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Clinical Cancer Research Vol. 11, 2597-2605, April 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Prognostic Significance of Fascin Overexpression in Human Esophageal Squamous Cell Carcinoma

Yosuke Hashimoto1, Tetsuo Ito1, Harutaka Inoue1, Tomoyuki Okumura1, Eiji Tanaka1, Shigeru Tsunoda1, Motoshige Higashiyama1, Go Watanabe1, Masayuki Imamura2 and Yutaka Shimada1

Authors' Affiliations: 1 Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan and
2 Department of Surgery, Saiseikai Noe Hospital, Osaka, Japan

Requests for reprints: Yutaka Shimada, Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawara-Cho, Sakyo-Ku, Kyoto 606-8507, Japan. Phone: 81-75-751-3445; Fax: 81-75-751-3219; E-mail: shimada{at}kuhp.kyoto-u.ac.jp.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Fascin, an actin bundling protein, induces membrane protrusions and increased cell motility in various transformed cells. The expression of fascin in epithelial neoplasms has been described only recently, and the role of fascin in esophageal squamous cell carcinoma (ESCC) is still unknown.

Experimental Design: Paraffin sections of 200 patients with ESCC were immunohistochemically investigated. The expression levels of fascin mRNA in 20 ESCC tissues were compared with that in corresponding normal esophageal epithelium by semiquantitative reverse transcription-PCR. We also examined fascin protein expression in 33 ESCC cell lines. The role of fascin in cell motility and invasiveness in ESCC cells was assessed by the vector-based small interfering RNA.

Results: In immunohistochemical study, the intensity of fascin expression was usually increased in the tumor compared with that in normal epithelium. Fascin overexpression was significantly associated with a poor prognosis (immunoreactive rate, P = 0.033; immunoreactive intensity, P = 0.031). The fascin immunoreactive rate was associated with extent of the tumor (P = 0.002) and lymph node metastasis (P = 0.003). Multivariate analysis showed that fascin expression intensity was an independent prognostic factor, but the immunoreactive rate was not.

In addition, up-regulation of fascin mRNA was found in 60% (12 of 20) of patients. In vitro study revealed that all 33 ESCC cell lines expressed fascin protein at a certain level. KYSE170, one of the fascin-overexpressed cells, decreased its motile and invasive properties after down-regulation of fascin expression.

Conclusion: Our findings suggest that fascin overexpression may play an important role in the progression of ESCC.

Key Words: Lymph node metastasis • tumor invasion • vector-based siRNA

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies in the world, including Japan, with a 5-year survival rate of 20% to 30% after curative surgery (1, 2). One of the reasons for the poor prognosis is that ESCC exhibits extensive local invasion or frequent regional lymph node metastasis even at initial diagnosis. Tumor invasion and metastasis are the results of several sequential steps and in part are caused by the highly motile properties of tumor cells to overcome cell-cell and cell-matrix adhesion and to invade surrounding tissue (3–5). Invasive tumor cells often show specific morphologic features, such as the appearance of membrane protrusions as well as loss of cell-cell adhesion and loss of junctional communications, and these are thought to result from rearrangements of the cytoskeletal microfilaments by the action of actin cross-linking proteins (6–8).

Among these actin cross-linking proteins, this study focused on fascin, an actin bundling protein originally found in the extracts of unfertilized sea urchin eggs and localized to the microfilament bundles within microvilli cores and within filopodia on the surface of fertilized sea urchin eggs (9–13). In mammalian cells, fascin is present in membrane ruffles, microspikes, and other motility-associated cell fibers (6, 12, 14, 15). The expression of fascin is markedly increased in many transformed cells. A morphologic characteristic seen in the cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present (16).

