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Carcinogenesis Advance Access originally published online on December 20, 2006
Carcinogenesis 2007 28(5):947-956; doi:10.1093/carcin/bgl247
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Insulin-like growth factor-I receptor as a marker for prognosis and a therapeutic target in human esophageal squamous cell carcinoma

Arisa Imsumran{dagger}, Yasushi Adachi*,{dagger}, Hiroyuki Yamamoto, Rong Li, Yu Wang, Yongfen Min, Wenhua Piao, Katsuhiko Nosho, Yoshiaki Arimura, Yasuhisa Shinomura, Masao Hosokawa1, Choon-Taek Lee2, David P. Carbone2 and Kohzoh Imai

First Department of Internal Medicine, Sapporo Medical University, South-1, West-16, Chuo-ku, Sapporo 060–8543, Japan
1 Department of Surgery, Keiyukai Sapporo Hospital, Sapporo 003-0027, Japan
2 Vanderbilt-Ingram Cancer Center and Departments of Medicine and Cell Biology, Vanderbilt University, Nashville, TN 37232-6838, USA

* To whom correspondence should be addressed. Tel: +81 11 611 2111; Fax: +81 11 611 2282; Email: yadachi{at}sapmed.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Insulin-like growth factor (IGF)-I receptor (IGF-Ir) signaling is required for tumorigenicity and progression of many tumors but this pathway has not been well studied as a prognostic factor or potential therapeutic target in esophageal squamous cell carcinoma (ESCC). In this paper, the association between the expression of IGF-Ir and IGF-II ligand and prognosis was investigated immunohistochemically in 100 surgically resected ESCC. We then assessed the therapeutic effect of blocking IGF receptor signaling using dominant negative IGF-Ir (IGF-Ir/dn) in ESCC in vitro. Expression of IGF-Ir and IGF-II were detected in 60 and 50% of tumors, respectively, and were associated with invasion depth, metastasis, advanced tumor stage and recurrence. Patients with tumors expressing both IGF-Ir and IGF-II had a significantly shorter survival than those expressing either alone or neither in both single and multivariate analysis. IGF-Ir/dn suppressed proliferation and motility as well as upregulating chemotherapy-induced apoptosis through blocking ligand-induced Akt activation. We propose that detection of IGF-Ir/IGF-II in ESCC may be useful for the prediction of recurrence and poor prognosis and for selecting patients for IGF-Ir-targeted therapy. Therapeutic blockade of IGF-Ir may be a useful anticancer therapeutic for ESCC.

Abbreviations: Ad-IGF-Ir/482st, adenoviruses expressing IGF-Ir/482st; Ad-IGF-Ir/950st, adenoviruses expressing IGF-Ir/950st; des(1-3)IGF-I, NH2-terminally truncated IGF-I; dn, dominant negative; ELISA, enzyme-linked immunosorbent assay; IGF, insulin-like growth factor; IGFBP, IGF-binding protein; IGF-Ir, IGF-I receptor; IGF-Ir/dn, dominant negative form of IGF-Ir; IGF-Ir/482st, truncated IGF-Ir of 482 amino acid long; IGF-Ir/950st, truncated IGF-Ir of 950 amino acid long; IR, insulin receptor; MAPK, mitogen-activated protein kinase; moi, multiplicity of infection; PBS, phosphate-buffered saline; TNM, tumor node metastasis; UV, ultraviolet


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Esophageal cancer is one of the least studied and deadliest cancers worldwide (1). At the time of diagnosis, >50% of patients have either unresectable tumors or radiographically visible metastases. Even after curative-intent surgical resection, the 5 year survival is still very low (2), and for unresectable esophageal squamous cell carcinoma (ESCC) therapy is typically minimally effective.

Recently, advances in molecular research in cancer have brought new therapeutic strategies into clinical usage. One group of new targets is the tyrosine kinase receptors, including epidermal growth factor receptor, Her2/neu and c-Kit. These can be blocked by small molecule tyrosine kinase inhibitors, for example gefitinib and imatinib, targeting epidermal growth factor receptor and c-kit, respectively, or trastuzuamb, a monoclonal antibody for Her2. None of these has demonstrated significant activity in esophageal cancer, underscoring the importance of finding other receptors that might be better targets in this disease. The insulin-like growth factor (IGF) family is a promising candidate (3,4). Agents targeting the insulin-like growth factor-I receptor (IGF-Ir) pathway are moving into the clinic, but it is clear that rational selection of clinical populations will be key in their clinical development. Toward that end, we have studied this pathway in ESCC.

