Fascin,aNovelTargetof B-Catenin-TCFSignaling ... · Fascin,aNovelTargetofB-Catenin-TCFSignaling, IsExpressedattheInvasiveFrontof HumanColonCancer DanijelaVignjevic,1 MarieSchoumacher,1

Fascin, a Novel Target of B-Catenin-TCF Signaling,

Is Expressed at the Invasive Front of

Human Colon Cancer

Danijela Vignjevic,1Marie Schoumacher,

1Nancy Gavert,

3Klaus-Peter Janssen,

4

Gloria Jih,3Marick Lae,

2Daniel Louvard,

1Avri Ben-Ze’ev,

3and Sylvie Robine

1

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; and4Department of Surgery, Technical University of Munich, Munich, Germany

Abstract

Cancer cells become metastatic by acquiring a motile andinvasive phenotype. This step requires remodeling of the actincytoskeleton and the expression of exploratory, sensoryorganelles known as filopodia. Aberrant B-catenin-TCF targetgene activation plays a major role in colorectal cancerdevelopment. We identified fascin1, a key component offilopodia, as a target of B-catenin-TCF signaling in colorectalcancer cells. Fascin1 mRNA and protein expression wereincreased in primary cancers in a stage-dependent manner.Fascin1 was exclusively localized at the invasive front oftumors also displaying nuclear B-catenin. Forced expressionof fascin1 in colorectal cancer cells increased their migrationand invasion in cell cultures and caused cell disseminationand metastasis in vivo , whereas suppression of fascin1expression by small interfering RNA reduces cell invasion.Although expression of fascin1 in primary tumors correlatedwith the presence of metastases, fascin1 was not expressed inmetastases. Our studies show that fascin1 expression is tightlyregulated during development of colon cancer metastases andis a novel target of B-catenin-TCF signaling. We propose thattransient up-regulation of fascin1 in colorectal cancerpromotes the acquisition of migratory and invasive pheno-types that lead to metastasis. Moreover, the expression offascin1 is down-regulated when tumor cells reach theirmetastatic destination where migration ceases and prolifera-tion is enhanced. Although metastasis to vital organs is oftenthe cause of mortality, only limited success has been attainedin developing effective therapeutics against metastatic dis-ease. We propose that genes involved in cell migration andinvasion, such as fascin1 , could serve as novel targets formetastasis prevention. [Cancer Res 2007;67(14):6844–53]

Introduction

Colorectal carcinomas carry mutations in a variety of oncogenesand tumor suppressor genes that contribute to the pathogenesis ofcancer. Loss of function mutation in the adenomatosis polyposiscoli (APC) tumor suppressor gene is an early event in colorectal

carcinogenesis leading to activation of the Wnt/h-catenin signalingpathway (1). Later in tumorigenesis, there is an accumulation ofadditional mutations, in K-ras, p53, Rb , and genes encodingcomponents of the transforming growth factor h signaling pathway(2). Although the effect of such mutations on cell cycle control andcell proliferation was extensively studied, much less is known aboutmutations that contribute to the formation of metastases.h-Catenin is a central player in the Wnt pathway having a dual

function in epithelial cells. First, it is a component of adherensjunctions that is essential to link the cytoplasmic tail of cadherinsto the cytoskeleton (3). A process mediated by the APC/Axin/glycogen synthase kinase-3h complex efficiently degrades un-bound, cytoplasmic h-catenin. However, on activation of the Wntpathway, or by aberrations in the h-catenin degradation machinery,h-catenin accumulates in the nucleus where it does a secondtranscriptional role by interacting with the family of TCF/LEFfactors (4, 5). Mutations in components of the h-catenin pathwaygenerally occur early in colon cancer progression consistent withthe ability of h-catenin to activate target genes that are involved incell proliferation, such as cyclin D1 (6, 7) and c-myc (8). At thisstage, tumor cells are still adherent to each other in an epithelialstructure. h-Catenin accumulates to higher levels in the nuclei ofcells at the tumor-host interface at later stages of tumorprogression (9). At this stage, h-catenin is believed to activatethe expression of genes involved in invasion and metastasis, suchas matrix metalloproteinases (MMP) and the cell adhesionmolecule L1 (9–11).

A critical hallmark of the invasive phenotype in cancer cells isthe abundant expression of exploratory, sensory organelles knownas filopodia. Efficient bundling of actin filaments within filopodia isessential for filopodia formation both in vitro (12) and in culturedcells (13, 14). Fascin1 is currently the only actin bundling proteinlocalized along the entire length of filopodia and its depletion bysmall interfering RNA (siRNA) leads to a substantially reducednumber of filopodia (13). Moreover, several studies showed thatfascin1 significantly increases cell migration in transfilter assays(15–17). Thus, by participating in filopodia formation, fascin1 maypromote cell migration. Fascin1 is expressed predominantly inneuronal tissue and is absent from normal epithelial cells. However,high levels of fascin1 expression were reported in many types ofcancer cells (refs. 18–25 and reviewed in ref. 26), including coloncancer (16, 27, 28). Fascin1 was also identified in a set of genes thatmediate breast cancer metastasis to the lung and clinicallycorrelated with the development of lung metastasis whenexpressed in primary breast cancer tissue (29). The role of fascin1up-regulation in cancer and the mechanisms involved are currentlyunknown.