The expression of fascin in epithelial neoplasms has been described only recently. In normal epithelial cells, fascin expression is usually absent or very low but is often up-regulated in several types of human neoplasms, such as ovarian, breast, pancreatic, colon, lung, and skin tumors (17–24). There are two reports on the malignancy of the squamous epithelium, and both indicated that fascin might have some role in the malignant portion of the squamous cell epithelium (24). We found previously that fascin immunoreactivity in gastric carcinoma was associated with lymph node metastasis (25). However, the clinicopathologic features of fascin expression in ESCC are still unclear. To determine whether fascin plays a role in ESCC, we examined a large number of surgical specimens of ESCC for the expression of fascin. We also evaluated the role of fascin protein in ESCC cell lines.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and surgical specimens. Frozen tumor tissues and corresponding normal tissues were obtained for semiquantitative reverse transcription-PCR from 20 patients, and paraffin-embedded sections were acquired from 200 patients for immunohistochemistry with primary ESCC who underwent surgery at Kyoto University Hospital from 1990 to 2001. Twenty-eight of these 200 patients for immunohistochemistry were women and 172 were men. The median age of the patients was 65 years with a range of 43 to 90 years. The median follow-up time of survival was 40.1 months, with a range of 2 to 146 months. Information on gender, age, stage of disease, and histopathologic factors was abstracted from the medical records. Patients' data are summarized in Table 1. All of the tumors were confirmed as ESCC by the clinicopathologic department of the hospital. All of the cases were classified according to the pathologic tumor-node-metastasis classification (26). Of the 200 patients, 54 (27.0%) had T1, 45 (22.5%) had T2, 67 (33.5%) had T3, and 34 (17.0%) had T4. Specimens were fixed in a 10% formaldehyde solution and embedded in paraffin blocks, cut in 4 µm thick sections, and mounted on glass slides. Written informed consent was obtained from the patients for surgery and to use resected samples for research. The study was approved by the Institutional Review Board of Kyoto University.


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  		Table 1. Patients and tumor characteristics

 
Immunohistochemical staining. Immunohistochemical staining was done using the avidin-biotin method as described previously (25). Briefly, antigen retrieval was carried out by incubation in 0.01 mol/L citrate buffer (pH 6.0) at 95°C for 40 minutes in a water bath. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide for 30 minutes. Next, sections were incubated with 5% normal horse serum in PBS for 30 minutes at room temperature to block nonspecific antibody reaction. After a wash with TBS plus 0.1% Tween 20, slides were incubated overnight at 4°C with anti-human fascin monoclonal antibody clone 55k-2 (1:50 dilution, DAKO, Osaka, Japan). After being rinsed in TBS plus 0.1% Tween 20, tissue sections were incubated for 40 minutes at room temperature with biotinylated anti-mouse IgG followed by avidin-biotin-peroxidase complex (Vecstatin Elite ABC kit, Vector Laboratories, Inc., Burlingame, CA) for 40 minutes. Subsequently, they were stained with 0.003% 3,3-diaminobenzide tetrahydrochloride and 0.005% hydrogen peroxide in 0.05 mol/L Tris-HCl (pH 7.2), counterstained with Mayer's hematoxylin, dehydrated, and mounted.

Evaluation of immunohistochemical staining. A section without the primary antibody was used as a negative control in each case. A metastatic breast carcinoma shown previously to have immunoreactivity was used as a positive control to confirm the fascin immunoreactivity in each series of experiments. Fascin immunoreactivity in each tumor was verified by the labeling of endothelial cells of microvessels in every specimen. Fascin-positive samples were defined as those showing a cytoplasmic pattern of lesional tissue.

The distribution of fascin labeling was measured using the following scale according to the percentage of fascin-positive cells: <25% (1+), 25% to 50% (2+), 50% to 75% (3+), and >75% (4+). Fascin staining intensity was graded into three groups standardized by the staining level of endothelial cells of the microvessels: weak (less intense than endothelial cells), moderate (same intensity as endothelial cells), and intense (more intense than endothelial cells).

All slides were evaluated independently by two investigators (Y.H. and H.I.) without any prior knowledge of each patient's clinical information. When the opinions of the two evaluators were different, agreement was reached by careful discussion.

Statistical analysis. Fascin expression levels and clinicopathologic factors were analyzed using {chi}2. The overall survival was defined as that from the date of the operation to the date of death due to cancer. The Kaplan-Meier method was used to determine the probability of survival, and data were analyzed with the log-rank test. Multivariate analysis was done using the Cox regression model to study the effects of different variables on overall survival, and six factors (fascin status, gender, age, extent of primary tumor, nodal status, and metastasis) were studied. A score was assigned to each variable for the regression analysis. P < 0.05 was considered significant.

