Efficacy of and resistance to anti-IGF-1R therapies in Ewing's sarcoma is dependent on insulin receptor signaling

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Abstract

Identification of patient selection criteria and understanding of the potential mechanisms involved in the development of resistance are crucial for an appropriate and successful design of clinical trials with anti-insulin-like growth factor (IGF)-1R therapies. Few Ewing's sarcomas are highly sensitive to IGF-1R targeting and understanding the reason why, may hold the secret to improve successful treatments. In this paper, we show that a major mechanism of resistance to highly specific inhibitors of IGF-1R, either antibodies or tyrosine kinase inhibitors may involve enhanced insulin receptor (IR)-A homodimer formation and IGF-2 production. Resistant cells are able to switch from IGF-1/IGF-1R to IGF-2/IR-A dependency to maintain sustained activation of AKT and ERK1/2, proliferation, migration and metastasis. These cells also showed higher proliferative response to insulin, in keeping with a switch towards insulin pathways sustaining proliferation and malignancy, rather than metabolism. Our findings demonstrate a role for IR-A in eliciting intrinsic and adaptive resistance to anti-IGF-1R therapies. Thus, we indicate that tumors with low IGF-1R:IR ratio are unlikely to greatly benefit from anti-IGF-1R therapies and that the efficacy of anti-IGF-1R therapies should be evaluated in relationship to the IR-A:IGF-1R ratio in cancer cells. Moreover, we provide evidences supporting IR-A as an important target in sarcoma therapy.

Introduction

Insulin-like growth factor (IGF) signaling has been implicated as a critical contributor to malignant transformation and tumor resistance to cytotoxic chemotherapy, targeted therapies, hormonal therapy and radiation (for reviews see Casa et al., 2008; Chitnis et al., 2008). As a result, IGF signaling has become an attractive target for development of novel anticancer agents, and a large number of compounds, including blocking antibodies and tyrosine kinase inhibitors (TKIs) disrupting IGF-I receptor (IGF-1R) functions are in preclinical and clinical development. In general, phase I and II studies in different tumors have provided evidences suggesting safety of anti-IGF-1R antibodies and a few anecdotes of impressive single-agent activity particularly in Ewing's sarcoma (Pollak, 2008; Wahner Hendrickson et al., 2009; Buck et al., 2010) indicated anti-IGF-1R drugs as an effective clinical possibility. However, these early studies also clearly pointed out that only a minority of patients really benefit from IGF-1R targeting. Evidences so far accumulated have in fact revealed that, although most tumors express the IGF-1R, expression alone is unlikely to be sufficient for sensitivity to IGF-targeted treatments. Deeper understanding of the IGF signaling system and its downstream effectors is thus necessary to identify patients that may really benefit from this new targeted therapy.

Ewing's sarcoma is one of the few tumors in which the efficacy of IGF-1R targeting in preclinical models has been partly predictive of clinical activity (Scotlandi and Picci, 2008). However, we still do not know exactly the mechanisms responsible of the exquisite efficacy of anti-IGF-1R therapy in this tumor. It is possible that the particular sensitivity of Ewing's sarcoma cells to anti-IGF-1R therapies derives from the direct connection established between the genetic hallmark of Ewing's sarcoma, the oncogene fusion EWS/ETS transcripts (Delattre et al., 1994) and the IGF system (Toretsky et al., 1997; Prieur et al., 2004; Cironi et al., 2008). Autocrine production of IGF-1 (Scotlandi et al., 1996) is in fact sustained by abnormal transcription activity of EWS/ETS hybrid products (Cironi et al., 2008) and induces constitutive IGF-1R activation. However, this is not enough to explain why anti-IGF-1R therapy gave extraordinary results as single-agent in heavily pretreated few patients but not in others. Moreover, autocrine activation of IGF-1R and association with oncogenic events is a common feature of other sarcomas, which did not show the same levels of sensitivity towards anti-IGF-1R drugs as Ewing's sarcoma (Benini et al., 1999; Chitnis et al., 2008; Kolb et al., 2008). In this paper, we contributed to identify indicators of efficacy and resistance to the anti-IGF-1R monoclonal antibody AVE1642, the humanized counterpart of EM164 (Maloney et al., 2003; Sachdev et al., 2006) or to the anti-IGF-insulin receptor small-TKI NVP-AEW541. We developed an experimental model of resistance to these drugs starting from two highly sensitive Ewing's sarcoma cell lines and demonstrate that insulin receptor (IR) pathway is heavily involved in native and acquired resistance to anti-IGF-1R targeted drugs.