The IGF-Ir is a heterodimer of {alpha}- and ß-chains (5). After the binding of ligands IGF-I or IGF-II, the receptor is autophosphorylated and then activates multiple signaling pathways, including mitogen-activated protein kinase (MAPK) and Akt-1 (6). Elevation of serum IGF-I increases the risk of developing several cancers (7–9) and IGF-Ir is essential for both malignant transformation and progression (3,4). Reduction of IGF-Ir has been shown to induce apoptosis in tumors, but produce only growth reduction in untransformed cells, suggesting that it might be an excellent target for therapeutic intervention (3). The potential for a good therapeutic index is supported by the finding that IGF-Ir knockout mice completely lacking the receptor are viable (though physically small), indicating that relatively normal development and differentiation can occur in its absence (10). This is not true for most other candidate molecular targets for cancer therapy. These findings suggest a potential basis for tumor selectivity in therapeutic applications.

Human esophageal epithelial cells express IGF-Ir, and IGF-I is known to stimulate both thymidine incorporation and proliferation in these cells (11–13). Salivary IGF-I continuously bathes the esophageal lumen and, unlike the serum pool, is in a free form (not bound to IGF-binding protein, IGFBP), a fact that dramatically enhances its ability to bind to receptors on the esophageal mucosal cells (14). These data indicate that the IGF/receptor may play important roles in homeostasis and esophageal premalignancy (13).

IGF-Ir is overexpressed in the esophageal carcinoma cell line CE48T/VGH, and IGF-I promotes the proliferation and ligand-dependent IGF-Ir autophosphorylation (15). Both IGF-Ir and the IGFs are overexpressed in cancer tissues compared with the normal ones (16–18). In addition, IGFBP3 and an antibody for IGF-Ir suppress cancer cell proliferation (15,19). Moreover, our preliminary studies with cDNA microarray analysis in 20 cases with ESCC show that IGF-Ir and IGF-II are two of the four most significantly upregulated gene in tumors from patients with short survivals (Hiroyuki Yamamoto, unpublished data). However, the role of the IGF axis in ESCC has not been adequately studied.

There are several possible approaches to blocking IGF-Ir signaling with therapeutic intent (20), including blocking the ligand or receptor using antibodies (21,22), or small molecular receptor kinase inhibitors (23,24). All of these are complicated by the high homology of this receptor to the insulin receptor (IR). An approach that is intrinsically specific for IGF-Ir is to use dominant negative or soluble IGF-Ir receptor approaches to specifically inhibit the function of the wild-type receptor (25,26). We have constructed two different adenoviruses expressing dominant negative IGF-Ir (Ad-IGF-Ir/dn) to directly test the anticancer effects of IGF-signaling blockade (27–30). Ad-IGF-Ir/482st, adenoviruses expressing IGF-Ir/482st, encodes a truncated extracellular domain of IGF-Ir (without the transmembrane domain) and thus should result in a secreted form that affects neighboring cells in addition to the transduced cells (a bystander effect). Another Ad-IGF-Ir/950st, adenoviruses expressing IGF-Ir/950st, encodes a receptor that lacks the tyrosine kinase domain and thus remains on the membrane of the transduced cells to form non-functional receptor heterodimers with endogenous wild-type receptor. In experiments with other tumor types, we have reported that Ad-IGF-Ir/dn may be a useful therapeutic strategy and was more effective than the antisense approach (27,29,31).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Patients and tissue samples
One hundred formalin-fixed, paraffin-embedded tumor specimens were obtained from patients who had undergone curative surgical treatment at a Sapporo Medical University affiliated hospital, Keiyukai Sapporo Hospital. Histopathological features of the specimens were classified according to the pathological tumor node metastasis (TNM) classification system of the American Joint Committee on Cancer and the Union International Contre Cancer. Informed consent was obtained from each patient and the Institutional Review Committee approved the experiments.

Standard surgical methods were used. In brief, esophagectomies with lymph node dissection were performed by a right thoracotomy, and subsequent reconstructions were carried out by an esophagogastrostomy using a gastric tube through the retrosternal route. No patients received preoperative chemotherapy or radiotherapy. Fifty patients received postoperative irradiation (45 Gy) and 15 patients received postoperative chemotherapy with two cycles of concurrent fluorouracil and cisplatin. An analysis of the effect of postoperative therapy for recurrence or prognosis in this group of patients showed no significant effect on recurrence or survival (data not shown). This is the same result observed in a recent meta-analysis by Malthaner et al. (32).

Materials, cell lines and recombinant adenovirus vectors
Anti-Akt1 (c-20), anti-ERK1 (K-23), antiphospho-ERK1 (E-4), anti-IGF-I (G-17) and anti-IGF-Ira (2C8, H78) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and antiphospho-Akt (Ser473) was from Cell-Signaling Technology (Beverly, MA). Anti-IGF-Ir (Ab-4) was from Oncogene Research Products (Cambridge, MA) and anti-IGF-II was from Peninsula Laboratories (San Carlos, CA). Cisplatin (CDDP) and 5-fluorouracil were purchased from Sigma (St Louis, MO), Recombinant human IGF-I and IGF-II were purchased from R&D Systems (Minneapolis, MN) and NH2-terminally truncated IGF-I (des(1-3)IGF-I) from GroPep (Adelaide, Australia). All human esophageal carcinoma cell lines (Figure 1) were obtained from the Japanese Cancer Collection of Research Bioresources Cell Bank (Tokyo, Japan). Cells were passaged in RPMI1640 and DMEM both with 10% fetal bovine serum.


