CRC that involves endogenous CIN and cellular transformation arising through a microbiome-driven bystander effect.
authors.affiliation: The Muchmore Laboratories for Infectious Diseases Research, Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma, USA Department of Radiation Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Xingmin
authors.initials: X
authors.lastname: Wang
authors.affiliation: The Muchmore Laboratories for Infectious Diseases Research, Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma, USA Department of Radiation Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Yang
authors.affiliation: The Muchmore Laboratories for Infectious Diseases Research, Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma, USA Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Mark M
authors.initials: MM
authors.lastname: Huycke
mesh_terms: D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D024201:Bystander Effect / Q000502:physiology; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D003106:Colon / Q000378:metabolism / Q000382:microbiology*; D003110:Colonic Neoplasms / Q000209:etiology* / Q000378:metabolism; D013293:Enterococcus faecalis; D005260:Female; D016908:Gram-Positive Bacterial Infections / Q000150:complications* / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007413:Intestinal Mucosa / Q000378:metabolism / Q000382:microbiology; D008264:Macrophages / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D016688:Mice, Inbred NOD; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000378:metabolism*
chemical_list: D000447:Aldehydes; C027576:4-hydroxy-2-nonenal
keywords: Colon Carcinogenesis; Colonic Bacteria; Genetic Instability; Macrophages; Stem Cells