Results

Insulin receptor signaling is a major mediator of resistance to anti-IGF-1R drugs

Starting from the sensitive TC-71 and IOR/BRZ 71 Ewing's sarcoma cell lines, we obtained cell variants stably resistant to HAb AVE1642 (named TC/AVE and BRZ/AVE). These resistant cells were selected and maintained with 10 μg/ml of the antibody, a dose 100-fold higher than the one able to induce around 50% of growth inhibition in parental cells. As shown in Figure 1a, cells maintained their resistance at least up to 100 μg/ml of HAb AVE1642. Biological characterization of these resistant cells indicated that their malignant behavior did not change compared with parental cell lines, as shown by the similar in vitro growth abilities (Figure 1b) as well as metastasis spread (Figure 1c). Consistent with maintenance of proliferative abilities, cells resistant to HAb AVE1642 preserved the sensitivity of parental cell lines to conventional drugs, such as doxorubicin, vincristine and ifosfamide (Figure 1d), indicating that they may still be treatable with conventional chemotherapy. Thus, these cell variants apparently do not show other phenotypic differences apart from being insensitive to HAb AVE1642 and represent a good experimental model to highlight molecular mechanisms of resistance to anti-IGF-1R agents. We did not observe any changes in the expression of some other growth receptors, including members of the human epidermal growth factor receptor (HER) family that were recently shown to be associated with resistance to anti-IGF-1R agents (Desbois-Mouthon et al., 2009) (Supplementary Figure 1). Thus, we analyzed the gene expression profile of resistant cells, and compared these cells with parental, sensitive cells using HG-U133 Plus 2.0 Array. We considered together the genetic profiles of TC-71 and IOR/BRZ cells (Supplementary Table 2) and panther pathway analysis is reported in Table 1. Among other pathways, involvement of IGF/insulin signaling was suggested and also confirmed by GeneGo network analysis (Figure 2a). Immunoblotting experiments validated gene expression profiling information. Resistant variants showed lower levels of expression of IGF-1R, enhanced IR expression and higher levels of phosphorylation of pAKT, pERK and pSTAT3 than parental cell lines in basal conditions (Figure 2b). No changes were observed in the expression of the two major mediators, IRS1 and Shc. The sequencing analysis of IGF-1R did not reveal mutations in the receptor gene (data not shown). Evaluation of the two isoforms of IR by RT–PCR indicated that only IR-A is expressed in our cell lines and overexpressed in resistant variants (Figure 2c). Higher expression of IR compared with IGF-1R in resistant cells was also confirmed by quantitative PCR (Figure 2d).

Figure 1
figure1

Biological features of Ewing's sarcoma cells resistant to AVE1642. (a) Lack of effects of AVE1642 in TC/AVE or BRZ/AVE cells. Cell growth was assessed by MTT assay after 72 h exposure to AVE1642. Columns represent mean values of three independent experiments and bars represent s.e. (b) Anchorage-independent growth of TC/AVE and BRZ/AVE compared with parental cell lines. (Left) Number of colonies in triplicate plates was determined after 7 days of growth in IMDM 10% FBS. Columns represent mean values of three independent experiments and bars represent s.e. (Right) Representative pictures are shown (magnification, × 40). (c) Metastasis ability of TC/AVE cells in nude mice. Data obtained from groups of seven mice. (d) Efficacy of doxorubicin, vincristine and ifosfamide in TC/AVE and BRZ/AVE. Cell growth was assessed by MTT assay after 72 h exposure to AVE1642 and displayed as a percentage of growth inhibition. Columns represent mean values of three independent experiments and bars represent s.e.

Table 1 Significantly enriched pathways as determined by Panther analysis on genes upregulated in Ewing's sarcoma cell lines resistant to IGF-1R inhibition (P<0.05)
Figure 2
figure2

Increased activation of IR pathway in resistant variants. (a) Insulin network identified by GeneGo Pathways analysis for the genes most differentially expressed in TC/AVE cells compared with parental cell line (P<0.005). (b) Western blotting expression of IGF-1R/IR signaling mediators in parental and resistant cells. Total proteins were used as controls. Blots are representative of two independent experiments. (c) IR isoforms evaluated by RT–PCR. Amplicons were resolved on 1% agarose gel. (d) mRNA quantification of IGF-1R and IR in parental and AVE1642 resistant cells. Columns are representative of mRNA copy number ratio between two receptors. (e) mRNA expression level of IGF-1, IGF-2 and IGFBP-3, expressed as 2−ΔΔCT, in TC/AVE respect to parental cells. TC-71 was used as calibrator (2−ΔΔCT=1).

