Carcinogenesis

Carcinogenesis Advance Access originally published online on January 7, 2006
Carcinogenesis 2006 27(5):962-971; doi:10.1093/carcin/bgi336

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Insulin-like growth factor 1 is a potent stimulator of cervical cancer cell invasiveness and proliferation that is modulated by _vß₃ integrin signaling

Meng-Ru Shen ^1, 2, 5, Yueh-Mei Hsu ³, Keng-Fu Hsu ², Yih-Fung Chen ³, Ming-Jer Tang ^4, 5 and Cheng-Yang Chou ^2, 5, *

¹ Department of Pharmacology, ² Department of Obstetrics and Gynecology, ³ Institute of Basic Medical Sciences, ⁴ Department of Physiology, College of Medicine and ⁵ Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan 704, Taiwan

^* To whom correspondence should be addressed at: Dr Cheng-Yang Chou, Department of Obstetrics and Gynecology, National Cheng Kung University Hospital, 138 Sheng-Li Road, Tainan 704, Taiwan Tel: +886 6 2353535 ext. 5608; Fax: +886 6 2766185; Email: chougyn{at}mail.ncku.edu.tw

	Abstract

Top Abstract Introduction Materials and methods Results Discussion References

 
Insulin-like growth factor 1 (IGF-1) has been implicated in promoting mitogenic, metastatic and antiapoptotic phenotypes in several types of cancer. But little is known about the signal interaction of IGF-1 and integrin in the regulation of cervical cancer development and progression. This study is to investigate the regulatory mechanism of IGF-1 receptor (IGF-1R) signaling and its importance in cervical cancer formation. The growth and invasiveness of cervical cancer cells (SiHa and CaSki) were dose-dependently stimulated by IGF-1, whereas those of normal cervical epithelial cells were not. The immunoblot showed that IGF-1R proteins were abundant in cervical cancer cell lines. In contrast, IGF-1R protein was nearly undetectable in normal cervical epithelial cells. IGF-1-stimulated invasion and proliferation were abolished by functional-blocking monoclonal antibody against IGF-1R, whereas these cellular functions were unaffected by either IgG or monoclonal antibody to insulin receptor. Functional-blocking monoclonal antibody against integrins _vß₃, but not _{2, 3}, _{4, 6,} ß₁, ß₄ or ₂ß₁, inhibited the IGF-1-stimulated invasion and proliferation in cervical cancer cells. _vß₃ integrin modulated IGF-1R phosphorylation by altering the rate of Src homology 2-containing phosphotyrosine phosphatase (SHP-2) recruitment to the activated IGF-1R. The modulation of _vß₃ occupancy also affected the activation of IGF-1R downstream-signaling elements, including activation of Akt and extracellular signal-regulated protein kinases 1/2 (Erk1/2). The treatment of blocking antibody of _vß₃ integrin or IGF-1R significantly inhibited tumor growth and caused tumor regression in SCID mice model. Immunoblots of tumor tissues confirmed that the phosphorylation of IGF-1R and downstream targets of Akt and Erk1/2 were remarkably decreased in SCID mice treated with blocking antibodies of _vß₃ or IGF-1R. Thus, these data suggest that the signal interaction between IGF-1R and _vß₃ integrin plays an important role in promoting the development and progression of cervical cancer.

Abbreviations: EGF, epidermal growth factor; Erk1/2, extracellular signal-regulated protein kinases 1/2; IGF-1R, insulin-like growth factor 1 receptor; IRS, insulin receptor substrate; KSFM, keratinocyte serum-free medium; MAP, mitogen-activated protein; PI3K, phosphatidylinositol-3 kinase; SHP-2, Src homology 2-containing phosphotyrosine phosphatase

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
Cervical cancer is a major woman health problem worldwide (1). A growing body of evidence has accumulated to indicate that oncogenic types of human papillomavirus (HPV) serve as an important factor in the development of the precursors of cervical cancer (2). However, only a small fraction of those infected by HPV develop cancer, indicating that other factors contribute to the progression to cervical cancer (3,4). Although prognostic factors such as pelvic lymph node metastasis affects the outcome of cervical cancer, the variability in progression-free and overall survival among patients with similar clinical and pathological characteristics makes it difficult to predict the outcome reliably (5).

Specific growth factors significantly enhance the metastatic and invasive properties of cancer cells, which pose serious problems to successful cancer treatment. The insulin-like growth factor 1 (IGF-1) system has been implicated in promoting mitogenic, metastatic and antiapoptotic phenotypes in several types of cancer (6,7). Each of these properties contributes to IGF-1-mediated maintenance and progression of cancer. Integrins are adhesion receptors that function as cell adhesion and signaling receptors regulating cell death, proliferation, migration and tissue remodeling (8). One mechanism by which integrin family influence tumor cell progression is through the modulation of growth factor signaling (9). For example, ₁ß₁ and ₂ß₁ integrin are the key regulators of hepatocarcinoma cell invasion across the fibrotic matrix microenvironment in response to the stimulation of basic fibroblast growth factor and epidermal growth factor (EGF) (10). Furthermore, _vß₃ integrin is known to influence growth factor signaling when bound to its ligands, that is, interaction of _vß₃ integrin with tenascin-c modifies the EGF growth response and results in enhanced EGF receptor activation and downstream signaling (11).

It has been reported that downregulation of IGF-1 receptor (IGF-1R) by antisense RNA can reverse the transformed phenotype of human cervical cancer cell lines (12). But the regulatory mechanism of IGF-1R signalings in cervical cancer cells is not clear. In addition, little is known about the signal interaction of integrin and IGF-1 in the regulation of cervical cancer development and progression. This study is aimed at investigating the regulatory mechanism of IGF-1R signaling and its importance in the formation of cervical cancer. The results show that IGF-1R signaling cooperates with _vß₃ integrin to promote cervical cancer cell proliferation and invasion.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

 
Cell cultures, transfection and animal models
Cultures of normal human cervical epithelial cells and cervical cancer SiHa and CaSki cell lines were prepared as described previously (13). In some experiments, SiHa cells were transfected with an inactive Src homology 2-containing phosphotyrosine phosphatase (SHP-2) (a gift from Dr Ohnishi at Gunma University, Japan) that was generated by the replacement of cysteine with serine (SHP-2-C>S) in the catalytic site of SHP-2. This single substitution generates a dominant negative SHP-2 mutant (14). The dominant negative SHP-2 mutant was subcloned into the eukaryotic expression vector pCDNA3 (Invitrogen, Carlsbad, CA, USA) and transfected into SiHa cells by lipofection, and stable lines were selected as described (15). In the animal model, 10⁷ cervical cancer SiHa cells were inoculated subcutaneously into the left thigh of female BALB/c SCID mice aged 6–8 weeks (The Jackson Laboratory, ME). The tumor xenografts were measured in two dimensions by caliper twice per week, starting 10 days after inoculation. Tumor volume was calculated by the following equation: width² x length x 1/2 (15). When tumor had reached a volume of 80 mm³ (Day 10), the mice were randomized by tumor volume into three groups of five animals each. The three groups were injected subcutaneously in the right thigh with control IgG, blocking antibody of _vß₃ integrin (15 µg) or IGF-1R (3 µg), respectively. Experimental treatments with antibodies were performed by subcutaneous injection of antibody in 100 µl sterile saline solution at indicated time point. Control mice were treated with 100 µl sterile saline solution containing IgG. The animal experiments were performed according to ethical guidelines for laboratory animal use and approved by the institutional ethical committee.

Invasion and proliferation assay
Cell migration was assayed in the Boyden chamber as an index of invasive activity of tumor cells (16). Matrigel was applied to the upper surface of the filter, and fibronectin (10 µg/ml), vitronectin (2.5 µg/ml), laminin (100 µg/ml) or type IV collagen (100 µg/ml) was used as the chemoattractant in the lower compartment of the chamber. To study the stimulatory effects of growth factors, cervical cancer SiHa and CaSki cells were at first incubated with different concentrations of various growth factors in serum-free DMEM for 24 h. After incubation, invasion assays were run for 6 h in serum-free culture media (DMEM) at 37°C. The invasive ability of cancer cells incubated with DMEM alone for 24 h was used as the control. We also compared the invasive ability between normal cervical epithelial cells and cervical cancer cells. The invasion assays were performed in normal and cancer cells that had been grown in keratinocyte serum-free medium (KSFM) with or without IGF-1 for 24 h. The invasion assays were subsequently run for 6 h in KSFM at 37°C. The invasive ability of normal cells incubated with KSFM alone for 24 h was used as the control. In some experiments, cells were preincubated with blocking antibodies to IGF-1R, insulin receptor (IR) or integrins _vß₃, _{2, 3}, _{4, 6,} ß₁, ß₄ or ₂ß₁ (Chemicon, Temecula, CA) for 30 min at 37°C before invasion assay. After invasion assay, cells were fixed with paraformaldehyde, stained with crystal violet and counted immediately after staining. To assess proliferation, cells were plated at the density of 1.2 x 10⁵ per dish on 60 mm dishes and the medium was changed every 2 days. Counts were performed with the aid of a hemocytometer using trypan blue exclusion (0.08%) to monitor viability. For comparison, normal or cancer cells were grown in KSFM containing different concentration of IGF-1, or supplement for keratinocytes (bovine pituitary extract plus recombinant EGF) or without any growth factor (control). We used a flow cytometry apoptosis detection kit (Clontech, Palo Alto, CA) to identify programmed cell death according to the manufacturer's protocol. In brief, cells cultured in 6-well plates were treated with IgG or blocking antibodies of _vß₃ (15 µg/ml) or IGF-1R (3 µg/ml) for 18 h and then harvested and stained with Annexin V to detect phosphatidyl serine expression on cells during early apoptotic phases and with propidium iodide to exclude dead cells. The reading was done with a Becton Dickinson FACScan (Rutherford, NJ) and data were analyzed with the Cell Quest program.

Immunoblot and immunoprecipitation
Subconfluent cervical cancer SiHa cells were serum starved for 24 h and stimulated with or without 50 ng/ml IGF-1 for 10 min at 37°C. Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and then scraped into lysis buffer (1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EGTA, 150 mM NaCl, 50 mM Tris–HCl pH 7.5, 1 mM sodium vanadate, 1.0 mM NaF, 1.0 mM PMSF, 1 µg/ml pepstatin, 1 µg/ml leupeptin and 1.0 µg/ml aprotinin) termed RIPA buffer. Protein concentrations were determined by Lowry's protein assay. Equal amounts of proteins were immunoprecipitated with anti-IGF-1R, anti-insulin receptor substrate 1 (IRS-1) or anti-IRS-2 antibody (Santa Cruz, CA) for 4 h at 4°C. The immunoprecipitates were incubated in the presence of protein A-G agarose, washed twice with lysis buffer, and subjected to western blotting analysis. Immunoblotting for IGF-1R phosphorylation was carried out by using anti-phospho-IGF-1R (Tyr1131) (Cell Signaling, Beverly, MA). To determine whether the p85 subunit of phosphatidylinositol-3 kinase (PI3K) would bind to IRS, the IRS-1 or IRS-2 immunoprecipitates were analyzed for the presence of p85 by incubating the immunoblots of the immunoprecipitates with anti-p85 antibody (Santa Cruz). To study the phosphorylation of mitogen-activated protein (MAP) kinase and Akt, immunoblots were first probed with phospho-specific antibodies against anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) or phospho-Akt (Cell Signaling) and then stripped and reprobed with the antibody against the corresponding protein.

Statistics
All values in the present study were reported as mean ± SEM (standard error of the mean). Student's paired or unpaired t-test was used for statistical analyses. Differences between values were considered significantly when P < 0.05.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
IGF-1 is a potent stimulator of cellular invasion and proliferation
We studied in cell culture systems to test whether IGF-1 is an important modulator of cervical cancer cell invasion and proliferation. As shown in Figure 1A and B, two cervical cancer cell lines (SiHa and CaSki) migrated in response to the stimulation of IGF-1, EGF and transforming growth factor beta. Dose–response assays done with both cell types established that 50 ng/ml IGF-1 was an optimal concentration for maximal stimulation of tumor invasion, when vitronectin was used as chemoattractor in the lower Boyden's chamber. In addition, cervical cancer cell lines presented the better capability of invasion than that of normal epithelial cells in the absence of any stimulation of growth factors (Figure 2). Although IGF-1 modestly stimulated the invasive ability of normal cervical epithelial cells, the invasive capability of SiHa and CaSki cervical cancer cells was markedly enhanced by IGF-1 stimulation, when vitronectin or fibronectin was used as chemoattractor (Figure 2A and B). In contrast, type IV collagen and laminin did not significantly synergize with IGF-1 in increasing the invasiveness of cervical cancer cells (Figure 2C and D).

View larger version (10K): [in this window] [in a new window]   Fig. 1. IGF-1 is a potent stimulator of cervical cancer cell invasion. The invasion assays were performed in cervical cancer SiHa (A) and CaSki (B) cells that had been incubated with different concentrations of various growth factors for 24 h. Invasion assays were run for 6 h in serum-free culture media (DMEM) at 37°C, when vitronectin was used as chemoattractor in the lower Boyden's chamber. The invasive ability of cells incubated with DMEM alone for 24 h was used as the control. Each column represents mean ± SEM (n = 6). TGFß: transforming growth factor beta; HGF: hepatocyte growth factor; IGF-1: insulin-like growth factor 1; EGF: epidermal growth factor; IGF-2: insulin-like growth factor 2.

 

View larger version (19K): [in this window] [in a new window]   Fig. 2. Comparison of the invasive migration between normal cervical epithelial cells and cervical cancer cells. The invasion assays were performed in normal and cancer cells (SiHa and CaSki) that had been grown in KSFM with or without 50 ng/ml IGF-1 for 24 h. The invasion assays were subsequently run for 6 h in KSFM at 37°C. The invasive ability of normal cervical cells incubated with KSFM alone for 24 h was used as the control. The invasion assays were performed when 2.5 µg/ml vitronectin (A), 10 µg/ml fibronectin (B), 100 µg/ml laminin (C) or 100 µg/ml type IV collagen (D) was used as chemoattractor in the lower Boyden's chamber. Each column represents mean ± SEM (n = 6).

 
In proliferation assay, the growth of cervical cancer cells (SiHa and CaSki) was dose-dependently stimulated by IGF-1 (Figure 3A), whereas that of normal cervical epithelial cells was not (Figure 3B). The immunoblot showed that IGF-1R proteins were abundant in cervical cancer cell lines. In contrast, IGF-1R protein was nearly undetectable in normal cervical epithelial cells (Figure 3C). This might explain the differential response of normal cervical cells and cancer cells to IGF-1 stimulation.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Different growth responses to IGF-1 stimulation between normal cervical epithelial cells and cervical cancer cells. The growth of cervical cancer SiHa and CaSki cells (A) was dose-dependently stimulated by IGF-1, whereas that of normal cervical epithelial cells (B) was not. Control: cells cultured with KSFM alone. Supplement for keratinocytes: bovine pituitary extract (20 µg/ml) plus recombinant EGF (5 ng/ml). Each point represents mean ± SEM (n = 6). (C) The differential expression of IGF-1R in normal cervical epithelial cells (Normal) and cervical cancer cells (SiHa and CaSki), detected by western immunoblots with antibodies to ß-subunit of IGF-1R and ß-actin.

 
As shown in Figure 4A–C, IGF-1-stimulated invasion and proliferation were abolished by functional-blocking monoclonal antibody against -subunit of IGF-1R, whereas the invasion and proliferation were unaffected by either IgG or monoclonal antibody to IR. This indicates that IGF-1-stimulated invasion and proliferation was specific via IGF-1R signalings. Although epithelial cells express a variety of cell-adhesion molecules, adhesion receptors of integrins are the most important extracellular matrix receptors known to play a major role in the invasion and proliferation of most types of cancers (8,17). Expressions of integrin molecules, such as _vß₃, ₆ß₄, ₂ß₁, ₃ß₁ and ₄ß₁, have been reported in human cervical cancer cells (15,18,19). We therefore investigated if integrins participate in IGF-1-mediated cellular invasion and proliferation. Functional-blocking monoclonal antibody against integrins _vß₃, but not _{2, 3}, _{4, 6,} ß₁, ß₄ or ₂ß₁, abolished the IGF-1-stimulated invasion and proliferation of cervical cancer SiHa cells (Figure 4A and C) and CaSki cells (Figure 4B). This indicates that _vß₃ integrin is the key regulator of cervical cancer cell invasiveness across the matrix microenvironment in response to IGF-1 stimulation. Furthermore, Figure 4D shows the effect of blocking antibodies of IGF-1R and _vß₃ integrin on Annexin V binding in SiHa cells, as assessed by fluorescence-activated cell sorter analysis. Pictured by the increase in Annexin V binding, both IGF-1R and _vß₃ integrin blocking antibodies induced an increase in externalization of phosphatidyl serine from the inner leaflet of the plasma membrane, a typical early sign for apoptotic cell death. This indicates that IGF-1R and _vß₃ integrin mediate the survival signalings that inhibit apoptosis. Figure 5 further demonstrated that blocking antibodies of IGF-1R and _vß₃ integrin inhibited cervical cell invasiveness and proliferation in a dose-dependent manner.

View larger version (24K): [in this window] [in a new window]   Fig. 4. IGF-1-stimulated invasion and proliferation were abolished by blocking antibody against -subunit of IGF-1R or vß3 integrin. (A and B) IGF-1-stimulated invasion was abolished by blocking antibody against -subunit of IGF-1R or vß3 integrin. SiHa (A) and CaSki (B) cells were incubated without or with IGF-1 (50 ng/ml) for 24 h at 37°C. Then, cells were treated with IgG or blocking antibodies against IGF-1R (3 µg/ml), insulin receptor (IR, 3 µg/ml) or integrins (15 µg/ml) vß3, 2, 3, 4, 6, ß1, ß4 or 2ß1 for 30 min at 37°C before invasion assay. The invasive ability of cells incubated without IGF-1 for 24 h was used as the control. Invasion assays were run for 6 h in serum-free culture media (DMEM) at 37°C when vitronectin was used as chemoattractor in the lower Boyden's chamber. Each column represents mean ± SEM (n = 6). (C) The antagonistic monoclonal antibodies of IGF-1R and vß3 integrin abolished IGF-1-stimulated cervical cancer SiHa cell growth. To assess proliferation, cervical cancer SiHa cells were initially plated at the density of 1.2 x 105 per dish and cultured with or without 50 ng/ml IGF-1. The blocking antibodies against IGF-1R (3 µg/ml), insulin receptor (IR, 3 µg/ml) or integrins (15 µg/ml) vß3, 2, 3, 4 or 6 were added into the culture medium. The cell counts were determined 48 h later. Each column represents mean ± SEM (n = 6). (D) IGF-1 mediates the survival signalings that inhibit apoptosis. Cervical cancer SiHa cells were cultured with 50 ng/ml IGF-1 in the presence of control IgG (Control) or blocking antibody of vß3 (15 µg/ml) or IGF-1R (3 µg/ml) for 18 h and then harvested. Cells were then subjected to simultaneous Annexin V and propidium iodide staining followed by fluorescence-activated cell sorter analysis. A representative of five similar experiments.