Figure 1
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  		Fig. 1. The expression of IGF-I, IGF-II, and IGF-Ir in 10 human ESCC lines. (A) reverse transcription–PCR analyses revealed that ESCC lines frequently express mRNAs of IGF-II (468 bp) and IGF-Ir (755 bp) but not IGF-I message (396 bp). (B) ELISA assay showed that ESCC lines seldom produce detectable IGF-I protein in the media. However, all ESCC lines secreted high levels of IGF-II protein. Expression of IGF-Ir protein varied in these cells. Both ELISA and western blot (C) identified that the concentrations of IGF-Ir protein are related to their message levels.

 
Recombinant adenoviruses expressing IGF-Ir/dn (482 and 950 amino acids long, IGF-Ir/482st and IGF-Ir/950st, Ad-IGF-Ir/482st and Ad-IGF-Ir/950st, respectively) were generated as described previously by homologous recombination. An adenovirus expressing the ß-galactosidase gene was used as a control (Ad-LacZ).

Immunohistochemistry
Formalin-fixed, paraffin-embedded specimens of 5 µm in thickness were dewaxed in xylene and rehydrated in alcohol, and then heated to 105°C in an autoclave for 10 min. Endogenous peroxidase activity was suppressed by a solution of 3% hydrogen peroxide in methanol for 5 min. After being rinsed twice in phosphate-buffered saline (PBS), the sections were treated for 18 h with a mouse antihuman IGF-Ir (Ab-4) or a rabbit antihuman IGF-II antibody (1:500 dilution). Normal mouse or rabbit immunoglobulins were substituted for each primary antibody as negative controls. After washing three times in PBS, the sections were treated with biotinylated antirabbit or antimouse immunoglobulin (Dako, Glostrup, Denmark) for 10 min and then with horseradish peroxidase–avidin complex, diluted as recommended by the manufacturer, for 10 min. The slides were then washed in PBS and developed in 0.05 M Tris–HCl (pH 7.5) containing 0.6 mg/ml 3-3' diaminobenzidine at room temperature. The sections were counterstained in Mayer's hematoxylin and mounted. Immunostaining signals were scored by two independent observers. As described previously (33), staining intensity was rated on a four-point scale: 1+, none or minimal; 2+, light; 3+, moderate; 4+, heavy. Scales of 3+ and 4+ were considered positive for IGF-Ir and IGF-II. According to the positivity of IGF-Ir and IGF-II, we generated an ‘IGF score’ as follows: score 0, both are negative; score 1, either IGF-II or IGF-Ir is positive; score 2, both are positive.

Reverse transcription–PCR
Total RNA from cells was isolated by the acid guanidinium thiocyanate–phenol–chloroform method. Primer sets for the amplification of IGF-I cDNA sequences were 5'-CACTGTCACTGCTAAATTCA-3' and 5'-CTGTGGGCTTGTTGAAATAA-3' (34). Primers for IGF-II cDNA were 5'-AGTCGATGCTGGTGCTTCTCA-3' and 5'-GTGGGCGGGGTCTTGGGTGGGTAG-3' (35). Primers for IGF-Ir were 5'-ATTGAGGAGGTCACAGAGAAC-3' and 5'-TTCATATCCTGTTTTGGCCTG-3' (35). Randomly primed cDNAs were prepared from 1 mg of total RNA by Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Takara, Otsu, Shiga, Japan) and amplified by PCR. For amplification of these sequences, 35 cycles of PCR was programmed as follows: 94°C, 30 s; 60°C, 30 s; 72°C, 30 s.

Western blotting
Cells were treated as indicated in the text. Cell lysates were prepared as described previously (27). Equal aliquots of lysate (100 µg) were separated by 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted onto polyvinylidene Hybond-P membrane (Amersham, Arlington Heights, IL). Analysis was performed using indicated antibodies, and bands were visualized by ECL (Amersham).

ELISA
TE1 was infected with ad-lacZ or Ad-IGF-Ir/482st [100 multiplicity of infection (moi)] for 48 h and then the medium was changed to 0.1% BSA (without fetal calf serum (FCS)) overnight. The cells were treated with IGF-I (0, 1, 10, 100 nM) for 5 h, then media were collected and concentrated (50 times) and the cells were separately lysed. Concentrations of IGF-Ir and the ligands were measured using enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems).

Assessment of the effect on in vitro cell growth
Four thousand TE1 cell were seeded into wells of a 96 well plate and each was infected with adenovirus (100 moi) or control. Cell growth was measured everyday using WST-1 reagent (Roche, Basel, Switzerland) (36). Cells were incubated with WST-1 reagent for 3 h and then were measured by ELISA plate reader.