It is well known that IR-A homodimers can be activated by IGFs, particularly IGF-2, beside insulin and induce proliferative signals. Consistently, although autocrine production of IGF-1 is prevalent in Ewing's sarcoma with respect to IGF-2 (Scotlandi et al., 1996; Cironi et al., 2008), cells resistant to anti-IGF-1R HAb AVE1642 showed increased mRNA expression of IGF-2 (Figure 2e), thus inducing formation of an IGF-2/IR-A loop that may sustain cell proliferation of resistant cells independently of blockage induced by HAb AVE1642. Expression of IGFBP-3, the major local regulator of IGFs, was also considered (Figure 2e) but we did not observe remarkable variations in resistant cells, as previously indicated for cells resistant to the TKI BMS-536924 (Huang et al., 2009).

Upregulation of IR-A and increased expression of IGF-2 appeared as a more general mechanism. Indeed, formation of an IGF-2/IR-A loop was also observed in TC-71 cells that we made resistant to the anti-IGF-1R TKI NVP-AEW541. These cells were 50-fold more resistant to the TKI, they grew in soft-agar similarly to parental cells and displayed upregulation of IR-A and IGF-2 (Figures 3a–c). In addition, these cells also down-modulated IGF-1R (Figures 3c and d), with consequent switch of IGF-1R:IR ratio in favor of IR. According to the IGF-1R decrease, a common event in TC/AVE and TC/AEW cells, TC/AEW cells are completely resistant to AVE1642 and similarly, TC/AVE cells are resistant to NVP-AEW541 (data not shown). In addition, resistant cells were sensitized to proliferative effects of insulin, as indicated by higher growth response to insulin exposure in vitro (Figure 3e) while silencing of IR in TC/AVE cells induced inhibition of cell growth, thus supporting the proliferative role of IR-A in these cells (Figure 3f).

Figure 3
figure3

Resistance to NVP-AEW541 is mediated by IR-A/IGF-2 loop. (a) Lack of efficacy of NVP-AEW541 in TC/AEW resistant cells. Cell growth was assessed by MTT assay after 72 h exposure to AVE1642. Columns represent mean values of three independent experiments and bars represent s.e. (b) Anchorage-independent growth of TC/AEW compared with parental cell lines. Number of colonies was determined after 7 days of growth in IMDM 10% FBS. Columns represent mean values of three independent experiments and bars represent s.e. (c) Expression of IGF-1R, IR, IGF-1 and IGF-2, expressed as 2−ΔΔCT, in NVP-AEW541 resistant cells (TC/AEW) with respect to parental cells. TC-71was used as calibrator (2−ΔΔCT=1). (d) Western blotting expression of IGF-1R and IR in TC/AEW cells. (e) Proliferative effects of insulin in resistant cells. Cell growth was assessed by trypan blue assay after 72 h exposure to insulin 10 nM, and shown as percentage of growth increase over untreated controls. (f) Silencing of IR in TC/AVE cells induced inhibition of cell growth. Cells were exposed to siRNA-IR 75 nM for 72 h and cell growth was assessed as described in Materials and methods and shown as percentage of inhibition over untransfected controls. Expression of IR was evaluated by western blotting.

Native resistance to IGF-1R drugs is mediated by IGF-1R:IR ratio

The ratio IGF-1R:IR seems to be responsible also for the native resistance of Ewing's sarcoma cells to anti-IGF-1R HAb AVE1642 and to NVP-AEW541. We evaluated the efficacy of HAb AVE1642 and NVP-AEW541 on cell proliferation of seven Ewing's sarcoma cell lines (Figure 4a). Either HAb AVE1642 or NVP-AEW541 showed variably effectiveness. All seven Ewing's sarcoma cell lines here considered showed exclusive or prevalent expression of IR-A (Table 2). Cells that did not respond to anti-IGF-1R drugs or that showed modest levels of sensitivity had higher expression of IR-A with respect to IGF-1R and indeed the ratio IGF-1R:IR positively correlates with the percentage of growth inhibition induced by the antibody (Spearman's test, r=0.99, P<0.001 with respect to HAb AVE1642; r=0.90, P<0.001, with respect to NVP-AEW541) (Table 2).