 

View larger version (11K): [in this window] [in a new window]   Fig. 5. (A and B) Dose–response curves of blocking antibodies to IGF-1R or vß3 integrin in the inhibition of invasion and proliferation of cervical cancer SiHa cells. For invasion assays, SiHa cells were incubated with IGF-1 (50 ng/ml) for 24 h at 37°C. Then, cells were treated with IgG or different concentrations of blocking antibodies against IGF-1R or vß3 integrin for 30 min at 37°C before invasion assay. The invasive ability of cells incubated with IgG was used as the control. To assess proliferation, cervical cancer SiHa cells were initially plated at the density of 1.2 x 105 per dish and cultured with 50 ng/ml IGF-1. Different concentrations of blocking antibodies against IGF-1R or vß3 integrin were added into the culture medium. The cell counts were determined 48 h later. Each point represents mean ± SEM (n = 5).

 
Mechanisms by which _vß₃ integrin regulates IGF-1R signalings
Blocking _vß₃ integrin occupancy results in attenuation of cellular invasion and proliferation in response to IGF-1 stimulation (Figures 4 and 5). The interactions between IGF-1R and integrin receptors were therefore analyzed either by attempting to determine that there was a direct binding of the two components using co-immunoprecipitation or showing a change in binding of a known interacting signaling intermediate. We failed to demonstrate a direct physical association between ß₃ integrin and IGF-1R in cervical cancer SiHa cells (Figure 6A). To further define the mechanism by which this interaction occurred, we exposed SiHa cells to IGF-1 or IGF-1 plus blocking antibody of _vß₃ integrin and then immunoprecipitated IGF-1R from cell lysates. We compared the time course of IGF-1R phosphorylation in response to IGF-1 stimulation in the presence or absence of _vß₃ integrin blocking antibody (Figure 6B and D). Following incubation of cervical cancer SiHa cells with IGF-1, there is a rapid increase in IGF-1R phosphorylation by 5 min. This increase is sustained through 10 min, and is subsequently decreased by 20 min (Figure 6B). In contrast, if SiHa cells are preincubated with _vß₃ integrin blocking antibody, there is an equivalent increase in IGF-1R phosphorylation after 5 min. But this phosphorylation is not sustained, in which IGF-1R phosphorylation is significantly decreased after 10 min IGF-1 treatment (Figure 6C). Similar results were obtained from three independent experiments and were summarized in Figure 6D.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Molecular mechanisms by which vß3 integrin regulates IGF-1R signalings. (A) No physical association of IGF-1R with vß3 integrin in response to IGF-1 (50 ng/ml) stimulation. P: positive control from whole cell lysates of SiHa cells. (B and C) Time courses of IGF-1R phosphorylation in response to IGF-1 stimulation (50 ng/ml) in the absence (B) or presence (C) of blocking antibody of vß3 integrin (15 µg/ml). IP: immunoprecipitation; IB: immunoblot. (D) Effect of blocking antibody of vß3 integrin on the time course of IGF-I-stimulated receptor phosphorylation. IGF-1-stimulated receptor phosphorylation in the presence or absence of blocking antibody over the time course indicated was measured by scanning densitometry. The time at which maximum phosphorylation was achieved was designated 100% and then the relative level of receptor phosphorylation at the other time points was determined. Each point represents mean ± SEM (n = 3).

