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Clinical Cancer Research Vol. 12, 1308-1316, February 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

An Inducible Short-Hairpin RNA Vector against Osteopontin Reduces Metastatic Potential of Human Esophageal Squamous Cell Carcinoma In vitro and In vivo

Tetsuo Ito1, Yosuke Hashimoto1, Eiji Tanaka1, Takatsugu Kan2, Shigeru Tsunoda1, Fumiaki Sato2,3, Motoshige Higashiyama1, Tomoyuki Okumura1 and Yutaka Shimada1

Authors' Affiliations: 1 Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan and Departments of 2 Medicine and 3 Pathology, University of Maryland School of Medicine, Baltimore, Maryland

Requests for reprints: Yutaka Shimada, Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Kawaracho 54 Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Phone: 81-75-751-3626; Fax: 81-75-751-4390; E-mail: shimada{at}kuhp.kyoto-u.ac.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: To elucidate the clinical significance of osteopontin and the effect of conditional down-regulation of osteopontin in esophageal squamous cell carcinoma (ESCC), we investigated osteopontin expression in tumors and tested an inducible osteopontin-short-hairpin RNA (shRNA) expression vector in an ESCC cell line.

Experimental Design: Osteopontin mRNA expression was extracted from gene expression profiles of 23 tumors determined by cDNA microarray and was analyzed. Paraffin sections of 144 tumors were immunohistochemically investigated. Osteopontin protein expression in 34 cell lines was examined by Western blot. A doxycycline-inducible osteopontin-shRNA vector was stably transfected into HSA/c cells to assess the role of osteopontin in cell motility, invasion in vitro, tumor formation, and lymph node metastasis in nude mice.

Results: cDNA microarray revealed that high osteopontin mRNA expression was associated with poor survival of ESCC patients (P = 0.029). In immunohistochemistry, osteopontin protein expression was associated with poor prognosis (P < 0.001), distant lymph node metastasis (P = 0.0004), tumor staging (P = 0.027), and histologic grade (P = 0.024). Multivariate analysis showed that osteopontin overexpression was the strongest independent prognostic factor among nine clinicopathologic variables (P < 0.001). Among cell lines tested, 30 had overexpressed osteopontin protein compared with a normal esophageal epithelial cell line. An inducible shRNA vector against osteopontin successfully down-regulated osteopontin expression by 71% to 88% and repressed cell motility by 69% to 97%, cell invasion by 59% to 71%, tumor formation by 56% to 92%, and lymph node metastasis by 50% to 67% in HSA/c cells.

Conclusions: Our findings suggest that osteopontin overexpression may play an important role in progression of ESCC and osteopontin could be a potential target of ESCC therapy.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies in the world, including Japan, despite recent refinement of various therapeutic strategies, including surgery, chemotherapy, radiotherapy, or combined therapy (1, 2). The main reason for the poor outcome is the aggressive behavior of infiltrative and systemic spread, resulting in frequent local recurrence and regional or distant lymph node metastasis. Therefore, identifying and targeting genes associated with progression of ESCC is needed to improve the survival of patients with this type of refractory cancer.

Osteopontin is an integrin-binding secreted adhesive glycoprotein involved in a variety of physiologic cellular functions, including osteoblast differentiation and bone formation (3, 4). It has been shown to play an important role in tumorigenesis, tumor invasion, and metastasis in variety of cancers (5–7). The function of osteopontin in tumor pathophysiology is, however, complex and not fully understood. Investigators have identified several important downstream osteopontin signals that regulate tumor progression and invasive behavior (6). Osteopontin seems to regulate the activity of extracellular matrix–degrading proteins, such as matrix metalloproteinase-2 (MMP-2; ref. 8). Recent analyses of microarray results revealed that osteopontin expression was shown to have the most significant association with the metastatic potential of hepatocellular carcinoma (9), and osteopontin was identified as a leading marker of colon cancer progression using pooled sample expression profiling (10). In esophageal cancer, Coppola et al. and Casson et al. reported that osteopontin protein overexpression was observed in 70% (7 of 10) and 100% (6 of 6) of ESCC tumors, respectively (11, 12). Although these reports showed that osteopontin overexpression was frequent in ESCC tumors, the relationship between osteopontin expression and patients' prognosis has not been shown in their small-scale studies.