Figure 4
figure4

Ratio IR:IGF-1R is responsible for the level of sensitivity of Ewing's sarcoma cells to anti-IGF-1R therapies. (a) In vitro sensitivity of Ewing's sarcoma cell lines to AVE1642 or to NVP-AEW541. Cell growth was assessed by MTT assay after 72 h of treatment and displayed as a percentage of survival over controls. (b) Silencing of IGF-1R or IR modulates the sensitivity of SK-M-NC cells to AVE1642. Cell growth was determined as described in Materials and methods after 72 h of treatments and displayed as a percentage of survival over controls. (c) Silencing of IGF-1R induces compensatory increase in expression of IR and vice versa. Expression of IGF-1R or IR was evaluated by western blotting. (d) Transcriptional regulation of IR upon si-IGF-1R in SK-N-MC cells. Parental growing cells were used as calibrator (2−ΔΔCT=1).

Table 2 Expression of IGF-1R and IR by ELISA in Ewing's sarcoma cell lines

Expression of IGF-1R and IR was also evaluated in 109 biopsies of Ewing's sarcoma primary tumors. Positivity for IGF-1R was observed in 88% (96/109); positivity for IR was observed in 96% (105/109). High-expressing patients were 60% (65/109) for IGF-1R and 81% (88/109) for IR. The expression of IGF-1R and IR showed an inverse correlation (Spearman's test r=−0.23; P=0.01).

The high expression of IR in clinical samples may account for the limited effectiveness observed in phase II clinical trials of truly selective anti-IGF-1R agents (Wahner Hendrickson et al., 2009). Indeed, we could functionally confirm the cross-talk between IGF-1R and IR-A in mediating sensitivity to HAb AVE1642. We use a silencing interfering approach in SK-N-MC cells that co-express the IGF-1R and IR-A in almost equal proportion (Table 2). Either IGF-1R or IR-A knockdown resulted in decreased cell growth. The percentage of growth inhibition due to IGF-1R silencing was, however, double with respect to that observed after IR-A depletion (Figure 4b), suggesting that IR-A did not completely compensate for loss of IGF-1R in cells that produce primarily IGF-1. In contrast, when cells deprived of IGF-1R or IR-A were exposed to 10 ng/ml AVE1642 for 48 h we could appreciate the complementary role of the two receptors. Upon IR-A depletion, we observed increased sensitivity to the drug (−31%, with respect to cells treated with small-inhibitory RNA (siRNA)-IR-A alone), whereas upon IGF-1R depletion, we observed decreased sensitivity to AVE1642 (+20% with respect to cells treated with siRNA-IGF-1R alone). Consistent with this, upon IR-A depletion we found a concomitant enhancement of IGF-1R protein (Figure 4c) that explains the higher sensitivity of the cells towards a drug that specifically target IGF-1R. Instead upon IGF-1R depletion we observed a concomitant increase in IR-A expression, both at transcriptional and protein level (Figures 4c and d) that may compensate for IGF-1R, and result in increased cell viability. Overall, this experiment confirms that variations in IGF-1R:IR-A ratio are directly responsible for sensitivity to anti-IGF-1R targeted drugs.