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Requests for reprints: Danijela Vignjevic, Equipe de Morphogenese etSignalisation Cellulaires, UMR 144 Centre National de la Recherche Scientifique/Institut Curie, 25 rue d’Ulm, 75248 Paris Cedex 05, France. Phone: 33-1-42-34-63-61;Fax: 33-1-42-34-63-77; E-mail: [email protected].

I2007 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-07-0929

Cancer Res 2007; 67: (14). July 15, 2007 6844 www.aacrjournals.org

Research Article

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In the present study on the role of fascin1 in colon cancerprogression, we addressed three fundamental questions: (a) Doesfascin1 have a role in cell migration and tumor cell invasion? (b) Atwhat stage of human colon cancer progression is fascin1 expressioninduced and how does it contribute to progression of tumor cellstoward a more aggressive state? (c) What is/are the mechanism/sthat control fascin1 expression in colorectal cancer cells?

Materials and Methods

Plasmids. The mouse fascin1 promoter-enhanced green fluorescent

protein (EGFP) construct containing the transcription initiation site and

2.6 kb upstream sequence (pmFascin-EGFP; ref. 30) was obtained from Dr.

A. Reske-Kunz (University of Mainz, Mainz, Germany) and subcloned into

the pGL3 vector using NheI/KpnI restriction sites resulting in the pmFascin-

luc construct. Fascin-GFP is described by Vignjevic et al. (13). Fascin-

internal ribosome entry site (IRES)-EGFP was obtained by fascin1 excision

using BsrGI followed by Klenow modification and BamHI and subcloning

into pIRES2-EGFP (Clontech) linearized by EcoRI/Klenow and BamHI.

Plasmids containing wild-type (wt) h-catenin and h-catenin deletion of the

first 89 amino acids (D89) were provided by Dr. C. Perret (Institut Cochin,

Paris, France; ref. 31).

Cell lines and transfections. HT29, SW480, HCT116, CT26, and 293Tcell

lines obtained from the American Type Culture Collection were cultured in

standard conditions. Transient transfection of HT29 and SW480 cells was

carried out by Nucleofector (Amaxa) and of 293T cells using calcium

phosphate. In transactivation assays, 0.25 Ag h-galactosidase plasmid was

cotransfected with 1 Ag reporter plasmid and 2 Ag of either h-catenin or

cadherin tail constructs. Cells were plated in duplicate and lysed after 48 h,

and luciferase and h-galactosidase levels were determined by enzyme assay

kits (Promega). Luciferase activity was normalized to h-galactosidaseactivity as an internal transfection control. Induction levels of the fascin1

promoter were calculated using the empty reporter plasmid (pGL2).

Stable lines of HT29 cells expressing EGFP, fascin-EGFP, and fascin-IERS-

EGFP were transiently transfected, and GFP-positive cells were sorted by

fluorescence-activated cell sorting (FACS) and maintained as stable lineswith 0.2 mg/mL G418.

Transient transfection of HCT116 cells with 30 nmol/L siRNA against

fascin1 (5¶-CAAAGACUCCACAGGCAAAUU-3¶), produced by Dharmacon,was carried out using LipofectAMINE RNAiMAX (Invitrogen). Cells lysed

3 days after transfection were analyzed by Western blot analysis using NIH

ImageJ software. Reduction in protein levels was normalized to the loading

control (tubulin) and mock-transfected cells.Chromatin immunoprecipitation assay. Chromatin immunoprecipi-

tation (ChIP) assays were done using a ChIP assay kit according to the

manufacturer’s instructions (Upstate Biotechnology). Rabbit anti-TCF4 or

rabbit IgG were used to immunoprecipitate DNA-containing complexes.PCR was done with primers complementary to the fascin1 promoter region.

The primer sequences are given in Supplementary Table S2.

Immunofluorescence and immunohistochemistry. Evaluation offascin1 expression by immunohistochemistry was done on two tissue

arrays, containing 144 colon carcinoma samples, CO802 and CO641

(Euromedex), and 34 colon adenocarcinomas obtained from surgical

resection and/or biopsy specimens at The Curie Institute Hospital (Paris,France). Staining of >10% of tumor cells was scored as positive

immunoreactivity. Tumor staging was done using the tumor-node-

metastasis staging system according to the WHO protocol. Immunohisto-

chemistry for h-catenin and fascin1 was done as previously (16, 32).Immunostaining of cell culture samples was done as described (13). The

details of the procedure are given in Supplementary Data.

