title: Identification of Endothelin-1 and NR4A2 as CD133-regulated genes in colon cancer cells.
abstract: Several in vitro assays have been proposed to identify cancer stem cells (CSCs), including immunophenotyping, sphere assay and side population (SP) assay. CD133 antigen has been proposed as a CSC marker in colon cancer (CC). However, no functional data are available to date and conflicting results have been reported regarding its role as true CSC marker. Here we set out to identify a molecular signature associated with potential CSC. CD133(+) cells isolated from the CaCo-2 CC cell line were analysed by microarray molecular profiling compared to CD133(-) counterparts. Various differentially expressed genes were identified and the most relevant transcripts found to be over-expressed in CD133(+) cells were evaluated by quantitative RT-PCR in the CD133(+) fractions isolated from several CC cell lines. In the attempt to find a correlation between putative CSCs, isolated by means of CD133 immunophenotyping and the SP approach, we demonstrated a significant enrichment of CD133(+) cells within the SP fraction of CC cells, and comparison of the gene expression profiles revealed that Endothelin-1 (END-1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts are highly expressed in both CD133(+) and SP fractions of CC cells. Moreover, depletion of CD133 by siRNA induced a significant attenuation of END-1 and NR4A2 expression levels in CaCo-2 cells, while expression of all three molecules decreased during sodium butyrate-induced differentiation. In conclusion, we have identified a molecular signature associated with potential CSCs and showed for the first time the existence of a functional relationship between CD133, END-1 and NR4A2 expression in colon cancer cells.
authors.affiliation: Institute of Internal Medicine, Università Cattolica del Sacro Cuore, Rome, Italy. ausiliapuglisi@yahoo.it
authors.forename: Maria Ausiliatrice
authors.initials: MA
authors.lastname: Puglisi
authors.forename: Marta
authors.initials: M
authors.lastname: Barba
authors.forename: Maddalena
authors.initials: M
authors.lastname: Corbi
authors.forename: Maria Federica
authors.initials: MF
authors.lastname: Errico
authors.forename: Ezio
authors.initials: E
authors.lastname: Giorda
authors.forename: Nathalie
authors.initials: N
authors.lastname: Saulnier
authors.forename: Alma
authors.initials: A
authors.lastname: Boninsegna
authors.forename: Anna Chiara
authors.initials: AC
authors.lastname: Piscaglia
authors.forename: Rita
authors.initials: R
authors.lastname: Carsetti
authors.forename: Achille
authors.initials: A
authors.lastname: Cittadini
authors.forename: Antonio
authors.initials: A
authors.lastname: Gasbarrini
authors.forename: Alessandro
authors.initials: A
authors.lastname: Sgambato
mesh_terms: D000071916:AC133 Antigen; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D018938:Caco-2 Cells; D002469:Cell Separation; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism; D019332:Endothelin-1 / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016130:Immunophenotyping; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2 / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D019332:Endothelin-1; D006023:Glycoproteins; C539882:NR4A2 protein, human; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; C000606023:PROM1 protein, human; D010455:Peptides