Discussion

IGF-1R represents a promising therapeutic target, particularly in sarcomas (Scotlandi and Picci, 2008). However, evidences so far obtained have already clearly shown that expression of IGF-1R is not sufficient to a tumor to be responsive to IGF-targeted therapy. In sarcomas, the biological relevance of the IGF system has been extensively documented (Scotlandi and Picci, 2008). However, phase I and II clinical trials have shown efficacy only against few cases of Ewing's sarcoma, with remarkable results in heavily pretreated patients, but these represent around 10% of cases. These findings clearly indicated the need to come back to the laboratory to understand deeper the IGF pathway if enhancement of anti-IGF-1R therapy would be obtained. Several important recent reports offer a comprehensive preclinical evaluation of predictive biomarkers for antibodies or TKIs against IGF-1R (Cao et al., 2008; Huang et al., 2009; Mukohara et al., 2009; Wahner Hendrickson et al., 2009; Zha et al., 2009). These data indicated that expression of IGF-1R itself or other IGF system mediators as well as expression of other growth factor receptors, correlated with response to IGF-1R blockade. However, data are not uniform and differences emerged in different models and tumors. We contributed to the field by evaluating mechanisms of resistance to anti-IGF-1R drugs, HAb AVE1642 and NVP-AEW541, in Ewing's sarcoma. Starting from two cell lines, we developed resistant variants to obtain clues on the molecular mechanisms that regulate sensitivity to anti-IGF-1R agents. Molecular comparisons with parental cells exclude alterations in ErbB/HER receptor signaling as previously indicated (Desbois-Mouthon et al., 2009; Huang et al., 2009), while they clearly show upregulation of IR signaling. Ewing's sarcoma cells may adapt to anti-IGF-1R HAb by inducing compensatory inputs leading to activation of an IR-A-dependent pathway. IR-A has a higher affinity for IGF-2 than for IGF-1 (ED50=2.5 nM for IGF-2 vs ED50 >30 nM for IGF-1) (for details see the review Belfiore et al., 2009). Indeed, increased expression of IGF-2 was observed together with increased expression of IR-A in resistant cells. Our data supported the idea that suppression of IGF-1R leads to altered transcription of IR-A and IGF-2. Thus, resistant cells are able to switch from IGF-1/IGF-1R to IGF-2/IR-A dependency to maintain proliferation, migration and metastasis. These cells also showed higher proliferative response to insulin, thus confirming the upregulation of IR signaling. The dogma indicates that IR is mainly metabolic, while IGF-1R is mitogenic. However, there is mounting evidence to suggest that the IR may also be involved in the pathogenesis of cancer (Belfiore et al., 2009; Wahner Hendrickson et al., 2009; Brierley et al., 2010; Ulanet et al., 2010; Zhang et al., 2010). The IR is expressed at two isoforms that differ at the carboxy terminus of the α subunits by 12 amino acids (Belfiore et al., 2009). The IR-B is the classic IR that regulates glucose uptake and binds insulin with high affinity but binds IGFs poorly. Conversely, the IR-A binds both insulin and IGF-2 with high affinity but IGF-1 with low affinity. In some conditions like fetal growth, cancer and diabetes, IR may display some non-metabolic effects like cell proliferation and migration and may affect metastasis and tumor progression. The molecular mechanisms underlying this functional switch of IR have been attributed to several factors including predominant expression of IR-A isoform over IR-B (Belfiore et al., 2009). In our cells we observed exclusive expression of the IR-A isoform and increased activation of ERK1/2, AKT and STAT-3, all potentially associated with the conserved malignant phenotype of these resistant cells and associated to insulin signaling proliferation pathways (Zhou et al., 2004). Thus, cells resistant to IGF-1R HAb show a switch towards insulin pathways sustaining proliferation and malignancy, rather than metabolism. Activation of IR-A by IGF-2 has been demonstrated to induce mitogenic effects in fibroblast null for IGF-1R (R-cells) transfected to express IR-A. More recent data have indicated that increased insulin signaling is observed on downregulation of IGF-1R (Zhang et al., 2007; Buck et al., 2010; Dinchuk et al., 2010) and autocrine proliferative loop has been demonstrated in several cancer cells including sarcomas (Avnet et al., 2009; Belfiore et al., 2009). Involvement of IR signaling in eliciting resistance has very recently been reported also in other experimental models (Buck et al., 2010; Ulanet et al., 2010). Ratio of IGF-1R:IR in favor of IR seems to be responsible also for native resistance of some Ewing's sarcoma to anti-IGF-1R therapy and may also explain the lower level of sensitivity of other sarcomas, such as osteosarcoma and rhabdomyosarcoma. In fact, these tumors express higher levels of IR-A than IGF-1R together with IGF-2 (Sciacca et al., 2002; Avnet et al., 2009). Similarly high IR:IGF-1R ratios conveyed resistance to IMC-A12 in breast cancer cells (Ulanet et al., 2010), whereas higher levels of IGF-1R were seen in squamous cell carcinoma of patients responding to figitumumab-containing therapy as compared with non-responders (Gualberto et al., 2010). As HAbs AVE1642, IMC-A12 and figitumumab, as well as NVP-AEW541, show high specificity for IGF-1R and all display lack of or limited interaction with IR homodimers, the mechanisms of resistance may be overlapping.

