title: Expression of LGR-5, MSI-1 and DCAMKL-1, putative stem cell markers, in the early phases of 1,2-dimethylhydrazine-induced rat colon carcinogenesis: correlation with nuclear β-catenin.
abstract: BACKGROUND
  Colon cancer stem cells may drive carcinogenesis and account for chemotherapeutic failure. Although many markers for these cells have been proposed, there is no complete agreement regarding them, nor has their presence in the early phases of carcinogenesis been characterized in depth.
  
  
  METHODS
  The expression of the putative markers LGR-5 (leucine-rich-repeat-containing G-protein-coupled receptor 5), MSI-1 (Musashi-1) and DCAMKL-1 (doublecortin and calcium/calmodulin-dependent protein kinase-like-1) was studied in normal colon mucosa (NM), in the precancerous lesions Mucin Depleted Foci (MDF) and in macroscopic tumours (adenomas) of 1,2-dimethylhydrazine-treated rats. Co-localization between these markers and nuclear β-catenin (NBC), an attributed feature of cancer stem cells, was also determined. Moreover, since PGE2 could increase NBC, we tested whether short-term treatment with celecoxib, a COX-2 inhibitor (2 weeks, 250 ppm in the diet) could reduce the expression of these markers.
  
  
  RESULTS
  LGR-5 expression in NM was low (Labelling Index (LI): 0.22 ± 0.03 (means ± SE)) with positive cells located mainly at the base of the crypts. Compared to NM, LGR-5 was overexpressed in MDF and tumours (LI: 4.7 ± 2.0 and 2.9 ± 1.0 in MDF and tumours, respectively, P<0.01 compared to NM). DCAMKL-1 positive cells, distributed along the length of normal crypts, were reduced in MDF and tumours. Nuclear expression of MSI-1, located mainly at the base of normal crypts, was not observed in MDF or tumours. In both MDF and tumours, few cells co-expressed LGR-5 and NBC (LI: 1.0 ± 0.3 and 0.4 ± 0.2 in MDF and tumours, respectively). Notwithstanding the lower expression of DCAMKL-1 in tumours, the percentage of cells co-expressing DCAMKL-1 and NBC was higher than in NM (LI: 0.5 ± 0.1 and 0.04 ± 0.02 in tumours and NM, respectively). MSI-1 and NBC co-localization was not observed. Celecoxib did not reduce cells co-expressing LGR-5 and NBC.
  
  
  CONCLUSIONS
  Based on its prevalent localization at the base of normal crypts, as expected for stem cells, and on the overexpression in precancerous lesions and tumours, we support LGR-5, but not MSI-1 or DCAMKL-1, as putative neoplastic stem cell marker. In both MDF and tumours, we identified LGR-5-positive cells co-expressing NBC which could be a subpopulation with the highest stem cell features.
authors: Department of Pharmacology, University of Florence, 6 Viale Pieraccini, 50139, Florence, Italy.
authors: Angelo Pietro
authors: AP
authors: Femia
authors: Piero
authors: P
authors: Dolara
authors: Maddalena
authors: M
authors: Salvadori
authors: Giovanna
authors: G
authors: Caderni
mesh_terms: D019813:1,2-Dimethylhydrazine; D000236:Adenoma / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism*; D000068579:Celecoxib; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002471:Cell Transformation, Neoplastic / Q000378:metabolism / Q000473:pathology; D003110:Colonic Neoplasms / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D051546:Cyclooxygenase 2 / Q000378:metabolism; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism; D000089662:Doublecortin Protein; D000089742:Doublecortin-Like Kinases; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000378:metabolism / Q000473:pathology; D008297:Male; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011230:Precancerous Conditions / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D051176:beta Catenin / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; C495268:Ctnnb1 protein, rat; D052246:Cyclooxygenase 2 Inhibitors; C000714848:Dcx protein, rat; D000089662:Doublecortin Protein; C576179:Lgr5 protein, rat; C491296:Msi1 protein, rat; D009419:Nerve Tissue Proteins; D011720:Pyrazoles; D016601:RNA-Binding Proteins; D043562:Receptors, G-Protein-Coupled; D013449:Sulfonamides; D051176:beta Catenin; D051546:Cyclooxygenase 2; C489691:Ptgs2 protein, rat; D000089742:Doublecortin-Like Kinases; C485526:Dclk1 protein, rat; D017346:Protein Serine-Threonine Kinases; D019813:1,2-Dimethylhydrazine; D000068579:Celecoxib; D015232:Dinoprostone
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