We recently reported that the plasma osteopontin levels were associated with lymph node metastasis of ESCC patients and the overall survival of the patients with high osteopontin levels was worse than that of those with low osteopontin levels (13). We hypothesized that osteopontin expression in tumor is also associated with the clinicopathologic factors or the prognoses of the patients with ESCC. Our purpose of this study is to examine the clinical significance of osteopontin and evaluate the role of osteopontin in progression of ESCC. Firstly, we analyzed osteopontin mRNA expression from gene expression profiles of 23 ESCC tumors determined by cDNA microarray. Then, we examined osteopontin protein expression in 144 ESCC tumors by immunohistochemistry. A plasmid vector-mediated doxycycline-inducible short-hairpin RNA (shRNA) expression system was used to evaluate the role of osteopontin using a previously established mouse model for lymph node metastasis (14).


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and surgical specimens. Frozen tumor tissues were obtained for cDNA microarray analysis from 23 patients with primary ESCC who underwent surgery at Kyoto University Hospital from 1990 to 1995. Four of these 23 patients were women and 19 were men. The median age of the patients was 65.4 years (range, 43-81 years). The median follow-up time of survival was 61.4 months (range, 3-89 months). Paraffin-embedded sections were acquired for immunohistochemistry from 144 patients with primary ESCC who underwent surgery at Kyoto University Hospital from 1991 to 2001. Twenty of these 144 patients were women and 124 were men. The median age of the patients was 63.7 years (range, 43-84 years). The median follow-up time of survival was 40.8 months (range, 3-134 months). Information on gender, age, stage of disease, and histopathologic factors was abstracted from the medical records. All of the tumors were confirmed as ESCC by the clinicopathologic department of the hospital. All of the cases were classified according to the sixth edition of the pathologic tumor-node-metastasis classification of 2002 (15). Of the 23 patients for cDNA microarray, 8 (34.8%) had T1, 2 (8.7%) had T2, 7 (30.4%) had T3, and 5 (21.7%) had T4. Of the 144 patients for immunohistochemistry, 42 (29.1%) had T1, 45 (31.3%) had T2, 57 (39.6%) had T3, and 20 (13.9%) had T4. Written informed consent was obtained from all the patients for surgery and to use resected samples for research. (The approval numbers of the Institutional Review Board of Kyoto University were 232 and G48).

Purification of mRNA. Tissue samples were frozen in liquid nitrogen immediately after resection. Frozen samples were crushed into pieces and immediately lysed in a buffer containing guanidium isothiocyanate, and total RNA was extracted by a modified AGPC method. mRNA was purified using a polyadenylic acid purification kit (Oligotex; Takara Bio, Shiga, Japan) according to the manufacturer's instructions. The quality of mRNA was assessed by A260/280 ratios and mRNA was used only when the ratio was >1.9.

cDNA microarray. Gene expression profiles of all 23 primary ESCC tumors were obtained by cDNA microarray analysis. Detailed procedures were described previously (16, 17). In brief, 1.2 mg of each extracted mRNAs from cancer tissues and a pooled reference mRNA from eight normal esophageal epithelia (correspond to the cancer tissues) were used for fluorescent labeling with Cy5-dUTP or Cy3-dUTP, respectively. Each of the labeled first-strand cDNAs was mixed and hybridized to cDNA microarray chips containing 1,153 genes (Human Cancer Chip version 2.0, customized, Takara Bio). After 16 hours of hybridization, fluorescent images were scanned with an Array Scanner 428 (Affymetrix, Santa Clara, CA), and the signal intensities were calculated with ImaGene 4.0 (BioDiscovery, Marina Del Rey, CA). Obtained data were normalized with intensity-dependent normalization.