Purification of total cellular RNA and semiquantitative reverse transcription-PCR. Total cellular RNA was purified from frozen stored tissues of ESCC patients by the TRIzol reagent (Invitrogen, Carlsbad, CA) method (27, 28). Reverse transcription of total cellular RNA (5 µg) was done using a First-Strand cDNA Synthesis kit (Amersham, Buckinghamshire, United Kingdom), and cDNA was subjected to PCR for 25 cycles of amplification. Amplification was done for 1 minute at 94°C and 1 minute at 60°C, and the final extension step was carried out for 1 minute at 72°C. The amplification products were separated on 1.5% agarose gels and visualized by ethidium bromide staining. The PCR primers used for fascin were as follows: forward primer 5'-AGGCGGCCAACGAGAGGAAC-3' and reverse primer 5'-ACGATGATGGGGCGGTTGAT-3'. According to the fascin gene structure, a PCR product of 364 bp was obtained. For glyceraldehyde-3-phosphate dehydrogenase, the forward primer was 5'-TGGTATCGTGGAAGGACTCATGAC-3' and the reverse primer was 5'-ATGCCAGTGAGCTTCCCGTTCAGC-3'. A single 189-bp band amplified with primers specific for glyceraldehyde-3-phosphate dehydrogenase with the same cDNA was detected and used as an internal control under identical conditions.

Cell cultures. Human esophageal squamous carcinoma cell lines KYSE series, Sum/c, and HSA were established in our department and cultured in Ham's F-12/RPMI 1640 with 2% fetal bovine serum according to the method reported previously (29). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air.

Western blot analysis. Cells were lysed in a sample buffer [2% SDS, 10% glycerol, 50 mmol/L Tris-HCl (pH 6.8)] at room temperature. Cell lysates were sonicated and the protein concentration was estimated by the Bradford method using BCA Protein Assay Reagent (Pierce, Rockford, MA). Cell lysates (20 µg) were electrophoresed on a 2% to 15% gradient polyacrylamide gel (Daiichi Pure Chemicals, Tokyo, Japan) and transferred to polyvinylidene difluoride membranes (Immobilon, Millipore, Bedford, MA). The membranes were then blocked with TBS [20 mmol/L Tris, 150 mmol/L NaCl (pH 7.6)] containing 5% skim milk (Difco, Detroit, MI) and 0.1% Tween 20 for 1 hour and incubated at room temperature for 1 hour with the primary antibody described above. The membrane was subsequently incubated at room temperature for 1 hour with horseradish peroxidase–linked goat anti-mouse IgG (EY Laboratories, Inc., San Mateo, CA) and analyzed using Western Blotting Luminol Reagent (Santa Cruz Biotechnology, San Diego, CA).

Quantitative analysis was done on a Macintosh computer using the public domain NIH Image program version 1.61 (developed at NIH and available on at http://rsb.info.nih.gov/nih-image/).

Construction of fascin-small interfering RNA expression vector. To construct a vector for fascin-small interfering RNA (siRNA), the pSilencer2.1-U6 hygro (Ambion, Inc., Austin, TX) was digested with BglII and HindIII. A chemically synthesized oligonucleotide encoding a fascin-short hairpin siRNA that included a loop motif was inserted into downstream of the U6 promoter of the plasmid using DNA ligation kit (Takara Bio, Inc., Shiga, Japan) and cloned. Sequences of the oligonucleotide targeted to fascin are 5'-GCCUGAAGAAGAAGCAGAU-3' corresponding to positions 116 to 125 within the fascin exon 1.

Transfections. An ESCC cell line KYSE170 was stably transfected with the fascin-siRNA expression vector or the negative control vector (pSilencer2.1-U6 hygro) using FuGene6 reagent (Roche Diagnostics, Basel, Switzerland), and cell clones were selected against 100 µg/mL hygromycin (Nacalai Tesque, Kyoto, Japan).

