Unbiased Screening for Transcriptional Targets of ZKSCAN3 Identifies Integrin β4 and Vascular Endothelial Growth Factor as Downstream Targets

Abstract

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-Metrelated tyrosine kinase (MST1R), MEK2; the guanine nucleotide exchanger RasGRP2, insulin-like growth factor-2, integrin ␤4), cell migration (MST1R), angiogenesis (vascular endothelial growth factor), and proteolysis (MMP26; cathepsin D; PRSS3 (protease serine 3)). We pursued integrin ␤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 ␤4 expression, confirming the array analysis. Moreover, ZKSCAN3 bound to the integrin ␤4 promoter in vitro and in vivo, and the integrin ␤4-derived ZKSCAN3 motif fused upstream of a tk-Luc reporter conferred ZKSCAN3 sensitivity. Integrin ␤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 ␤4.

ZNF667 Serves as a Putative Oncogene in Human Hepatocellular Carcinoma

“…The researchers found that down-regulation of ZKSCAN3 significantly inhibited tumor growth in colon cancer cells, whereas overexpression of ZKSCAN3 produced the opposite effect. In addition, ZKSCAN3 was found to be highly expressed in multiple myeloma [23, 24] and prostate cancer [25]. Through transcriptional activation of cyclin D2 expression, ZKSCAN3 enhanced tumor cell proliferation.…”

Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

“…In a recent study, we found that expression of the mesotrypsinogen/trypsinogen IV gene PRSS3 is up-regulated with advancing malignancy in a cell series modeling breast cancer progression and that suppression of PRSS3 expression inhibits malignant growth of breast cancer cells, whereas treatment with recombinant mesotrypsin stimulates the malignant phenotype (37). PRSS3 is also transcriptionally up-regulated with progression in lung, colon, and prostate cancers (32,34,36) and has been correlated with increased metastasis and poorer survival in non-small cell lung cancer (32). Engineered Kunitz domain inhibitors have recently entered development as therapeutics designed to target serine proteases instrumental in cancer progression (91,92), and it is worth considering whether the possible presence of mesotrypsin in the tumor microenvironment may serve as a mechanism of inactivation or clearance of these drugs.…”

“…Mesotrypsin, a human trypsin encoded by the PRSS3 gene, is produced and secreted as a digestive zymogen by the pancreas (29); a major splice isoform of mesotrypsinogen termed "trypsinogen 4" and lacking a classical secretion signal is highly expressed in brain tissue (30,31) and in some epithelial cell lines and tumors (32)(33)(34)(35)(36)(37). The splice isoforms differ only at the N terminus, and processing of either form by removal of the prodomain results in active mesotrypsin of identical amino acid sequence (38).…”

Evidence of a role for the novel zinc-finger transcription factor ZKSCAN3 in modulating Cyclin D2 expression in multiple myeloma

“…Unbiased screening by Cyclic Amplification and Selection of Targets (CAST) identified the DNA recognition motif (Yang et al , 2008b). ECgene analysis indicated ZKSCAN3 was predominantly expressed in malignant bone marrow cells, and sequence analysis showed several binding sites upstream of the cyclin D2 promoter.…”