Overall, these studies suggest that therapies targeting specifically IGF-1R may have unwanted consequence of enhancing IR signaling, which may provide an important mechanism of resistance to IGF-1R-targeted therapies and may favor activated IR-A. Efficacy of anti-IGF-1R-specific agents may be limited in settings where IR-A homodimers are the predominant IGF-2 receptor. If so, future trials with these highly selective drugs should involve preselection of patients with tumors that express low levels of IR-A and IGF-2. In the event of high IR-A expression in an IGF-2 expressing cancer, the approach of cotargeting IGF-1R and IR with dual specific inhibitors such as BMS-7548097 (Carboni et al., 2009) or OSI-906 (Buck et al., 2010) that are being tested in phase I clinical trial, would be preferable.

Materials and methods

Drugs

The anti-IGF-1R human antibody (HAb) AVE1642 was kindly provided by Sanofi Aventis/Immunogen (Waltham, AM, USA). The small-molecule NVP-AEW541 was kindly obtained from Novartis (Basel, Switzerland). Doxorubicin and vincristine were purchased from Sigma (St Louis, MO, USA). D-18851, an ifosfamide analog not requiring metabolic activation, was kindly provided by Baxter Oncology GmbH (Frankfurt, Germany). Insulin was purchased from Novo Nordisk (Bagsvaerd, Denmark). Working dilutions of all drugs were prepared immediately before use.

Cell culture, condition and treatments

A panel of seven Ewing's sarcoma cell lines were analyzed. The cell lines have been obtained as previously specified (Manara et al., 2010). Most of them have been authenticated recently (Ottaviano et al., 2010). All have been tested for mycoplasma contamination by PCR Mycoplasma detection Set (Takara Bio Inc, Shiga, Japan) and verified to be mycoplasma free (last control May 2010). Cells resistant to anti-IGF-1R agents were obtained from TC-71 or IOR/BRZ71 Ewing's sarcoma cell lines by exposure to increasing concentrations of the anti-IGF-1R HAb AVE1642 (up to 10 μg/ml) or the TKI NVP-AEW541 (up to 5 μM) for 6 months. Resistant variants were here referred as TC/AVE or BRZ/AVE or as TC/AEW. In experimental conditions, cells were maintained in standard medium (Iscove Modified Dulbecco's medium (IMDM) plus 10% fetal bovine serum (FBS)) without selecting agents for at least 48 h, in order to avoid effects of direct exposition to HAb or TKI.

To assess cell growth, MTT assay (Roche, Indianapolis, IN, USA) was used according to the manufacturer's instructions. Cells were plated into 96-well plates (2500 cells/well) in IMDM plus 10% FBS. After 24 h, various concentrations of AVE1642 (0.1–10 μg/ml), doxorubicin (1–100 ng/ml), vincristine (0.1–3 ng/ml), ifosfamide (0.1–6 μg/ml) or AEW541 (0.03–5 μM) were added and cells were exposed up to 72 h.

In silencing experiments, cell growth was evaluated on harvested cells by trypan blue vital cell count.

Transfection

For transient silencing of IGF-1R and IR, by using siRNA, cells were transfected in 6-cm dishes at 50% confluence with 25–75 nM siRNA using Lipofectamine (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocols. After 24 h, media was removed, cultures were grown in fresh full-growth medium for 48–72 h with or without AVE1642 10 ng/ml. siRNA sequences are the following: si IGF-1R: (Ambion, Austin, TX, USA) sense strand 5′-GGAUUGAGAAAAAUGCUGA-3′ and antisense strand 5′-UCAGCAUUUUUCUCAAUCC-3; si-IR: (Invitrogen) sense strand 5′-CUAGUCCUGCAGAGGAUUU-3′ and antisense strand 5′-AAAUCCUCUGCAGGACUAG-3′ (Brierley et al., 2010). Negative control siRNA was purchased from Invitrogen and has the following sequence sense strand 5′-UUCUCCGAACGUGUCACGU-3′ and antisense strand 5′-ACGUGACACGUUCGGAGAA-3′.

Cytofluorometry

The following primary mouse monoclonal antibodies were used: anti-NGF-R clone 8211 (Boehringer Ingelheim, Germany); anti-human HER-1 (EGF-R) clone 528 (Oncogene Research Products, Uniondale, NY, USA); anti-human HER-2 clone MGR-2 (Alexis Biochemicals, Lausen, Switzerland); anti-human HER-3 clone SGP1 (NeoMarkers, Fremont, CA, USA) and c-KIT clone YB5.B8 (Pharmingen, San Diego, CA, USA). The secondary antibody was Alexa Fluor 488 (Invitrogen). Cells were subjected to cytofluorometric analysis with a FACSCalibur (Becton Dickinson, St Jose, CA, USA).

