The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells.
BACKGROUND
There is growing evidence that the Wilms' tumor 1 suppressor gene (WT1) behaves as an oncogene in some forms of breast cancer. Previous studies have demonstrated that the N-terminal domain of WT1 can act as a dominant negative through self-association. In the studies presented here we have explored the potential for the zinc finger domain (ZF) of WT1 to also have dominant-negative effects, and thus further our understanding of this protein.


METHODS
Using full-length and ZF-only forms of WT1 we assessed their effect on the WT1 and c-myc promoter using luciferase and chromatin immunoprecipitation assays. The gene expression levels were determined by quantitative real-time RT-PCR, northern blot and western blot. We also assessed the effect of the ZF-only form on the growth of breast cancer cell lines in culture.


RESULTS
Transfection with WT1-ZF plasmids resulted in a stronger inhibition of WT1 promoter than full-length WT1 in breast cancer cells. The WT1-ZF form lacking the lysine-threonine-serine (KTS) insert (ZF - KTS) can bind to the majority of WT1 consensus sites throughout the WT1 promoter region, while the ZF containing the insert (ZF + KTS) form only binds to sites in the proximal promoter. The abundances of endogenous WT1 mRNA and protein were markedly decreased following the stable expression of ZF - KTS in breast cancer cells. The expressions of WT1 target genes, including c-myc, Bcl-2, amphiregulin and TERT, were similarly suppressed by ZF - KTS. Moreover, WT1-ZF - KTS abrogated the transcriptional activation of c-myc mediated by all four predominant isoforms of WT1 (including or lacking alternatively spliced exons 5 and 9). Finally, WT1-ZF - KTS inhibited colony formation and cell division, but induced apoptosis in MCF-7 cells.


CONCLUSIONS
Our observations strongly argue that the WT1-ZF plasmid behaves as a dominant-negative regulator of the endogenous WT1 in breast cancer cells. The inhibition on proliferation of breast cancer cells by WT1-ZF - KTS provides a potential candidate of gene therapy for breast cancer.
Han
Youqi
Y
Department of Cellular and Molecular Biology, Ontario Cancer Institute, University Health Network, Toronto, Ontario M5G 2M9, Canada.
San-Marina
Serban
S
Yang
Lin
L
Khoury
Haytham
H
Minden
Mark D
MD
D066258:Amphiregulin; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000517:prevention & control; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D047369:Chromatin Immunoprecipitation; D003114:Colony-Forming Units Assay; D066255:EGF Family of Proteins; D024202:Electrophoretic Mobility Shift Assay; D005799:Genes, Dominant / Q000502:physiology*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D008156:Luciferases; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000378:metabolism; D014407:Tumor Cells, Cultured; D025721:WT1 Proteins / Q000235:genetics* / Q000378:metabolism; D016335:Zinc Fingers
C587848:AREG protein, human; D066258:Amphiregulin; D066255:EGF Family of Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D025721:WT1 Proteins; D008156:Luciferases; C509186:TERT protein, human; D019098:Telomerase