Identification of a DNA-binding site and transcriptional target for the EWS-WT1(+KTS) oncoprotein.
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.
Reynolds
Paul A
PA
Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, MA 02129, USA.
Smolen
Gromoslaw A
GA
Palmer
Rachel E
RE
Sgroi
Dennis
D
Yajnik
Vijay
V
Gerald
William L
WL
Haber
Daniel A
DA
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*
C106032:EWS1-WT1 fusion protein, human; D015514:Oncogene Proteins, Fusion; D004247:DNA