Fascin, a Novel Target of B-Catenin-TCF Signaling, Is Expressed at the Invasive Front of Human Colon Cancer

Research Article

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.

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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 carried out by Nucleofector (Amaxa) and of 293T cellsusingcalcium of 10 (4 weeksold) mice were usedfor each cell type. HT29 cells(10 6) stably phosphate. In transactivation assays, 0.25 Ag h-galactosidase plasmid was expressing either EGFP or fascin-EGFP in 100 AL PBS were injected into the cotransfected with 1 Ag reporter plasmid and 2 Ag of either h-catenin or tail vein. Mice were sacrificed 3 weeks after injection, the left lung lobes cadherin tail constructs. Cells were plated in duplicate and lysed after 48 h, were embedded, and one section every 100 Am wasstained with H&E and luciferase and h-galactosidase levels were determined by enzyme assay according to standard protocols. Metastasis was defined as a group of more kits(Promega). Luciferaseactivity wasnormalized to h-galactosidase than five enterocytes. The intestinal origin of cells was confirmed by villin activity as an internal transfection control. Induction levels of the fascin1 immunostaining. 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 Results fluorescence-activated cell sorting (FACS) and maintained as stable lines Fascin1 expression promotes cell migration, invasion, and with 0.2 mg/mL G418. cell dissemination. Fascin1 is expressed in highly aggressive Transient transfection of HCT116 cells with 30 nmol/L siRNA against carcinomas of various origins. Because fascin1 is required for ¶ ¶ fascin1 (5 -CAAAGACUCCACAGGCAAAUU-3 ), produced by Dharmacon, filopodia formation (13) and these organelles are considered was carried out using LipofectAMINE RNAiMAX (Invitrogen). Cells lysed necessary for directional cell movement, we investigated whether 3 days after transfection were analyzed by Western blot analysis using NIH ImageJ software. Reduction in protein levels was normalized to the loading fascin1 plays a role in colorectal cancer metastasis by promoting control (tubulin) and mock-transfected cells. migratory and invasive capabilities in tumor cells. We expressed Chromatin immunoprecipitation assay. Chromatin immunoprecipi- fascin1 in HT29 human colon cancer cells that do not normally tation (ChIP) assays were done using a ChIP assay kit according to the express fascin1, to levels as high as those found in another human manufacturer’s instructions (Upstate Biotechnology). Rabbit anti-TCF4 or colorectal cancer cell line, SW480 (Fig. 1A). On the dorsal side of rabbit IgG were used to immunoprecipitate DNA-containing complexes. the cells, fascin1 was recruited to microvilli-like structures (Fig. 1A) PCR wasdone with primerscomplementary to the fascin1 promoter region. that were also present in nontransfected cells (Supplementary Fig. The primer sequences are given in Supplementary Table S2. S1A). However, on the ventral side of HT29 cells, the expression of Immunofluorescence and immunohistochemistry. Evaluation of fascin1 induced filopodia formation (Fig. 1A), which were not fascin1 expression by immunohistochemistry was done on two tissue A arrays, containing 144 colon carcinoma samples, CO802 and CO641 present in wt cells (Supplementary Fig. S1 ). (Euromedex), and 34 colon adenocarcinomasobtained from surgical We also examined the proliferation of cells overexpressing fascin1 resection and/or biopsy specimens at The Curie Institute Hospital (Paris, and found that fascin1-expressing cells and control cells have a France). Staining of >10% of tumor cellswasscored aspositive similar growth rate (population doubling time of 31.2 F 7.2 h for immunoreactivity. Tumor staging was done using the tumor-node- HT29 cells, 28.8 F 4.8 h for HT29 cells expressing GFP,28.8 F 7.2 h for metastasis staging system according to the WHO protocol. Immunohisto- cells expressing fascin1-GFP, and 31.2 F 9.6 h for cells expressing chemistry for h-catenin and fascin1 was done as previously (16, 32). fascin1-IRES-GFP). In addition, fascin1-expressing and control cells Immunostaining of cell culture samples was done as described (13). The were s.c. injected into SCID mice and tumor size was measured every detailsof the procedure are given in Supplementary Data. 2 to 3 daysover a 4-week period, but no significantdifferencesin Quantitative real-time PCR. Tissue samples for quantitative real-time tumor volume were detected (Supplementary Fig. S1B). Thus, forced PCR (qRT-PCR) analysis of fascin1 expression were obtained at Klinikum RechtsDer Isar(Munich, Germany). cDNA preparation wasdone according expression of fascin1 does not affect cell proliferation. to standard procedures. Expression of fascin1 was determined using Next, we examined the effect of fascin1 expression on the hypoxanthine phosphoribosyltransferase as internal reference by ABI Prism motility of HT29 cellstoward medium containing growth factorsin 7300 (Applied Biosystems) and SYBR Green I. The values were calibrated to a Transwell filter assay. We found that fascin1-expressing cells were www.aacrjournals.org 6845 Cancer Res 2007; 67: (14). July 15, 2007

Figure 1. Expression of fascin1 enhances cell invasion and metastasis to the lung in experimental animal models. A, top, expression of fascin1 in SW480 and HT29 cells was compared (by Western blotting) with that in HT29 cells stably expressing GFP, fascin1 tagged with GFP, or fascin1 and GFP expressed individually (fascin-IRES-GFP). Bottom, HT29 cells expressing fascin1-GFP develop filopodia at the ventral side and numerous ‘‘spikes’’ at the dorsal side. B, invasion potential of colon cancer HT29 cells stably expressing fascin1 (fascin1-GFP or fascin1-IRES- GFP) or GFP alone and nontransfected HT29 cells was tested in Transwell analysis. Fascin1-expressing HT29 cells displayed enhanced invasion through Matrigel-coated filters. Columns, mean; bars, SE. C, left, the ability of HT29 cells (expressing GFP or fascin1-GFP) to form lung metastases was determined by tail vein injection of mice with 106 cells per mouse and by counting the number of metastatic lesions in the lungs after 4 wks. Columns, mean (n = 10); bars, SE. Right, histologic staining (H&E) of serial sections from lungs of animals injected with HT29 cells expressing fascin1-GFP and immunostained with anti-villin and anti-fascin1 antibodies. Bar, 25 Am. D, invasion potential of nontransfected and fascin1-depleted colon cancer HCT116 cells was tested in Transwell analysis. Suppression of fascin1 expression was analyzed by Western blot. Fascin1- depleted cells displayed reduced invasion property through Matrigel-coated filters. Columns, mean; bars, SE.

six times more motile compared with untransfected cells or to cells suggesting that fascin1-transfected cells gained extensive extrava- transfected with GFP alone (Supplementary Fig. S1C) in agreement sation ability. In addition, 50% of the mice injected with fascin1- with previousobservations(15–17). In addition, we examined expressing cells became severely paralysed compared with only 1 of whether fascin1 has also a role in invasion and metastasis. Fascin1- 10 mice injected with cells expressing GFP alone. X-ray analysis transfected cells were about thrice more invasive through Matrigel- revealed that the paralysis was not caused by bone metastasis but coated filters(Fig. 1 B). Moreover, cellsi.v. injected into the tail vein rather by the development of tumors along the spine with invasion of SCID mice displayed a 10 times higher ability to disseminate and to the back muscle (data not shown). Next, we took the opposite form metastases in the lungs of mice (Fig. 1C). These tumors were strategy: using siRNA, we suppressed fascin1 levels in HCT116 cells positive for fascin1 and villin (a marker for intestinal cells) (which normally express fascin1) and tested their invasion confirming that they were derived from the injected cells(Fig. 1 C). potential in transfilter assays (Fig. 1D). We found that in cells Histologic analysis of such micrometastases confirmed that the where fascin1 levels were suppressed by about 90%, their invasion metastatic lesions replaced large areas of the lung parenchyma, capacity wasreduced three-fold.

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Together, these results imply that fascin1 expression in human (Fig. 2C), its expression became prominent in ‘‘high-grade’’ tumors colon cancer cellsplaysan important role in their motile, invasive, that are characterized by loss of normal cellular differentiation and metastatic capacities. (anaplasia) and poorly defined margins, or diffuse spread, which Fascin1 is overexpressed in high-grade and late-stage often precluded complete surgical resection. In addition, we found human colon carcinoma. Fascin1 is absent from normal that fascin1 was expressed in a high percentage of stage III and IV epithelial cells, whereas high levels of fascin1 expression were tumors(Fig. 2 B). Both stages were characterized by extensive reported in many typesof cancer cells,including colorectal cancer invasion of the primary tumor into the submucosa, muscularis cells (16, 27, 28). Because the treatment and prognosis of colorectal propria, or through the wall of the colon, and also by invasion to cancer patientsdependson tumor grade (the degree of primary nearby lymph nodes at stage III and to distant metastases in stage tumor differentiation) and tumor-node-metastasis stage (how IV tumors. A correlative analysis between fascin1 expression in widespread the cancer is at the time of diagnosis), we first primary tumors and formation of metastases in the corresponding determined if, and at which stage, fascin1 expression is induced in patientsrevealed that in patientswith lymph node and/or liver human colon cancer tissue. We examined fascin1 RNA expression metastases, the primary tumors were more often fascin1 positive in human colon carcinomasusingqRT-PCR. We found that (64% and 67%, respectively) compared with primary tumors of whereas normal tissue was essentially negative for fascin1 RNA patients without metastases at surgery (28% and 40%, respectively; (n = 10; with mean expression of 1.8 F 2.2 RU), the expression of Fig. 2D; Supplementary Table S1). fascin1 was significantly elevated in carcinoma tissue when We conclude that fascin1 expression correlates with aggressive- compared with normal epithelium, and thisincreasewastumor ness of human colorectal cancer and that the presence of fascin1 in stage dependent (Fig. 2A). Fascin1 RNA levels remained unchanged primary tumorshaspredictive value in determining the incidence in benign lesions and increased slightly in early tumor stages (2 of of metastasis. 10 tumors were fascin1 positive, 3.8 F 3.8 RU) but were elevated Fascin1 is expressed at the invasive front of human colon significantly in stage II and III tumors [50% of the tumors were carcinomas but is absent in metastases. Most solid tumors, fascin1 positive in each group; 19.4 F 14.9 RU (n = 10), 27.7 F 31.9 including colorectal cancer, are not homogeneousoften maintain- RU (n = 10), and 7.2 F 9.3 RU (n = 16), respectively], correlating ing a central more differentiated part and an invasive front that with a more invasive phenotype and metastasis to lymph nodes. differsin cell morphology and molecular composition(reviewed in To determine fascin1 protein levels in tumors, we did ref. 11). Asa model for such differencesin cell morphology and immunohistochemical analyses of paraffin-embedded tissue sec- gene regulation, we used the density-dependent phenotypic tions of two tissue arrays containing 118 cases of human colon conversion displayed by the SW480 colon cancer cell line (10). cancer derived from surgical resections (Supplementary Table S1). These cells mimic the changes in h-catenin localization associated Although fascin1 was expressed in a subset of ‘‘low-grade’’ tumors with the position of the cells in the tumor (10, 33, 34). h-Catenin is

Figure 2. Fascin1 expression correlates with the aggressiveness of human colon carcinoma. A, fascin1 expression in normal colon tissue, adenoma, and carcinoma at different stages was evaluated by qRT-PCR. Each dot represents one patient. The threshold was set at the mean expression level in normal tissue plus thrice the SD (expression level equal to 8.3). B and C, fascin1 expression was evaluated by immunohistochemistry (IH) at different tumor stages (I–IV; B) and different tumor grades (G1–G4; C). The total number of carcinomas analyzed was 82 and 148, respectively. D, fascin1 expression in the primary tumor correlated with the presence of metastases in lymph nodes and in the liver. The total number of carcinomas analyzed was 79. Black columns, primary tumors from patients who did not have metastases in lymph nodes (N0) and/or the liver (M0). Gray columns, primary tumors from patients who had metastases in lymph node/s (N1 and N2) and/or the liver (M1).

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Figure 3. Fascin is expressed in tumor cells at the invasive front of colon carcinomas. Immunohistochemical staining (brown) for fascin1 (first column) and h-catenin (second column) in adjacent serial sections of human colon cancer tissue, with nuclear counterstaining (blue). A, overviewof the normal tissue showing no fascin1 expression in epithelial cells of the mucosa displaying membranal h-catenin. Stromal cells were positive for fascin1 staining. Bar, 50 Am. B, overviewof the invasive front of colon carcinoma showing restricted fascin1 expression in carcinomatous cells located at the invasive border of tumor and stroma (arrowhead), whereas the tumor cells located at the center of the tumor (star) were fascin1 negative. Note the stronger staining for h-catenin in the membrane and in the cytoplasm of these cells. Bar, 50 Am. C, higher magnification of the boxed area at the center of the tumor in (B). In the tumor center, h-catenin is present at the membrane, but no fascin1 expression was observed in epithelial tumor cells. Bar, 25 Am. D, higher magnification of the boxed area at the invasive front of the tumor shown in (B). h-Catenin was detected in the membrane, cytoplasm, and also in some nuclei (arrows) and fascin1 was expressed in tumor cells. Bar, 25 Am.

mostly present at cell-cell contacts in dense cell cultures series of 34 patients, to evaluate the expression and localization of (Supplementary Fig. S2A), similar to cells in the more differentiated fascin1 in primary human colon cancers. Serial sections of these central area of tumors. In sparse cultures of SW480 cells because tumors were immunostained with antibodies against h-catenin and they have mutant APC, wt h-catenin accumulatesin the nuclei of fascin1. In normal colon epithelium, h-catenin staining was the cells(Supplementary Fig. S2 A) mimicking h-catenin localiza- observed mostly at cell-cell contact sites (9, 10), whereas fascin1 tion in cellsat the invasivefront of tumors(33). When comparing staining was only detected in infiltrating stromal cells, but not in the expression of fascin1 in sparse and dense SW480 cell cultures, enterocytes(Fig. 3 A). In the central more differentiated areasof we found that in sparse cell cultures displaying nuclear h-catenin, tumors, h-catenin staining was detected in the membrane and in fascin1 expression was about twice higher than in dense cultures the cytoplasm with no nuclear staining (Fig. 3B and C). Fascin1 was (Supplementary Fig. S2B). detected in only 22% of differentiated colon carcinomas(Fig. 3 B Next, we did immunohistologic staining of sections derived from and C). In contrast, the invasive front of tumors displayed surgical resections of adenocarcinoma tumors from an unselected cytoplasmic and nuclear h-catenin localization (Fig. 3D, arrows)

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. The Role of Fascin in the Colon Cancer and was associated with strong fascin1 expression (Fig. 3B and D). metastases of one patient. The fascin1-negative metastases were all This phenotype was apparent in 61% of the tumors. Fascin1 well differentiated and displayed glandular morphology (Fig. 5A), expression was strong in sheets of invading tumor cells and also in recapitulating the organization characteristic to the differentiated disseminating single tumor cells. center of the primary tumor, where h-catenin islocalized at the Disruption of cell-cell contacts and the gain of cell motility in membrane and in the cytoplasm. The single fascin1-positive liver invasive tumors is suggested to be achieved by a process metastasis seemed undifferentiated and h-catenin accumulated in reminiscent of epithelial to mesenchymal transition (EMT; ref. these cells in the cytoplasm as well as in the nuclei of cells (Fig. 5B). 35). This includes down-regulation of epithelial-specific proteins, We conclude that fascin1 is expressed at the invasive front of including E-cadherin and cytokeratins, and expression of mesen- human colon carcinomas, suggesting that fascin1 may participate chymal-specific molecules, such as vimentin and fibronectin. We in tumor cell invasion. However, fascin1 was mostly absent from immunostained colorectal cancer tissue samples (n = 10) with distant metastases, suggesting a tight regulation of fascin1 various EMT markers and found strong E-cadherin staining at the expression during tumorigenesis. membrane of most tumor cells, including fascin1-positive cells Fascin1 expression is induced by B-catenin-TCF signaling. (Fig. 4). The tumor cellswere alsovillin and cytokeratin 20 ( CK20) The association of fascin1 expression with nuclear localization of positive, whereas vimentin was only present in stromal cells h-catenin at the invasive front of colon cancer tissue and in (Fig. 4A). Thus, fascin1 was expressed at the invasive front of cultured colon cancer cellsprompted usto investigatewhether the colorectal cancer tissue that did not display EMT. fascin1 gene isa novel target of h-catenin signaling. The activity of We also detected fascin1 enrichment in the stromal compart- the fascin1 gene promoter was shown previously to correlate with ment, in the extracellular matrix, mature dendritic cells, fibroblasts, fascin1 protein expression: whereas the fascin1 promoter issilent and blood vessels (Fig. 4B), in agreement with a previousreport in keratinocytesand in immature dendritic cells,which do not (27). This staining was strongest in the stroma adjacent to the express fascin1 protein, it is highly active in fascin1-positive mature invading tumor front but wasindependent of whether the dendritic cells and neuroblastoma (30). Cotransfection of 293 cells epithelial cells in the tumor itself were fascin1 positive (Fig. 4B) with the fascin1 promoter luciferase reporter plasmid, or the or fascin1 negative (Fig. 4B) and thisphenotype wasobserved in synthetic TCF reporter plasmid TOPFLASH, and a point mutant >90% of invasive colon carcinomas. (S33Y) stabilized h-catenin construct resulted in a 12-fold Because fascin1 expression in primary tumors correlated with activation of the fascin1 promoter reporter and a close to 50-fold the presence of metastases in the respective patients, we examined activation of TOPFLASH (Fig. 6A). TOPFLASH served as control in whether fascin1 was also expressed in metastatic tissue of colon this experiments because it behaves similarly to the promoters of carcinoma patients. Using qRT-PCR analysis, we found that all previously characterized h-catenin target genessuch as cyclin D1 human liver tissue samples from colon cancer patients were (6), Nr-CAM (36), or L1-CAM (10). The involvement of TCF/LEF negative for fascin1, independently of whether the patients had or factors in fascin1 transactivation is suggested by the inhibition of lacked liver metastasis (fascin1 expression was 1.29 F 0.57 RU for fascin1 promoter activation using dominant-negative TCF4 normal liver and 0.58 F 0.46 RU for liver containing metastases, (DNTCF), whereas a dominant-positive LEF construct, containing with 10 samples analyzed in each group). We also examined by the DNA-binding domain of LEF linked to the transactivation immunohistochemistry tissue samples from seven patients (that domain of viral VP16 (LEF/VP16), activated the fascin1 promoter included 7 primary tumors, 4 lymph nodes, and 5 liver metastases) (Fig. 6A). Transactivation of the fascin1 promoter by the and detected fascin1 at the invasive front of six primary tumors, endogenous h-catenin/TCF complex in SW480 colon cancer cells but fascin1 was only detected in the lymph node and liver wasenhanced 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, immunofluorescent staining for fascin1 in fascin1-positive (top) and fascin1-negative (bottom) epithelial compartments of colon cancer tissue. Epithelial cells, blood vessels, and dendritic cells. www.aacrjournals.org 6849 Cancer Res 2007; 67: (14). July 15, 2007

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 colon cancer epithelial cells of well-differentiated liver metastases. Bar, 25 Am. B, poorly differentiated areas of a liver metastasis were fascin1 positive and contained cells with nuclear h-catenin (arrows).

TCF4 (DNTCF; Fig. 6B). We also investigated whether the fascin1 shown that the migration of certain cells could be blocked by gene promoter is active in colon cancer cells using a fascin1 antibodies that perturb actin-fascin1 interactions (38), but fascin1 promoter-GFP reporter plasmid. In SW480 cells that express overexpression in other cell types did not display a clear correlation endogenousfascin1,the promoter washighly active, whereasthis with the rate of cell locomotion (16). However, several studies promoter reporter wassilentin HT29 cellsthat normally do not reported that fascin1 significantly increases cell migration in express fascin1 (Fig. 6C). To test whether the fascin1 gene is transfilter haptotactic assays (refs. 15–17 and this study), suggest- activated by h-catenin signaling, we also cotransfected HT29 cells ing that fascin1 can enhance the directional motility of cells. We with wt, or the activated stable form of h-catenin in which the first showed here that the expression of fascin1 promotes the invasive 89 amino acidswere deleted ( D89-h-cat), together with the fascin1 capability of colon cancer cellsby showingthat ( a) cultured promoter-GFP reporter plasmid. A GFP signal was detected in colorectal cancer cells expressing fascin1 degraded Matrigel, a f50% of cells expressing D89h-cat, but not in wt h-catenin– basement membrane matrix and penetrated through filters more b expressing cells (Fig. 6C). We identified five putative TCF-binding efficiently than control cells; ( ) fascin1-expressing cells had a sites in the 5-untranslated region of the mouse fascin1 promoter higher ability to disseminate to the lungs and form metastases on c (Fig. 6D). In ChIP analyses, we PCR amplified three fascin1-specific injection into the tail vein of mice; and ( ) fascin1 was expressed at promoter sequences from chromatin immunoprecipitates using high levels in human colon carcinomas, most significantly in the mouse colon cancer CT26 cell line using TCF4-specific cancer cells at the invasive front of tumors. antibodies(Fig. 6 D). No fascin1-specific promoter sequences were The dissemination of tumor cells requiring cell migration and amplified when control IgG wasusedfor precipitation. Taken invasion capacities is a prerequisite for metastasis. Such change/s together, these data indicate that the fascin1 gene isa direct in cell phenotype could be achieved either by loss of epithelial transcriptional target of h-catenin/TCF signaling. characteristics and migration of individual cells, as during EMT These results suggest that h-catenin-TCF signaling is involved in (11, 39), or by the migration of sheets of attached cells, in a process the regulation of fascin1 gene transcription in human colorectal known ascollective cell migration (40). Although EMT-like changes cancer cells. are strongly implicated in metastasis, a significant number of cancers(41), including colon carcinomas(analyzed in thisstudy), which were characterized by all pathologic criteria (stage and Discussion grade) as invasive and malignant, did not display the molecular The role of fascin1 in metastasis of colorectal cancer cells signatures of EMT. Analysis of human colorectal cancer biopsies in involves the promotion of a migratory and invasive phenotype our study revealed a phenotype of large, invading tumor fronts with through filopodia formation. The actin bundling protein fascin1 rare microinvasion, corresponding best to the phenotype of is expressed in human colon carcinomas in a grade- and stage- collective cell migration. Thiscould explain the difference between dependent manner but isabsentfrom normal colonic epithelium. the observed decrease in E-cadherin reported in a previous study, In addition, consistent with our results, fascin1 up-regulation in where mostly small cell aggregates were observed at the invasive many other typesof cancer wasfound to correlate with poor front (33) and our study detecting E-cadherin expression. Moreover, prognosis and with decreased survival (19, 27, 28, 37). a recent study showed that the molecule podoplanin can induce The role of fascin1 in cell migration on planar substrata in the tumor cell invasion using collective cell migration, without the absence of a gradient of chemoattractant is unclear. It has been need for an EMT-like process (41). Interestingly, both podoplanin

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. The Role of Fascin in the Colon Cancer and fascin1, besides being localized at the invasive front of tumors, endogenous fascin1, whereas it was silent in HT29 cells that also confer similar cellular characteristics when overexpressed in normally do not express fascin1. Although Wnt signaling is up- cells leading to increased cell migration (15–17, 41) and invasion regulated in both cell lines(owing to mutationsin APC; ref. 42), the (ref. 41 and this study). In addition, podoplanin overexpression activity of the synthetic TOPFLASH reporter plasmid in SW480 induces filopodia formation (41) and fascin1 is an essential cellsis25-fold higher than in HT29 cells(43). structural protein required for building filopodia (13). We showed that the fascin1 gene promoter wasactivated by the Together, these studies suggest that fascin1 may promote the h-catenin-TCF signaling complex in 293 cells transfected with invasion and metastasis of cancer cells during the process of activated h-catenin and also by the endogenous h-catenin-TCF collective cell migration by participating in the formation of filopodia, complex of colon cancer cell lines. These results agree with the which are guidance organellesfor directional cell migration. observation that depletion of a coactivator of the h-catenin-TCF Fascin1 is transiently induced in aggressive colon cancer complex, CBP, in NT2 neuronal cellsleadsto a significantreduction cells by the B-catenin-TCF signaling pathway. In the present in fascin1 levels (44), and imply that fascin1 isa novel target gene study, we found that expression of fascin1 in human colon of h-catenin-TCF signaling in colon cancer cells. carcinoma and also in sparse cultures of colon cancer cells We found that fascin1 levels were higher in human primary correlateswith the presenceof h-catenin in the nuclei of cells, tumors that developed into distant metastases. Surprisingly, indicative of itsactivity in h-catenin-TCF signaling. In addition, the however, distant metastases were fascin1 negative, indicating that fascin1 promoter was highly active in SW480 cells that express fascin1 expression is tightly regulated in time and space.

Figure 6. Activation of the fascin1 gene promoter by h-catenin-TCF signaling in colon cancer cells. A and B, the fascin1 promoter and TOPFLASH reporter plasmids were transfected into 293 cells (A) or SW80 cells (B) together with h-galactosidase that served to normalize for transfection efficiency. Cells were cotransfected with either the stabilized, activated S33Y mutant h-catenin (b-cat), DNTCF4 lacking the h-catenin–binding domain, or wt TCF4. Cells were also cotransfected with these gene reporter plasmids and a dominant-positive LEF1/ VP16 construct containing the DNA- binding domain of LEF1 and the transactivation domain of a viral transcription factor (VP16). Fold activation for TOPFLASH was determined after dividing promoter activity by the values obtained with the mutant TOPFLASH construct. For the fascin1 promoter, the values were divided by those obtained with empty vector. C, the fascin1 gene promoter-GFP reporter plasmid (pFas-GFP) was transfected into SW480 and HT29 cells either alone or together with wt h-catenin or with a stabilized, activated h-catenin deletion mutant (D89-b-cat). Activity of the promoter was determined as the level of GFP expressed in the transfected cells. Bar, 30 Am. D, schematic representation of putative TCF-binding sites in the fascin1 gene promoter (left) and ChIP analysis of TCF4 binding to the fascin1 promoter (right). Anti-TCF4 antibodies or IgG were used to immunoprecipitate DNA-containing complexes. Subsequent PCR was done with primers complementary to the fascin1 promoter region containing the TCF4 binding sites. DNA samples were analyzed by agarose gel electrophoresis.

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Interestingly, in the two-phase model for h-catenin target gene cancer development istightly regulated in a spatiotemporal activation suggested by Brabletz et al. (11), h-catenin drivesthe manner, and fascin1 most probably belongs to the special group expression of target genes in a temporally and spatially controlled of ‘‘second phase’’ h-catenin-TCF target genes. manner. In phase I, early in carcinogenesis, low levels of nuclear h- Cancer metastasis is the least understood aspect of this disease catenin might be sufficient for the persistent activation of and remainsa tremendouschallenge for drug discovery. Key proliferation-associated genes, which are expressed throughout molecules involved in filopodia formation, such as fascin1, and tumor progression. During progression from adenoma to carcino- those involved in the regulation of its expression could serve as ma, activation of phase II in tumor development (possibly by potential novel targets for prognosis and treatment of metastatic aberrant ‘‘environmental’’ signals) drives an increase in nuclear h- colorectal cancer. catenin, reaching maximal levelsin cellsat the invasivefront of h carcinomas(32). Such very high -catenin levelscould lead to Acknowledgments transient induction of metastasis-associated genes, such as L1- CAM MMP14 S100A4 Received 3/9/2007; revised 5/4/2007; accepted 5/9/2007. (10), (45), and (46). These genes are down- Grant support: Human FrontiersScience Program Organization (D. Vignjevic), regulated later, once cellsreach their destinationin the target Institut National du Cancer PL043 and Association pour la Recherche sur le Cancer organ (by metastasis), when migration and invasion are no longer 2976 (S. Robine), and The Israel Science Foundation and The Delores and Eugene M. Zemsky Weizmann-Johns Hopkins Research Program (A. Ben-Ze’ev). required and the cellsresumeproliferation and redifferentiation The costs of publication of this article were defrayed in part by the payment of page (11). Thislater step ischaracterized by reduction in nuclear h- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. catenin levelsand signaling,and itsrelocalization to adherens We thank Drs. A. Reske-Kunz (University of Mainz, Mainz, Germany), M. Arpin junctionstogether with E-cadherin. In our current study, we have (Institut Curie, Paris, France), C. Perret (Institut Cochin, Paris, France), M.A. Buendia shown that h-catenin can regulate fascin1 expression and this (Institut Pasteur, Paris, France), and H. Clevers (Hubrecht Institut, Utrecht, The h Netherlands) for generous gifts of reagents; J. Peloquin, G. Montagnac, and R. Zaarour expression follows the activation status of -catenin. Our for careful reading of the manuscript; Z. Maciorowski for help with FACS sorting; F. observations suggest that fascin1 expression during colorectal Valenty for tissue sample processing; and C. Marthen for qRT-PCR sample preparation.

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