A relatively new view of colorectal cancer is that its development/progression reflects the contribution of a large set of altered gene products in varying combinations, each providing a "fitness advantage." In searching for novel contributing gene products using Unigene cluster data mining, we found overrepresentation of expressed sequence tags corresponding to a previously uncharacterized gene (ZKSCAN3) in colorectal tumors. ZKSCAN3 was pursued for several reasons: (a) its sequence similarity with bowl required for Drosophila hindgut development; (b) it lies in a chromosomal region (6p22.1) amplified in colorectal cancer; and (c) its coding sequence predicts tandem C(2)H(2) zinc finger domains present in a class of proteins gaining attention for their role in oncogenesis/tumor progression. Reverse transcription-PCR confirmed overexpression in colorectal tumor tissue compared with adjacent nonmalignant mucosa due in part to gene amplification determined by Southern blotting. Further, immunohistochemistry with an antibody generated to the predicted protein sequence revealed higher ZKSCAN3 expression in invasive compared with noninvasive tumors. Intriguingly, the ZKSCAN3 protein was also expressed in tumors wild-type for genes (APC, p53, K-Ras) commonly targeted in colorectal cancer. ZKSCAN3 knockdown in two independent colon cancer cell lines impaired anchorage-independent growth and orthotopic tumor growth, whereas overexpression in a third cell line had the opposite effect and increased 5-fluorouracil resistance. Liposomal delivery of a ZKSCAN3-targeting small interfering RNA reduced tumorigenicity of orthotopic colon cancer. Thus, the hitherto uncharacterized ZKSCAN3 adds to an expanding set of encoded products contributing to the progression of colorectal cancer.