CITED1 confers stemness to Wilms tumor and enhances tumorigenic responses when enriched in the nucleus
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Andrew J. Murphy1, Janene Pierce1, Christian de Caestecker1, Gregory D. Ayers2, Alex Zhao2, Hernan Correa6,Jonathan R. Krebs1, Vincente Kenyi Saito-Diaz3, Ethan Lee3, Alan O. Perantoni4, Mark P. de Caestecker5, and Harold N. Lovvorn III 1
1 Department of Pediatric Surgery, Monroe Carell Jr. Children’s Hospital at Vanderbilt, Nashville, Tennessee
2 Department of Biostatistics, Vanderbilt University Medical Center, Nashville, Tennessee
3 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee
4 Cancer and Developmental Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland
5 Departments of Medicine and Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee
6 Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee
Harold N. Lovvorn III, email:
Keywords: CITED1, Wilms tumor, WNT, WiT49, cancer stem cell
Received: November 2, 2013 Accepted: December 28, 2013 Published: December 28, 2013
Wilms tumor (WT) is the most common childhood kidney cancer and retains gene expression profiles reminiscent of the embryonic kidney. We have shown previously that CITED1, a transcriptional regulator that labels the self-renewing, multipotent nephron progenitor population of the developing kidney, is robustly expressed across all major WT disease and patient characteristics. In this malignant context, CITED1 becomes enriched in the nucleus, which deviates from its cytosolic predominance in embryonic nephron progenitors. We designed the current studies to test the functional and mechanistic effects of differential CITED1 subcellular localization on WT behavior. To mimic its subcellular distribution observed in clinical WT specimens, CITED1 was misexpressed ectopically in the human WT cell line, WiT49, as either a wild-type (predominantly cytosolic) or a mutant, but transcriptionally active, protein (two point mutations in its nuclear export signal, CITED1ΔNES; nuclear-enriched). In vitro analyses showed that CITED1ΔNES enhanced WiT49 proliferation and colony formation in soft agar relative to wild-type CITED1 and empty vector controls. The nuclear-enriched CITED1ΔNES cell line showed the greatest tumor volumes after xenotransplantation into immunodeficient mice (n=15 animals per cell line). To elucidate CITED1 gene targets in this model, microarray profiling showed that wild-type CITED1 foremost upregulated LGR5 (stem cell marker), repressed CDH6 (early marker of epithelial commitment of nephron progenitors), and altered expression of specific WNT pathway participants. In summary, forced nuclear enrichment of CITED1 in a human WT cell line appears to enhance tumorigenicity, whereas ectopic cytosolic expression confers stem-like properties and an embryonic phenotype, analogous to the developmental context.
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