 
In smooth muscle cells, the interaction of SHP2 with IGF-1R is associated with receptor dephosphorylation (20,21). Accordingly, we examined whether _vß₃ integrin altered SHP-2 association in cervical cancer cells. In the presence of blocking antibody of _vß₃ integrin, there is an increase in the basal association of SHP-2 with IGF-1R before IGF-1 exposure and a remarkable increase in its association with IGF-1R following a 5 min exposure to IGF-1 (Figure 6C). In contrast, in cells that were not incubated with the blocking antibody, the association of IGF-1R with SHP-2 was not significant until 20 min after IGF-1 stimulation (Figure 6B). This implies that the acceleration in the rate of IGF-1R dephosphorylation observed in the presence of blocking antibody of _vß₃ integrin was due to an early recruitment of SHP-2.

We cloned cervical cancer SiHa cells expressing a catalytically inactive SHP-2. The function of SHP-2 has been reported as part of a positive signaling pathway mediating IGF-1 activation of MAP kinase (22,23). To analyze whether cervical cancer SiHa cells transfected with SHP-2 mutant are in fact deficient in SHP-2 activity, we measured the phosphorylation level of MAP kinase in response to IGF-1 stimulation. Figure 7 shows the characterization of SHP-2-deficient clones. SiHa cells transfected with empty vector show normal extracellular signal-regulated protein kinases 1/2 (Erk1/2) activation in response to IGF-1. In contrast, SiHa cells transfected with SHP-2 mutant show a substantial decrease in IGF-1-triggered Erk1/2 phosphorylation. We further studied the time course of IGF-1-stimulated receptor phosphorylation in SiHa cells expressing a catalytically inactive SHP-2. As shown in the right panel of Figure 8B, when SiHa cells expressing SHP-2 mutant were exposed to blocking antibody of _vß₃ integrin, it does not result in a decrease of IGF-1R phosphorylation as was seen in cells expressing wild-type SHP-2 (Figure 8A, right panel). This change is not due to the absence of SHP-2 recruitment, because cells expressing SHP-2 mutant that were preincubated with blocking antibody still showed the association of SHP-2 with IGF-1R at 0, 5 and 10 min (Figure 8B, right panel). This contrasts with the cells expressing only endogenous SHP-2, in which IGF-1R phosphorylation is significantly reduced in the presence of blocking antibody at 10 min, and this reduction is associated with an increase of SHP-2 association with IGF-1R (Figure 8A, right panel).

View larger version (13K): [in this window] [in a new window]   Fig. 7. Characterization of SHP-2-deficient clones in cervical cancer SiHa cells. Western immunoblot analysis of Erk1/2 activation. Equal amounts of cell lysates (30 µg) of empty vector (mock) or mutant (SHP-2-C>S) transfected cells, stimulated with 50 µg/ml IGF-1 at the times indicated, were resolved by SDS–PAGE and immunoblotted with anti-phospho-Erk1/2-specific (upper panel) or anti-Erk1/2 antibodies (lower panel). A representative figure of three similar experiments.

 

View larger version (25K): [in this window] [in a new window]   Fig. 8. (A and B) Effect of blocking vß3 integrin antibody in SiHa cells expressing a catalytically inactive form of SHP-2. SiHa cells expressing a catalytically inactive form of SHP-2 (B) and SiHa cells transfected with empty vector (A, mock transfection) were grown to 80% confluency, serum starved for 24 h and then incubated with or without blocking vß3 integrin antibody (15 µg/ml) for 30 min at 37°C. Cells were then exposed to IGF-1 (50 ng/ml) for various time durations as indicated. Following cell lysis and immunoprecipitation, IGF-1R phosphorylation and SHP-2 recruitment were visualized by western immunoblotting. A representative of three similar experiments. IP: immunoprecipitation; IB: immunoblots.

 
To determine whether modulation of _vß₃ occupancy would affect the IGF-1R-mediated signal transduction, we studied the binding capacity of IGF-1R immediate downstream substrate (IRS) with p85, the regulatory subunit of PI3K. SiHa cells were exposed to IGF-1 alone or the combination of IGF-1 plus antibody to _vß₃ integrin or IGF-1R for 10 min. Antibody was added 30 min before the addition of IGF-1. After a 10-min incubation at 37°C, the cells were lysed and immunoprecipitated by IRS-1 or IRS-2, and then the immunoprecipitated proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotted for p85 subunit of PI3K. The addition of antibody to _vß₃ integrin or IGF-1R almost abolished the binding of p85 with IRS-2. In contrast, no association between p85 and IRS-1 was noted (Figure 9A).

