1. Wilms tumor cells with WT1-mutations have characteristic features of mesenchymal stem cells
Maike Busch1, Constanze Duhme1, Artur Brandt1, Manfred Beier1, Carmen de Torres2, Jaume Mora2, Hans-Dieter Royer1+3, Brigitte Royer-Pokora1
1Institute of Human Genetics and Anthropology, Heinrich-Heine University, Düsseldorf , 2Department of Oncology, Hospital Sant Joan de Deu, Barcelona, 3Center of Advanced European Studies and Research, CAESAR, Bonn
Introduction
Wilms tumor (WT) is a genetically heterogeneous, embryonic kidney tumor resulting from abnormalities in the mesenchymal to epithelial transition (MET) during kidney development. The cells of origin for WT are still unknown. About 10-15% of the Wilms tumors habour mutations of the tumor suppressor gene WT1 and show loss of the corresponding wildtype allele (LOH). These tumors are often correlated with stromal histology and ectopic mesenchymal elements like striated and smooth muscle cells, chondrocytes, osteocytes and adipocytes. This tumor subgroup also frequently has associated mutations in the CTNNB1 gene.
Results
Here we describe the differentiation potential of five primary Wilms tumor cell lines, established from five individual stromal-predominant Wilms Tumors with WT1-mutations. In in vitro differentiation experiments we show that the cells have a limited capacity for myogenic, chondrogenic, osteogenic and adipogenic differentiation. The differentiation potential varies between the WT cell lines with different genetic backgrounds, depending on WT1- and CTNNB1-mutations and their LOH status. In addition the Wilms tumor cell lines show high similarity to human mesenchymal stem cells (hMSC) in gene expression profiles, which confirm the stem cell like nature of the Wilms tumor cells. In conclusion we could show that the Wilms tumor cell lines resemble human mesenchymal stem cells.
WT1-mutation status of patient in blood DNA
WT1-mutation status in cell lines
LOH
CTNNB1-mutation
Wilms1 germ-line c.149 C>A, p.S50X
Homozygous, p.S50X
11p11-11pter
Heterozygous p.S45F
Wilms2 germ-line c.1084 C>T, p.R362X
Homozygous, p.R362X
Herterozygous p.S45Y
Wilms3 wild-type
Homozygous, c insA, p.V432fsX87
Wild-type
Wilms4 WAGR,
Del 11p13
WT1-deletion and hemizygous c insC, p.H438Pfs79
No LOH
Heterozygous p.∆S45
Wilms5 germ-line c.1168 C>T, p.R390X
Homozygous, c delGC, pR433Pfs84
11p11-11pter loss of R390X
Wilms-cell line CLS1, unknown
Wilms3
hMSC
Figure 2: Hierarchical cluster analysis of Wilms tumor cell lines with WT1-mutations and mesenchymal stem cells (MSC) vs. CLS1 and normal kidney (NK).
Table 1: Mutation status of the analyzed Wilms tumor cell lines.
Figure 1: Phase contrast microscopy of one Wilms tumor cell line and hMSC.
Figure 3: Mesenchymal differentiation potential of Wilms tumor cell lines. A: Oil Red O stain for adipocytes and Safranin O stain for chondrocytes after induction of differentiation in Wilms tumor cell lines. Immunfluorescence with an antibody against titin of uninduced and induced cells after muscle differentiation. B: Semiquantitative RT-PCR analysis of FABP4 mRNA expression after adipogenic induction of the Wilms tumor cell lines and hMSC as positive control. C: Osteogenic differentiation potential of Wilms tumor cell lines and hMSC. Identified by the calcium concentration in the supernatant of the cell culture after induction.
Supported by the Deutsche Forschungsgemeinschaft [RO 501/12-1, RO 501/12-2].
Publication: Royer-Pokora et al.: Human Molecular Genetics 2010; doi: /hmg/ddq042