Research Article Fascin, a Novel Target of B-Catenin-TCF Signaling, Is Expressed at the Invasive Front of Human Colon Cancer Danijela Vignjevic,1 Marie Schoumacher,1 Nancy Gavert,3 Klaus-Peter Janssen,4 Gloria Jih,3 MarickLae´,2 Daniel Louvard,1 Avri Ben-Ze’ev,3 and Sylvie Robine1 1UMR 144 Centre National de la Recherche Scientifique and 2Department of Pathology, Institut Curie, Paris, France; 3Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel; and 4Department of Surgery, Technical University of Munich, Munich, Germany Abstract carcinogenesis leading to activation of the Wnt/h-catenin signaling Cancer cells become metastatic by acquiring a motile and pathway (1). Later in tumorigenesis, there is an accumulation of ras, p53, Rb invasive phenotype. This step requires remodeling of the actin additional mutations, in K- , and genesencoding h cytoskeleton and the expression of exploratory, sensory componentsof the transforminggrowth factor signaling pathway organelles known as filopodia. Aberrant B-catenin-TCF target (2). Although the effect of such mutations on cell cycle control and gene activation plays a major role in colorectal cancer cell proliferation was extensively studied, much less is known about development. We identified fascin1, a key component of mutations that contribute to the formation of metastases. h filopodia, as a target of B-catenin-TCF signaling in colorectal -Catenin isa central player in the Wnt pathway having a dual cancer cells. Fascin1 mRNA and protein expression were function in epithelial cells. First, it is a component of adherens increased in primary cancers in a stage-dependent manner. junctions that is essential to link the cytoplasmic tail of cadherins Fascin1 was exclusively localized at the invasive front of to the cytoskeleton (3). A process mediated by the APC/Axin/ h tumors also displaying nuclear B-catenin. Forced expression glycogen synthase kinase-3 complex efficiently degradesun- h of fascin1 in colorectal cancer cells increased their migration bound, cytoplasmic -catenin. However, on activation of the Wnt h and invasion in cell cultures and caused cell dissemination pathway, or by aberrationsin the -catenin degradation machinery, h and metastasis in vivo, whereas suppression of fascin1 -catenin accumulatesin the nucleuswhere it doesa second expression by small interfering RNA reduces cell invasion. transcriptional role by interacting with the family of TCF/LEF h Although expression of fascin1 in primary tumors correlated factors(4, 5). Mutationsin componentsof the -catenin pathway with the presence of metastases, fascin1 was not expressed in generally occur early in colon cancer progression consistent with h metastases. Our studies show that fascin1 expression is tightly the ability of -catenin to activate target genesthat are involved in cyclin D1 c-myc regulated during development of colon cancer metastases and cell proliferation, such as (6, 7) and (8). At this is a novel target of B-catenin-TCF signaling. We propose that stage, tumor cells are still adherent to each other in an epithelial transient up-regulation of fascin1 in colorectal cancer structure. h-Catenin accumulatesto higher levelsin the nuclei of promotes the acquisition of migratory and invasive pheno- cells at the tumor-host interface at later stages of tumor types that lead to metastasis. Moreover, the expression of progression (9). At this stage, h-catenin isbelieved to activate fascin1 is down-regulated when tumor cells reach their the expression of genes involved in invasion and metastasis, such metastatic destination where migration ceases and prolifera- asmatrix metalloproteinases(MMP) and the cell adhesion tion is enhanced. Although metastasis to vital organs is often molecule L1 (9–11). the cause of mortality, only limited success has been attained A critical hallmark of the invasive phenotype in cancer cells is in developing effective therapeutics against metastatic dis- the abundant expression of exploratory, sensory organelles known ease. We propose that genes involved in cell migration and asfilopodia. Efficient bundling of actin filamentswithin filopodia is invasion, such as fascin1, could serve as novel targets for essential for filopodia formation both in vitro (12) and in cultured metastasis prevention. [Cancer Res2007;67(14):6844–53] cells(13, 14). Fascin1iscurrently the only actin bundling protein localized along the entire length of filopodia and itsdepletion by Introduction small interfering RNA (siRNA) leads to a substantially reduced number of filopodia (13). Moreover, several studies showed that Colorectal carcinomascarry mutationsin a variety of oncogenes fascin1 significantly increases cell migration in transfilter