Antibodies. Anti-human osteopontin rabbit polyclonal antibody clone O-17 (IBL, Gunma, Japan) was used as a primary antibody for immunohistochemistry and Western blot (diluted 1:50 and 1:100, respectively). Anti-human ß-actin mouse monoclonal antibody clone AC-15 (diluted 1:10,000, Sigma, St. Louis, MO), anti-tetracycline repressor (TetR) monoclonal mouse antibody (TET03; diluted 1:1,000, MoBiTec, Goettingen, Germany), and anti-human MMP-2 purified mouse IgG monoclonal antibody clone F-68 (diluted 1:1,000, Daiichi Fine Chemical, Toyama, Japan) were used as primary antibodies for Western blot. Horseradish peroxidase–labeled anti-rabbit or anti-mouse IgG (Zymed, San Francisco, CA) was used as a secondary antibody for Western blot.

Immunohistochemical staining. Resected esophageal specimens, which included both tumor and normal epithelia, were fixed in 10% formaldehyde solution, embedded in paraffin blocks, cut in 4-µm-thick sections, and mounted on APS-coated glass slides. Immunohistochemistry was carried out retrospectively using an Envision kit (DakoCytomation, Glostrup, Denmark) as described previously (18). Unmasking of antigens was done by microwave heating of sections in citrate buffer (pH 6.0) five times for 2 minutes each. Sections were incubated overnight at 4°C with antibody for osteopontin and then incubated with the secondary antibody for 30 minutes at room temperature. After rinsing in TBS thrice for 10 minutes each, the sections were incubated with 3,3'-diaminobenzidine liquid for 5 minutes, counterstained with Mayer's hematoxylin, dehydrated, and then mounted. As the negative control, the primary antibody was replaced with normal mouse IgG.

Evaluation of immunohistochemical staining. The osteopontin immunoactivity was evaluated in five areas of each slide for correlation and confirmation of the tissue analysis, and the osteopontin expression was classified in three groups as follows: – or +, negative or weak positive expression; ++, moderate or focal strong positive expression; +++, strong expression. All slides were evaluated independently by two investigators (T.I. and Y.S.) 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. Osteopontin overexpression was defined as ++ or +++.

Cell cultures. Human esophageal squamous carcinoma cell lines, KYSE series, were all established in our department and cultured in Ham's F-12 (Nissui Pharmaceutical, Tokyo, Japan)/RPMI 1640 (Invitrogen, Carlsbad, CA) with 2% fetal bovine serum according to the method reported previously (19). SUm/c and HSA/c were also established in our department from the cancer cells floating in the lymph of the thoracic duct of patients with ESCC and maintained in Ham's F-12/RPMI 1640 with 5% fetal bovine serum (14). NEK2, a human normal esophageal epithelial cell line, was also established in our department and maintained in keratinocyte serum-free medium containing 2.5 µg epidermal growth factor and 25 µg bovine pituitary extract (Invitrogen) as described previously (20, 21). 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 (Nakalai Tesque, Kyoto, Japan) and 0.1% Tween 20 for 1 hour and incubated at room temperature for 1 hour with the primary antibody described above. The membranes were subsequently incubated at room temperature for 1 hour with secondary antibody and analyzed using enhanced chemiluminescence plus reagent (Amersham, Buckinghamshire, United Kingdom). Quantitative analysis was done using the ImageJ program version 1.34 (developed at the NIH and available at http://rsb.info.nih.gov/ij/index.html).

Construction of inducible osteopontin-shRNA expression plasmid vector and TetR expression plasmid vector. To construct an inducible vector for osteopontin-shRNA, the pSUPERIOR-puro (Oligoengine, Seattle, WA) was digested with BglII and HindIII (Takara Bio). A chemically synthesized oligonucleotide encoding an osteopontin-shRNA that included a loop motif was inserted into downstream of the inducible H1 promoter of the plasmid using DNA ligation kit (Takara Bio) and cloned. The sequence of the oligonucleotide targeted to osteopontin is 5'-ACAGGCUGAUUCUGGAAGU-3', corresponding to positions 223 to 241 within the osteopontin mRNA sequence (accession no. NM_000582). For the negative control vector, the empty vector of the pSUPERIOR-puro was used. The TetR expression plasmid vector (pcDNA6/TR) was purchased from Invitrogen.