Quantitative real-time PCR. Tissue samples for quantitative real-time

PCR (qRT-PCR) analysis of fascin1 expression were obtained at KlinikumRechts Der Isar (Munich, Germany). cDNA preparation was done according

to standard procedures. Expression of fascin1 was determined using

hypoxanthine phosphoribosyltransferase as internal reference by ABI Prism7300 (Applied Biosystems) and SYBR Green I. The values were calibrated to

median expression values in normal colonic mucosa [1.0 relative unit (RU)].An arbitrary threshold was defined by expression of fascin1 at the mean of

expression of normal tissue plus thrice the SD (corresponding to 8.3 RU). All

measurements were done in duplicate in at least two independent

experiments.The details of the procedure are given in Supplementary Data and primer

sequences in Supplementary Table S2.

Transfilter migration and invasion assays. Transfilter assays were

done with 8.0-Am pore inserts in 24-well BioCoat Chambers (Becton

Dickinson) using 105 cells in serum-free DMEM. Conditioned medium from

HT29 cells was placed in the lower chambers as chemoattractant. For

invasion assays, control or Matrigel-coated inserts were used. After 6 and

24 h in culture, for migration and invasion assays, respectively, cells were

removed from the upper surface of the filter by scraping with a cotton swab.

Cells that migrated through the filter were fixed with formaldehyde

followed by extraction with Triton X-100 and stained with Texas red-

phalloidin. The number of cells in nine randomly chosen fields was scored.

Assays were done thrice in triplicates and the mean values F SE are

presented. The invasion index was expressed as the ratio of ‘‘percentage

invasion’’ of a test cell over percentage invasion of a control cell. Percentage

invasion is calculated as invasion through the Matrigel-coated filters

relative to the migration through the control filter.Animal experiments. Severe combined immunodeficient (SCID) female

mice were obtained from Charles Rivers maintained in a specific pathogen-

free environment and all the experiments were carried out with the

approval of the local authorities. For experimental metastasis assays, groupsof 10 (4 weeks old) mice were used for each cell type. HT29 cells (106) stably

expressing either EGFP or fascin-EGFP in 100 AL PBS were injected into the

tail vein. Mice were sacrificed 3 weeks after injection, the left lung lobeswere embedded, and one section every 100 Am was stained with H&E

according to standard protocols. Metastasis was defined as a group of more

than five enterocytes. The intestinal origin of cells was confirmed by villin

immunostaining.

Results

Fascin1 expression promotes cell migration, invasion, andcell dissemination. Fascin1 is expressed in highly aggressivecarcinomas of various origins. Because fascin1 is required forfilopodia formation (13) and these organelles are considerednecessary for directional cell movement, we investigated whetherfascin1 plays a role in colorectal cancer metastasis by promotingmigratory and invasive capabilities in tumor cells. We expressedfascin1 in HT29 human colon cancer cells that do not normallyexpress fascin1, to levels as high as those found in another humancolorectal cancer cell line, SW480 (Fig. 1A). On the dorsal side ofthe cells, fascin1 was recruited to microvilli-like structures (Fig. 1A)that were also present in nontransfected cells (Supplementary Fig.S1A). However, on the ventral side of HT29 cells, the expression offascin1 induced filopodia formation (Fig. 1A), which were notpresent in wt cells (Supplementary Fig. S1A).

We also examined the proliferation of cells overexpressing fascin1and found that fascin1-expressing cells and control cells have asimilar growth rate (population doubling time of 31.2 F 7.2 h forHT29 cells, 28.8F 4.8 h for HT29 cells expressing GFP, 28.8F 7.2 h forcells expressing fascin1-GFP, and 31.2 F 9.6 h for cells expressingfascin1-IRES-GFP). In addition, fascin1-expressing and control cellswere s.c. injected into SCIDmice and tumor size wasmeasured every2 to 3 days over a 4-week period, but no significant differences intumor volume were detected (Supplementary Fig. S1B). Thus, forcedexpression of fascin1 does not affect cell proliferation.

Next, we examined the effect of fascin1 expression on themotility of HT29 cells toward medium containing growth factors ina Transwell filter assay. We found that fascin1-expressing cells were

The Role of Fascin in the Colon Cancer

www.aacrjournals.org 6845 Cancer Res 2007; 67: (14). July 15, 2007



six times more motile compared with untransfected cells or to cellstransfected with GFP alone (Supplementary Fig. S1C) in agreementwith previous observations (15–17). In addition, we examinedwhether fascin1 has also a role in invasion and metastasis. Fascin1-transfected cells were about thrice more invasive through Matrigel-coated filters (Fig. 1B). Moreover, cells i.v. injected into the tail veinof SCID mice displayed a 10 times higher ability to disseminate andform metastases in the lungs of mice (Fig. 1C). These tumors werepositive for fascin1 and villin (a marker for intestinal cells)confirming that they were derived from the injected cells (Fig. 1C).Histologic analysis of such micrometastases confirmed that themetastatic lesions replaced large areas of the lung parenchyma,

suggesting that fascin1-transfected cells gained extensive extrava-sation ability. In addition, 50% of the mice injected with fascin1-expressing cells became severely paralysed compared with only 1 of10 mice injected with cells expressing GFP alone. X-ray analysisrevealed that the paralysis was not caused by bone metastasis butrather by the development of tumors along the spine with invasionto the back muscle (data not shown). Next, we took the oppositestrategy: using siRNA, we suppressed fascin1 levels in HCT116 cells(which normally express fascin1) and tested their invasionpotential in transfilter assays (Fig. 1D). We found that in cellswhere fascin1 levels were suppressed by about 90%, their invasioncapacity was reduced three-fold.

Figure 1. Expression of fascin1 enhancescell invasion and metastasis to the lung inexperimental animal models. A, top,expression of fascin1 in SW480 and HT29cells was compared (by Western blotting)with that in HT29 cells stably expressingGFP, fascin1 tagged with GFP, orfascin1 and GFP expressed individually(fascin-IRES-GFP). Bottom, HT29 cellsexpressing fascin1-GFP develop filopodiaat the ventral side and numerous ‘‘spikes’’at the dorsal side. B, invasion potential ofcolon cancer HT29 cells stably expressingfascin1 (fascin1-GFP or fascin1-IRES-GFP) or GFP alone and nontransfectedHT29 cells was tested in Transwellanalysis. Fascin1-expressing HT29 cellsdisplayed enhanced invasion throughMatrigel-coated filters. Columns, mean;bars, SE. C, left, the ability of HT29 cells(expressing GFP or fascin1-GFP) to formlung metastases was determined by tailvein injection of mice with 106 cells permouse and by counting the number ofmetastatic lesions in the lungs after4 wks. Columns, mean (n = 10); bars, SE.Right, histologic staining (H&E) of serialsections from lungs of animals injected withHT29 cells expressing fascin1-GFP andimmunostained with anti-villin andanti-fascin1 antibodies. Bar, 25 Am.D, invasion potential of nontransfected andfascin1-depleted colon cancer HCT116cells was tested in Transwell analysis.Suppression of fascin1 expressionwas analyzed by Western blot. Fascin1-depleted cells displayed reduced invasionproperty through Matrigel-coated filters.Columns, mean; bars, SE.

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Together, these results imply that fascin1 expression in humancolon cancer cells plays an important role in their motile, invasive,and metastatic capacities.

Fascin1 is overexpressed in high-grade and late-stagehuman colon carcinoma. Fascin1 is absent from normalepithelial cells, whereas high levels of fascin1 expression werereported in many types of cancer cells, including colorectal cancercells (16, 27, 28). Because the treatment and prognosis of colorectalcancer patients depends on tumor grade (the degree of primarytumor differentiation) and tumor-node-metastasis stage (howwidespread the cancer is at the time of diagnosis), we firstdetermined if, and at which stage, fascin1 expression is induced inhuman colon cancer tissue. We examined fascin1 RNA expressionin human colon carcinomas using qRT-PCR. We found thatwhereas normal tissue was essentially negative for fascin1 RNA(n = 10; with mean expression of 1.8 F 2.2 RU), the expression offascin1 was significantly elevated in carcinoma tissue whencompared with normal epithelium, and this increase was tumorstage dependent (Fig. 2A). Fascin1 RNA levels remained unchangedin benign lesions and increased slightly in early tumor stages (2 of10 tumors were fascin1 positive, 3.8 F 3.8 RU) but were elevatedsignificantly in stage II and III tumors [50% of the tumors werefascin1 positive in each group; 19.4 F 14.9 RU (n = 10), 27.7 F 31.9RU (n = 10), and 7.2 F 9.3 RU (n = 16), respectively], correlatingwith a more invasive phenotype and metastasis to lymph nodes.

To determine fascin1 protein levels in tumors, we didimmunohistochemical analyses of paraffin-embedded tissue sec-tions of two tissue arrays containing 118 cases of human coloncancer derived from surgical resections (Supplementary Table S1).Although fascin1 was expressed in a subset of ‘‘low-grade’’ tumors

(Fig. 2C), its expression became prominent in ‘‘high-grade’’ tumorsthat are characterized by loss of normal cellular differentiation(anaplasia) and poorly defined margins, or diffuse spread, whichoften precluded complete surgical resection. In addition, we foundthat fascin1 was expressed in a high percentage of stage III and IVtumors (Fig. 2B). Both stages were characterized by extensiveinvasion of the primary tumor into the submucosa, muscularispropria, or through the wall of the colon, and also by invasion tonearby lymph nodes at stage III and to distant metastases in stageIV tumors. A correlative analysis between fascin1 expression inprimary tumors and formation of metastases in the correspondingpatients revealed that in patients with lymph node and/or livermetastases, the primary tumors were more often fascin1 positive(64% and 67%, respectively) compared with primary tumors ofpatients without metastases at surgery (28% and 40%, respectively;Fig. 2D ; Supplementary Table S1).

We conclude that fascin1 expression correlates with aggressive-ness of human colorectal cancer and that the presence of fascin1 inprimary tumors has predictive value in determining the incidenceof metastasis.

Fascin1 is expressed at the invasive front of human coloncarcinomas but is absent in metastases. Most solid tumors,including colorectal cancer, are not homogeneous often maintain-ing a central more differentiated part and an invasive front thatdiffers in cell morphology and molecular composition (reviewed inref. 11). As a model for such differences in cell morphology andgene regulation, we used the density-dependent phenotypicconversion displayed by the SW480 colon cancer cell line (10).These cells mimic the changes in h-catenin localization associatedwith the position of the cells in the tumor (10, 33, 34). h-Catenin is

Figure 2. Fascin1 expression correlateswith the aggressiveness of human coloncarcinoma. A, fascin1 expression innormal colon tissue, adenoma, andcarcinoma at different stages wasevaluated by qRT-PCR. Each dotrepresents one patient. The thresholdwas set at the mean expression levelin normal tissue plus thrice the SD(expression level equal to 8.3). B and C,fascin1 expression was evaluated byimmunohistochemistry (IH ) at differenttumor stages (I–IV; B) and different tumorgrades (G1–G4; C). The total number ofcarcinomas analyzed was 82 and 148,respectively. D, fascin1 expression in theprimary tumor correlated with the presenceof metastases in lymph nodes and in theliver. The total number of carcinomasanalyzed was 79. Black columns, primarytumors from patients who did not havemetastases in lymph nodes (N0) and/or theliver (M0). Gray columns, primary tumorsfrom patients who had metastases inlymph node/s (N1 and N2) and/or theliver (M1).





mostly present at cell-cell contacts in dense cell cultures(Supplementary Fig. S2A), similar to cells in the more differentiatedcentral area of tumors. In sparse cultures of SW480 cells becausethey have mutant APC, wt h-catenin accumulates in the nuclei ofthe cells (Supplementary Fig. S2A) mimicking h-catenin localiza-tion in cells at the invasive front of tumors (33). When comparingthe expression of fascin1 in sparse and dense SW480 cell cultures,we found that in sparse cell cultures displaying nuclear h-catenin,fascin1 expression was about twice higher than in dense cultures(Supplementary Fig. S2B).

Next, we did immunohistologic staining of sections derived fromsurgical resections of adenocarcinoma tumors from an unselected

series of 34 patients, to evaluate the expression and localization offascin1 in primary human colon cancers. Serial sections of thesetumors were immunostained with antibodies against h-catenin andfascin1. In normal colon epithelium, h-catenin staining wasobserved mostly at cell-cell contact sites (9, 10), whereas fascin1staining was only detected in infiltrating stromal cells, but not inenterocytes (Fig. 3A). In the central more differentiated areas oftumors, h-catenin staining was detected in the membrane and inthe cytoplasm with no nuclear staining (Fig. 3B and C). Fascin1 wasdetected in only 22% of differentiated colon carcinomas (Fig. 3Band C). In contrast, the invasive front of tumors displayedcytoplasmic and nuclear h-catenin localization (Fig. 3D, arrows)

Figure 3. Fascin is expressed in tumorcells at the invasive front of coloncarcinomas. Immunohistochemical staining(brown ) for fascin1 (first column) andh-catenin (second column ) in adjacentserial sections of human colon cancertissue, with nuclear counterstaining (blue ).A, overview of the normal tissue showingno fascin1 expression in epithelial cellsof the mucosa displaying membranalh-catenin. Stromal cells were positive forfascin1 staining. Bar, 50 Am. B, overview ofthe invasive front of colon carcinomashowing restricted fascin1 expression incarcinomatous cells located at the invasiveborder of tumor and stroma (arrowhead),whereas the tumor cells located at thecenter of the tumor (star ) were fascin1negative. Note the stronger staining forh-catenin in the membrane and in thecytoplasm of these cells. Bar, 50 Am.C, higher magnification of the boxed areaat the center of the tumor in (B ). In thetumor center, h-catenin is present at themembrane, but no fascin1 expression wasobserved in epithelial tumor cells. Bar,25 Am. D, higher magnification of theboxed area at the invasive front of thetumor shown in (B ). h-Catenin wasdetected in the membrane, cytoplasm, andalso in some nuclei (arrows ) and fascin1was expressed in tumor cells. Bar, 25 Am.

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and was associated with strong fascin1 expression (Fig. 3B and D).This phenotype was apparent in 61% of the tumors. Fascin1expression was strong in sheets of invading tumor cells and also indisseminating single tumor cells.

Disruption of cell-cell contacts and the gain of cell motility ininvasive tumors is suggested to be achieved by a processreminiscent of epithelial to mesenchymal transition (EMT; ref.35). This includes down-regulation of epithelial-specific proteins,including E-cadherin and cytokeratins, and expression of mesen-chymal-specific molecules, such as vimentin and fibronectin. Weimmunostained colorectal cancer tissue samples (n = 10) withvarious EMT markers and found strong E-cadherin staining at themembrane of most tumor cells, including fascin1-positive cells(Fig. 4). The tumor cells were also villin and cytokeratin 20 (CK20)positive, whereas vimentin was only present in stromal cells(Fig. 4A). Thus, fascin1 was expressed at the invasive front ofcolorectal cancer tissue that did not display EMT.

