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A. K. Silva, H. Yi, G. M. Seigel, A. S. Hackam; Wnt Signaling Regulates Stem Cells in Retinoblastoma. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1700.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate whether active Wnt signaling is involved in maintaining retinoblastoma (RB) stem cells and whether Sfrp2, a gene associated with deactivated Wnt signaling and differentiation, is involved in regulating the RB stem cell phenotype.
To determine if RB stem cell phenotype is related to active Wnt signaling, WERI-RB27 (W27) RB cells were sorted into stem and non-stem cell populations by FACS using an antibody against ABCG2. Activated Wnt signaling was measured in the sorted cells by quantifying levels of nuclear B-catenin (nB-cat) using Western blotting. Also, immunohistochemistry (IHC) on WERI-RB1 (W1) cells was used to determine the number of cells immunoreactive for stem cell marker Mushasi-1 (Msi-1), nB-cat, or differentiation marker Ki67. Additionally, to determine whether Wnt signaling regulated stem marker expression, W1 and Y79 RB cells were treated with Wnt agonist LiCl for 72 hrs and assessed by quantitative PCR (QPCR). Sfrp2 expression was measured by QPCR in RB cells treated with Wnt inducers Wnt3a and LiCl and Wnt inhibitor Dkk1. Finally, to determine whether Sfrp2 regulates stem cell marker expression, RB cells were treated with Sfrp2 siRNA and expression was measured by QPCR.
Western blot demonstrated that W27 ABCG2+ cells had 5.4x more nB-cat than ABCG2- cells. Also, IHC showed Msi-1 positive RB cells more often co-localized with cells positive for n-Bcat (27%) than for Ki67 (5%). RB cells treated with LiCl showed an increase in stem markers Nanog and Oct3/4. RB cells treated with LiCl and Wnt3a had reduced Sfrp2 expression, whereas Dkk1 increased Sfrp2 expression. RB cells with Sfrp2 expression blocked using siRNA showed elevation of stem markers CD133, Msi-1, and Oct3/4.
We demonstrated a relationship between active Wnt signaling and RB stem marker expression. We also showed an inverse relationship between Sfrp2 expression and active Wnt signaling in RB cells and that when Sfrp2 expression is knocked out in RB cells, stem marker expression increases. The relationship between Sfrp2 and Wnt signaling in RB provides insight into the mechanisms involved in RB stem cell expression, which will help lead to better RB treatment options.
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