Immunofluorescent staining. Cells were seed on a coverslip and incubated for 24 hours. After being washed with PBS, cells were fixed with 10% trichloroacetic acid in distilled water at 4°C (for fascin and 4',6-diamidino-2-phenylindole) or with 4% paraformaldehyde in PBS at room temperature (for ß-actin) for 15 minutes and treated with 0.2% Triton X-100 in PBS for 10 minutes. Cells were subsequently incubated with a blocking solution (1% bovine serum albumin in PBS) for 1 hour and incubated with primary antibody for 1 hour at room temperature. The cells were washed and incubated with fluorescein anti-mouse IgG (Vector Laboratories) and rhodamine phalloidin (dilution 1:1,000, Molecular Probes, Inc., Eugene, OR) as a secondary antibody for 30 minutes at room temperature. The cells were washed and nuclei were counterstained using 4',6-diamidino-2-phenylindole (Vector Laboratories), mounted in glycerol, and viewed with a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany).

Cell migration assay. The migration was determined by a micropore chamber assay. Cells (2.5 x 104) were seeded onto the top chamber of a 24-well micropore polycarbonate membrane filter with 8 µm pores (Becton Dickinson Labware, Lincoln Park, NJ), and the bottom chamber was filled with Ham's F-12/RPMI 1640 containing 10% fetal bovine serum as a chemoattractant. After 22 hours of incubation in a 5% CO2 humidified incubator at 37°C, the membranes were fixed and stained by DiffQuik reagent (International Reagents, Inc., Kobe, Japan), and the cells on the upper surface were carefully removed with a cotton swab. Migration was quantified by counting the average migrated cells in five random high-powered fields per filter.

Cell invasion assay. The invasion was determined by an invasion chamber assay. Cells (2.5 x 104) were seeded onto the top chamber of a 24-well Matrigel-coated micropore membrane filter with 8 µm pores (Becton Dickinson Labware), and the bottom chamber was filled with RPMI 1640 and Ham's F-12 mixed medium containing 10% fetal bovine serum as a chemoattractant. After 22 hours of incubation in a 5% CO2 humidified incubator at 37°C, the membranes were fixed and stained by DiffQuik reagent, and the cells on the upper surface were carefully removed with a cotton swab. Invasion was quantified by counting all of the cells that had migrated through the membrane.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Expression of fascin protein in esophageal squamous cell carcinoma. We first examined the distribution of fascin in the normal esophageal epithelium by antibody staining. Positive staining was apparent in the basal layer and lower spinous layer of the esophageal epithelium. The intensity of this staining pattern decreased progressively from moderate to weak, moving from the basal layer to the granular layer (Fig. 1). Positive staining was observed in endothelial cells, lymphocytes, and stromal cells in the underlying lamia propria, and fascin stained mainly in the cytoplasm of the cells. In the carcinomatous portion, heterogeneous labeling of the tumor was found. Tumor cells exhibited moderate to intense diffuse labeling by fascin. Intense staining was identified in tumor cells at the infiltrative margins. Areas exhibiting squamous pearl formation were negative for fascin. Of the 200 patients with ESCC, 145 (71.1%) patients were classified as 4+, >75% of positive staining, whereas 55 (28.9%) patients were classified as positive 1+, 2+, and 3+, <75% staining for fascin (Table 2). For fascin immunoreactive intensity, 82 (41%) patients were classified as weak, 85 (42.5%) as moderate, and 33 (16.5%) as intense.



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  		Fig 1. A, normal esophageal epithelium fascin is expressed in basal and lower spinous layers (x200). B, ESCC demonstrating intense immunoreactivity for fascin especially at the edge of the tumor (x100). C, another sample with high manifestation. Endothelial cells underlying the tumor (arrows) are positive for fascin (x400). D, the other case of ESCC demonstrating weak immunoreactivity for fascin from a different patient (x200).

 

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  		Table 2. Relationship between fascin expression and clinicopathologic characteristics in all patients

 
Correlation between fascin protein immunoreactivity and clinicopathologic findings. The correlation between fascin protein expression and various prognostic factors, such as pathologic tumor-node-metastasis classification, histopathologic grading, and stage grouping, was investigated (Tables 3 and 4). According to the fascin immunoreactive rates, fascin expression was correlated with gender (P = 0.016), extent of the tumor (P = 0.002), positive lymph node metastasis (P = 0.003), and tumor-node-metastasis staging (P = 0.034). There was no significant correlation between fascin expression and other factors, such as age, distant metastasis, histology, and tumor location. In contrast, the fascin immunoreactive intensity was not correlated with these clinicopathologic factors. The multivariate analysis showed that pT, pN, and fascin expression intensity were independent poor prognostic factors, but fascin immunoreactive rate was not (Table 5).