Transfections. An ESCC cell line HSA/c was stably transfected with pcDNA6/TR using FuGene6 (Roche Diagnostics, Basel, Switzerland). Each clone was selected against 10 µg/mL blasticidin (Nacalai Tesque) and TetR expression was examined by Western blot using an anti-TetR antibody. One of the clones, which expressed the strongest TetR, was chosen for further study. Subsequently, the cells were stably transfected with the inducible osteopontin-shRNA expression vector or the empty pSUPERIOR-puro vector using Lipofectin reagent (Invitrogen), and the cell clones were selected against 1.0 µg/mL puromycin (Nacalai Tesque). Doxycycline (0-10 µg/mL; Nacalai Tesque) was given to each clone for 24 hours and the expression of osteopontin in each clone was confirmed by Western blot in triplicate.

Cell growth assay. Cells were plated at a density of 1 x 104 per well onto six-well plates (Corning, Corning, NY) at day 0 and counted every other day until day 6. Doxycycline (0 or 10 µg/mL) was treated with the cells for 24 hours before the in vitro experiments, including the cell growth assay, migration assay, and invasion assay.

Transwell migration assay. The migration was determined by a Transwell chamber assay as described previously (22, 23). 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 5% fetal bovine serum as a chemoattractant. After 24 hours of incubation in a 5% CO2 humidified incubator at 37°C, the membranes were fixed and stained by DiffQuik reagent (International Reagents, 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.

Matrigel invasion assay. The invasive capacity was determined by a Matrigel invasion assay. Cells (3 x 104) were seeded into the upper chamber of a 24-well Matrigel-coated micropore membrane filter with 8-µm pores (Becton Dickinson Labware), and the lower chamber was filled with Ham's F-12/RPMI 1640 containing 10% fetal bovine serum as a chemoattractant. After 30 hours of incubation at 37°C, the membranes were fixed and stained by DiffQuik reagent. Then, all the cells that had invaded through the membrane were counted under a light microscope. Control uncoated membranes were used to determine the invasion probability under an identical condition with the Matrigel insert membranes.

Tumor formation assay in nude mice. Suspensions of 2 x 106 HSA/c-derived cells in PBS (60 µL) were injected s.c. into the left flanks of 5-week-old male BALB/c nu/nu mice (Japan SLC, Shizuoka, Japan) at day 0, and the tumor growth was estimated by the average volume of tumors at five sites. The tumor volume was calculated by the formula: 1/2 x L2 x W, where L is length and W is width of the tumor. Doxycycline was given by oral route with concentration of 2 mg/mL in natural mineral water from days 0 to 26 as described previously (24). At 26 days after injection, all the mice were sacrificed and the s.c. tumors were resected and fixed in 10% formaldehyde/PBS. All the tumors were paraffin-embedded, stained with H&E, and osteopontin. All the animal experiments were done in accordance with institutional guidelines.

Lymph node metastasis assay in nude mice. Suspensions of 2 x 106 HSA/c-derived cells in PBS (60 µL) were injected into the right footpads of 5-week-old male BALB/c nu/nu mice (Japan SLC) at day 0. Doxycycline was given by an oral route as mentioned above. At 26 days after injection, all the mice were sacrificed and visible popliteal lymph nodes and footpad tumors were resected and fixed in 10% formaldehyde/PBS. All the lymph nodes were paraffin-embedded, stained with H&E and osteopontin, and examined for the existence of metastasis. Each footpad tumor was used as a positive control for the staining. All the animal experiments were done in accordance with institutional guidelines.