Western blotting

Cell lysates were prepared and processed as previously described (Scotlandi et al., 2005). Membranes were incubated overnight with the following primary antibodies: anti-IGF1-Rβ clone C-20, anti-IRβ clone C-19, anti-Shc clone PG-797 (Santa Cruz Biotechnology, San Diego, CA, USA), anti-phospho-Akt (Ser473) clone 736E11, anti-Akt, anti-phospho-STAT3 (Ser727), anti-STAT3, anti-ERK (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-ERK (Tyr202/Tyr204) (Covance, Princeton, NJ, USA), anti-IRS-1 (Upstate Biotechnology, Temecula, CA, USA), anti-rabbit or anti-mouse antibodies conjugated to horseradish peroxidase (GE Healthcare, Piscataway, NJ, USA) were used as secondary antibody.

Real-time PCR

Total RNA (2 mg) was extracted by TRIzol and reverse transcribed by ThermoScript RT (Invitrogen) and Oligo dT primers (Applied Biosystems, Carlsbad, CA, USA) Quantitative real-time PCR was performed on an ABI Prism 7500 (Applied Biosystems, Carlsbad, CA, USA) using SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). Primer Express software (Applied Biosystems, Carlsbad, CA, USA) was used to design appropriate primers pairs for target genes (IGF-1R, IR, IGF-1 and IGF-2) as well as for the reference gene (GAPDH) (Supplementary Table 1). Amplification reactions were checked for the presence of non-specific products by dissociation curves analysis and agarose gel electrophoresis. Relative quantitative determination of target gene levels was performed by comparing the comparative threshold cycle (Ct) method (DCt) (Ginzinger, 2002). Total IR and IGF-1R was also measured by using the absolute quantification assay by serial dilutions (ranging from 1 × 108 to 1 × 101 copies/ml) of separate plasmid DNA encoding the genes of interest. Copy number of each plasmid was calculated using the following formula (DNA concentration (g/ml)/plasmid length (bp) × 600) × 6022 × 1023=molecules/ml. IR isoforms relative abundance was measured by RT–PCR analysis using primers to flanking exons 10 and 12 and electrophoretically resolved on 1% agarose gel. Electrophoretic analysis revealed the 167 and 131-bp DNA fragments representing Ex 11+ (IR-B isoform) and Ex 11− (IR-A isoform), respectively. Gel was stained with SYBR Safe and bands density was quantified by scanning densitometry.

IGF-1R sequencing

DNA from cell lines was isolated with a Migration System 12GC robot (Precision System Science Co Ltd, Worrstadt, Germany) using the Migration-MegaZorbDNA common kit (Precision System Science Co Ltd). DNA was quantified with a Nanodrop-1000 spectrophotometer (Thermo Scientific, West Palm Beach, FL, USA). Fifty nanograms of DNA was used for each PCR amplification. Specific PCR primers were designed using the Primer Express Software v3.0 (Applied Biosystems, Carlsbad, CA, USA) for the complete exon sequences codifying the extracellular juxtamembrane domain (exons 12 and 13), intracellular juxtamembrane domain (exon 15) and tyrosine kinase domain (exons 16–21) of IGF1R (Supplementary material). Purified PCR products were then sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) and electrophoresed on an ABI 3130xl Genetic Analyzer (Applied Biosystems Carlsbad, CA, USA). Sequencing analysis was performed with the Sequencing Analysis 5.2 Software (Applied Biosystems Carlsbad, CA, USA). The sequence reference used for identifying genetic variants of IGF1R was IGF1R-001 ENST00000268035 from Ensembl (http://www.ensembl.org/index.html).