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  		Table 3. Relationship between fascin intensity and clinicopathologic characteristics in all patients

 

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  		Table 4. Relationship between fascin positivity and clinicopathologic characteristics in all patients

 

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  		Table 5. Cox multivariate analysis

 
Association between fascin status and patient prognosis. Calculations of survival by the Kaplan-Meier method revealed that both fascin immunoreactive intensity and immunoreactive rate were significant prognostic factors (Fig. 2).



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  		Fig. 2. A, cumulative survival curves of the patients. The survival rate of patients with a high fascin-positive rate (>75%) was significantly lower than that of patients with fascin-negative tumors (P = 0.033). B, prognosis of patients with intense fascin immunoreactivity is shorter than that of patients with weak or moderate fascin immunoreactivity (P = 0.031).

 
Semiquantitative reverse transcription-PCR. Immunohistochemical data of fascin overexpression raised us the question whether this phenomenon was transcriptional or post-transcriptional. To assess this question, we did semiquantitative reverse transcription-PCR by using frozen samples of ESCC patients. At first, the signal intensity of each sample was calculated by using NIH image and the ratio of fascin to glyceraldehyde-3-phosphate dehydrogenase was scored. Next, fascin expression in each specimen was evaluated by the ratio of the tumor to the corresponding normal epithelial part (T/N ratio). Parts of the results are shown in Fig. 1. The distribution of fascin mRNA expression among 20 patients is as follows: five cases had a T/N ratio of 0-1.0, three cases had 1.0-1.5, three cases had 1.5-2.0, and nine cases had >2.0 (Fig. 3).



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  		Fig. 3. Semiquantitative reverse transcription-PCR. Fascin mRNA expression of tumor samples (T) and corresponding normal epithelium (N) were investigated. At first, the signal intensity of each sample was calculated using NIH image software, and the ratio of fascin/glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was scored. Next, fascin expression in each specimen was evaluated by the ratio of the tumor to the corresponding normal epithelial part (T/N ratio) and finally divided into two groups according to the T/N ratio.

 
Western blot analysis in esophageal squamous cell carcinoma cell lines. Next, we examined fascin expression in 33 ESCC cell lines. All cell lines tested expressed fascin protein. Of these cell lines, 29 (87.9%) expressed fascin protein at the same level as or a higher level than positive control HeLa cells (Fig. 4).



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  		Fig. 4. Expression of fascin protein in 33 ESCC cell lines. Equal amounts of total protein (20 µg) were loaded in all lanes. Immunoblots were probed with anti-fascin monoclonal antibody and anti-ß-actin monoclonal antibody. HeLa cell line was included as a positive control of fascin expression. Expression of ß-actin served to confirm the equivalent loading of total protein.

 
Vector-based small interfering RNA decreased motile and invasive properties in esophageal squamous cell carcinoma cell lines. To assess the role of fascin overexpression in ESCC cells, we firstly established subclones by the transfection of vector-based siRNA for fascin in KYSE170 cells (30–32). Fascin expression level was efficiently reduced by 67% in stable subclones 170F1, 94% in 170F2, 94% in 170F3, and 96% in 170F4. Transfection of the vector of nonspecific siRNA (170mock) did not reduce fascin expression level (Fig. 5A and B). Stable transfectants of siRNA for fascin tended to show less filopoidal formation (Fig. 5B). Next, we examined the ability of fascin siRNA treatment to affect cell migration and invasion. Fascin down-regulation dramatically reduced cell motile and invasive properties compared with parental cells and/or mock cells; cell migration was decreased 50% to 90%, and cell invasion was decreased 80% to 93% (Fig. 5C and D).