Statistical analysis. The ages of the patients were compared by the Kruskal-Wallis test. Survival curves were calculated by the Kaplan-Meier method and differences were analyzed by the log-rank test. Multivariate analysis was done using the Cox's proportional hazard model. The correlation between osteopontin expression and each clinicopathologic factor was evaluated using the Pearson test. The Tukey-Kramer multiple comparison test was used for the evaluation of malignant phenotypes of each stable cell line. The Fisher's exact test was used to analyze the relationship between osteopontin status and lymph node metastasis. The software StatView for Windows version 5 (SAS Institute, Cary, NC) was used for the analyses. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Microarray analysis. The signal intensities of osteopontin were extracted from gene expression profiles of 23 ESCC tumors determined by cDNA microarray and analyzed. The osteopontin mRNA expression in tumors was ~7.9-fold higher on average than that in normal epithelia (Supplementary Fig. S1A; P = 0.042). The patients with ESCC (n = 23) were divided into two subgroups: patients with high osteopontin expression in tumors (n = 10) and patients with low osteopontin expression in tumors (n = 13). The mean value of osteopontin signal intensities was provided as a cutoff value of expression. The patients with high osteopontin tumors had a poorer prognosis than those with low osteopontin tumors (Supplementary Fig. S1B; P = 0.029).

Immunohistochemical staining. Osteopontin protein expression in tumors examined by immunohistochemistry was usually increased compared with that in normal epithelia. Osteopontin stained mainly in the cytoplasm of the cells. As shown in Fig. 1A (a-c), we classified 144 ESCC tumors in following three subgroups: 72 tumors with negative or weak expression (50%; –/+), 50 tumors with moderate and focal strong expression (35%; ++), and 22 tumors with strong expression (15%; +++). Macrophages and inflammatory cells in tumors were stained strongly, but immunoactivities of these cells were excluded in this study (Fig. 1A, d). The correlation between osteopontin protein expression and various prognostic factors, such as pTNM pathologic classification or histopathologic grade, was investigated. Osteopontin protein expression was correlated with distant lymph node metastasis (pM), tumor staging (pTNM), and histologic grade (Table 1; P = 0.0004, 0.027, and 0.024, respectively). Overall survival analysis using the Kaplan-Meyer method revealed that the prognosis of patients with tumors expressing high or moderate osteopontin was significantly poorer than that with tumors expressing negative or weak osteopontin (Fig. 1B; P < 0.001). The multivariate analysis showed that primary tumor (>pT1), regional lymph node metastasis (pN1), and osteopontin overexpression (++ or +++) were independent poor prognostic factors; however, age (>64 years), gender (male), distant lymph node metastasis (pM1), histologic grade (>G2), postoperative chemotherapy, and postoperative radiotherapy were not (Table 2). Osteopontin overexpression was the strongest survival factor among these nine variables.


Figure 1
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  		Fig. 1. A, immunohistochemistry of osteopontin. We classified 144 esophageal tumors in the following three subgroups: a, tumors with negative or weak expression (– or +). Magnification, x200. b, tumors with moderate and focal strong expression (++). Magnification, x200. c, tumors with strong expression (+++). Magnification, x200. d, macrophages and inflammatory cells in tumors were stained strongly. Magnification, x200. Immunoactivities of inflammatory cells were excluded in this study. B, overall survival of patients determined by the immunoactivity of osteopontin. Overall survival analysis using the Kaplan-Meyer method revealed that the prognosis of patients with tumors expressing strong or moderate osteopontin were significantly poorer than that with tumors expressing weak osteopontin.

 

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  		Table 1. Relationship between osteopontin immunoactivity and clinicopathologic characteristics in 144 patients

 

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  		Table 2. Cox's proportional hazard analysis

 
Osteopontin expression in ESCC cell lines. As shown in Fig. 2A, osteopontin protein expression in 34 esophageal cancer cell lines was determined by Western blot. Osteopontin overexpression was observed in 30 of the 34 (88%) cell lines compared with NEK2, a normal esophageal epithelial cell line.