Receptor measurement by enzyme-linked immunosorbent assay

The characteristics and specificity of IR or IGF-1R enzyme-linked immunosorbent assays have been previously described (Pandini et al., 1999). Receptors were captured by incubating lysates (0.5–60 μg/well) in Maxisorp immunoplates (Nunc Int., Rochester, NY, USA) precoated with anti-IR antibody MA-20 (2 μg/ml) and detected with biotinylated anti-IR antibody CT-1 (Forsayeth et al., 1987; Ganderton et al., 1992) or with anti-IGF-1R antibody αIR-3 and detected with biotinylated antibody 17–69 (Kull et al., 1983; Soos et al., 1992). Reaction was amplified with peroxidase-conjugated streptavidin. Peroxidase activity was determined colorimetrically by adding 100 μl of 3,3′,5,5′-tetramethylbenzidine (0.4 mg/ml in 0.1 M citrate/phosphate buffer (pH 5.0) with 0.4 μl/ml 30% H2O2). The reaction was stopped by the addition of 1.0 M H3PO4, and the absorbance was measured at 450 nm.

Experimental metastasis

Female athymic 4–5-weeks old Crl:CD-1-nu/nu BR mice (Charles River Italia, Calco, Lecco, Italy) were used. Metastasis were determined by injection of 2 × 106 viable cells in a tail lateral vein. Animals were checked with X-ray for the presence of bone metastases. Care of mice and experimental protocols were in accordance with the European Community and Italian guidelines. Experiments were authorized by the institutional review board of the University of Bologna, Italy.

Microarray analysis

Experimental models (TC-71 and IOR/BRZ71 cells, either parental or resistant to IGF-1R inhibitor AVE1642) were profiled by using HG-U133 Plus 2.0 Array following the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA, USA). Two independent experiments were performed for each cell line analyzed. Gene expression data were quantified by the rma algorithm, filtered and analyzed with supervised techniques by Limma modified t-test and corrected for multiple testing by Benjamini and Hochberg method for the detection of differentially expressed genes. Genes were considered differentially expressed at the 0.05 P-value cutoff level. Hierarchical clustering using Pearson correlation was performed using MeV TM4 software (http://www.tm4.org). The Kyoto Encyclopedia of Genes and Genomes pathways were analyzed by GeneGo software and Panther pathway analysis (http://www.pantherdb.org). Microarray data will be available at GeneExpressionOmnibus.

Immunohistochemistry

Representative samples of 110 paraffin-embedded Ewing's sarcomas, at least three cores per patients, were included in tissue microarray. Tissue samples were collected from the tissue bank of the Laboratory of Experimental Oncology, Rizzoli Institute. Histology was reviewed by a pathologist panel. All samples included were diagnosed as conventional Ewing's sarcoma, according to histological and molecular diagnostic criteria (Machado et al., 2009). Sample sets were handled in a coded manner. The ethical committee of the Rizzoli Institute approved the studies and informed consent was obtained from all subjects involved. To assess IGF-1R and IR expression, avidin–biotin–peroxidase procedure was used for immunostaining, as previously described (Manara et al., 2010). Sections were pretreated with a citrate buffer solution (0.01 mol/l citric acid and 0.01 mol/l sodium citrate (pH 6.0)) in a microwave oven at 750 W for three cycles of 5 min each to ensure antigen retrieval. The following primary antibodies were used: anti-IGF-IRβ antibody, diluted 1:50 (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) or anti-IR, diluted 1:10. Samples were classified on the basis of the positivity score as follows: ‘low-expressors’, when no staining or low positivity was observed (intensity of the staining scored as +/−, +−− by pathologists); ‘high-expressors’, when a diffused immunostaining was present (intensity of the staining scored as ++− or +++).

Statistical analysis

Differences among mean values were analyzed using a two-sided Student's t-test. When data were not normally distributed, the non-parametric Mann–Whitney rank-sum test was used. IC50 values were calculated from linear transformation of dose–response curves. Correlations were analyzed by Spearman's correlation test.

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Acknowledgements

We thank Cristina Ghinelli and Alba Balladelli for their help in editing the manuscript. Studies are supported by grants from the Italian Association for Cancer Research (KS and AB), the Italian Ministry of Health (Strategico Oncologia RFPS-2006-3-340280; RF2008 to KS); the Italian Ministry of Research and Instruction (PRIN 2009) and the EU project Eurobonet (#018814).

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Correspondence to K Scotlandi.

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Supplementary Information accompanies the paper on the Oncogene website

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Garofalo, C., Manara, M., Nicoletti, G. et al. Efficacy of and resistance to anti-IGF-1R therapies in Ewing's sarcoma is dependent on insulin receptor signaling. Oncogene 30, 2730–2740 (2011) doi:10.1038/onc.2010.640

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Keywords

  • sarcomas
  • IGF-1R
  • insulin signaling
  • drug resistance

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