Molecular and Cellular Pathobiology c-Kit Is Suppressed in Human Colon Cancer Tissue and Contributes to L1-Mediated Metastasis Nancy Gavert 1 , Anna Shvab 1 , Michal Sheffer 2 , Amir Ben-Shmuel 1 , Gal Haase 1 , Eszter Bakos 1 , Eytan Domany 2 , and Avri Ben-Ze'ev 1 Abstract The transmembrane neural cell adhesion receptor L1 is a Wnt/b-catenin target gene expressed in many tumor types. In human colorectal cancer, L1 localizes preferentially to the invasive front of tumors and when overexpressed in colorectal cancer cells, it facilitates their metastasis to the liver. In this study, we investigated genes that are regulated in human colorectal cancer and by the L1-NF-kB pathway that has been implicated in liver metastasis. c-Kit was the most highly suppressed gene in both colorectal cancer tissue and the L1-NF-kB pathway. c-Kit suppression that resulted from L1-mediated signaling relied upon NF-kB, which directly inhibited the transcription of SP1, a major activator of the c-Kit gene promoter. Reconstituting c-Kit expression in L1- transfected cells blocked the biological effects conferred by L1 overexpression in driving motility and liver metastasis. We found that c-Kit expression in colorectal cancer cells is associated with a more pronounced epithelial morphology, along with increased expression of E-cadherin and decreased expression of Slug. Although c-Kit overexpression inhibited the motility and metastasis of L1-expressing colorectal cancer cells, it enhanced colorectal cancer cell proliferation and tumorigenesis, arguing that separate pathways mediate tumorigenicity and metastasis by c-Kit. Our findings provide insights into how colorectal cancer metastasizes to the liver, the most common site of dissemination in this cancer. Cancer Res; 73(18); 5754–63. Ó2013 AACR. Introduction Aberrant activation of Wnt/b-catenin signaling is a hallmark in the majority of human colorectal cancers (1–3). A central question in human colorectal cancer development is the identification and role of downstream b-catenin-T-cell factor (TCF) target genes. We identified members of the neural immunoglobulin-like cell adhesion receptors L1 and Nr-CAM (4, 5) as b-catenin-TCF target genes in colorectal cancer cells and showed that L1 is exclusively expressed in cells at the invasive front of human colorectal cancer tissue (5). Over- expression of L1 in human colorectal cancer cells conferred enhanced motility and metastasis to the liver in a mouse metastasis model (6). We further showed that the mechanisms whereby L1 confers such metastatic capacities involve an association of the L1 juxtamembrane cytoplasmic domain with the cytoskeletal linker protein ezrin and with the inhibitor of kB (IkB) component of the NF-kB signaling pathway (7). This association results in ezrin phosphorylation/activation by Rho-associated protein kinase and an enhanced phosphoryla- tion and proteasomal degradation of IkB leading to the release, nuclear localization, and increased activation of NF-kB-medi- ated transcription (8). In this study we wished to determine the target genes of the L1-NF-kB pathway that are also regulated in a large set of human colorectal cancer tissue samples com- pared with normal colon tissue. Surprisingly, we found that the expression of the tyrosine kinase growth factor receptor c-Kit is suppressed in human colorectal cancer tissue when compared with normal colon tissue, as well as in L1-mediated NF-kB signaling that promotes colorectal cancer cell metastasis. We report on the mechanisms whereby the L1-NF-kB pathway inhibits c-Kit expression and the means by which the metas- tasis suppressive effects are conferred by c-Kit in colorectal cancer cells. Materials and Methods Cell culture, proliferation, artificial wound closure, and transfections HEK 293T, Ls174T, and DLD-1 cells were grown as described (6). Ls174T-L1, Ls174T-p65, and Ls174T-control cells were maintained in medium containing neomycin (800 mg/mL), Ls174T-L1þshp65, Ls174-L1þIkB-SR, and Ls174T-L1þc-Kit cells in medium with both neomycin (800 mg/mL) and puro- mycin (10 mg/mL). For cell growth assays, 10 4 cells/well were seeded in 96-well dishes and cell number determined in triplicates for 5 days. An artificial wound was introduced into confluent cell cultures using a micropipette. The medium was replaced with fresh medium containing 0.35 mg/mL mitomy- cin-C to inhibit cell proliferation. Pictures were taken at 0 and Authors' Affiliations: Departments of 1 Molecular Cell Biology; and 2 Phys- ics and Complex Systems, The Weizmann Institute of Science Rehovot, Israel Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Avri Ben-Ze'ev, Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel. Phone: 972-8-9342422; Fax: 972-8-9465261; E-mail: avri.ben-zeev@weizmann.ac.il doi: 10.1158/0008-5472.CAN-13-0576 Ó2013 American Association for Cancer Research. Cancer Research Cancer Res; 73(18) September 15, 2013 5754