assays and tumor suppressor genes that contribute to the pathogenesis of (15–17). Thus, by participating in filopodia formation, fascin1 may cancer. Loss of function mutation in the adenomatosis polyposis promote cell migration. Fascin1 is expressed predominantly in coli (APC) tumor suppressor gene is an early event in colorectal neuronal tissue and is absent from normal epithelial cells. However, high levels of fascin1 expression were reported in many types of cancer cells(refs.18–25 and reviewed in ref. 26), including colon Note: Supplementary data for thisarticle are available at Cancer ResearchOnline cancer (16, 27, 28). Fascin1 was also identified in a set of genes that (http://cancerres.aacrjournals.org/). mediate breast cancer metastasis to the lung and clinically Requests for reprints: Danijela Vignjevic, Equipe de Morphogene`se et Signalisation Cellulaires, UMR 144 Centre National de la Recherche Scientifique/ correlated with the development of lung metastasis when Institut Curie, 25 rue d’Ulm, 75248 Paris Cedex 05, France. Phone: 33-1-42-34-63-61; expressed in primary breast cancer tissue (29). The role of fascin1 Fax: 33-1-42-34-63-77; E-mail: [email protected]. I2007 American Association for Cancer Research. up-regulation in cancer and the mechanisms involved are currently doi:10.1158/0008-5472.CAN-07-0929 unknown. Cancer Res 2007; 67: (14). July 15, 2007 6844 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. The Role of Fascin in the Colon Cancer In the present study on the role of fascin1 in colon cancer median expression values in normal colonic mucosa [1.0 relative unit (RU)]. progression, we addressed three fundamental questions: (a) Does An arbitrary threshold was defined by expression of fascin1 at the mean of fascin1 have a role in cell migration and tumor cell invasion? (b)At expression of normal tissue plus thrice the SD (corresponding to 8.3 RU). All what stage of human colon cancer progression is fascin1 expression measurements were done in duplicate in at least two independent experiments. induced and how does it contribute to progression of tumor cells c The detailsof the procedure are given in Supplementary Data and primer toward a more aggressive state? ( ) What is/are the mechanism/s sequences in Supplementary Table S2. that control fascin1 expression in colorectal cancer cells? Transfilter migration and invasion assays. Transfilter assays were done with 8.0-Am pore inserts in 24-well BioCoat Chambers (Becton 5 Materials and Methods Dickinson) using 10 cellsin serum-free DMEM. Conditioned medium from HT29 cellswasplaced in the lower chambersaschemoattractant. For Plasmids. The mouse fascin1 promoter-enhanced green fluorescent invasion assays, control or Matrigel-coated inserts were used. After 6 and protein (EGFP) construct containing the transcription initiation site and 24 h in culture, for migration and invasion assays, respectively, cells were 2.6 kb upstream sequence (pmFascin-EGFP; ref. 30) was obtained from Dr. removed from the upper surface of the filter by scraping with a cotton swab. A. Reske-Kunz (University of Mainz, Mainz, Germany) and subcloned into Cellsthat migrated through the filter were fixed with formaldehyde the pGL3 vector using NheI/KpnI restriction sites resulting in the pmFascin- followed by extraction with Triton X-100 and stained with Texas red- luc construct. Fascin-GFP is described by Vignjevic et al. (13). Fascin- phalloidin. The number of cells in nine randomly chosen fields was scored. internal ribosome entry site (IRES)-EGFP was obtained by fascin1 excision Assays were done thrice in triplicates and the mean values F SE are using BsrGI followed by Klenow modification and BamHI and subcloning presented. The invasion index was expressed as the ratio of ‘‘percentage into pIRES2-EGFP (Clontech) linearized by EcoRI/Klenow and BamHI. invasion’’ of a test cell over percentage invasion of a control cell. Percentage Plasmids containing wild-type (wt) h-catenin and h-catenin deletion of the invasion is calculated as invasion through the Matrigel-coated filters first 89 amino acids (D89) were provided by Dr. C. Perret (Institut Cochin, relative to the migration through the control filter. Paris, France; ref. 31). Animal experiments. Severe combined immunodeficient (SCID) female Cell lines and transfections. HT29, SW480, HCT116, CT26, and 293T cell mice were obtained from CharlesRiversmaintained in a specificpathogen- linesobtained from the American Type Culture Collection were cultured in free environment and all the experimentswere carried out with the standard conditions. Transient transfection of HT29 and SW480 cells was approval of the local authorities. For experimental metastasis assays, groups