We also detected fascin1 enrichment in the stromal compart-ment, in the extracellular matrix, mature dendritic cells, fibroblasts,and blood vessels (Fig. 4B), in agreement with a previous report(27). This staining was strongest in the stroma adjacent to theinvading tumor front but was independent of whether theepithelial cells in the tumor itself were fascin1 positive (Fig. 4B)or fascin1 negative (Fig. 4B) and this phenotype was observed in>90% of invasive colon carcinomas.

Because fascin1 expression in primary tumors correlated withthe presence of metastases in the respective patients, we examinedwhether fascin1 was also expressed in metastatic tissue of coloncarcinoma patients. Using qRT-PCR analysis, we found that allhuman liver tissue samples from colon cancer patients werenegative for fascin1, independently of whether the patients had orlacked liver metastasis ( fascin1 expression was 1.29 F 0.57 RU fornormal liver and 0.58 F 0.46 RU for liver containing metastases,with 10 samples analyzed in each group). We also examined byimmunohistochemistry tissue samples from seven patients (thatincluded 7 primary tumors, 4 lymph nodes, and 5 liver metastases)and detected fascin1 at the invasive front of six primary tumors,but fascin1 was only detected in the lymph node and liver

metastases of one patient. The fascin1-negative metastases were allwell differentiated and displayed glandular morphology (Fig. 5A),recapitulating the organization characteristic to the differentiatedcenter of the primary tumor, where h-catenin is localized at themembrane and in the cytoplasm. The single fascin1-positive livermetastasis seemed undifferentiated and h-catenin accumulated inthese cells in the cytoplasm as well as in the nuclei of cells (Fig. 5B).

We conclude that fascin1 is expressed at the invasive front ofhuman colon carcinomas, suggesting that fascin1 may participatein tumor cell invasion. However, fascin1 was mostly absent fromdistant metastases, suggesting a tight regulation of fascin1expression during tumorigenesis.

Fascin1 expression is induced by B-catenin-TCF signaling.The association of fascin1 expression with nuclear localization ofh-catenin at the invasive front of colon cancer tissue and incultured colon cancer cells prompted us to investigate whether thefascin1 gene is a novel target of h-catenin signaling. The activity ofthe fascin1 gene promoter was shown previously to correlate withfascin1 protein expression: whereas the fascin1 promoter is silentin keratinocytes and in immature dendritic cells, which do notexpress fascin1 protein, it is highly active in fascin1-positive maturedendritic cells and neuroblastoma (30). Cotransfection of 293 cellswith the fascin1 promoter luciferase reporter plasmid, or thesynthetic TCF reporter plasmid TOPFLASH, and a point mutant(S33Y) stabilized h-catenin construct resulted in a 12-foldactivation of the fascin1 promoter reporter and a close to 50-foldactivation of TOPFLASH (Fig. 6A). TOPFLASH served as control inthis experiments because it behaves similarly to the promoters ofpreviously characterized h-catenin target genes such as cyclin D1(6), Nr-CAM (36), or L1-CAM (10). The involvement of TCF/LEFfactors in fascin1 transactivation is suggested by the inhibition offascin1 promoter activation using dominant-negative TCF4(DNTCF), whereas a dominant-positive LEF construct, containingthe DNA-binding domain of LEF linked to the transactivationdomain of viral VP16 (LEF/VP16), activated the fascin1 promoter(Fig. 6A ). Transactivation of the fascin1 promoter by theendogenous h-catenin/TCF complex in SW480 colon cancer cellswas enhanced by wt TCF4 and inhibited by dominant-negative

Figure 4. Fascin1-mediated tumor cell invasion does not involve an EMT. A, immunohistochemical staining (brown ) of E-cadherin (E-cad ), CK20, and vimentin (Vim )with nuclear counterstaining (blue ) at the invasive front of human colon carcinoma in a region of tumor as shown by H&E staining. Bar, 25 Am. B, immunofluorescentstaining for fascin1 in fascin1-positive (top ) and fascin1-negative (bottom ) epithelial compartments of colon cancer tissue. Epithelial cells, blood vessels, anddendritic cells.





TCF4 (DNTCF; Fig. 6B). We also investigated whether the fascin1gene promoter is active in colon cancer cells using a fascin1promoter-GFP reporter plasmid. In SW480 cells that expressendogenous fascin1, the promoter was highly active, whereas thispromoter reporter was silent in HT29 cells that normally do notexpress fascin1 (Fig. 6C). To test whether the fascin1 gene isactivated by h-catenin signaling, we also cotransfected HT29 cellswith wt, or the activated stable form of h-catenin in which the first89 amino acids were deleted (D89-h-cat), together with the fascin1promoter-GFP reporter plasmid. A GFP signal was detected inf50% of cells expressing D89h-cat, but not in wt h-catenin–expressing cells (Fig. 6C). We identified five putative TCF-bindingsites in the 5-untranslated region of the mouse fascin1 promoter(Fig. 6D). In ChIP analyses, we PCR amplified three fascin1-specificpromoter sequences from chromatin immunoprecipitates usingthe mouse colon cancer CT26 cell line using TCF4-specificantibodies (Fig. 6D). No fascin1-specific promoter sequences wereamplified when control IgG was used for precipitation. Takentogether, these data indicate that the fascin1 gene is a directtranscriptional target of h-catenin/TCF signaling.