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  		Fig. 5. Vector-based siRNA for fascin down-regulation in ESCC cells. A, Western blot analysis for fascin expression in parental KYSE170 cells and stable transfectant with nonspecific siRNA or with siRNA for fascin. B, immunofluorescent staining of each clone. Top, anti-fascin antibody; middle, 4',6-diamidino-2-phenylindole (DAPI); bottom, phalloidin. C, transwell migration assay; D, Matrigel invasion assay.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this report, we showed that fascin overexpression frequently occurred in ESCC and was correlated with several aspects of tumor progression summarized in the tumor-node-metastasis classification. Recently, the expression of fascin in various kinds of malignant tumors has been reported. However, only a few reports, including our previous article, showed the prognostic relevance of fascin expression in neoplasms. Pelosi et al. reported that they observed fascin expression in 98% of 116 squamous cell carcinomas of stage I non–small cell lung cancer and 78% of 96 adenocarcinomas of this disease. In their study, fascin immunoreactivity was associated with high tumor grade. Patients with tumors showing diffuse (>60% immunoreactive neoplastic cells) and/or strong immunoreactivity for fascin had a shorter survival. Especially squamous cell carcinoma, patients with strong immunoreactivity for fascin had a shorter survival, and fascin intensity was an independent prognostic factor. We showed previously the prognostic relevance of fascin expression in a large series of gastric carcinoma and its significant relation with clinicopathologic findings (25). These results indicated that fascin overexpression might be an outcome of some important genetic and/or epigenetic change in cancer progression; however, it remains to be seen whether in these carcinomas fascin expression represents merely a surrogate marker for prognosis or whether it plays a pathogenic role in carcinogenesis and tumor progression.

By using the vector-based siRNA, we succeeded fascin down-regulation and decreased motility and invasiveness in an ESCC cell line. The mechanism by which fascin increases motile and invasive potential in cancer cells are not fully understood. Recent report showed overexpression of fascin in colon cancer cells with a decreased capacity for normal glandular differentiation as well as increased cell motility and invasiveness (22). Another report showed that the ectopic constitutive expression of mouse fascin in rat Con8 cells disrupted the dexamethasone-induced localization of the tight junction protein occludin and the adherens junction protein ß-catenin to the cell periphery and prevented the rearrangement of the actin cytoskeleton (33). These reports support the present data, and fascin overexpression in ESCC ultimately affects the changes in cell motility and aggregation status.

ESCC cell lines all expressed fascin protein in the Western blot analysis, and 29 (87.9%) expressed fascin protein at the same level as or a higher level than HeLa cells, a fascin-overexpressed cell line widely used as a positive control (16). Only a few reports are available to concern about fascin overexpression in cancer cell lines. Compared with breast carcinoma cell lines (19) or gastric carcinoma cell lines (22), the rate of fascin overexpression is relatively high in ESCC cell lines. This finding is partly explained by the finding that fascin was also expressed in normal esophageal epithelium. However, fascin staining intensity is usually increased in the tumor portion compared with the normal epithelium. In addition, fascin mRNA expression level in the tumor is higher than that in the normal epithelium in 60% of 20 samples. These data supported our view of fascin overexpression in the tumor. There still remains the question of which pathway contributes to the overexpression of fascin protein in ESCC. A previous report suggested that a pathway for fascin up-regulation is dependent on amplification or overexpression of c-erbB-2/HER-2 (19). Others showed a possible influence of wnt (wingless-type) signaling on fascin activity, suggesting that anomalies of this pathway, such as stabilizing ß-catenin mutations or inactivation of the APC gene, may up-regulate fascin expression in cancer cells (34). Neither c-erbB-2 amplification nor wnt signaling abnormalities are particularly common in ESCC (35, 36), and an alternative pathway of transactivation may exist in these tumors.

According to our data, fascin is usually overexpressed in ESCC cells compared with normal esophageal epithelia, and increased expression of fascin was associated with a poor prognosis. Moreover, down-regulation of fascin reduced cell motility and invasiveness in the ESCC cell line. Thus, our findings suggest that fascin overexpression may play an important role in the progression of ESCC. Tumor-specific fascin down-regulation may become a novel therapeutic strategy to ESCC patients.


   Acknowledgments
 
We thank Sakiko Shimada for kind assistance in immunohistochemical staining and Dr. Josephine C. Adams (Lerner Research Institute, Cleveland, OH) for helpful discussion.


   Footnotes
 
Grant support: Japanese Ministry of Education, Culture, Sports, Science and Technology grant-in-aid 14370385.

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

Received 7/13/04; revised 12/27/04; accepted 1/13/05.


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