Measurement of the effect of IGF-Ir/dn on apoptosis
The DNA fragmentation assay was performed as follows: low molecular weight DNA was extracted with 0.5% Triton X-100, 10 nM ethylenediaminetetraacetic acid (EDTA), and 10 mM Tris–HCl, pH 7.4; treated with 400 mg/ml RNase A and then Proteinase K for 1 h at 37°C, ethanol precipitated, and subjected to 1% agarose gel electrophoresis. The gels were stained with 1 mg/ml ethidium bromide. Early apoptosis was quantified by staining with Annexin-V-FITC, according to the manufacturer's protocol (BD Biosciences, Franklin Lakes, New Jersey) and measured by flow cytometry. Cells undergoing apoptosis showed an increase in Annexin-V binding but excluded propidium iodide. TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assays were performed with in situ apoptosis detection kit (Takara) following the manufacturer's protocol. Caspase-3 colorimetric protease assay was performed following the manufacturer's protocol (Caspase-3 Colorimetric Protease Assay Kit; MBL, Nagoya, Aichi, Japan). In brief, 3 x 106 cells were lysed in 100 ml of chilled cell lysis buffer, and total cell lysates (100 mg) were incubated with 4 mM VETD-pNA substrate (200 mM final concentration) at 37°C for 1 h. Caspase-3 activity was measured by colorimetric reaction at 405 nm.

First, cancer cells infected with Ad-IGF-Ir/dns or Ad-LacZ were induced with 10 mJ/cm2 ultraviolet (UV) light. To assess the efficacy of IGF-Ir/dn on chemotherapy-induced apoptosis, tumor cells were treated 24 h with 1 mM 5-fluorouracil or 50 µM cisplatin.

Migration assay
Wounding assays were performed using a modification of the procedure described by Pennisi et al. (37). Briefly, six-well chambers were prepared by scratching registration marks onto the slide surface. TE1 cells (infected with adenoviruses) were plated, grown normally for 48 h and starved overnight. Cells were cut with a cell scraper and five images were captured along the cut surface on an Olympus IX 71S1F-2 microscope (Tokyo, Japan) using a 20x objective. Additional images were captured 24 h later. For each experiment, the number of migrating cells was counted by two independent observers (37).

Statistical analysis
Expression of IGF-Ir and IGF-II was assessed for associations with clinicopathological characteristics using the following statistical tests: Student's t-test, the Mann–Whitney test, the chi-square two-tailed test and Fisher's exact test. Cumulative survival rates were calculated by the Kaplan–Meier method. The difference between the survival curves was analyzed by the log-rank test. Factors related to survival were analyzed by Cox's proportional hazards regression model.

The results of in vitro experiments are presented as means ± SE for each sample. The statistical significance of differences was determined by one-way analysis of variance or two-factor factorial analysis of variance. P values of <0.05 were considered to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
First, we evaluated the mRNA expression of both IGF-Ir and its ligands in 10 esophageal cancer cell lines using reverse transcription–PCR (Figure 1A). IGF-Ir message was identified in all. IGF-II mRNA was expressed strongly in two and weakly in eight. However, only TE4 expressed IGF-I message faintly. Then we assessed those protein expression levels using ELISA (Figure 1B). IGF-I protein was weakly expressed in four cell lines. All the cell lines expressed high levels of IGF-II, although this message showed some differences between the cell lines. The protein expression of IGF-Ir generally reflects the results of the reverse transcription–PCR, which was also confirmed by western blot analysis (Figure 1C). Importantly, there was striking concordance of ligand and receptor expression, especially for those cells with high receptor expression, suggesting an autocrine loop in these cells. These data indicate that IGF-Ir and IGF-II may play important roles in human ESCC.

According results with cell lines and our preliminary cDNA microarray study, the expression of both IGF-Ir and IGF-II were then analyzed immunohistochemically in 100 human-resected ESCC specimens (Figure 2A–F). Weak immunoreactivity for both antibodies was only occasionally observed in the normal epithelium. However, there were strong and specific IGF-Ir cytoplasmic and membranous expression and IGF-II cytoplasmic staining in tumor cells. Expressions of IGF-Ir and IGF-II were observed in 60 and 50% of the tumors, respectively (Figure 1). The expression of either was significantly correlated with the depth of invasion, lymph node metastasis, distant metastasis, advanced pTNM stage, recurrence and recurrence within the first postoperative year (Table I). These results indicate that ESCCs expressing both IGF-Ir and IGF-II tend to be more aggressive than those expressing only one or neither. Patients with IGF-Ir- or IGF-II-positive carcinomas had significantly shorter overall and disease-free survival periods than those with negative (P = 0.0002 and 0.0001 for IGF-Ir, and 0.0020 and 0.0009 for IGF-II, respectively, Figure 3). The prognosis of patients with ESCC also was progressively worse with increasing IGF immunohistochemical scores, a composite of the expression of both IGF-Ir and IGF-II (see Methods, Figure 4). In the univariate analysis, significant prognostic variables for predicting both overall and disease-free survival were IGF-Ir, IGF-II, IGF score, invasion depth, lymph node metastasis, distant metastasis and pTNM stage (Table II). In the multivariate analysis, IGF score, depth of invasion and lymph node metastasis retained independent significant predictive value for survival.