These results suggest that h-catenin-TCF signaling is involved inthe regulation of fascin1 gene transcription in human colorectalcancer cells.

Discussion

The role of fascin1 in metastasis of colorectal cancer cellsinvolves the promotion of a migratory and invasive phenotypethrough filopodia formation. The actin bundling protein fascin1is expressed in human colon carcinomas in a grade- and stage-dependent manner but is absent from normal colonic epithelium.In addition, consistent with our results, fascin1 up-regulation inmany other types of cancer was found to correlate with poorprognosis and with decreased survival (19, 27, 28, 37).

The role of fascin1 in cell migration on planar substrata in theabsence of a gradient of chemoattractant is unclear. It has been

shown that the migration of certain cells could be blocked byantibodies that perturb actin-fascin1 interactions (38), but fascin1overexpression in other cell types did not display a clear correlationwith the rate of cell locomotion (16). However, several studiesreported that fascin1 significantly increases cell migration intransfilter haptotactic assays (refs. 15–17 and this study), suggest-ing that fascin1 can enhance the directional motility of cells. Weshowed here that the expression of fascin1 promotes the invasivecapability of colon cancer cells by showing that (a) culturedcolorectal cancer cells expressing fascin1 degraded Matrigel, abasement membrane matrix and penetrated through filters moreefficiently than control cells; (b) fascin1-expressing cells had ahigher ability to disseminate to the lungs and form metastases oninjection into the tail vein of mice; and (c) fascin1 was expressed athigh levels in human colon carcinomas, most significantly incancer cells at the invasive front of tumors.

The dissemination of tumor cells requiring cell migration andinvasion capacities is a prerequisite for metastasis. Such change/sin cell phenotype could be achieved either by loss of epithelialcharacteristics and migration of individual cells, as during EMT(11, 39), or by the migration of sheets of attached cells, in a processknown as collective cell migration (40). Although EMT-like changesare strongly implicated in metastasis, a significant number ofcancers (41), including colon carcinomas (analyzed in this study),which were characterized by all pathologic criteria (stage andgrade) as invasive and malignant, did not display the molecularsignatures of EMT. Analysis of human colorectal cancer biopsies inour study revealed a phenotype of large, invading tumor fronts withrare microinvasion, corresponding best to the phenotype ofcollective cell migration. This could explain the difference betweenthe observed decrease in E-cadherin reported in a previous study,where mostly small cell aggregates were observed at the invasivefront (33) and our study detecting E-cadherin expression. Moreover,a recent study showed that the molecule podoplanin can inducetumor cell invasion using collective cell migration, without theneed for an EMT-like process (41). Interestingly, both podoplanin

Figure 5. Fascin1 is only observed in poorly differentiated areas of human colon carcinoma liver metastases. Immunohistochemical staining (brown ) of fascin1(first row ) and h-catenin (second row) with counterstaining (blue ). Insets, enlarged areas on the right. Bar, 100 Am. A, no fascin1 expression was observed in coloncancer epithelial cells of well-differentiated liver metastases. Bar, 25 Am. B, poorly differentiated areas of a liver metastasis were fascin1 positive and containedcells with nuclear h-catenin (arrows ).

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and fascin1, besides being localized at the invasive front of tumors,also confer similar cellular characteristics when overexpressed incells leading to increased cell migration (15–17, 41) and invasion(ref. 41 and this study). In addition, podoplanin overexpressioninduces filopodia formation (41) and fascin1 is an essentialstructural protein required for building filopodia (13).

Together, these studies suggest that fascin1 may promote theinvasion and metastasis of cancer cells during the process ofcollective cellmigration by participating in the formation of filopodia,which are guidance organelles for directional cell migration.

Fascin1 is transiently induced in aggressive colon cancercells by the B-catenin-TCF signaling pathway. In the presentstudy, we found that expression of fascin1 in human coloncarcinoma and also in sparse cultures of colon cancer cellscorrelates with the presence of h-catenin in the nuclei of cells,indicative of its activity in h-catenin-TCF signaling. In addition, thefascin1 promoter was highly active in SW480 cells that express

endogenous fascin1, whereas it was silent in HT29 cells thatnormally do not express fascin1. Although Wnt signaling is up-regulated in both cell lines (owing to mutations in APC; ref. 42), theactivity of the synthetic TOPFLASH reporter plasmid in SW480cells is 25-fold higher than in HT29 cells (43).

We showed that the fascin1 gene promoter was activated by theh-catenin-TCF signaling complex in 293 cells transfected withactivated h-catenin and also by the endogenous h-catenin-TCFcomplex of colon cancer cell lines. These results agree with theobservation that depletion of a coactivator of the h-catenin-TCFcomplex, CBP, in NT2 neuronal cells leads to a significant reductionin fascin1 levels (44), and imply that fascin1 is a novel target geneof h-catenin-TCF signaling in colon cancer cells.

