Analysis of an alternative human CD133 promoter reveals the implication of Ras/ERK pathway in tumor stem-like hallmarks.
BACKGROUND
An increasing number of studies support the presence of stem-like cells in human malignancies. These cells are primarily responsible for tumor initiation and thus considered as a potential target to eradicate tumors. CD133 has been identified as an important cell surface marker to enrich the stem-like population in various human tumors. To reveal the molecular machinery underlying the stem-like features in tumor cells, we analyzed a promoter of CD133 gene using human colon carcinoma Caco-2 and synovial sarcoma Fuji cells, which endogenously express CD133 gene.


RESULTS
A reporter analysis revealed that P5 promoter, located far upstream in a human CD133 gene locus, exhibits the highest activity among the five putative promoters (P1 to P5). Deletion and mutation analysis identified two ETS binding sites in the P5 region as being essential for its promoter activity. Electrophoretic mobility shift assays demonstrated the specific binding between nuclear factors and the ETS binding sequence. Overexpression of dominant-negative forms of Ets2 and Elk1 resulted in the significant decrease of P5 activity. Furthermore, treatment of Fuji cells with a specific MEK/ERK inhibitor, U0126, also markedly decreased CD133 expression, but there was no significant effect in Caco-2 cells, suggesting cell type-specific regulation of CD133 expression. Instead, the side population, another hallmark of TSLCs, was dramatically diminished in Caco-2 cells by U0126. Finally, Ras-mediated oncogenic transformation in normal human astrocytes conferred the stem-like capability to form neurosphere-like colonies with the increase of CD133 mRNA expression.


CONCLUSIONS
In conclusion, the Ras/ERK pathway at least in part contributes to the maintenance and the acquisition of stem-like hallmarks, although the extent of its contribution is varied in a cell type-specific manner. These findings could help our comprehensive understanding of tumor stemness, and also improve the development of eradicative therapies against human malignancies.
Tabu
Kouichi
K
Laboratory of Cancer Research, Department of Pathology, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-ku, Sapporo, Japan.
Kimura
Taichi
T
Sasai
Ken
K
Wang
Lei
L
Bizen
Norihisa
N
Nishihara
Hiroshi
H
Taga
Tetsuya
T
Tanaka
Shinya
S
D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D001253:Astrocytes / Q000378:metabolism; D001665:Binding Sites; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000473:pathology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D005799:Genes, Dominant / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D016513:Mice, SCID; D014411:Neoplastic Stem Cells / Q000201:enzymology* / Q000473:pathology*; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D050783:Proto-Oncogene Proteins c-ets / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D018631:ras Proteins / Q000378:metabolism*
D000071916:AC133 Antigen; D015703:Antigens, CD; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases; D018631:ras Proteins