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  		Table I. Expression of IGF-Ir or IGF-II and clinicopathological characteristics in patients with ESCC

 


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  		Table II. Univariate and multivariate analyses of overall and disease-free survival in patients with ESCC

 


Figure 2
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  		Fig. 2. Immunohistochemistry in resected human ESCC tumors. (A) Expression of IGF-Ir. (Original magnification x40). (B) Weak cytoplasmic expression of IGF-Ir in normal esophageal epithelial cells in the parabasal layer (x200). (C) Strong cytoplasmic and membranous expression of IGF-Ir in carcinoma cells (x100). (D) Expression of IGF-II in a serial section of A (x40). (E) Weak cytoplasmic expression of IGF-II in normal esophageal epithelial cells in the parabasal layer (x200). (F) Strong cytoplasmic expression of IGF-II in carcinoma cells in a serial section of C (x100).

 


Figure 3
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  		Fig. 3. Kaplan–Meier life-table analyses of the survival rate of patients with ESCC. Overall (A,C) and disease-free (B,D) survival curves of patients with ESCC according to IGF-Ir (A,B) or IGF-II (C,D) expression show significant differences (P = 0.0002 and 0.0001 for IGF-Ir, and 0.0020 and 0.0009 for IGF-II, respectively).

 


Figure 4
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  		Fig. 4. Kaplan–Meier life-table analyses of the survival rate of patients with ESCC. Overall (A) and disease-free (B) survival curves of patients with ESCC according to the IGF score show significant differences (P < 0.0001). The IGF score is a composite of the expression of IGF-Ir and IGF-II as follows: score 0, both negative; score 1, either alone positive; score 2, both positive.

 
Since our immunohistochemical studies indicated a striking prognostic effect of IGF/IGF-Ir in ESCC, we then studied the effect of IGF-signaling blockade by Ad-IGF-Ir/dn. For these studies, we first selected the cell line TE1 that expresses both IGF-II and IGF-Ir (Figure 1). As expected, the expression of the truncated receptor increased with increasing viral dose of the two Ad-IGF-Ir/dns (10–100 moi) (Figure 5A,B). In addition, as expected, fluorescence activated cell sorting (FACS) analysis revealed that IGF-Ir/950st was detected on the tumor cell surface and IGF-Ir/482st was detectable in the culture media by western blotting. Ad-IGF-Ir/482st also significantly suppressed cell growth (Figure 5C), but as previous studies have observed it had a much more profound effect on anchorage-independent growth and tumorigenicity than on in vitro growth on plastic (Figure 5D).


Figure 5
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  		Fig. 5. The effects of adenoviruses expressing IGF-Ir/dn (100 moi) on the human ESCC cell line TE1. (A) These cells expressed {alpha}-chains of IGF-Ir after infection with Ad-IGF-Ir/950st in a dose-dependent manner. (B) The same amounts of cultured medium were collected and concentrated 50 times and loaded on a 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Western blotting showed that the cell line infected with Ad-IGF-Ir/482st secreted a short IGF-Ir (arrow) into the medium. (C) Growth of TE1 cells (open squares) and those infected with Ad-IGF-Ir/482st (closed circles) or control virus (open circles) on plastic plates was evaluated by the WST-1 assay. Although the control virus did not affect proliferation, Ad-IGF-Ir/482st dramatically reduced growth (P < 0.0001). (D) Soft agar clonogenicity assay, which better reflects the ability to form tumors in vivo, revealed that both IGF-Ir/482st and IGF-Ir/950st reduced colony formation (P = 0.0046 and 0.0123, respectively). (E) DNA fragmentation assay shows that UV-induced apoptosis was increased in the short receptor-expressing cells. (F) The apoptotic fraction after induction by 50 mM CDDP was increased by expression of the IGF-Ir/482st (P = 0.0002) as detected by the annexin-V assay. (G) Tunnel assays demonstrated that CDDP-induced apoptosis was upregulated in cells infected by Ad-IGF-Ir/482st (P = 0.0142). (H,I) Caspase-3 colorimetric protease assays demonstrated that IGF-Ir/950st upregulates both CDDP- and 1 mM 5FU-induced apoptosis markedly (P = 0.0138 and 0.0047 compared with cells infected with control virus and treated with each drug, respectively). (J) Scratching assays showed that the ability of TE1 to migrate into the wound after 24 h culture was 122.4 ± 6.0 and that this was dramatically inhibited by both IGF-Ir/dns (*,P < 0.0001 compare with the cells infected with Ad-LacZ). Bars show means and SE.