Figure 2
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  		Fig. 2. A, expression of osteopontin protein in 34 ESCC cell lines. Equal amounts of total protein (20 µg) were loaded in all lanes. Immunoblots were probed with the anti-osteopontin antibody and the anti-ß-actin antibody. The HeLa cell line was included as a positive control of osteopontin expression. The osteopontin expression of NEK2, a normal esophageal epithelial cell line, was included to compare with that of the ESCC cell lines. Expression of ß-actin confirmed the equivalent loading of total protein. B, DNA sequence of the osteopontin-shRNA insert subcloned into pSUPERIOR-puro. BamHI and HindIII restriction enzyme sequences flank the insert. The sense and antisense sequences are separated by a loop sequence (loop). A RNA polymerase III terminator sequence (RNA poly-T) is positioned 3' to the antisense sequence to terminate transcription. Asterisks, intentional mismatches (converted from G to C) within the sense strand sequence. C, suppression of osteopontin protein expression by an inducible osteopontin-shRNA vector and the association between osteopontin and MMP-2 protein expression. HSA/c cells expressing TetR were stably transfected with an empty vector (Hsa/TR/mock) or an inducible osteopontin-shRNA vector (Hsa/TR/OPN1 or Hsa/TR/OPN2). After administering 0 to 10 µg/mL doxycycline for 24 hours, the expression of osteopontin protein in these clones was examined by Western blot. The lower two indicate protein expression of ß-actin and TetR. D, quantitative analysis of osteopontin and MMP-2 protein expression in the stable transfectants of the inducible osteopontin-shRNA vector. Black columns or white columns, average ratio of osteopontin or MMP-2 to ß-actin, respectively.

 
Establishment of cell clones with the inducible shRNA vector against osteopontin. Our clinicopathologic findings lead us to speculate that osteopontin could be a promising target of therapy for ESCC. To assess the effect of osteopontin conditional down-regulation in ESCC cells, we established subclones by transfection of the TetR vector and the inducible shRNA vector against osteopontin into HSA/c, which is an aggressive ESCC cell line with an ability to metastasize from footpad to lymph nodes. The DNA sequence of the osteopontin-shRNA insert was shown in Fig. 2B. These cell clones were given 0 to 10 µg/mL doxycycline, and osteopontin expression was confirmed by Western blot. Two stable clones (Hsa/TR/OPN1 and Hsa/TR/OPN2) had down-regulated osteopontin expression in a dose-dependent manner by doxycycline (Fig. 2C and D). Especially, 10 µg/mL doxycycline down-regulated osteopontin expression (osteopontin/ß-actin) by 67% and 87% in Hsa/TR/OPN1 and Hsa/TR/OPN2 cells, respectively, but not in mock-transfected cells (Hsa/TR/mock). We then examined MMP-2 expression in the same samples to evaluate the relationship between osteopontin and MMP-2, showing that osteopontin suppression was associated with MMP-2 down-regulation. On the other hand, ß-actin and TetR expression in these clones were almost the same.

Effect of osteopontin down-regulation on cell motility, invasion in vitro, tumor formation, and lymph node metastasis in vivo. We examined the effect of the inducible osteopontin-shRNA vector on cell motility and invasion in vitro by a Transwell chamber assay and a Matrigel invasion assay, respectively. Cell migrations were suppressed by 69% in Hsa/TR/OPN1 (P < 0.05) and 97% in Hsa/TR/OPN2 (P < 0.01) by administration of 10 µg/mL doxycycline (Fig. 3A). In contrast, those in mock-transfected cells (Hsa/TR/mock) were not changed by the same concentration of doxycycline. The invasion probabilities were calculated by the invasion through the Matrigel matrix and membrane relative to the migration through the control uncoated membrane. Cell invasions were suppressed by 59% in Hsa/TR/OPN1 (P < 0.05) and 71% in Hsa/TR/OPN2 (P < 0.01) by administration of 10 µg/mL doxycycline (Fig. 3B). Thus, these results suggest that osteopontin suppression reduces both cell migration and invasion in vitro in HSA/c cells according to the degree of suppression. We also examined cell growth in vitro; however, cell growth of each group was not changed (Supplementary Fig. S2).


Figure 3
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  		Fig. 3. A, Transwell migration assay in stable transfectants of the inducible osteopontin-shRNA vector. White columns or black columns, average migrated cells per field without [Dox(–)] or with [Dox(+)] doxycycline, respectively. B, Matrigel invasion assay. White columns or black columns, invasion probability in Dox(–) or Dox(+) group, which was calculated as the ratio of number of cells invading through the Matrigel insert membrane relative to the mean number of cells migrating through the control insert membrane. C, tumor formation assay in nude mice. White columns or black columns, average tumor volume at day 26 after inoculation into the flanks in Dox(–) or Dox(+) group, respectively (n = 5). *, P < 0.05; **, P < 0.01, significant difference versus Dox(–) in each group (Tukey-Kramer test).

 
To examine the possible activity of osteopontin-shRNA on tumorigenesis in vivo, a s.c. tumor formation assay in nude mice was done. As shown in Fig. 3C, the average tumor volumes of the mice at day 26 were suppressed by 56% in Hsa/TR/OPN1 and 92% in Hsa/TR/OPN2 (P < 0.05) by oral administration of doxycycline (2 mg/mL) but were not in Hsa/TR/mock. All of the tumors were stained with H&E and osteopontin (Fig. 4A and B). Osteopontin protein expression in the xenograft was reduced when doxycycline was given to the mice inoculated with osteopontin-shRNA vector but were not with the mock vector.


Figure 4
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  		Fig. 4. Models for tumor formation and lymph node metastasis in vivo. A and B, osteopontin immunohistochemical staining of s.c. tumors on the flanks of mice with Hsa/TR/OPN2 cells with or without doxycycline. Magnification, x400. C, a footpad tumor at day 26 after inoculation. White arrowheads, the inoculation site and the expected site of popliteal lymph node. D, representative H&E staining of footpad tumor. Magnification, x400. E and F, representative H&E staining of popliteal lymph nodes with Hsa/TR/OPN2 cells with or without doxycycline. Magnification, x100. A metastasized lesion is shown inside red arrowheads. All the metastasized lesions were located at the cortical sinus of the lymph nodes.

 
Finally, we examined the effect of the inducible osteopontin-shRNA vector in a lymph node metastasis model of nude mice (Fig. 4C-F). As shown in Table 3, the number of mice with metastasis from the footpads to the popliteal lymph nodes at day 26 was decreased by 50% (from 4 to 2) in Hsa/TR/OPN1 and 67% (from 3 to 1) in Hsa/TR/OPN2 when doxycycline was given by an oral route in each group but was not changed in Hsa/TR/mock (from 4 to 4). Moreover, the mice with down-regulated osteopontin [OPN(–)], which include Hsa/TR/OPN1 Dox(+) and Hsa/TR/OPN2 Dox(+), had significantly fewer lymph node metastases than those with sufficient osteopontin [OPN(+)], which include Hsa/TR/mock, Hsa/TR/OPN1 Dox(–), and Hsa/TR/OPN2 Dox(–) (Table 4; P = 0.028). Each mouse had one popliteal lymph node, except one mouse with Hsa/TR/mock-Dox(+) and one mouse with Hsa/TR/OPN1-Dox(+) (Table 3, numbers in parentheses). The total number of lymph node metastasis was 18 in all the mice. There was no animal death during the experiment.


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  		Table 3. Lymph node metastasis assay in nude mice

 

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  		Table 4. Relationship between osteopontin down-regulation and lymph node metastasis

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Although osteopontin overexpression is associated with tumor progression in various cancers (10, 25–33), the role of osteopontin was not shown in esophageal cancer previously. In this report, we found that high osteopontin expression in 23 ESCC tumors was associated with poor prognosis of the patient by microarray analysis, which was done previously in our laboratory. The microarray result led us to examine the significance of osteopontin expression in ESCC. Our clinicopathologic analysis by immunohistochemistry of 144 ESCC tumors showed that osteopontin overexpression in tumors was significantly associated with poor patient survival. Moreover, osteopontin overexpression was the strongest prognostic factor of the patients among nine major variables. A similar result was shown by Rudland et al. that overexpression of osteopontin in 222 patients with operable stage I and II breast cancer was the strongest prognostic factor in their multivariate analysis (7). These results suggest that osteopontin overexpression could be a useful prognostic marker for the patients with ESCC. Osteopontin overexpression in the tumors was shown to be correlated with high histologic grade, distant lymph node metastasis, and tumor staging in this report, suggesting that its expression might be important for the acquirement of malignant potential in ESCC.

The mechanisms by which osteopontin may enhance malignancy are still unclear, although several mechanisms have been suggested through studies in cultured cells. Our in vitro cell growth assay using osteopontin-shRNA transfectants did not exhibit growth inhibition compared with mock-transfected cells even when doxycycline was given. However, the in vivo tumor formation assay using osteopontin-shRNA transfectants exhibited significant growth inhibition compared with mock-transfected cells when doxycycline was given, suggesting that some interactions between osteopontin expression in tumors and surrounding stromal tissues may exist. The ability of cells to migrate may be tied to their tumorigenicity and osteopontin clearly participates in pathways regulating migration in diverse cell types, including osteoclasts, fibroblasts, macrophages, and tumor cells (34, 35). Philip et al. have shown that osteopontin up-regulates pro-MMP-2 expression in a nuclear factor-{kappa}B–dependent fashion during extracellular matrix invasion (8). We also show that inhibition of osteopontin expression attenuates MMP-2 expression and suggest that osteopontin mediates ESCC invasion or lymph node metastasis by enhancing tumor cell migration and invasion through the extracellular matrix by regulating MMP-2 expression.

We established two independent stable cell clones with different suppressed osteopontin expression in the presence of doxycycline. The several malignant phenotypes tested in this study were according to the osteopontin expression in these two clones, suggesting that the level of osteopontin expression may be important for control of the phenotypes. The osteopontin protein levels in Hsa/TR/OPN1 and Hsa/TR/OPN2 cells in the absence of doxycycline were slightly lower than that in Hsa/TR/mock cells. This phenomenon may be caused by a transgene leakage in a doxycycline-inducible expression system as described previously (36). Nonetheless, the osteopontin level in Hsa/TR/OPN1 and Hsa/TR/OPN2 in the absence of doxycycline preserves 73% and 85% of the expression to Hsa/TR/mock, respectively. Thus, these cell lines we established is thought to be a useful model for functional analysis of osteopontin inhibition in spite of the small amount of the transgene leakage. Although our shRNA target sequence of osteopontin was effective enough for suppressing the phenotypes, complete suppression of osteopontin expression in cancer cells may be more effective to control the progression of ESCC. Experiments evaluating tumorigenesis in osteopontin-knockout mice have yielded disparate results, possibly because osteopontin expressed by normal tissues and tumors may have differential functional effects (5). Thus, we speculate that if we could control tumor-derived osteopontin adequately and locally by advanced inducible shRNA systems, cancer therapy using short interfering RNA might be more efficient in the future.

In summary, we have shown that osteopontin overexpression in ESCC tumors was associated with the poor survival of patients, distant lymph node metastasis, histologic grade, and tumor staging. Conditional down-regulation of osteopontin in an ESCC cell line using an inducible shRNA vector decreased cell motility, invasion in vitro, tumor formation, and lymph node metastasis in vivo. Our findings suggest that osteopontin overexpression may play an important role in progression of ESCC and osteopontin could be a promising target for ESCC therapy.


   Acknowledgments
 
We thank Sakiko Shimada for kind assistance in immunohistochemistry and cell culture, Takako Murai, Kumi Kodama, Akane Iwase, and Fumie Uemura for technical assistance, Drs. Junichiro Kawamura, Yukiko Mori, and Go Watanabe for providing the clinical samples, Dr. Susan Rittling (Rutgers University, Piscataway, NJ) for helpful discussion, and Dr. John M. Abraham (University of Maryland, Baltimore, MD) for his support in proofreading the article.


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

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

Received 7/26/05; revised 11/28/05; accepted 12/ 9/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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