We previously established a highly metastatic subline, LNM35, from the NCI-H460 lung cancer line (Cancer Res. 60: 2535-40, 2000), and demonstrated up-regulation of a novel gene, CLCP1 (CUB, LCCL-homology, coagulation factor V/VIII homology domains protein), in LNM35 and lung cancer specimens (Oncogene 21: 2822-8, 2002). The purpose of this study is to clarify the potential roles of that gene in cancer metastasis. First, using RNAi constructs expressing short hairpin RNAs targeting CLCP1, we established stable CLCP1-knochdown clones derived from LNM35. In these stable clones, CLCP1 expression was significantly suppressed, and their motility was also significantly reduced, though growth rates were not changed. Concurrently, we searched for cell surface molecules specific to LNM35 using in vitro selection of a phage display library in order to identify the molecular mechanisms of the high motility/invasion ability of LNM35. We found that a phage clone displaying a peptide SAYIPDS similar to a sequence SAYIPES within the Sema domain of semaphorin 4B (SEMA4B) interacted with LNM35. Immunoprecipitation experiments confirmed interaction of CLCP1 with SEMA4B. In addition, we found regulation of CLCP1 protein by ubiquitination, and proteasome degradation enhanced in the presence of SEMA4B. These results are the first to indicate that CLCP1 plays a role in cell motility, while they also showed that at least one of its ligands is SEMA4B and that their interaction mediates proteasome degradation by CLCP1. Although the physiological role of the interaction between CLCP1 and SEMA4B remains to be investigated, this novel gene may become a target of therapy to inhibit metastasis of lung cancers.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA