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Fascin Protein Is Critical for Transforming Growth Factor β Protein-induced Invasion and Filopodia Formation in Spindle-shaped Tumor Cells *

Fascin Protein Is Critical for Transforming Growth Factor β Protein-induced Invasion and... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 45, pp. 38865–38875, November 11, 2011 © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Fascin Protein Is Critical for Transforming Growth Factor Protein-induced Invasion and Filopodia Formation in Spindle-shaped Tumor Cells Received for publication, June 13, 2011, and in revised form, September 12, 2011 Published, JBC Papers in Press, September 13, 2011, DOI 10.1074/jbc.M111.270413 ‡§1 ‡§1 ¶ ‡§ ‡§ ‡§ Jianwei Sun , Huifang He , Yin Xiong , Shuang Lu , Junling Shen , Anna Cheng , Wei-Chiao Chang**, ¶ ‡‡‡ ‡§2 Ming-Feng Hou**, Johnathan M. Lancaster , Minjung Kim , and Shengyu Yang ‡ § ¶ From the Comprehensive Melanoma Research Center, Department of Tumor Biology, Department of Women’s Oncology, and ‡‡ Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, the Qingdao Agricultural University, Qingdao 266109, China,and the **Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan Background: Fascin is a pro-metastasis actin-bundling protein overexpressed in metastatic tumors. Results: TGF induced fascin expression in spindle tumor cells through Smads. Conclusion: Fascin is a TGF target gene essential for the pro-invasion activity of TGF. Significance: Our data shed new light on how TGF dysregulates actin cytoskeleton to promote tumor metastasis. Fascin, an actin-bundling protein overexpressed in all carci- protrusions, it is crucial to cross-link actin filaments into bun- nomas, has been associated with poor prognosis, shorter sur- dles as individual actin filaments are flexible. Cross-linking by vival, and more metastatic diseases. It is believed that fascin bundling protein provides the essential rigidity to counter the facilitates tumor metastasis by promoting the formation of inva- compressive forces from the plasma membrane (3). sive membrane protrusions. However, the mechanisms by Fascin is an actin-bundling protein critical for tumor metas- which fascin is overexpressed in tumors are not clear. TGF is a tasis (4–7). Expression levels of fascin are very low or not cytokine secreted by tumor and mesenchymal cells and pro- detected in normal epithelia, but are highly elevated in malig- motes metastasis in many late stage tumors. The pro-metastasis nant tumors (4). Overexpression of fascin protein is associated mechanisms of TGF remain to be fully elucidated. Here we with poor prognosis in patients (7–12). Knockdown of fascin demonstrated that TGF induced fascin expression in spindle- expression inhibited tumor cell migration and invasion in vitro shaped tumor cells through the canonical Smad-dependent and decreased tumor metastasis in mouse models. Moreover, pathway. Fascin was critical for TGF-promoted filopodia for- ectopic expression of fascin promoted tumor cell invasion and mation, migration, and invasion in spindle tumor cells. More metastasis (5, 6, 13). The causal role of fascin overexpression in importantly, fascin expression significantly correlates with tumor metastasis is well established; however, the molecular TGF1 and TGF receptor I levels in a cohort of primary breast mechanisms underlying elevated fascin level in metastatic tumor samples. Our results indicate that elevated TGF level in tumors are not clear. the tumor microenvironment may be responsible for fascin TGF is a cytokine in the tumor microenvironment that reg- overexpression in some of the metastatic tumors. Our data also ulates various tumor progressions in a context-dependent suggest that fascin could play a central role in TGF-promoted manner (14). In early stage tumors, TGF is a potent prolifera- tumor metastasis. tion inhibitor that deters tumor growth; however, late stage tumors are often able to evade the growth inhibition and secrete elevated levels of TGF to promote metastasis (15). The mech- One essential characteristic of metastatic cancer cells is anisms by which late stage tumors use TGF signaling to pro- enhanced motility, which facilitates the infiltration of meta- mote tumor spreading are largely unknown. static cells into lymphatic and blood vessels and extravasation Accompanying the loss of capacity to differentiate during out of the circulation (1). The forces that drive tumor cell tumor progression, tumor cells undergo a transition in gene migration and invasion are provided by the actin cytoskeleton expression, reorganization of cytoskeleton, and acquisition of underlying the critical membrane protrusions in migrating spindle cell morphology (16). Tumors with spindle cell mor- tumor cells (2). To efficiently drive the formation of membrane phology were characterized as highly malignant and invasive (17). In breast cancer, fascin is overexpressed in the estrogen * This work was supported in part by an Institutional Research Grant from the American Cancer Society (IRG-93-032-16), a Career Development Award receptor-negative, basal-like subgroup (11), a highly metastatic from the Donald A. Adams Comprehensive Melanoma Research Center, group of breast cancers typically with spindle cell morphology and a Milestone Award from Miles for Moffitt Foundation Center (to S. Y.). □ S (18). In melanoma, elongated, spindle-like tumor cells showed The on-line version of this article (available at http://www.jbc.org) contains supplemental supplemental Movies 1 and 2 and Figs. S1–S5. intense fascin staining, whereas rounded, amoeboid-like mela- Both authors contributed equally to this work. noma cells were generally fascin-negative (19). To whom correspondence should be addressed: 12902 Magnolia Dr. SRB-2, Here, we demonstrate that TGF elevates fascin protein Tampa, FL 33612. Tel.: 813-745-8371; Fax: 813-745-3829; E-mail: shengyu. yang@moffitt.org. expression and promotes invasion and filopodia formation in NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 38865 This is an Open Access article under the CC BY license. Fascin in TGF-induced Tumor Cell Invasion tumor cells with spindle morphology, but not in tumor cells were photographed, and the cells on the lower surface of the with epithelial-like, polygonal morphology. We also show that insert were counted. transcription of fascin mRNA induced by TGF is independent Immunofluorescence Microscopy—A549 cells cultured on of de novo protein synthesis but relies on the canonical Smad- collagen-coated glass coverslips were fixed with 3.7% para- dependent pathway. Furthermore, fascin is essential for TGF formaldehyde in PBS for 10 min at room temperature, per- to promote invasion and filopodia formation in spindle tumor meabilized with 0.1% Triton X-100 for 5 min, and then cells. Therefore, our data suggest that fascin is an immediate washed with PBS three times. To block nonspecific binding, TGF target gene essential for its pro-invasion activity. Our the cells were incubated with a solution of PBS containing 1% data also suggest that TGF might be responsible for the fascin bovine serum albumin for 30 min and then incubated with overexpression in some metastatic tumors. Alexa Fluor 594-labeled phalloidin (Invitrogen). The cover- slips were then fixed onto slides and imaged using a Zeiss EXPERIMENTAL PROCEDURES fluorescence microscope. Cell Culture Media—The cell culture media used were Live Cell Imaging—A549 cells with or without TGF pre- DMEM (for MDA-MB-231 and MCF-7), F-12K (for A549), and treatment were plated on collagen-coated glass-bottomed RPMI (for CHL-1, WM115, and H1299). All cell culture media 35-mm tissue culture dishes (MatTek) overnight. The mem- were supplemented with 10% fetal bovine serum and brane protrusion dynamics and cell movement were recorded penicillin/streptomycin. with a differential interference contrast microscopy under a Antibodies—The following antibodies were used in this 40 objective using a Zeiss inverted microscope equipped with study: fascin, Santa Cruz Biotechnology antibody number sc- a live imaging chamber. The temperature and CO concentra- 21743; phospho-ERK1/2, Cell Signaling antibody number 9101; tion in the chamber were maintained at 37 °C and 5%, respec- phospho-c-Jun(Ser-63), Cell Signaling antibody number 9261; tively. Time-lapse images were recorded at 10-s intervals. Smad3, Cell Signaling antibody number 9523; phospho- qRT-PCR—Total RNA was extracted from cultured cells Smad3(Ser-423/425), Cell Signaling antibody number 9520; using TRIzol reagent (Invitrogen), and the reverse transcription GAPDH, Sigma product number G8795. was performed using the iScript cDNA synthesis kit (Bio-Rad). RNA Interference—RNAi of Smad2, Smad3, Smad4, and fas- The quantitative real-time PCR (qRT-PCR) assay was carried cin was performed using pSUPER.Retro.puro vector (Oligoen- out with the Applied Biosystems 7900HT fast real-time PCR gine) encoding small hairpin RNA. The previously reported tar- system using Applied Biosystems SYBR Green PCR master mix. get sequences were used: GGTGGGCAAAGATGAGCTC Primers for qRT-PCR are shown in supplemental Table S1. All (Fascin) (6), GGTGGGCAAAGATGAGCTC (Smad2) (20), reactions were performed in triplicate, and the experiment was GGACGAGGTCTGCGTGAAT (Smad3) (20), and GGTGTG- repeated three times. CAGTTGGAATGTA (Smad4) (20). RESULTS TGF and Inhibitor Treatment—Unless stated otherwise, all TGF Promotes the Migration of Spindle-shaped Tumor Cells— cells were treated with 10 ng/ml TGF1 (Peprotech, Rocky Hill, NJ) in growth medium for 2 days before being used for assays. It was recently reported that exposure to TGF within the Inhibitors, when used, were added together with TGF to tumor microenvironment might predispose tumor cells for metastasis to distant organs (14). To evaluate the effects of growth medium. TGF exposure on the migration of tumor cells, we treated a Transwell Cell Migration Assay—Cells (1  10 ) suspended panel of tumor cell lines with TGF. We used two breast in starvation medium were added to the upper chamber of an insert (6.5-mm diameter, 8-m pore size, BD Biosciences), and cancer lines (MDA-MB-231, and MCF-7), two non-small cell lung cancer lines (A549 and H1299), and two melanoma the insert was placed in a 24-well dish containing starvation lines (CHL-1 and WM115). Three of the six cell lines (MDA- medium with or without 10% FBS (21, 22). Migration assays were carried out for 4–6 h for spindle tumor cells and 12–24 h MB-231, A549, and CHL-1) have spindle cell morphology, for polygonal tumor cells. Cells were fixed with 3.7% formalde- whereas the other three (MCF-7, H1299, and WM115) have polygonal, epithelial-like morphology (Fig. 1A). After 48 h of hyde and stained with crystal violet staining solution, and cells treatment, we found that TGF induced morphology change on the upper side of the insert were removed with a cotton swab. Three randomly selected fields (10 objectives) on the in the three spindle-shaped cell lines, with cells becoming lower side of the insert were photographed, and the migrated more elongated and disperse. Extremely long and finger-like protrusions were also observed in some TGF-pretreated cells were counted. The migration was expressed as the average cells. However, the effects of TGF on the morphology of the number of migrated cells in a field. Cell Invasion Assay—Cells (1 10 three polygonal, epithelial-like tumor cells were much less ) suspended in starvation medium were added to the upper chamber of a Matrigel-coated noticeable (Fig. 1A). Next, we examined the effects of TGF pretreatment on cell insert (6.5-mm diameter, 8-m pore size, BD Biosciences), and migration using the Transwell migration assay. Interestingly, the insert was placed in a 24-well dish containing medium with or without serum. Invasion assays were carried out for 16 h, and TGF priming significantly increased the migration of the cells were fixed with 3.7% formaldehyde. Cells were stained with crystal violet staining solution, and cells on the upper side The abbreviations used are: qRT-PCR, quantitative real-time PCR; CHX, of the insert were removed with a cotton swab. Three randomly RI, TGF receptor I; TRII, TGF receptor II; CREB, cAMP- cycloheximide; T selected fields (10 objectives) on the lower side of the insert response element-binding protein. 38866 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Fascin in TGF-induced Tumor Cell Invasion FIGURE 1. TGF promoted cell migration in spindle-shaped tumor cells. A, morphology changes induced by TGF. Spindle-shaped tumor cells (MDA-MB-231, A549, and CHL-1) became more elongated and scattered after TGF treatment. Tumor cells were cultured in growth medium with or without 10 ng/ml TGF. The cell morphologies were recorded with phase contrast microscopy using a 10 objective. Scale bar,50 m. B, the effects of TGF pretreatment on tumor cell migration. TGF pretreatment promoted cell migration in spindle-shaped tumor cells, but not in polygon-shaped tumor cells. The data presented are mean  S.D. of migrated cells per field from three randomly selected 10 fields. C–F, kymograph analysis of membrane protrusion dynamics in control A549 cell (C and D) and TGF-pretreated A549 cells (E and F); C and E, individual frames from time-lapse movies (supplemental Movies 1 and 2) used to generate the kymographs (D and F). Descending and ascending contours in the kymographs indicate membrane protrusion and withdrawal events, respectively. Arrowheads in D and F indicate new membrane protrusions. G, the extension of membrane protrusions in control and TGF-treated cells. three spindle-shaped tumor cells by about 3–10-fold (Fig. 1B). lamellipodia, turnover of focal adhesions, contraction of cell In sharp contrast, TGF had little effect on (MCF-7 and H1299) body, and retraction of trailing tail (23). To investigate the or slightly inhibited (WM115) the migration of the three poly- mechanisms underlying the enhanced cell migration in TGF- gon-shaped tumor cell lines (Fig. 1B). pretreated spindle tumor cells, we used live cell imaging to To characterize the functional characteristics of spindle- study the membrane protrusion dynamics at the leading shaped and polygon-shaped tumor cells in the absence of edge of migrating cells. As shown in supplemental Movies 1 TGF, we used the Transwell assay to evaluate their mobility and 2, TGF pretreatment induced hyperactive membrane and invasiveness. Tumor cells were allowed to migration/in- protrusion in A549 cells. The membrane protrusion dynam- vade for 6 and 12 h, respectively. As shown in supplemental Fig. ics in the TGF-pretreated cells followed a protrusion-ruf- S1, the three spindle-shaped tumor cell lines migrate and fle-protrusion pattern (supplemental Fig. S2). New lamelli- invade much faster than the three polygonal tumor cells, con- podia extended from the edge of membrane ruffles, sistent with the notion that spindle-shaped tumor cells are sometimes expanding in the space between adjacent filopo- more invasive and metastatic. dia. At the end of the lamellipodium expansion, the edge of TGF Induces Hyperactive Membrane Protrusions—Cell lamellipodia pulled back and formed membrane ruffles. New migration is a multistep process involving the formation of filopodia or lamellipodia would extend from the edge of NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 38867 Fascin in TGF-induced Tumor Cell Invasion membrane ruffles and begin the next protrusion cycle. In contrast, lamellipodia usually pulled back without mem- brane ruffle formation in control cells (supplemental Fig. S2 and supplemental Movies 1 and 2). Next, we employed kymography to analyze the membrane protrusion kinetics in control cells and TGF-pretreated cells. A line was drawn at the leading edge of a migrating cell; inten- sity values along the defined line region in each image of a time-lapse series were extracted and assembled together side- by-side to generate a kymograph montage (Fig. 1, C–F). As shown in the kymograph in Fig. 1, C and D, transient protru- sions frequently extended out from the lamellipodium region of the control A549 cells and quickly withdrew to the starting point, generating spike-like structures on a flat basal line on the kymograph. The leading edge of the control A549 cells barely moved forward during the recording period (Fig. 1, D and G). In contrast, the kymographs of TGF-pretreated cells appeared as stair-like structures, indicating that the lamellipodia in these cells extended in “bursts”; in addition, the newly formed protrusions in TGF-treated cells were able to hold their posi- tions for a period of time until the next extension, instead of withdrawing to the starting point in the control cell. Conse- quently, the leading edges moved steadily forward following each extension (Fig. 1, F and G). The hyperactive membrane FIGURE 2. TGF elevated fascin expression level in spindle tumor cells. A, staining of F-actin in A549 cells with fluorescence-labeled phalloidin. The protrusions may contribute to increased mobility in TGF- weak and wavy filopodia in control cells were indicated by the gray arrow- treated cells. heads. The long filopodia in TGF-treated cells were indicated by the white TGF Induces Actin Cytoskeleton Remodeling—The mor- arrowheads. The scale bar is 10 m. B, quantification of cells with long filopo- dia in A549 cells with TGF or control treatment. Results are mean S.D. from phology changes and hyperactive membrane protrusions sug- 3 experiments. C, TGF treatment increased the protein expression of fascin gested remodeling of the actin cytoskeleton in TGF-treated in spindle tumor cells (MDA-MB-231, A549, and CHL-1), but not in polygonal spindle tumor cells. Therefore, we used fluorescence micros- tumor cells (MCF-7, H1299, and WM115). The protein levels of fascin in control or TGF-treated tumor cells were determined by Western blotting. Results copy to visualize actin filaments in the cells. As shown in Fig. are representatives from 3 independent experiments. D, quantification of the 2A, the phalloidin staining in control A549 cells showed typical Western blotting results in A by densitometry. Results are mean S.D. of 2–5 experiments. E, effects of TGF treatment on the mRNA expression of fascin in cortical actin staining, with the majority of F-actin in the corti- MDA-MB-231, A549, and MCF-7 cells. Results are mean  S.D. of triplicates. cal region. On the outer periphery of the strong actin staining, RU, relative units. numerous weak and wavy filopodia-like protrusions were also observed in most of the control cells (Fig. 2A, gray arrowheads). treatment on fascin protein levels in these tumor cells were In contrast, stress fibers were noted in the majority of the evaluated with Western blotting using a fascin-specific anti- TGF-pretreated A549 cells, consistent with previous observa- body. TGF treatment elevated fascin protein levels by 2–6- tions that TGF promoted stress fiber formation (24, 25). Most fold in the three spindle tumor cells. In sharp contrast, no strikingly, TGF induced very long and straight filopodia TGF-induced fascin expression was observed in any of the (filopodia protruded more than 20 m out of the cell boundary) polygonal tumor cells (Fig. 2, C and D). (Fig. 2A). Typically, several such long filopodia extended To investigate whether TGF regulates the degradation of around the cells, rendering a distinct “spiky” morphology in fascin protein, we used cycloheximide (CHX), a protein syn- TGF-treated A549 cells. The extremely long filopodia were thesis inhibitor, to inhibit the new synthesis of fascin in A549 observed in 70% of TGF-treated cells (110 out of a total of 158 cells and used Western blotting to monitor fascin degrada- cells); in contrast, only less than 5% (8 out of a total of 228 cells) tion at different time points after CHX treatment. No degra- of control cells had such filopodia (Fig. 2B). dation of fascin protein in control cells or in TGF-treated Elevated Fascin Expression in Spindle Tumor Cells after cells was detected even 24 h after CHX treatment, indicating TGF Treatment—Filopodia are finger-like protrusions critical that fascin is a very stable protein (data not shown). Next, we for tumor cell invasion and metastasis (26). Cross-linking of investigated whether TGF regulated fascin expression at parallel actin filaments is considered to be crucial for filopodia the mRNA level in two spindle tumor cell lines (A549 and formation as individual filaments lack the rigidity required to MDA-MB-231 cells) and one polygonal cell line (MCF-7 overcome the compressive forces from plasma membrane (3). cells) using qRT-PCR. Consistent with our Western blot Fascin is a key actin-bundling protein in filopodia (27), and results, we found that TGF treatment elevated fascin fascin overexpression has been reported in all carcinomas mRNA levels in A549 and MDA-MB-231 cells, but not in examined to date (4). The long filopodia in TGF-pretreated MCF-7 cells, suggesting that TGF promoted the transcrip- cells prompted us to examine the possibility that TGF might tion of fascin only in spindle tumor cells, but not in polygonal regulate fascin expression in tumor cells. The effects of TGF tumor cells (Fig. 2E). 38868 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Fascin in TGF-induced Tumor Cell Invasion FIGURE 3. Fascin is a TGF target gene regulated by Smad3 and Smad4. A, inhibition of TGF-induced fascin expression by TRI kinase inhibitor SB431542 in MDA-MB-231 cells and A549 cells. B–D, the effects of MEK inhibitor UO126 (10 M) and JNK inhibitor SP600125 (20 M)onTGF-induced fascin expression at the protein level (B and C) and the mRNA level (D). MDA-MB-231 cells were treated with TGF and the two inhibitors as indicated for6h(B and C)or24h(D), respectively; the mRNA and protein levels were determined through qRT-PCR and Western blotting. Results are mean S.D. from 3 experiments. p-ERK1/2, phospho-ERK1/2; p-c-Jun, phospho-c-Jun; RU, relative units. E and F, the role of Smads in TGF-induced fascin expression. MDA-MB-231 and A549 cells stably expressing Smad3, Smad4, and Smad2 shRNA or control shRNA were treated with 10 ng/ml TGF, and the expression of fascin mRNA was determined using qRT-PCR. Smad3 and Smad4 shRNA inhibited TGF-induced fascin expression, whereas Smad2 shRNA had no effect. Results are mean S.D. of triplicates. G, Western blots showing that Smad3 and Smad4 knockdown inhibited TGF-induced expression of fascin protein in MDA-MB-231 cells and A549 cells. Results are representative of 3 independent experiments. H, relative TRI and TRII mRNA levels in the six tumor cell lines as determined by qRT-PCR. Results are mean  S.D. from 3 experiments. I, TGF-induced Smad3 phosphorylation in breast cancer and non-small cell lung cancer cells. Cells were treated with 10 ng/ml TGF1 for 3 h, and phospho-Smad3 (p-Smad3) levels were detected through Western blotting. Induction of Fascin Expression by TGF Requires Smad3 and receptor complex. Treatment with SB-431542 completely Smad4—To explore the mechanisms through which TGF ele- blocked the overexpression of fascin in TGF-treated MDA- vates fascin expression, we first used SB-431542, a TGF recep- MB-231 cells and A549 cells, suggesting that TRI was essential torI(TRI) kinase inhibitor, to inhibit the activity of the TGF to mediating the TGF effects (Fig. 3A). NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 38869 Fascin in TGF-induced Tumor Cell Invasion Cip Kip TGF could regulate gene transcription either through the ing p21 , p27 , N-cadherin, and vimentin, and we noted Smad-dependent pathways or through the Smad-independent  failed to induce the transcription of any of these that TGF pathways by activation of Erk, JNK, and p38 MAPK (28). We genes in MCF-7 cells (supplemental Fig. S5). Therefore, the first examined the role of Erk and JNK because it was recently global inhibition of TGF response in some cells might at least reported that the core promoter region of fascin contains a partially explain the lack of TGF-induced fascin expression. binding site for AP-1/CREB, which could be activated by Erk Induction of Fascin Expression by TGF Requires No de Novo and JNK through phosphorylation (29, 30). The addition of the Protein Synthesis—To explore the kinetics of TGF-induced MEK inhibitor UO126 and JNK inhibitor SP600125 signifi- fascin mRNA expression, we used qRT-PCR to determine fas- cantly reduced the amount of phospho-Erk 1/2 and phospho- cin mRNA levels in MDA-MB-231 cells after treatment with c-Jun, respectively, in both control cells and TGF-treated TGF for different periods of time. As shown in Fig. 4A (upper cells, indicating the successful inhibition of MAPK and JNK panel), there was a 3-h lag in TGF-induced fascin transcrip- activity (Fig. 3, B and C). However, neither UO126 nor tion. Fascin mRNA levels increased steadily after the lag, reach- SP600125 had any effect on TGF-induced fascin expression ing the plateau level (6.4-fold) after 24 h and remaining at this (Fig. 3, B–D). We further examined the role of p38 MAPK using plateau for at least another 24 h. Next, we further evaluated the p38 kinase inhibitor SB203580 (supplemental Fig. S3). Treat- effects of TGF on the protein expression kinetics of fascin. As ment with p38 inhibitor failed to inhibit TGF-induced fascin shown in Fig. 4A (lower panel), TGF significantly elevated the expression at the mRNA level or the protein level, suggesting fascin protein level after 24 h, and the fascin protein level that ERK, JNK, and p38 MAPK were not involved (supplemen- remained high for up to at least 72 h. A similar time course was tal Fig. S3 and Fig. 3, B–D). also observed in A549 cells (data not shown), suggesting that To explore whether TGF-induced fascin expression is TGF was able to induce and maintain elevated fascin expres- mediated through the TGF-Smad pathway, we employed sion in spindle tumor cells. small hairpin RNA (shRNA) to knock down the expression of We further examined the kinetics of Smad3 phosphorylation Smad4. The knockdown of Smad4 was confirmed with qRT- in MDA-MB-231 cells. Smad3 phosphorylation was increased PCR (supplemental Fig. S4C). MDA-MB-231 and A549 cells by more than 10-fold 3 h after TGF treatment. Phospho- stably expressing control shRNA or Smad4 shRNAs were cul- Smad3 level decreased gradually after 3 h but remained at a tured in TGF or in control medium, and the expression of fairly high level for at least 26 h (Fig. 4B). fascin was evaluated with qRT-PCR and Western blotting. As Next, we investigated whether TGF-induced fascin tran- shown in Fig. 3, E and G, Smad4 shRNA almost completely scription required de novo protein synthesis. TGF regulates inhibited TGF-induced fascin expression at both the mRNA the expression of a plethora of genes. Some are immediately and the protein level, arguing that fascin is regulated by TGF downstream of the TGF signaling cascade, whereas others are through the canonical TRI-Smad pathway. To investigate indirectly modulated through TGF-regulated transcription whether either or both of the two receptor-regulated Smads factors (31, 32). To determine whether the induction of fascin (R-Smads) are involved, we further used shRNA to decrease the gene transcription by TGF was immediately downstream of expression of Smad2 and Smad3 (supplemental Fig. S4, A and the TGF signaling cascade, we used CHX to block de novo B). Smad3 shRNA, but not Smad2 shRNA, reduced the TGF- protein synthesis. CHX treatment did not change the TGF- induced expression in both MDA-MB-231 cells and A549 cells, induced expression of fascin mRNA in MDA-MB-231 cells and suggesting that fascin expression is regulated by TGF through A549 cells, indicating that elevated fascin expression does not the TRI-Smad3-Smad4 pathway, whereas Smad2 is not require new protein synthesis (Fig. 4C). As controls, CHX treat- required (Fig. 3, E and F). ment successfully blocked the inhibition of E-cadherin tran- Characterization of TGF Signaling in Spindle-shaped and scription and the induction of N-cadherin mRNA expression Polygon-shaped Tumor Cells—To gain insight into the differ- by TGF (Fig. 4D), confirming that gene transcriptions indi- ential regulation of fascin by TGF in spindle- and polygon- rectly regulated by TGF through other transcription factors shaped tumor cells, we used qRT-PCR to examine the expres- required new protein synthesis. sion levels TRI and TRII in the six cell lines (Fig. 3H). As It has been previously reported that TGF signaling acceler- shown in Fig. 3H, TGF receptor I and receptor II mRNA levels ates the degradation of Smad co-repressors such as Ski and were higher in the spindle-shaped tumor cells (MDA-MB-231, SnoN through the ubiquitin-proteasome pathway (33). To A549, and CHL-1)than in the polygonal cells (MCF7, H1299 determine whether the ubiquitin-proteasome pathway might and WM115). We further examined the total Smad3 and be required of TGF-induced fascin transcription, we used phoso-Smad3 level in the two breast cancer cell lines (MDA- proteasome inhibitor MG132 to treat MDA-MB-231 cells. As MB-231 and MCF7) and the two non-small cell lung cancer cell shown in Fig. 4E, treatment with MG132 significantly lines (A549 and H1299). As shown in Fig. 3I, both the total decreased TGF-induced fascin transcription, indicating that Smad3 and the TGF-induced phosho-Smad3 levels are higher degradation of co-suppressors might be involved in the regula- in the MDA-MB-231 and A549 cells when compared with tion of fascin expression. MCF7 and H1299 cells, respectively. The lower TRI, TRII, Fascin Is Required for TGF-induced Invasion and Filopodia and phospho-Smad3 levels in the polygonal tumor cells may Formation—To explore the role of fascin in TGF-induced lead to global inhibition of TGF responses. To examine this actin cytoskeleton remodeling and cell migration, we employed possibility, we examined the effect of TGF treatment on the shRNA to knock down fascin expression in spindle tumor cells. transcription of several other genes regulated by TGF, includ- The successful knockdown of fascin protein was confirmed by 38870 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Fascin in TGF-induced Tumor Cell Invasion FIGURE 4. TGF regulation of fascin transcription requires no de novo protein synthesis. A, upper panel, the kinetics of TGF-induced expression of fascin mRNA in MDA-MB-231 cells. MDA-MB-231 cells were treated with TGF, and the mRNA levels in the cells at the indicated time points were determined through qRT-PCR. Results are mean S.D. of triplicates. Lower panel, the kinetics of TGF-induced expression of fascin protein in MDA-MB-231 cells. Cells were treated with TGF and lysed at different time points as indicated. The levels of fascin protein were determined through Western blotting. RU, relative units. B, MDA-MB-231 cells were treated with TGF for different time points as indicated, and phospho-Smad3 (p-Smad3) levels were determined through Western blotting. C, TGF was able to induce fascin mRNA expression in the presence of protein synthesis inhibitor CHX (10 M). Results are mean S.D. of triplicates. D, CHX (10 M) inhibited the regulation of E-cadherin and N-cadherin transcription by TGF. Results are mean S.D. of triplicates. E, treatment with MG132 (10 M) inhibited TGF-induced fascin transcription. Results are mean  S.D. of triplicates. Western blotting (Fig. 5A). To assess the role of fascin in TGF on the invasion ability of spindle tumor cells stably TGF-induced filopodia formation, A549 cells stably expressing fascin or control shRNA. TGF pretreatment pro- expressing control shRNA or fascin shRNA were treated moted the invasion of control shRNA-expressing spindle with TGF and stained for F-actin. TGF induced long filop- tumor cells by about 3–5-fold, consistent with the pro-metas- odia and spiky morphology in control shRNA cells (Fig. 5B). tasis function of TGF. Expression of fascin shRNA dramati- TGF-induced long filopodia were noted in about 67% of cally inhibited TGF-promoted invasion in all three of the spin- A549 cells expressing control shRNA (183 out of a total of dle tumor cells. In contrast to the 3–5-fold invasion increase in 274 cells examined) (Fig. 5C). In contrast, TGF-induced control shRNA cells, TGF was only able to promote invasion long filopodia were only observed in roughly 20% of A549 by about 1.2–1.5-fold in fascin shRNA cells (Fig. 5E). fascin shRNA cells (60 out of a total of 294 cells examined), To further evaluate the hypothesis that fascin is critical for suggesting that fascin was critical for TGF-induced filopo- TGF-induced tumor cell migration and invasion, we overex- dia formation (Fig. 5, B and C). pressed fascin in tumor cells expressing Smad4 shRNA. As Next, we investigated the role of fascin in TGF-promoted shown in Fig. 5, F and G, Smad4 knockdown nearly completely spindle tumor cell migration. Using the Transwell migration abolished TGF-induced migration and invasion in MDA-MB- assay, we found that TGF-promoted cell migration was signif- 231 cells, consistent with the observation that Smad4 was icantly lower in fascin shRNA-treated cells when compared essential for TGF-induced fascin expression; ectopic fascin with the control shRNA-expressing cells, suggesting that fascin expression restored shSmad4 cell migration and invasion to was important for TGF-induced cell migration. (Fig. 5D). levels similar to TGF-primed control cells, consistent with the To further assess the role of fascin in TGF-promoted notion that fascin is critical for TGF-induced migration and metastasis in spindle cell tumors, we evaluated the effects of invasion in metastatic tumor cells. NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 38871 Fascin in TGF-induced Tumor Cell Invasion 38872 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Fascin in TGF-induced Tumor Cell Invasion FIGURE 6. Fascin expression levels correlate with TGF1 and TGF receptor 1 levels in primary breast tumors. A, correlation between fascin (FSCN1), TGF1, and TGF receptor 1 probe sets in 99 primary tumor samples in the Sloan cohort. Pearson correlation coefficient values (r) and probability values (p) were calculated using Excel. B, fascin expression levels in the 99 breast tumor samples. Tumor samples were sorted according to TGF1 levels. RU, relative units. C, fascin expression levels in the TGF1 low and TGF1 high groups. p value was calculated using two-tailed Student’s t test. Correlation between TGF1, TRI, and Fascin Expression TGF1 low group (p 0.002) (Fig. 6C), suggesting that elevated Levels in Primary Breast Tumor Samples—To validate our cell TGF1 levels in primary breast tumors contributed to fascin culture-based discoveries in breast cancer patients, we exam- overexpression. ined the correlation between fascin levels and TGF as well as DISCUSSION TGF receptor levels in a cohort of 99 primary breast tumor samples collected at the Memorial Sloan-Kettering Cancer Fascin Expression Is Regulated by TGF—Although there are Center (34). Pearson correlation coefficient (r) and probability a plethora of studies about fascin overexpression in various car- (p) values between the two fascin probe sets and each of the cinomas (4, 5, 7, 8, 11, 12), the factors contributing to fascin probe sets for TGF1, TGF2, and TGF3 and TGF receptor overexpression in metastatic tumors remain largely unknown. I and II were calculated. Fascin expression significantly corre- Binding sites for several transcription factors, including lates with TGF1 and TGF receptor I, but not with TGF2 -catenin-TCF (T-cell factor), CREB, and AP-1, in the FSCN1 and TGF3orTGF receptor II (Fig. 6A and data not shown). promoter region have been previously reported (5, 29, 30, 35). Next, tumor samples were sorted according to TGF1 expres- However, it is not clear whether and how factors in the tumor sion level (Fig. 6B). The 50 tumor samples with TGF1 level at microenvironment contribute to fascin overexpression in met- or below median level were assigned to the “TGF low” group, astatic tumor cells. Our results here demonstrated that TGF and the other 49 samples with TGF1 level above the median promoted fascin expression in spindle tumor cells, implicating level were assigned to the “TGF1 high” group. Fascin (FSCN1) that cytokines in the tumor microenvironment could affect fas- levels in the TGF1 high group were about 2-fold as high as the cin expression. FIGURE 5. Fascin is required for TGF-induced filopodia formation, cell migration, and invasion. A, Western blots showing that fascin shRNA decrease the protein level of fascin in MDA-MB-231, A549, and CHL-1 cells. B, F-actin staining showing that TGF induced long filopodia in A549 cells expressing control shRNA, but not in cells expressing fascin shRNA. The scale bar is 10 m. Results are representative of 3 experiments. C, quantification of cells with long filopodia in A549 cells expressing control shRNA or fascin shRNA, with or without TGF treatment, respectively. D and E, the effects of TGF pretreatment on tumor cell migration (D) and invasion (E) in spindle tumor cells expressing control shRNA or fascin shRNA, respectively. Fascin knockdown with shRNA inhibited TGF- promoted tumor cell migration and invasion. F and G, ectopic fascin overexpression restore migration (F) and invasion (G) in MDA-MB-231 stably expressing Smad4 shRNA. The data in C–G are representative of either 3–5 similar experiments or mean S.D. of 3 experiments. *, p  0.05. NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 38873 Fascin in TGF-induced Tumor Cell Invasion The up-regulation of fascin by TGF was surprising in a found impacts on tumor progression and metastasis (14, 15, sense because TGF target genes have been studied previously  signaling in primary tumor was linked to 17). Activated TGF in various epithelial and tumor cell lines (including MDA-MB- lung metastasis in estrogen receptor-negative breast tumors 231), and fascin was not among the TGF response genes iden- (14). Our data demonstrated that TGF induced fascin expres- tified through microarray screening (14, 36). We noticed that sion and promoted migration/invasion in MDA-MB-231 breast previous screenings focused on early response genes and that tumor cells. More importantly, TGF1 and TGF receptor I tumor cells were treated with TGF for no more than 3 h (14, significantly correlate with fascin (FSCN1) level levels in a 36). Therefore, we explored the kinetics of TGF-induced tran- cohort of primary breast tumors, suggesting that activation of scription of fascin. Our data revealed a 3-h lag in TGF-in- TGF signaling may contribute to fascin overexpression in pri- duced fascin expression (Fig. 3D), which may explain the dis- mary tumors. Interestingly, fascin overexpression in breast crepancy between our data and previous studies. The lag might tumor has been associated with lung metastasis, and fascin is be due to the requirement for TGF-induced degradation of one of the top performing lung metastasis signature genes (34). Smad co-suppressors as proteasome inhibitor MG132 inhib- It is possible that elevated fascin expression is critical for TGF ited TGF-induced fascin transcription. Despite the lag, fascin to promote tumor cell migration, invasion, and lung metastasis expression is likely directly downstream of TGF signaling in estrogen receptor-negative breast tumors. Indeed, TGF because TGF was able to promote the expression of fascin promoted migration and invasion in all three spindle tumor cell mRNA in the presence of CHX, a protein synthesis inhibitor. lines with elevated fascin expression, but not in the three polyg- Fascin Is Regulated by TGF through the Canonical TRI- onal tumor cells, in which TGF-induced fascin expression was Smad Pathway—Our data further demonstrated that fascin absent. expression was regulated through the canonical TRI-Smad More importantly, knockdown of fascin expression with pathway. Smads in the TGF pathway recognized the consen- shRNA significantly inhibited migration and invasion in TGF- sus DNA sequence CAGAC (37). To determine whether the treated spindle tumor cells. The near complete inhibition of human fascin 1 gene contains Smad-binding sites, we examined TGF-promoted invasion by fascin shRNA was quite remark- the promoter region of FSCN1 and discovered that there were able considering that TGF also up-regulates the expression of two CAGAC sequences at 381 and 1225 positions, respec- other pro-invasion genes such as matrix metalloproteases 2 and tively. It is possible that activated Smad3 and Smad4 induced 9 (17, 39). During invasion, tumor cells use protrusion struc- fascin expression by binding to one or both of the Smad-bind- tures termed invadopodia to secrete matrix metalloproteases ing sites. and to coordinate the degradation of extracellular matrix (40). It is intriguing to note that TGF only induced fascin expres- The formation and elongation of invadopodia requires filopo- sion in spindle tumor cells, but not in polygonal cells. One way dia-associated protein components (41). Fascin is the major for tumor cells to overcome growth inhibition effects exerted actin-bundling protein in filopodia protrusions (27). It was by TGF is through loss of expression or functional inactiva- recently reported that fascin was critical to stabilize actin in tion of TRI and TRII (38). We noted that that TRI and invadopodia (26, 41, 42). Depletion of fascin with shRNA desta- TRII levels in the three spindle tumor cells were higher than bilized invadopodia and impaired extracellular matrix degrada- the three polygonal cells. The reduced expression of TGF tion (42). Given the similar components and regulatory mech- receptors might partially explain the lack of TGF-induced fas- anism between invadopodia and filopodia, it has been cin expression in polygonal cells. suggested that invadopodia are “invasive filopodia” (26, 42). The Smad co-activator context in cells may also contribute to Our data showed that fascin was required for long filopodia the regulation of fascin by TGF. Although Smad3 and Smad4 induced by TGF. It is possible that fascin played a central role can directly bind to DNA to mediate gene transcription, inter- in the invasive machinery mobilized by TGF in spindle tumor action between Smads and their target sequence is of low affin- cells. By stabilizing actin cytoskeleton within invadopodia and ity, and DNA-binding co-factors are required to provide spe- filopodia, fascin coordinates the invasion and metastasis pro- cific regulation of gene transcriptions (37). Consequently, the moted by TGF. transcriptomic output of activated Smads is highly dependent on the cellular context of Smad co-factors (15). The conversion Acknowledgments—We thank Drs. Srikumar Chellappan and Xin- from polygonal morphology to spindle morphology during Yun Huang for critically reading the manuscript, Dr. Steven Enkeman tumor progression is a recessive event resulting from the for consultation on microarray data mining, the Analytic Microscopy change of gene expressions that control epithelial differentia- Core at Moffitt Cancer center for image acquisition assistance, and Rasa Hamilton for editorial assistance. tion (16). It is possible that the transition from epithelial-like to mesenchymal-like tumor cells provides the cellular co-factor context required for the induction of fascin expression by acti- REFERENCES vated Smads. We noted that some polygonal tumor cells (e.g. 1. Mareel, M., and Leroy, A. (2003) Physiol. 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Fascin Protein Is Critical for Transforming Growth Factor β Protein-induced Invasion and Filopodia Formation in Spindle-shaped Tumor Cells *

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American Society for Biochemistry and Molecular Biology
Copyright
Copyright © 2011 Elsevier Inc.
ISSN
0021-9258
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1083-351X
DOI
10.1074/jbc.m111.270413
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 45, pp. 38865–38875, November 11, 2011 © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Fascin Protein Is Critical for Transforming Growth Factor Protein-induced Invasion and Filopodia Formation in Spindle-shaped Tumor Cells Received for publication, June 13, 2011, and in revised form, September 12, 2011 Published, JBC Papers in Press, September 13, 2011, DOI 10.1074/jbc.M111.270413 ‡§1 ‡§1 ¶ ‡§ ‡§ ‡§ Jianwei Sun , Huifang He , Yin Xiong , Shuang Lu , Junling Shen , Anna Cheng , Wei-Chiao Chang**, ¶ ‡‡‡ ‡§2 Ming-Feng Hou**, Johnathan M. Lancaster , Minjung Kim , and Shengyu Yang ‡ § ¶ From the Comprehensive Melanoma Research Center, Department of Tumor Biology, Department of Women’s Oncology, and ‡‡ Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, the Qingdao Agricultural University, Qingdao 266109, China,and the **Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan Background: Fascin is a pro-metastasis actin-bundling protein overexpressed in metastatic tumors. Results: TGF induced fascin expression in spindle tumor cells through Smads. Conclusion: Fascin is a TGF target gene essential for the pro-invasion activity of TGF. Significance: Our data shed new light on how TGF dysregulates actin cytoskeleton to promote tumor metastasis. Fascin, an actin-bundling protein overexpressed in all carci- protrusions, it is crucial to cross-link actin filaments into bun- nomas, has been associated with poor prognosis, shorter sur- dles as individual actin filaments are flexible. Cross-linking by vival, and more metastatic diseases. It is believed that fascin bundling protein provides the essential rigidity to counter the facilitates tumor metastasis by promoting the formation of inva- compressive forces from the plasma membrane (3). sive membrane protrusions. However, the mechanisms by Fascin is an actin-bundling protein critical for tumor metas- which fascin is overexpressed in tumors are not clear. TGF is a tasis (4–7). Expression levels of fascin are very low or not cytokine secreted by tumor and mesenchymal cells and pro- detected in normal epithelia, but are highly elevated in malig- motes metastasis in many late stage tumors. The pro-metastasis nant tumors (4). Overexpression of fascin protein is associated mechanisms of TGF remain to be fully elucidated. Here we with poor prognosis in patients (7–12). Knockdown of fascin demonstrated that TGF induced fascin expression in spindle- expression inhibited tumor cell migration and invasion in vitro shaped tumor cells through the canonical Smad-dependent and decreased tumor metastasis in mouse models. Moreover, pathway. Fascin was critical for TGF-promoted filopodia for- ectopic expression of fascin promoted tumor cell invasion and mation, migration, and invasion in spindle tumor cells. More metastasis (5, 6, 13). The causal role of fascin overexpression in importantly, fascin expression significantly correlates with tumor metastasis is well established; however, the molecular TGF1 and TGF receptor I levels in a cohort of primary breast mechanisms underlying elevated fascin level in metastatic tumor samples. Our results indicate that elevated TGF level in tumors are not clear. the tumor microenvironment may be responsible for fascin TGF is a cytokine in the tumor microenvironment that reg- overexpression in some of the metastatic tumors. Our data also ulates various tumor progressions in a context-dependent suggest that fascin could play a central role in TGF-promoted manner (14). In early stage tumors, TGF is a potent prolifera- tumor metastasis. tion inhibitor that deters tumor growth; however, late stage tumors are often able to evade the growth inhibition and secrete elevated levels of TGF to promote metastasis (15). The mech- One essential characteristic of metastatic cancer cells is anisms by which late stage tumors use TGF signaling to pro- enhanced motility, which facilitates the infiltration of meta- mote tumor spreading are largely unknown. static cells into lymphatic and blood vessels and extravasation Accompanying the loss of capacity to differentiate during out of the circulation (1). The forces that drive tumor cell tumor progression, tumor cells undergo a transition in gene migration and invasion are provided by the actin cytoskeleton expression, reorganization of cytoskeleton, and acquisition of underlying the critical membrane protrusions in migrating spindle cell morphology (16). Tumors with spindle cell mor- tumor cells (2). To efficiently drive the formation of membrane phology were characterized as highly malignant and invasive (17). In breast cancer, fascin is overexpressed in the estrogen * This work was supported in part by an Institutional Research Grant from the American Cancer Society (IRG-93-032-16), a Career Development Award receptor-negative, basal-like subgroup (11), a highly metastatic from the Donald A. Adams Comprehensive Melanoma Research Center, group of breast cancers typically with spindle cell morphology and a Milestone Award from Miles for Moffitt Foundation Center (to S. Y.). □ S (18). In melanoma, elongated, spindle-like tumor cells showed The on-line version of this article (available at http://www.jbc.org) contains supplemental supplemental Movies 1 and 2 and Figs. S1–S5. intense fascin staining, whereas rounded, amoeboid-like mela- Both authors contributed equally to this work. noma cells were generally fascin-negative (19). To whom correspondence should be addressed: 12902 Magnolia Dr. SRB-2, Here, we demonstrate that TGF elevates fascin protein Tampa, FL 33612. Tel.: 813-745-8371; Fax: 813-745-3829; E-mail: shengyu. yang@moffitt.org. expression and promotes invasion and filopodia formation in NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 38865 This is an Open Access article under the CC BY license. Fascin in TGF-induced Tumor Cell Invasion tumor cells with spindle morphology, but not in tumor cells were photographed, and the cells on the lower surface of the with epithelial-like, polygonal morphology. We also show that insert were counted. transcription of fascin mRNA induced by TGF is independent Immunofluorescence Microscopy—A549 cells cultured on of de novo protein synthesis but relies on the canonical Smad- collagen-coated glass coverslips were fixed with 3.7% para- dependent pathway. Furthermore, fascin is essential for TGF formaldehyde in PBS for 10 min at room temperature, per- to promote invasion and filopodia formation in spindle tumor meabilized with 0.1% Triton X-100 for 5 min, and then cells. Therefore, our data suggest that fascin is an immediate washed with PBS three times. To block nonspecific binding, TGF target gene essential for its pro-invasion activity. Our the cells were incubated with a solution of PBS containing 1% data also suggest that TGF might be responsible for the fascin bovine serum albumin for 30 min and then incubated with overexpression in some metastatic tumors. Alexa Fluor 594-labeled phalloidin (Invitrogen). The cover- slips were then fixed onto slides and imaged using a Zeiss EXPERIMENTAL PROCEDURES fluorescence microscope. Cell Culture Media—The cell culture media used were Live Cell Imaging—A549 cells with or without TGF pre- DMEM (for MDA-MB-231 and MCF-7), F-12K (for A549), and treatment were plated on collagen-coated glass-bottomed RPMI (for CHL-1, WM115, and H1299). All cell culture media 35-mm tissue culture dishes (MatTek) overnight. The mem- were supplemented with 10% fetal bovine serum and brane protrusion dynamics and cell movement were recorded penicillin/streptomycin. with a differential interference contrast microscopy under a Antibodies—The following antibodies were used in this 40 objective using a Zeiss inverted microscope equipped with study: fascin, Santa Cruz Biotechnology antibody number sc- a live imaging chamber. The temperature and CO concentra- 21743; phospho-ERK1/2, Cell Signaling antibody number 9101; tion in the chamber were maintained at 37 °C and 5%, respec- phospho-c-Jun(Ser-63), Cell Signaling antibody number 9261; tively. Time-lapse images were recorded at 10-s intervals. Smad3, Cell Signaling antibody number 9523; phospho- qRT-PCR—Total RNA was extracted from cultured cells Smad3(Ser-423/425), Cell Signaling antibody number 9520; using TRIzol reagent (Invitrogen), and the reverse transcription GAPDH, Sigma product number G8795. was performed using the iScript cDNA synthesis kit (Bio-Rad). RNA Interference—RNAi of Smad2, Smad3, Smad4, and fas- The quantitative real-time PCR (qRT-PCR) assay was carried cin was performed using pSUPER.Retro.puro vector (Oligoen- out with the Applied Biosystems 7900HT fast real-time PCR gine) encoding small hairpin RNA. The previously reported tar- system using Applied Biosystems SYBR Green PCR master mix. get sequences were used: GGTGGGCAAAGATGAGCTC Primers for qRT-PCR are shown in supplemental Table S1. All (Fascin) (6), GGTGGGCAAAGATGAGCTC (Smad2) (20), reactions were performed in triplicate, and the experiment was GGACGAGGTCTGCGTGAAT (Smad3) (20), and GGTGTG- repeated three times. CAGTTGGAATGTA (Smad4) (20). RESULTS TGF and Inhibitor Treatment—Unless stated otherwise, all TGF Promotes the Migration of Spindle-shaped Tumor Cells— cells were treated with 10 ng/ml TGF1 (Peprotech, Rocky Hill, NJ) in growth medium for 2 days before being used for assays. It was recently reported that exposure to TGF within the Inhibitors, when used, were added together with TGF to tumor microenvironment might predispose tumor cells for metastasis to distant organs (14). To evaluate the effects of growth medium. TGF exposure on the migration of tumor cells, we treated a Transwell Cell Migration Assay—Cells (1  10 ) suspended panel of tumor cell lines with TGF. We used two breast in starvation medium were added to the upper chamber of an insert (6.5-mm diameter, 8-m pore size, BD Biosciences), and cancer lines (MDA-MB-231, and MCF-7), two non-small cell lung cancer lines (A549 and H1299), and two melanoma the insert was placed in a 24-well dish containing starvation lines (CHL-1 and WM115). Three of the six cell lines (MDA- medium with or without 10% FBS (21, 22). Migration assays were carried out for 4–6 h for spindle tumor cells and 12–24 h MB-231, A549, and CHL-1) have spindle cell morphology, for polygonal tumor cells. Cells were fixed with 3.7% formalde- whereas the other three (MCF-7, H1299, and WM115) have polygonal, epithelial-like morphology (Fig. 1A). After 48 h of hyde and stained with crystal violet staining solution, and cells treatment, we found that TGF induced morphology change on the upper side of the insert were removed with a cotton swab. Three randomly selected fields (10 objectives) on the in the three spindle-shaped cell lines, with cells becoming lower side of the insert were photographed, and the migrated more elongated and disperse. Extremely long and finger-like protrusions were also observed in some TGF-pretreated cells were counted. The migration was expressed as the average cells. However, the effects of TGF on the morphology of the number of migrated cells in a field. Cell Invasion Assay—Cells (1 10 three polygonal, epithelial-like tumor cells were much less ) suspended in starvation medium were added to the upper chamber of a Matrigel-coated noticeable (Fig. 1A). Next, we examined the effects of TGF pretreatment on cell insert (6.5-mm diameter, 8-m pore size, BD Biosciences), and migration using the Transwell migration assay. Interestingly, the insert was placed in a 24-well dish containing medium with or without serum. Invasion assays were carried out for 16 h, and TGF priming significantly increased the migration of the cells were fixed with 3.7% formaldehyde. Cells were stained with crystal violet staining solution, and cells on the upper side The abbreviations used are: qRT-PCR, quantitative real-time PCR; CHX, of the insert were removed with a cotton swab. Three randomly RI, TGF receptor I; TRII, TGF receptor II; CREB, cAMP- cycloheximide; T selected fields (10 objectives) on the lower side of the insert response element-binding protein. 38866 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Fascin in TGF-induced Tumor Cell Invasion FIGURE 1. TGF promoted cell migration in spindle-shaped tumor cells. A, morphology changes induced by TGF. Spindle-shaped tumor cells (MDA-MB-231, A549, and CHL-1) became more elongated and scattered after TGF treatment. Tumor cells were cultured in growth medium with or without 10 ng/ml TGF. The cell morphologies were recorded with phase contrast microscopy using a 10 objective. Scale bar,50 m. B, the effects of TGF pretreatment on tumor cell migration. TGF pretreatment promoted cell migration in spindle-shaped tumor cells, but not in polygon-shaped tumor cells. The data presented are mean  S.D. of migrated cells per field from three randomly selected 10 fields. C–F, kymograph analysis of membrane protrusion dynamics in control A549 cell (C and D) and TGF-pretreated A549 cells (E and F); C and E, individual frames from time-lapse movies (supplemental Movies 1 and 2) used to generate the kymographs (D and F). Descending and ascending contours in the kymographs indicate membrane protrusion and withdrawal events, respectively. Arrowheads in D and F indicate new membrane protrusions. G, the extension of membrane protrusions in control and TGF-treated cells. three spindle-shaped tumor cells by about 3–10-fold (Fig. 1B). lamellipodia, turnover of focal adhesions, contraction of cell In sharp contrast, TGF had little effect on (MCF-7 and H1299) body, and retraction of trailing tail (23). To investigate the or slightly inhibited (WM115) the migration of the three poly- mechanisms underlying the enhanced cell migration in TGF- gon-shaped tumor cell lines (Fig. 1B). pretreated spindle tumor cells, we used live cell imaging to To characterize the functional characteristics of spindle- study the membrane protrusion dynamics at the leading shaped and polygon-shaped tumor cells in the absence of edge of migrating cells. As shown in supplemental Movies 1 TGF, we used the Transwell assay to evaluate their mobility and 2, TGF pretreatment induced hyperactive membrane and invasiveness. Tumor cells were allowed to migration/in- protrusion in A549 cells. The membrane protrusion dynam- vade for 6 and 12 h, respectively. As shown in supplemental Fig. ics in the TGF-pretreated cells followed a protrusion-ruf- S1, the three spindle-shaped tumor cell lines migrate and fle-protrusion pattern (supplemental Fig. S2). New lamelli- invade much faster than the three polygonal tumor cells, con- podia extended from the edge of membrane ruffles, sistent with the notion that spindle-shaped tumor cells are sometimes expanding in the space between adjacent filopo- more invasive and metastatic. dia. At the end of the lamellipodium expansion, the edge of TGF Induces Hyperactive Membrane Protrusions—Cell lamellipodia pulled back and formed membrane ruffles. New migration is a multistep process involving the formation of filopodia or lamellipodia would extend from the edge of NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 38867 Fascin in TGF-induced Tumor Cell Invasion membrane ruffles and begin the next protrusion cycle. In contrast, lamellipodia usually pulled back without mem- brane ruffle formation in control cells (supplemental Fig. S2 and supplemental Movies 1 and 2). Next, we employed kymography to analyze the membrane protrusion kinetics in control cells and TGF-pretreated cells. A line was drawn at the leading edge of a migrating cell; inten- sity values along the defined line region in each image of a time-lapse series were extracted and assembled together side- by-side to generate a kymograph montage (Fig. 1, C–F). As shown in the kymograph in Fig. 1, C and D, transient protru- sions frequently extended out from the lamellipodium region of the control A549 cells and quickly withdrew to the starting point, generating spike-like structures on a flat basal line on the kymograph. The leading edge of the control A549 cells barely moved forward during the recording period (Fig. 1, D and G). In contrast, the kymographs of TGF-pretreated cells appeared as stair-like structures, indicating that the lamellipodia in these cells extended in “bursts”; in addition, the newly formed protrusions in TGF-treated cells were able to hold their posi- tions for a period of time until the next extension, instead of withdrawing to the starting point in the control cell. Conse- quently, the leading edges moved steadily forward following each extension (Fig. 1, F and G). The hyperactive membrane FIGURE 2. TGF elevated fascin expression level in spindle tumor cells. A, staining of F-actin in A549 cells with fluorescence-labeled phalloidin. The protrusions may contribute to increased mobility in TGF- weak and wavy filopodia in control cells were indicated by the gray arrow- treated cells. heads. The long filopodia in TGF-treated cells were indicated by the white TGF Induces Actin Cytoskeleton Remodeling—The mor- arrowheads. The scale bar is 10 m. B, quantification of cells with long filopo- dia in A549 cells with TGF or control treatment. Results are mean S.D. from phology changes and hyperactive membrane protrusions sug- 3 experiments. C, TGF treatment increased the protein expression of fascin gested remodeling of the actin cytoskeleton in TGF-treated in spindle tumor cells (MDA-MB-231, A549, and CHL-1), but not in polygonal spindle tumor cells. Therefore, we used fluorescence micros- tumor cells (MCF-7, H1299, and WM115). The protein levels of fascin in control or TGF-treated tumor cells were determined by Western blotting. Results copy to visualize actin filaments in the cells. As shown in Fig. are representatives from 3 independent experiments. D, quantification of the 2A, the phalloidin staining in control A549 cells showed typical Western blotting results in A by densitometry. Results are mean S.D. of 2–5 experiments. E, effects of TGF treatment on the mRNA expression of fascin in cortical actin staining, with the majority of F-actin in the corti- MDA-MB-231, A549, and MCF-7 cells. Results are mean  S.D. of triplicates. cal region. On the outer periphery of the strong actin staining, RU, relative units. numerous weak and wavy filopodia-like protrusions were also observed in most of the control cells (Fig. 2A, gray arrowheads). treatment on fascin protein levels in these tumor cells were In contrast, stress fibers were noted in the majority of the evaluated with Western blotting using a fascin-specific anti- TGF-pretreated A549 cells, consistent with previous observa- body. TGF treatment elevated fascin protein levels by 2–6- tions that TGF promoted stress fiber formation (24, 25). Most fold in the three spindle tumor cells. In sharp contrast, no strikingly, TGF induced very long and straight filopodia TGF-induced fascin expression was observed in any of the (filopodia protruded more than 20 m out of the cell boundary) polygonal tumor cells (Fig. 2, C and D). (Fig. 2A). Typically, several such long filopodia extended To investigate whether TGF regulates the degradation of around the cells, rendering a distinct “spiky” morphology in fascin protein, we used cycloheximide (CHX), a protein syn- TGF-treated A549 cells. The extremely long filopodia were thesis inhibitor, to inhibit the new synthesis of fascin in A549 observed in 70% of TGF-treated cells (110 out of a total of 158 cells and used Western blotting to monitor fascin degrada- cells); in contrast, only less than 5% (8 out of a total of 228 cells) tion at different time points after CHX treatment. No degra- of control cells had such filopodia (Fig. 2B). dation of fascin protein in control cells or in TGF-treated Elevated Fascin Expression in Spindle Tumor Cells after cells was detected even 24 h after CHX treatment, indicating TGF Treatment—Filopodia are finger-like protrusions critical that fascin is a very stable protein (data not shown). Next, we for tumor cell invasion and metastasis (26). Cross-linking of investigated whether TGF regulated fascin expression at parallel actin filaments is considered to be crucial for filopodia the mRNA level in two spindle tumor cell lines (A549 and formation as individual filaments lack the rigidity required to MDA-MB-231 cells) and one polygonal cell line (MCF-7 overcome the compressive forces from plasma membrane (3). cells) using qRT-PCR. Consistent with our Western blot Fascin is a key actin-bundling protein in filopodia (27), and results, we found that TGF treatment elevated fascin fascin overexpression has been reported in all carcinomas mRNA levels in A549 and MDA-MB-231 cells, but not in examined to date (4). The long filopodia in TGF-pretreated MCF-7 cells, suggesting that TGF promoted the transcrip- cells prompted us to examine the possibility that TGF might tion of fascin only in spindle tumor cells, but not in polygonal regulate fascin expression in tumor cells. The effects of TGF tumor cells (Fig. 2E). 38868 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Fascin in TGF-induced Tumor Cell Invasion FIGURE 3. Fascin is a TGF target gene regulated by Smad3 and Smad4. A, inhibition of TGF-induced fascin expression by TRI kinase inhibitor SB431542 in MDA-MB-231 cells and A549 cells. B–D, the effects of MEK inhibitor UO126 (10 M) and JNK inhibitor SP600125 (20 M)onTGF-induced fascin expression at the protein level (B and C) and the mRNA level (D). MDA-MB-231 cells were treated with TGF and the two inhibitors as indicated for6h(B and C)or24h(D), respectively; the mRNA and protein levels were determined through qRT-PCR and Western blotting. Results are mean S.D. from 3 experiments. p-ERK1/2, phospho-ERK1/2; p-c-Jun, phospho-c-Jun; RU, relative units. E and F, the role of Smads in TGF-induced fascin expression. MDA-MB-231 and A549 cells stably expressing Smad3, Smad4, and Smad2 shRNA or control shRNA were treated with 10 ng/ml TGF, and the expression of fascin mRNA was determined using qRT-PCR. Smad3 and Smad4 shRNA inhibited TGF-induced fascin expression, whereas Smad2 shRNA had no effect. Results are mean S.D. of triplicates. G, Western blots showing that Smad3 and Smad4 knockdown inhibited TGF-induced expression of fascin protein in MDA-MB-231 cells and A549 cells. Results are representative of 3 independent experiments. H, relative TRI and TRII mRNA levels in the six tumor cell lines as determined by qRT-PCR. Results are mean  S.D. from 3 experiments. I, TGF-induced Smad3 phosphorylation in breast cancer and non-small cell lung cancer cells. Cells were treated with 10 ng/ml TGF1 for 3 h, and phospho-Smad3 (p-Smad3) levels were detected through Western blotting. Induction of Fascin Expression by TGF Requires Smad3 and receptor complex. Treatment with SB-431542 completely Smad4—To explore the mechanisms through which TGF ele- blocked the overexpression of fascin in TGF-treated MDA- vates fascin expression, we first used SB-431542, a TGF recep- MB-231 cells and A549 cells, suggesting that TRI was essential torI(TRI) kinase inhibitor, to inhibit the activity of the TGF to mediating the TGF effects (Fig. 3A). NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 38869 Fascin in TGF-induced Tumor Cell Invasion Cip Kip TGF could regulate gene transcription either through the ing p21 , p27 , N-cadherin, and vimentin, and we noted Smad-dependent pathways or through the Smad-independent  failed to induce the transcription of any of these that TGF pathways by activation of Erk, JNK, and p38 MAPK (28). We genes in MCF-7 cells (supplemental Fig. S5). Therefore, the first examined the role of Erk and JNK because it was recently global inhibition of TGF response in some cells might at least reported that the core promoter region of fascin contains a partially explain the lack of TGF-induced fascin expression. binding site for AP-1/CREB, which could be activated by Erk Induction of Fascin Expression by TGF Requires No de Novo and JNK through phosphorylation (29, 30). The addition of the Protein Synthesis—To explore the kinetics of TGF-induced MEK inhibitor UO126 and JNK inhibitor SP600125 signifi- fascin mRNA expression, we used qRT-PCR to determine fas- cantly reduced the amount of phospho-Erk 1/2 and phospho- cin mRNA levels in MDA-MB-231 cells after treatment with c-Jun, respectively, in both control cells and TGF-treated TGF for different periods of time. As shown in Fig. 4A (upper cells, indicating the successful inhibition of MAPK and JNK panel), there was a 3-h lag in TGF-induced fascin transcrip- activity (Fig. 3, B and C). However, neither UO126 nor tion. Fascin mRNA levels increased steadily after the lag, reach- SP600125 had any effect on TGF-induced fascin expression ing the plateau level (6.4-fold) after 24 h and remaining at this (Fig. 3, B–D). We further examined the role of p38 MAPK using plateau for at least another 24 h. Next, we further evaluated the p38 kinase inhibitor SB203580 (supplemental Fig. S3). Treat- effects of TGF on the protein expression kinetics of fascin. As ment with p38 inhibitor failed to inhibit TGF-induced fascin shown in Fig. 4A (lower panel), TGF significantly elevated the expression at the mRNA level or the protein level, suggesting fascin protein level after 24 h, and the fascin protein level that ERK, JNK, and p38 MAPK were not involved (supplemen- remained high for up to at least 72 h. A similar time course was tal Fig. S3 and Fig. 3, B–D). also observed in A549 cells (data not shown), suggesting that To explore whether TGF-induced fascin expression is TGF was able to induce and maintain elevated fascin expres- mediated through the TGF-Smad pathway, we employed sion in spindle tumor cells. small hairpin RNA (shRNA) to knock down the expression of We further examined the kinetics of Smad3 phosphorylation Smad4. The knockdown of Smad4 was confirmed with qRT- in MDA-MB-231 cells. Smad3 phosphorylation was increased PCR (supplemental Fig. S4C). MDA-MB-231 and A549 cells by more than 10-fold 3 h after TGF treatment. Phospho- stably expressing control shRNA or Smad4 shRNAs were cul- Smad3 level decreased gradually after 3 h but remained at a tured in TGF or in control medium, and the expression of fairly high level for at least 26 h (Fig. 4B). fascin was evaluated with qRT-PCR and Western blotting. As Next, we investigated whether TGF-induced fascin tran- shown in Fig. 3, E and G, Smad4 shRNA almost completely scription required de novo protein synthesis. TGF regulates inhibited TGF-induced fascin expression at both the mRNA the expression of a plethora of genes. Some are immediately and the protein level, arguing that fascin is regulated by TGF downstream of the TGF signaling cascade, whereas others are through the canonical TRI-Smad pathway. To investigate indirectly modulated through TGF-regulated transcription whether either or both of the two receptor-regulated Smads factors (31, 32). To determine whether the induction of fascin (R-Smads) are involved, we further used shRNA to decrease the gene transcription by TGF was immediately downstream of expression of Smad2 and Smad3 (supplemental Fig. S4, A and the TGF signaling cascade, we used CHX to block de novo B). Smad3 shRNA, but not Smad2 shRNA, reduced the TGF- protein synthesis. CHX treatment did not change the TGF- induced expression in both MDA-MB-231 cells and A549 cells, induced expression of fascin mRNA in MDA-MB-231 cells and suggesting that fascin expression is regulated by TGF through A549 cells, indicating that elevated fascin expression does not the TRI-Smad3-Smad4 pathway, whereas Smad2 is not require new protein synthesis (Fig. 4C). As controls, CHX treat- required (Fig. 3, E and F). ment successfully blocked the inhibition of E-cadherin tran- Characterization of TGF Signaling in Spindle-shaped and scription and the induction of N-cadherin mRNA expression Polygon-shaped Tumor Cells—To gain insight into the differ- by TGF (Fig. 4D), confirming that gene transcriptions indi- ential regulation of fascin by TGF in spindle- and polygon- rectly regulated by TGF through other transcription factors shaped tumor cells, we used qRT-PCR to examine the expres- required new protein synthesis. sion levels TRI and TRII in the six cell lines (Fig. 3H). As It has been previously reported that TGF signaling acceler- shown in Fig. 3H, TGF receptor I and receptor II mRNA levels ates the degradation of Smad co-repressors such as Ski and were higher in the spindle-shaped tumor cells (MDA-MB-231, SnoN through the ubiquitin-proteasome pathway (33). To A549, and CHL-1)than in the polygonal cells (MCF7, H1299 determine whether the ubiquitin-proteasome pathway might and WM115). We further examined the total Smad3 and be required of TGF-induced fascin transcription, we used phoso-Smad3 level in the two breast cancer cell lines (MDA- proteasome inhibitor MG132 to treat MDA-MB-231 cells. As MB-231 and MCF7) and the two non-small cell lung cancer cell shown in Fig. 4E, treatment with MG132 significantly lines (A549 and H1299). As shown in Fig. 3I, both the total decreased TGF-induced fascin transcription, indicating that Smad3 and the TGF-induced phosho-Smad3 levels are higher degradation of co-suppressors might be involved in the regula- in the MDA-MB-231 and A549 cells when compared with tion of fascin expression. MCF7 and H1299 cells, respectively. The lower TRI, TRII, Fascin Is Required for TGF-induced Invasion and Filopodia and phospho-Smad3 levels in the polygonal tumor cells may Formation—To explore the role of fascin in TGF-induced lead to global inhibition of TGF responses. To examine this actin cytoskeleton remodeling and cell migration, we employed possibility, we examined the effect of TGF treatment on the shRNA to knock down fascin expression in spindle tumor cells. transcription of several other genes regulated by TGF, includ- The successful knockdown of fascin protein was confirmed by 38870 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Fascin in TGF-induced Tumor Cell Invasion FIGURE 4. TGF regulation of fascin transcription requires no de novo protein synthesis. A, upper panel, the kinetics of TGF-induced expression of fascin mRNA in MDA-MB-231 cells. MDA-MB-231 cells were treated with TGF, and the mRNA levels in the cells at the indicated time points were determined through qRT-PCR. Results are mean S.D. of triplicates. Lower panel, the kinetics of TGF-induced expression of fascin protein in MDA-MB-231 cells. Cells were treated with TGF and lysed at different time points as indicated. The levels of fascin protein were determined through Western blotting. RU, relative units. B, MDA-MB-231 cells were treated with TGF for different time points as indicated, and phospho-Smad3 (p-Smad3) levels were determined through Western blotting. C, TGF was able to induce fascin mRNA expression in the presence of protein synthesis inhibitor CHX (10 M). Results are mean S.D. of triplicates. D, CHX (10 M) inhibited the regulation of E-cadherin and N-cadherin transcription by TGF. Results are mean S.D. of triplicates. E, treatment with MG132 (10 M) inhibited TGF-induced fascin transcription. Results are mean  S.D. of triplicates. Western blotting (Fig. 5A). To assess the role of fascin in TGF on the invasion ability of spindle tumor cells stably TGF-induced filopodia formation, A549 cells stably expressing fascin or control shRNA. TGF pretreatment pro- expressing control shRNA or fascin shRNA were treated moted the invasion of control shRNA-expressing spindle with TGF and stained for F-actin. TGF induced long filop- tumor cells by about 3–5-fold, consistent with the pro-metas- odia and spiky morphology in control shRNA cells (Fig. 5B). tasis function of TGF. Expression of fascin shRNA dramati- TGF-induced long filopodia were noted in about 67% of cally inhibited TGF-promoted invasion in all three of the spin- A549 cells expressing control shRNA (183 out of a total of dle tumor cells. In contrast to the 3–5-fold invasion increase in 274 cells examined) (Fig. 5C). In contrast, TGF-induced control shRNA cells, TGF was only able to promote invasion long filopodia were only observed in roughly 20% of A549 by about 1.2–1.5-fold in fascin shRNA cells (Fig. 5E). fascin shRNA cells (60 out of a total of 294 cells examined), To further evaluate the hypothesis that fascin is critical for suggesting that fascin was critical for TGF-induced filopo- TGF-induced tumor cell migration and invasion, we overex- dia formation (Fig. 5, B and C). pressed fascin in tumor cells expressing Smad4 shRNA. As Next, we investigated the role of fascin in TGF-promoted shown in Fig. 5, F and G, Smad4 knockdown nearly completely spindle tumor cell migration. Using the