While widespread metastasis is the major cause of human lung cancer-related deaths, its underlying mechanism remains largely unclear. Our genome-wide comparison of the expression profiles of a highly metastatic lung cancer cell line, NCI-H460-LNM35 (LNM35), and its parental clone NCI-H460-N15 (N15), resulted in the identification of a cancer metastasis signature composed of 45 genes. Through gene ontology analysis, our study also provided insights into how this 45-gene metastasis signature may contribute to the acquisition of metastatic potential. Furthermore, we were able to show that enforced expression of the DLX4 homeobox gene, which was identified as a gene with significant down-regulation in LNM35 as well as with significant association with favorable prognosis for lung cancer patients, markedly inhibited in vitro motility and invasion as well as in vivo metastasis via both hematogenous and lymphogenous routes. We further identified a novel gene, uncharacterized gene, which is associated with lung cancer metastasis. We confirmed upregulation of its gene expression in a significant fraction of human lung adenocarcinomas cases as well as lung cancer cell lines. Taken together, these findings indicate that our combined transcriptome analysis is an efficient approach to searching for genes possessing both clinical usefulness in terms of prognostic prediction in human cancer cases and clear functional relevance for studying cancer biology in relation to metastasis. © 2009 The Japan Lung Cancer Society.