 
Next, we assessed the effect of IGF-Ir/dn on the induction of apoptosis. TE1 was stressed by UV light after transduction, and then evaluated by DNA fragmentation assay. IGF-Ir/950st transduction enhanced UV-induced apoptosis (Figure 5E). Both IGF-Ir/dns also significantly enhanced cisplatin-induced apoptosis as assessed by Annexin-V, Tunnel and caspase-3 assays (Figure 5F–H). 5-Fluorouracil-induced caspase-3 activity was also enhanced significantly by IGF-Ir/dn (Figure 5I). These results indicate that Ad-IGF-Ir/dn enhances the efficacy of the chemotherapy agents clinically used for the treatment of ESCC.

To confirm the effect of Ad-IGF-Ir/dn on ESCC, we used another cell line T.Tn. The IGF axis expression of T.Tn is different from TE1, since it produces high levels of IGF-Ir (Figure 1). Expression of IGF-Ir/dn was confirmed (figures not shown). Ad-IGF-Ir/482st (100 moi) suppressed growth on the plastic of T.Tn (Figure 6A). Ad-IGF-Ir/950st upregulated both CDDP- and 5FU-induced apoptosis detected by caspase-3 assay (Figure 6B,C). The effect of IGF-Ir/dn on T.Tn is very similar to that on TE1, indicated that IGF-Ir/dn suppresses tumor cell growth of ESCC expressing different levels of IGF-Ir.


Figure 6
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  		Fig. 6. The effects of adenoviruses expressing IGF-Ir/dn on another human ESCC cell line T.Tn. (A) Growth of T.Tn cells (open squares) and those infected with Ad-IGF-Ir/482st (100 moi, black closed circles) or control virus (100 moi, open circles) on plastic was evaluated by WST-1 assay. Although the control did not affect proliferation, Ad-IGF-Ir/482st reduced growth (P = 0.0036 compare with the control). (B,C) Caspase-3 colorimetric assay shows that Ad-IGF-Ir/950st (100 moi) upregulates both CDDP- and 5FU-induced apoptosis markedly (P = 0.0161 and 0.0002 compared with cells infected with control virus and treated with each drug, respectively). Bars show means and SE.

 
Esophageal cancer is both highly locally invasive and metastatic. Recent studies have shown that IGF-Ir plays an important role in tumor cell motility (37), so we evaluated the effect of IGF-Ir/dn on tumor cell migration using a wound assay (Figure 5J). Both Ad-IGF-Ir/dns remarkably suppressed the ability of these cells to migrate and IGF-Ir/482st resulted in almost complete elimination of migration compared with control virus-transduced cells.

We then investigated the downstream signaling of the IGF-Ir/dns. Akt-1 was phosphorylated between 5 and 25 min after treatment with IGF-I (5–100 ng/ml) and IGF-II (10–100 ng/ml) (data not shown). The effect of 5 ng/ml IGF-I on Akt phosphorylation was equivalent to that of 10 ng/ml IGF-II, so these ligands were used at these concentrations for further studies. IGF-induced phosphorylated-Akt was inhibited after infection by either of Ad-IGF-Ir/dns (Figure 7A). MAPK phosphorylation induced by IGFs was influenced to a much lesser degree than Akt-1 in both cells infected with Ad-IGF-Ir/dns.


Figure 7
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  		Fig. 7. Downstream signals from IGF-Ir in the esophageal cancer cell line TE1, assessed by western blotting. One hundred multiplicities of infection of each adenoviruses vector were used. (A) Phosphorylated Akt was seen after stimulation with 5 ng/ml IGF-I or 10 ng/ml IGF-II. Akt phosphorylation was reduced in the presence of truncated-IGF-Irs. Although phosphorylated MAPK-1/2 was seen after stimulation with IGFs, Ad-IGF-Ir/dns did not influence MAPK phosphorylation to the same extent. pAkt, phosphorylated Akt; tAkt, total Akt; pMAPK, phosphorylated MAPK; tMAPK, total MAPK. (B) Five ng/ml des(1-3)IGF-I phosphorylated Akt to the same degree as IGF-I. Ad-IGF-Ir/dns reduced des(1-3)IGF-I-induced Akt phosphorylation. dIGF-I, des(1-3)IGF-I. (C) Insulin induces Akt phosphorylation, and this was not influenced by Ad-IGF-Ir/dns.

 
As the bioactivity of IGF is modulated by several high affinity soluble proteins, the IGFBPs (6,9), des(1-3)IGF-I, a truncated IGF-I with very low affinity for IGFBPs, was evaluated. IGF-Ir/dns also blocked des(1-3)IGF-I-induced Akt phosphorylation (Figure 7B), demonstrating that the effect of IGF-Ir/dn is not probably to be a consequence of altered levels of IGFBPs. Importantly, although insulin also induced Akt phosphorylation in these cells, the IGF-Ir/dns did not block this phosphorylation (Figure 7C). This indicates that IGF-Ir/dn does not affect signal transduction through the closely related IR, important for minimizing toxicity in potential clinical applications.

In order to investigate the mechanism of action of IGF-Ir/482st, we assayed the concentrations of IGF-Ir by ELISA in both cell lysates and the media of TE1 infected with adenoviruses (100 moi) and treated with several concentrations of IGF-I (0–100 nM) (Figure 8A). In the culture media, IGF-Ir was detected only in cells infected with Ad-IGF-Ir/482st and the concentration of IGF-Ir tended to increase with the increasing of IGF-I. In washed cells, IGF-Ir was greatly increased in Ad-IGF-Ir/482st-transduced cells. In Figure 8B, we show, as expected, that truncated IGF-Ir protein was detected by IP/western in both media and cell lysates of TE1 infected only after infection with Ad-IGF-Ir/482st. When we immunoprecipitated with an IGF ligand-specific antibody then blotted with a receptor-specific antibody (Figure 8B), we observed that short IGF-Ir was co-immunoprecipitated both in washed TE1 cells and in the medium after transduction with Ad-IGF-Ir/482st in the presence of IGF-I ligand, suggesting that short IGF-Ir formed at least part of a complex containing IGF-I ligand, both cell associated and in the medium.


Figure 8
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  		Fig. 8. TE1 cells were infected with adenoviruses and were treated with different concentrations of IGF-I. The concentration of IGF-Ir was detected by ELISA. The left panels show concentrations in the media and the right panels show those in the washed cells. In the culture media, IGF-Ir was detected only in the cells infected with Ad-IGF-Ir/482st. (B) Western blot revealed that the short IGF-Ir was only in both media and cell lysate of TE1 infected with Ad-IGF-Ir/482st, when those samples were immunoprecipitated with anti-IGF-Ir (H78). In the samples immunoprecipitated with anti-IGF-I (G17), short IGF-Ir was only identified in TE1 infected with Ad-IGF-Ir/482st and stimulated with IGF-I, suggesting that short IGF-Ir was at least part of a complex (perhaps a heterodimer) bound to IGF-I. (C) TE1 cells were infected with Ad-IGF-Ir/482st or ad-LacZ and cultured with serum-free medium. This medium was then transferred to untreated cultures of TE1 cells. ELISA showed that the amount of IGF-Ir protein in TE1 cells cultured with IGF-Ir/482st protein-containing media was higher than those with control, suggesting that IGF-Ir/482st protein is associated with the cells.

 
TE1 cells were infected with Ad-IGF-Ir/482st or ad-LacZ, washed with PBS and cultured with serum-free medium with 0.1% BSA for several hours. This medium was then transferred to untreated cultures of TE1 cells. ELISA showed that the amount of IGF-Ir in TE1 cells cultured with IGF-Ir/482st protein-containing media was higher than in those infected with control virus (Figure 8C), indicating that IGF-Ir/482st protein could be transferred to uninfected cells by cell-free media, and that this protein could associate with cells as seen with direct transduction. Thus, these data support the hypothesis that IGF-Ir/482st can form at least part of a complex containing ligands in both media and washed cells, with potential mechanistic implications for the observed bystander effect.


   Discussion
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 Methods
 Results
 Discussion
References
 
This is the first report demonstrating relating the IGF-II/IGF-Ir axis to clinicopathological parameters in human ESCC and demonstrating that their expression very strongly correlates with poor prognosis, confirming our preliminary microarray analysis. Several other biomarkers have been reported to be prognostic in patients with esophageal carcinoma. Vascular endothelial growth factor is one of the strongest of these prognostic factors (38), and matrix metalloproteinases (39,40) and Ets-1 (41) are also previously reported poor prognostic markers. Interestingly, all these have been reported to be regulated by IGF-Ir signaling (42–45). Thus, in addition to the direct effects we observed on apoptosis via Akt, the IGFs/IGF-Ir axis may affect the outcome of ESCC patients via regulation of other important gene products, such as vascular endothelial growth factor, matrix metalloproteinases and Ets-1.

It has also been reported that serum level of IGFs reflects prognosis in some group of patient with several malignancies (46,47). In this paper, we assessed both IGF-II and IGF-Ir expression in tissue, but it would also be interesting to analyze the prognostic impact of both IGF-II and truncated IGF-Ir in the serum of patients with ESCC at diagnosis. These studies are underway.

In this study, we analyzed IGF-II and IGF-Ir expression by immunohistochemistry, a readily available technique in clinical practice, using a novel scoring system combining ligand and receptor expression information into a single score. In addition to suggesting the importance of an IGF autocrine loop in this disease, if this evaluation and scoring system are confirmed in larger studies, it may have direct importance in the identification of a poor prognostic subset of ESCC patients that might be candidates for additional therapies. However, our study goes beyond the simple identification of a prognostic biomarker and shows therapeutic efficacy of targeted IGF pathway modulation in human ESCC cells in vitro. In this study, we have demonstrated that the IGF-Ir axis is not only frequently overexpressed in esophageal cancer and associated with poor outcome but also an exciting potential target for therapeutic intervention in this specific disease. In our study, Ad-IGF-Ir/dn suppressed in vitro tumorigenicity, survival and migration of human ESCC cells, and also enhanced chemotherapy-induced apoptosis. In two cancer cell lines that express different patterns of IGF-Ir and IGF ligand expression, the effects of Ad-IGF-Ir/dns were very similar, suggesting that this strategy might have therapeutic potency for a variety of patients with ESCC expressing different components and levels of IGF-Ir pathway components.