Transwell migration abolished TGF-induced migration and invasion in MDA-MB- assay, we found that TGF-promoted cell migration was signif- 231 cells, consistent with the observation that Smad4 was icantly lower in fascin shRNA-treated cells when compared essential for TGF-induced fascin expression; ectopic fascin with the control shRNA-expressing cells, suggesting that fascin expression restored shSmad4 cell migration and invasion to was important for TGF-induced cell migration. (Fig. 5D). levels similar to TGF-primed control cells, consistent with the To further assess the role of fascin in TGF-promoted notion that fascin is critical for TGF-induced migration and metastasis in spindle cell tumors, we evaluated the effects of invasion in metastatic tumor cells. NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 38871 Fascin in TGF-induced Tumor Cell Invasion 38872 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Fascin in TGF-induced Tumor Cell Invasion FIGURE 6. Fascin expression levels correlate with TGF1 and TGF receptor 1 levels in primary breast tumors. A, correlation between fascin (FSCN1), TGF1, and TGF receptor 1 probe sets in 99 primary tumor samples in the Sloan cohort. Pearson correlation coefficient values (r) and probability values (p) were calculated using Excel. B, fascin expression levels in the 99 breast tumor samples. Tumor samples were sorted according to TGF1 levels. RU, relative units. C, fascin expression levels in the TGF1 low and TGF1 high groups. p value was calculated using two-tailed Student’s t test. Correlation between TGF1, TRI, and Fascin Expression TGF1 low group (p 0.002) (Fig. 6C), suggesting that elevated Levels in Primary Breast Tumor Samples—To validate our cell TGF1 levels in primary breast tumors contributed to fascin culture-based discoveries in breast cancer patients, we exam- overexpression. ined the correlation between fascin levels and TGF as well as DISCUSSION TGF receptor levels in a cohort of 99 primary breast tumor samples collected at the Memorial Sloan-Kettering Cancer Fascin Expression Is Regulated by TGF—Although there are Center (34). Pearson correlation coefficient (r) and probability a plethora of studies about fascin overexpression in various car- (p) values between the two fascin probe sets and each of the cinomas (4, 5, 7, 8, 11, 12), the factors contributing to fascin probe sets for TGF1, TGF2, and TGF3 and TGF receptor overexpression in metastatic tumors remain largely unknown. I and II were calculated. Fascin expression significantly corre- Binding sites for several transcription factors, including lates with TGF1 and TGF receptor I, but not with TGF2 -catenin-TCF (T-cell factor), CREB, and AP-1, in the FSCN1 and TGF3orTGF receptor II (Fig. 6A and data not shown). promoter region have been previously reported (5, 29, 30, 35). Next, tumor samples were sorted according to TGF1 expres- However, it is not clear whether and how factors in the tumor sion level (Fig. 6B). The 50 tumor samples with TGF1 level at microenvironment contribute to fascin overexpression in met- or below median level were assigned to the “TGF low” group, astatic tumor cells. Our results here demonstrated that TGF and the other 49 samples with TGF1 level above the median promoted fascin expression in spindle tumor cells, implicating level were assigned to the “TGF1 high” group. Fascin (FSCN1) that cytokines in the tumor microenvironment could affect fas- levels in the TGF1 high group were about 2-fold as high as the cin expression. FIGURE 5. Fascin is required for TGF-induced filopodia formation, cell migration, and invasion. A, Western blots showing that fascin shRNA decrease the protein level of fascin in MDA-MB-231, A549, and CHL-1 cells. B, F-actin staining showing that TGF induced long filopodia in A549 cells expressing control shRNA, but not in cells expressing fascin shRNA. The scale bar is 10 m. Results are representative of 3 experiments. C, quantification of cells with long filopodia in A549 cells expressing control shRNA or fascin shRNA, with or without TGF treatment, respectively. D and E, the effects of TGF pretreatment on tumor cell migration (D) and invasion (E) in spindle tumor cells expressing control shRNA or fascin shRNA, respectively. Fascin knockdown with shRNA inhibited TGF- promoted tumor cell migration and invasion. F and G, ectopic fascin overexpression restore migration (F) and invasion (G) in MDA-MB-231 stably expressing Smad4 shRNA. The data in C–G are representative of either 3–5 similar experiments or mean S.D. of 3 experiments. *, p  0.05. NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 38873 Fascin in TGF-induced Tumor Cell Invasion The up-regulation of fascin by TGF was surprising in a found impacts on tumor progression and metastasis (14, 15, sense because TGF target genes have been studied previously  signaling in primary tumor was linked to 17). Activated TGF in various epithelial and tumor cell lines (including MDA-MB- lung metastasis in estrogen receptor-negative breast tumors 231), and fascin was not among the TGF response genes iden- (14). Our data demonstrated that TGF induced fascin expres- tified through microarray screening (14, 36). We noticed that sion and promoted migration/invasion in MDA-MB-231 breast previous screenings focused on early response genes and that tumor cells. More importantly, TGF1 and TGF receptor I tumor cells were treated with TGF for no more than 3 h (14, significantly correlate with fascin (FSCN1) level levels in a 36). Therefore, we explored the kinetics of TGF-induced tran- cohort of primary breast tumors, suggesting that activation of scription of fascin. Our data revealed a 3-h lag in TGF-in- TGF signaling may contribute to fascin overexpression in pri- duced fascin expression (Fig. 3D), which may explain the dis- mary tumors. Interestingly, fascin overexpression in breast crepancy between our data and previous studies. The lag might tumor has been associated with lung metastasis, and fascin is be due to the requirement for TGF-induced degradation of one of the top performing lung metastasis signature genes (34). Smad co-suppressors as proteasome inhibitor MG132 inhib- It is possible that elevated fascin expression is critical for TGF ited TGF-induced fascin transcription. Despite the lag, fascin to promote tumor cell migration, invasion, and lung metastasis expression is likely directly downstream of TGF signaling in estrogen receptor-negative breast tumors. Indeed, TGF because TGF was able to promote the expression of fascin promoted migration and invasion in all three spindle tumor cell mRNA in the presence of CHX, a protein synthesis inhibitor. lines with elevated fascin expression, but not in the three polyg- Fascin Is Regulated by TGF through the Canonical TRI- onal tumor cells, in which TGF-induced fascin expression was Smad Pathway—Our data further demonstrated that fascin absent. expression was regulated through the canonical TRI-Smad More importantly, knockdown of fascin expression with pathway. Smads in the TGF pathway recognized the consen- shRNA significantly inhibited migration and invasion in TGF- sus DNA sequence CAGAC (37). To determine whether the treated spindle tumor cells. The near complete inhibition of human fascin 1 gene contains Smad-binding sites, we examined TGF-promoted invasion by fascin shRNA was quite remark- the promoter region of FSCN1 and discovered that there were able considering that TGF also up-regulates the expression of two CAGAC sequences at 381 and 1225 positions, respec- other pro-invasion genes such as matrix metalloproteases 2 and tively. It is possible that activated Smad3 and Smad4 induced 9 (17, 39). During invasion, tumor cells use protrusion struc- fascin expression by binding to one or both of the Smad-bind- tures termed invadopodia to secrete matrix metalloproteases ing sites. and to coordinate the degradation of extracellular matrix (40). It is intriguing to note that TGF only induced fascin expres- The formation and elongation of invadopodia requires filopo- sion in spindle tumor cells, but not in polygonal cells. One way dia-associated protein components (41). Fascin is the major for tumor cells to overcome growth inhibition effects exerted actin-bundling protein in filopodia protrusions (27). It was by TGF is through loss of expression or functional inactiva- recently reported that fascin was critical to stabilize actin in tion of TRI and TRII (38). We noted that that TRI and invadopodia (26, 41, 42). Depletion of fascin with shRNA desta- TRII levels in the three spindle tumor cells were higher than bilized invadopodia and impaired extracellular matrix degrada- the three polygonal cells. The reduced expression of TGF tion (42). Given the similar components and regulatory mech- receptors might partially explain the lack of TGF-induced fas- anism between invadopodia and filopodia, it has been cin expression in polygonal cells. suggested that invadopodia are “invasive filopodia” (26, 42). The Smad co-activator context in cells may also contribute to Our data showed that fascin was required for long filopodia the regulation of fascin by TGF. Although Smad3 and Smad4 induced by TGF. It is possible that fascin played a central role can directly bind to DNA to mediate gene transcription, inter- in the invasive machinery mobilized by TGF in spindle tumor action between Smads and their target sequence is of low affin- cells. By stabilizing actin cytoskeleton within invadopodia and ity, and DNA-binding co-factors are required to provide spe- filopodia, fascin coordinates the invasion and metastasis pro- cific regulation of gene transcriptions (37). Consequently, the moted by TGF. transcriptomic output of activated Smads is highly dependent on the cellular context of Smad co-factors (15). The conversion Acknowledgments—We thank Drs. Srikumar Chellappan and Xin- from polygonal morphology to spindle morphology during Yun Huang for critically reading the manuscript, Dr. Steven Enkeman tumor progression is a recessive event resulting from the for consultation on microarray data mining, the Analytic Microscopy change of gene expressions that control epithelial differentia- Core at Moffitt Cancer center for image acquisition assistance, and Rasa Hamilton for editorial assistance. tion (16). It is possible that the transition from epithelial-like to mesenchymal-like tumor cells provides the cellular co-factor context required for the induction of fascin expression by acti- REFERENCES vated Smads. We noted that some polygonal tumor cells (e.g. 1. Mareel, M., and Leroy, A. (2003) Physiol. 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Journal

Journal of Biological ChemistryAmerican Society for Biochemistry and Molecular Biology

Published: Nov 11, 2011

Keywords: Cell Migration; Cytoskeleton; Invasion; Metastasis; Transforming Growth Factor Beta (TGFbeta); Actin Cytoskeleton; Fascin; Filopodia

References