title: Identification of a DNA-binding site and transcriptional target for the EWS-WT1(+KTS) oncoprotein.
abstract: Desmoplastic small round cell tumor (DSRCT) is defined by a chimeric transcription factor, resulting from fusion of the N-terminal domain of the Ewing's sarcoma gene EWS to the three C-terminal zinc fingers of the Wilms' tumor suppressor WT1. Although DNA-binding sites have been defined for the uninterrupted WT1 zinc finger domains, the most prevalent isoforms of both WT1 and EWS-WT1 have an insertion of three amino acids [lysine, threonine, and serine (KTS)], which abrogates binding to known consensus sequences and transactivation of known target genes. Here, we used cDNA subtractive hybridization to identify an endogenous gene, LRRC15, which is specifically up-regulated after inducible expression of EWS-WT1(+KTS) in cancer cell lines, and is expressed within primary DSRCT cells. The chimeric protein binds in vitro and in vivo to a specific element upstream of LRRC15, leading to dramatic transcriptional activation. Mutagenesis studies define the optimal binding site of the (+KTS) isoform of EWS-WT1 as 5'-GGAGG(A/G)-3'. LRRC15 encodes a leucine-rich transmembrane protein, present at the leading edge of migrating cells, the expression of which in normal tissues is restricted to the invasive cytotrophoblast layer of the placenta; small interfering (siRNA)-mediated suppression of LRRC15 expression in breast cancer cells leads to abrogation of invasiveness in vitro. Together, these observations define the consequence of (KTS) insertion within WT1-derived zinc fingers, and identify a novel EWS-WT1 transcriptional target implicated in tumor invasiveness.
authors.affiliation: Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, MA 02129, USA.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Reynolds
authors.forename: Gromoslaw A
authors.initials: GA
authors.lastname: Smolen
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Palmer
authors.forename: Dennis
authors.initials: D
authors.lastname: Sgroi
authors.forename: Vijay
authors.initials: V
authors.lastname: Yajnik
authors.forename: William L
authors.initials: WL
authors.lastname: Gerald
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Haber
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D004247:DNA / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D015514:Oncogene Proteins, Fusion / Q000378:metabolism*; D010534:Peritoneal Neoplasms / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*
chemical_list: C106032:EWS1-WT1 fusion protein, human; D015514:Oncogene Proteins, Fusion; D004247:DNA