We found that fascin1 levels were higher in human primarytumors that developed into distant metastases. Surprisingly,however, distant metastases were fascin1 negative, indicating thatfascin1 expression is tightly regulated in time and space.

Figure 6. Activation of the fascin1 genepromoter by h-catenin-TCF signaling incolon cancer cells. A and B, the fascin1promoter and TOPFLASH reporterplasmids were transfected into 293 cells(A) or SW80 cells (B ) together withh-galactosidase that served to normalizefor transfection efficiency. Cells werecotransfected with either the stabilized,activated S33Y mutant h-catenin (b-cat ),DNTCF4 lacking the h-catenin–bindingdomain, or wt TCF4. Cells were alsocotransfected with these gene reporterplasmids and a dominant-positive LEF1/VP16 construct containing the DNA-binding domain of LEF1 and thetransactivation domain of a viraltranscription factor (VP16). Fold activationfor TOPFLASH was determined afterdividing promoter activity by the valuesobtained with the mutant TOPFLASHconstruct. For the fascin1 promoter, thevalues were divided by those obtained withempty vector. C, the fascin1 genepromoter-GFP reporter plasmid(pFas-GFP ) was transfected into SW480and HT29 cells either alone or togetherwith wt h-catenin or with a stabilized,activated h-catenin deletion mutant(D89-b-cat ). Activity of the promoter wasdetermined as the level of GFP expressedin the transfected cells. Bar, 30 Am.D, schematic representation of putativeTCF-binding sites in the fascin1 genepromoter (left ) and ChIP analysis of TCF4binding to the fascin1 promoter (right ).Anti-TCF4 antibodies or IgG were usedto immunoprecipitate DNA-containingcomplexes. Subsequent PCR was donewith primers complementary to the fascin1promoter region containing the TCF4binding sites. DNA samples were analyzedby agarose gel electrophoresis.





Interestingly, in the two-phase model for h-catenin target geneactivation suggested by Brabletz et al. (11), h-catenin drives theexpression of target genes in a temporally and spatially controlledmanner. In phase I, early in carcinogenesis, low levels of nuclear h-catenin might be sufficient for the persistent activation ofproliferation-associated genes, which are expressed throughouttumor progression. During progression from adenoma to carcino-ma, activation of phase II in tumor development (possibly byaberrant ‘‘environmental’’ signals) drives an increase in nuclear h-catenin, reaching maximal levels in cells at the invasive front ofcarcinomas (32). Such very high h-catenin levels could lead totransient induction of metastasis-associated genes, such as L1-CAM (10), MMP14 (45), and S100A4 (46). These genes are down-regulated later, once cells reach their destination in the targetorgan (by metastasis), when migration and invasion are no longerrequired and the cells resume proliferation and redifferentiation(11). This later step is characterized by reduction in nuclear h-catenin levels and signaling, and its relocalization to adherensjunctions together with E-cadherin. In our current study, we haveshown that h-catenin can regulate fascin1 expression and thisexpression follows the activation status of h-catenin. Ourobservations suggest that fascin1 expression during colorectal

cancer development is tightly regulated in a spatiotemporalmanner, and fascin1 most probably belongs to the special groupof ‘‘second phase’’ h-catenin-TCF target genes.

Cancer metastasis is the least understood aspect of this diseaseand remains a tremendous challenge for drug discovery. Keymolecules involved in filopodia formation, such as fascin1, andthose involved in the regulation of its expression could serve aspotential novel targets for prognosis and treatment of metastaticcolorectal cancer.

Acknowledgments

Received 3/9/2007; revised 5/4/2007; accepted 5/9/2007.Grant support: Human Frontiers Science Program Organization (D. Vignjevic),

Institut National du Cancer PL043 and Association pour la Recherche sur le Cancer2976 (S. Robine), and The Israel Science Foundation and The Delores and Eugene M.Zemsky Weizmann-Johns Hopkins Research Program (A. Ben-Ze’ev).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. A. Reske-Kunz (University of Mainz, Mainz, Germany), M. Arpin(Institut Curie, Paris, France), C. Perret (Institut Cochin, Paris, France), M.A. Buendia(Institut Pasteur, Paris, France), and H. Clevers (Hubrecht Institut, Utrecht, TheNetherlands) for generous gifts of reagents; J. Peloquin, G. Montagnac, and R. Zaarourfor careful reading of the manuscript; Z. Maciorowski for help with FACS sorting; F.Valenty for tissue sample processing; and C. Marthen for qRT-PCR sample preparation.

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2007;67:6844-6853. Cancer Res Danijela Vignjevic, Marie Schoumacher, Nancy Gavert, et al. Expressed at the Invasive Front of Human Colon Cancer

-Catenin-TCF Signaling, IsβFascin, a Novel Target of

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Fascin,aNovelTargetof B-Catenin-TCFSignaling ... · Fascin,aNovelTargetofB-Catenin-TCFSignaling, IsExpressedattheInvasiveFrontof HumanColonCancer DanijelaVignjevic,1 MarieSchoumacher,1