We previously described the novel zinc finger
protein ZKSCAN3 as a new "driver" of
colon cancer progression. To investigate the underlying mechanism and because the predicted structural features (tandem zinc fingers) are often present in
transcription factors, we hypothesized that ZKSCAN3 regulates the expression of a gene(s) favoring
tumor progression. We employed unbiased screening to identify
a DNA binding motif and candidate downstream genes. Cyclic amplification and selection of targets using a random
oligonucleotide library and ZKSCAN3
protein identified KRDGGG as the
DNA recognition motif. In expression profiling, 204 genes were induced 2-29-fold, and 76 genes reduced 2-5-fold by ZKSCAN3. To enrich for direct targets, we eliminated genes under-represented (<3) for the ZKSCAN3 binding motif (identified by CAST-ing) in 2 kilobases of regulatory sequence. Up-regulated putative downstream targets included genes contributing to growth (c-Met-related
tyrosine kinase (MST1R), MEK2; the
guanine nucleotide exchanger RasGRP2,
insulin-like growth factor-2,
integrin beta 4), cell migration (MST1R), angiogenesis (
vascular endothelial growth factor), and proteolysis (MMP26;
cathepsin D; PRSS3 (
protease serine 3)). We pursued
integrin beta 4 (induced up to 6-fold) as a candidate target because it promotes
breast cancer tumorigenicity and stimulates phosphatidyl 3-kinase implicated in
colorectal cancer progression. ZKSCAN3 overexpression/silencing modulated
integrin beta 4 expression, confirming the array analysis. Moreover, ZKSCAN3 bound to the
integrin beta 4 promoter in vitro and in vivo, and the
integrin beta 4-derived ZKSCAN3 motif fused upstream of a tk-Luc reporter conferred ZKSCAN3 sensitivity.
Integrin beta 4 knockdown by
short hairpin RNA countered ZKSCAN3-augmented anchorage-independent colony formation. We also demonstrate
vascular endothelial growth factor as a direct ZKSCAN3 target. Thus, ZKSCAN3 regulates the expression of several genes favoring
tumor progression including
integrin beta 4.