As IGF-Ir is closely related to the IR (5), it is important that any strategy designed to block IGF-Ir has a high degree of specificity for IGF-Ir compared with IR. We show here that Ad-IGF-Ir/dn does not suppress insulin-induced Akt phosphorylation indicating a high degree of receptor selectivity. Thus, our Ad-IGF-Ir/dn strategy has the distinct potential advantage of blocking both IGF signals, being independent of IGFBPs, interrupting signaling between IGF-Ir and Akt-1, and not affecting IR signaling as well as having bystander effects.

This is the first attempt to assess the efficacy of Ad-IGF-Ir/dn on squamous cancer cell lines. We have shown previously that IGF-targeted therapies are effective in adenocarcinomas of the colon, stomach and pancreas, as well as bronchioloalveolar cell carcinoma (A549) and large cell carcinoma (NCI-H460) (27–30). Although the efficiency of adenovirus transduction is different for each cell line, IGF-Ir/dn suppressed tumorigenicity and cell survival and enhanced chemotherapy-induced apoptosis very effectively in almost all of these systems. This suggests that IGF-Ir-targeted therapy might have significant potency in a wide variety of malignancies.

In this paper, we also studied the mechanism of IGF-Ir/482st action. IGF-Ir/482st co-immunoprecipitates with IGF-I ligand not only in the conditioned media but also in the cellular fraction; this cellular interaction occurs both when IGF-Ir/482st is synthesized inside the cell or when it is presented to cells in cell-free conditioned medium from transduced cells. Another study has reported that a truncated IGF-Ir (486/STOP) in the medium can be taken up by cells and internalized (48), supporting our data that IGF-Ir/482 can function in association with the cell, not just in solution. After immunoprecipitation with anti-IGF-I ligand, immunoblot data with anti-IGF-Ir alpha clearly showed the co-immunoprecipation of IGF-Ir/482st with IGF-I both associated with cells and in the conditioned medium after transduction. Other reports have demonstrated that whereas the 1-462 fragment does not bind ligand (49), 1-719 binds with high affinity and specificity (50). Neither of these studies used exactly the same fragment present in our construct, but it appears that the carboxyl terminus of IGF-Ir alpha subunits is important for avid and specific binding and this region is lacking in IGF-Ir/482st. The three domains of IGF-1R (L1–Cys-rich–L2) surround a central space of sufficient size to accommodate a ligand molecule. Although the fragment studied by crystallography (residues 1–462) does not bind ligand, many of the determinants responsible for ligand binding and specificity map to this central site. On the other hand, the conformation of ectodomain of IR is very different from that of previously reported wild-type receptor and the ectodomain can bind ligand (51). The conformation of truncated 1-482 IGF-I receptor might allow homodimerization of the truncated receptors, or heterodimerization with the wild-type receptor. Thus, IGF-Ir/482st might form homodimers or heterodimers with wild-type receptors to explain the co-immunoprecipation with IGF ligand that we observed both on the cell surface and in solution, and this binding might be responsible for its ability to block signal transduction. Our data do not definitively establish whether it is ligand sequestration or direct interference with membrane wild-type receptor signaling that is responsible for the observed antitumor effects. However, since Ad-IGF-Ir/dn is more effective than an antibody against IGF-Ir (52), this suggests that direct interference with the endogenous wild-type receptor may be an important aspect of the mechanism.

Several humanized monoclonal antibodies for IGF-Ir have been generated and some of which are now in phase I clinical studies (22). New tyrosine kinase inhibitors are also under development (23,24). This study provides support for testing of these therapies in ESCC phase II studies. Although the approach has several practical hurdles to overcome, the clinical application of the adenoviral approach we used here may be especially effective for localized therapy for early lesions or perhaps even for premalignant lesions to prevent malignant conversion without systemic dosing or toxicities. In addition, although we have delivered truncated receptors with a recombinant adenovirus, it is possible that direct infusion of recombinant truncated forms of this receptor protein might be a more effective systemic therapy for metastatic disease than the recombinant adenovirus. Our data may allow the identification of that subset of primary ESCC most probably to benefit from IGF-targeted cancer treatment and conversely, those patients in whom other approaches may be more appropriate.

Thus, the expression of IGF-Ir/IGF-II may be useful for the prediction of recurrence and poor prognosis in ESCC and possibly for selecting patients for IGF axis-targeted therapies. Blockade of IGF-Ir signaling using truncated receptor fragments may also be a promising therapeutic approach for this and other malignancies.


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (H.Y. and K.I.) and from the Ministry of Health, Labour and Welfare (H.Y. and K.I.), Japan.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received April 18, 2006; revised November 24, 2006; accepted December 10, 2006.


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