View larger version (19K): [in this window] [in a new window]   Fig. 9. IGF-1R downstream signalings are modulated by vß3 integrin. (A) IGF-1 stimulates the association of PI3K with IRS-2, which is inhibited by blocking antibody of vß3 integrin or IGF-1R. IP: immunoprecipitation; IB: immunoblotting. (B) IGF-1 stimulates Akt phosphorylation, which is abolished by vß3 integrin antibody (15 µg/ml), LY294002 (20 µM) or wortmanin (50 nM). (C) IGF-1 stimulates Erk1/2 phosphorylation, which is abolished by vß3 integrin antibody (15 µg/ml). For these experiments, subconfluent SiHa cells were pre-incubated without or with antibody of vß3 integrin (15 µg/ml) or IGF-1R (3 µg/ml) for 30 min in serum-free DMEM. The cultures were then exposed to no IGF-1 or 50 ng/ml IGF-1 for 10 min at 37°C. The other details are described in Materials and methods.

 
One of the important downstream signaling molecules in PI3K pathway is Akt. As shown in Figure 9B, IGF-1 treatment resulted in Akt phosphorylation, which was abolished by _vß₃ integrin antibody and two structurally different PI3K inhibitors (LY294002 or wortmanin). Similarly, _vß₃ integrin occupancy also abolished IGF-1R-mediated signal transduction of Erk1/2 activation (Figure 9C).

Inhibition of cervical cancer growth and development in vivo
To test whether manipulation of IGF-1R signalings alters tumor growth and invasion in vivo, we inoculated SCID mice subcutaneously with cervical cancer SiHa cells. As shown in Figure 7, rapid tumor growth was obvious in the control groups. In contrast, the treatment of blocking antibody of _vß₃ integrin or IGF-1R inhibited tumor growth in a dose-dependent manner (Figure 10A and B). Immunoblots of tumor tissues confirmed that the phosphorylation of IGF-1R decreased as a function of time in SCID mice treated with blocking antibodies of _vß₃ or IGF-1R (Figure 10C). In addition, IGF-1R downstream targets of Akt and Erk1/2 were remarkably decreased in the groups with antibody treatment (Figure 11).

View larger version (21K): [in this window] [in a new window]   Fig. 10. Inhibition of cervical cancer growth and development in vivo. (A) Tumor growth curves of SCID mice subcutaneously inoculated with cervical cancer SiHa cells. The tumor xenografts were measured 10 days after inoculation. When tumor had reached a volume of 80 mm3 (Day 10), the mice were randomized by tumor volume into three groups. The three groups were treated with control IgG, blocking antibody of vß3 integrin (150 µg/ml) or IGF-1R (30 µg/ml), respectively. Each point represents mean ± SEM (n = 15 before Day 10, n = 12 before Day 13, n = 9 before Day 25, n = 6 before Day 30). Arrow indicates injection of antibodies. (B) Blocking antibody of vß3 integrin or IGF-1R inhibited tumor growth in a dose-dependent manner. Each column represents mean ± SEM (n = 6). (C) Immunoblots of tumor tissues suggest that the phosphorylation of IGF-1R decreased as a function of time in SCID mice treated with blocking antibodies of vß3 integrin (150 µg/ml) or IGF-1R (30 µg/ml). A representative of three similar experiments.

 

View larger version (20K): [in this window] [in a new window]   Fig. 11. IGF-1R downstream targets of Akt and Erk1/2 were remarkably decreased by antibody treatment. (A) Tissue lysates of tumor xenografts after 30-day inoculation (Figure 10) at first immunoblotted with anti-phospho-IGF-1R (Tyr1131), anti-phospho-Akt or anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) were stripped and reprobed with anti-IGF-1R or Akt or Erk1/2 as the internal control. Lanes 1–3: tumor tissues from mice treated with control IgG (lane 1), blocking antibody of 150 µg/mlvß3 integrin (lane 2) and 30 µg/ml IGF-1R (lane 3). A representative of six similar experiments. (B) Phosphorylated IGF-1R, Akt and Erk1/2 levels were analyzed by scanning densitometry and the results were expressed as the relative of control (IgG injection). Each column represents mean ± SEM (n = 6 independent experiments). * < 0.001; # < 0.01 by unpaired t-test.

 
We also did the experiments to study whether the blocking antibodies have a role in tumor regression. When tumor xenografts had reached a volume of 400 mm³, the mice were randomized by tumor volume into three groups of six animals each. The three groups were treated every 3 days with control IgG, blocking antibody of _vß₃ integrin or IGF-1R, respectively. As shown in Figure 12, the treatment of blocking antibodies significantly caused tumor regression that could sustain 10–12 days after antibody withdrawing.

View larger version (14K): [in this window] [in a new window]   Fig. 12. Blocking antibodies cause tumor regression. SCID mice were subcutaneously inoculated with cervical cancer SiHa cells. When tumor xenografts had reached a volume of 400 mm3, mice were randomized by tumor volume into three groups of six animals each. The three groups were treated every 3 days with control IgG, blocking antibody of vß3 integrin or IGF-1R, respectively. Arrow indicates injection of antibodies.

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
Here we show that IGF-1R signaling is important for cervical cancer cell invasiveness and proliferation. This conclusion is supported by the following findings: (i) IGF-1R proteins are abundant in cervical cancer cell lines. In contrast, IGF-1R protein was nearly undetectable in normal cervical epithelial cells. (ii) IGF-1 is a potent stimulator of cellular invasion and proliferation in cell culture systems. (iii) The IGF-1-stimulated effects are completely inhibited by antagonistic antibody against IGF-1R, whereas IgG or monoclonal antibody to IR has no effect. (iv) The manipulation of IGF-1R signalings by blocking antibody of IGF-1R inhibits tumor growth and causes tumor regression in SCID mice model.

This study identifies an important role of _vß₃ integrin that functions as a signal modulation of IGF-1R biochemical outputs in cervical malignancy. Inhibition of ligand occupancy of _vß₃ integrin by blocking antibody results in attenuation of cervical cancer cell response to IGF-1 stimulation, such as receptor phosphorylation, subsequent signal transduction and cellular function. The antagonistic antibody used here is a purified monoclonal blocking vß3 integrin antibody (clone LM609, Chemicon, CA) and IGF-1R antibody (catalog number MAB1122, Chemicon). Several studies have shown that these antibodies could inhibit _vß₃ integrin-dependent (24,25) or IGF-1R-dependent function (26,27), respectively. Accordingly, there is a specific linkage between the activation of _vß₃ integrin and IGF-1R-mediated signal transduction pathways in cervical cancer cells. The molecular mechanism by which _vß₃ actually functions to alter IGF-1R phosphorylation could be mediated through multiple types of interactions. Because these two proteins do not appear to co-immunoprecipitate, it is likely that blocking occupancy of _vß₃ integrin by antibody directly affects an intermediary protein that binds to IGF-1R. The dephosphorylation of IGF-1R caused by blockade of _vß₃ occupancy was accompanied by increasing SHP2 recruitment to IGF-1R. This is consistent with the findings in porcine aortic smooth muscle cells that show that _vß₃ integrin regulates IGF-1R phosphorylation by altering the rate of SHP-2 recruitment to the activated IGF-1R (20,21,28). Therefore, SHP-2 is a candidate protein responsible for the cross-talk between the activation of _vß₃ integrin and IGF-1R-mediated signal transduction pathways. Further experiments on cervical cancer cells expressing a catalytically inactive SHP-2 confirm that the acceleration in the rate of IGF-1R dephosphorylation observed in the presence of _vß₃ integrin antibody was due to an early recruitment of SHP-2.

It has been reported that blocking the adhesion receptor of integrin family would inhibit IGF-1 signalings. For example, inhibiting integrin function of ₅ß₁ blocks the IGF-1 stimulation of cell motility in a metastatic breast cancer cell line, MDA-231BO (29). In smooth muscle cells, blocking ligand occupancy of _vß₃ with the distintegrin echistatin reduces IGF-1-stimulated receptor phosphorylation, and it also inhibits cellular migration and DNA synthesis responses to IGF-1 (30). Blocking ß₁ integrin inhibits IGF-1-induced cell adhesion to fibronectin in human multiple myeloma cells (31). Here we identified the specific integrin, _vß₃, which is critically involved in IGF-1-mediated growth and invasion of cervical cancer cells. Although ₅ß₁ integrin is expressed in cervical cancer cells (M.R. Shen and C.Y. Chou's, unpublished data), its role in IGF-1-induced signalings is not clear and needs to be explored in the future.

Taken together, this study identifies an unexpected role for _vß₃ integrin in cervical cancer progression and development as a functional amplification of biochemical outputs rather than a mechanical adhesion. IGF-1 stimulates the invasion and proliferation in human cervical cells via the cooperation of _vß₃ integrin. Therefore, blockade of _vß₃ integrin and IGF-1R signal transduction may provide a novel strategy for the treatment of cervical cancer.

	Acknowledgments

 
This work was partly supported by Center of Excellence for Clinical Trial & Research (DOH-TD-B-111-004), Department of Health, Executive Yuan, Taiwan; National Science Council, Taiwan (NSC 94-2314-B-006-070 & NSC 94-2320-B-006-009 to M.R.S.), National Health Research Institutes (NHRI-EX94-9311BS to M.-R.S. & NHRI-EX94-9422BI to C.-Y.C.), Center for Bioscience and Biotechnology, National Cheng Kung University and Program for Promoting University Academic Excellence (91-B-FA09-1-4 to M.-R.S. and C.-Y.C.).

Conflict of Interest Statement: None declared.

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Received September 12, 2005; revised November 11, 2005; accepted January 3, 2006.

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