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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2008-0065
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 9 3524-3531
Copyright © 2008 by The Endocrine Society

Expression of Insulin-Like Growth Factor-II and Its Receptor in Pediatric and Adult Adrenocortical Tumors

Madson Q. Almeida, Maria Candida Barisson Villares Fragoso, Claudimara Ferini Pacicco Lotfi, Mariza Gerdulo Santos, Mirian Yumie Nishi, Marcia Helena Soares Costa, Antonio Marcondes Lerario, Carolina Canton Maciel, Gabriele Ebling Mattos, Alexander Augusto Lima Jorge, Berenice B. Mendonca and Ana Claudia Latronico

Unidade de Endocrinologia do Desenvolvimento (M.Q.A., M.C.B.V.F., M.G.S., M.Y.N., M.H.S.C., A.M.L., A.A.L.J., B.B.M., A.C.L.), Laboratório de Hormônios e Genética Molecular/LIM42 da Disciplina de Endocrinologia do Hospital das Clínicas da Faculdade de Medicina, and Laboratório de Estrutura e Função Celular (C.F.P.L., C.C.M., G.E.M.), Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05403-900 São Paulo, Brazil

Address all correspondence and requests for reprints to: Madson Queiroz Almeida, M.D., Unidade de Endocrinologia do Desenvolvimento e Laboratorio de Hormonios e Genetica Molecular LIM-42, Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo, Av. Dr. Eneas de Carvalho Aguiar, 155, 20 andar Bloco 6, 05403-900 Sao Paulo, SP, Brasil. E-mail: madsonalmeida{at}gmail.com; anacl{at}usp.br.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: Adrenocortical tumors are heterogeneous neoplasms with incompletely understood pathogenesis. IGF-II overexpression has been consistently demonstrated in adult adrenocortical carcinomas.

Objectives: The objective of the study was to analyze expression of IGF-II and its receptor (IGF-IR) in pediatric and adult adrenocortical tumors and the effects of a selective IGF-IR kinase inhibitor (NVP-AEW541) on adrenocortical tumor cells.

Patients: Fifty-seven adrenocortical tumors (37 adenomas and 20 carcinomas) from 23 children and 34 adults were studied.

Methods: Gene expression was determined by quantitative real-time PCR. Cell proliferation and apoptosis were analyzed in NCI H295 cells and a new cell line established from a pediatric adrenocortical adenoma.

Results: IGF-II transcripts were overexpressed in both pediatric adrenocortical carcinomas and adenomas. Otherwise, IGF-II was mainly overexpressed in adult adrenocortical carcinomas (270.5 ± 130.2 vs. 16.1 ± 13.3; P = 0.0001). IGF-IR expression was significantly higher in pediatric adrenocortical carcinomas than adenomas (9.1 ± 3.1 vs. 2.6 ± 0.3; P = 0.0001), whereas its expression was similar in adult adrenocortical carcinomas and adenomas. IGF-IR expression was a predictor of metastases in pediatric adrenocortical tumors in univariate analysis (hazard ratio 1.84; 95% confidence interval 1.28–2.66; P = 0.01). Furthermore, NVP-AEW541 blocked cell proliferation in a dose- and time-dependent manner in both cell lines through a significant increase of apoptosis.

Conclusion: IGF-IR overexpression was a biomarker of pediatric adrenocortical carcinomas. Additionally, a selective IGF-IR kinase inhibitor had antitumor effects in adult and pediatric adrenocortical tumor cell lines, suggesting that IGF-IR inhibitors represent a promising therapy for human adrenocortical carcinoma.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
Adrenocortical carcinomas account for only 0.05–0.2% of all cancers, with an estimated incidence of 0.5–2 per million per year in adults (1, 2, 3, 4). Nevertheless, a remarkably high annual incidence of adrenocortical tumors has been reported in children younger than 15 yr from southern Brazil, where a high frequency of a germline mutation (Arg337His) of the P53 tumor suppressor gene has been reported (5, 6, 7). Differently from adults, pediatric adrenocortical tumors with apparently poor prognosis based on the histopathological features often have a better clinical outcome (8, 9). To date, there are limited data to define histological or molecular markers that can reliably distinguish benign from malignant adrenocortical tumors, mainly in pediatric patients (10).

The molecular pathogenesis of adrenocortical tumors is still poorly understood. The IGF system has an essential role in normal adrenocortical cell growth and development (11, 12). In a series of comprehensive studies, Gicquel et al. (13, 14, 15) demonstrated that structural rearrangement of the 11p15 locus, typically uniparental paternal isodisomy, and IGF-II overexpression were found in the great majority of adult sporadic adrenocortical carcinomas. IGF-II exerts its mitogenic effects through interaction with IGF-I receptor (IGF-IR) (16). Thus, overexpression of IGF-II and/or IGF-IR may trigger a cascade of molecular events that can ultimately lead to malignancy (17). However, the role of IGF-IR in adult and pediatric adrenocortical tumorigenesis remains to be determined.

Cytotoxic chemotherapy has been extensively used for metastatic adrenocortical carcinoma, although response rates are generally poor (3, 18, 19). The overall response rate of the Berruti protocol (mitotane with etoposide, doxorubicin, and cisplatin) was 49%, including mainly partial responses (19). Therefore, it is clear that current treatment protocols are not effective and that new therapies are strongly needed (20). Two microarray studies identified that up-regulation of IGF-II expression was the dominant change in malignant adrenocortical tumors (21, 22). Consequently, IGF-IR inhibition has been proposed as the most appropriated target for adrenocortical carcinoma treatment (16, 20, 23). Recently IGF-IR kinase inhibitors have been considered a new therapeutic approach for hematologic and solid malignancies (17, 23, 24, 25). A selective IGF-IR kinase inhibitor (NVP-AEW541) was capable of effectively inhibiting ligand-mediated IGF-IR autophosphorylation as well as protein kinase B and MAPK phosphorylation (24).

In this study, we investigated IGF-II and IGF-IR expression in pediatric and adult adrenocortical tumors. In addition, the effects of the NVP-AEW541 on proliferation and apoptosis were analyzed in NCI H295 cells and a new cell line that was established from a pediatric adrenocortical adenoma of our cohort.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
The study was approved by the Ethics Committee of Hospital das Clinicas, Sao Paulo, Brazil, and informed written consent was obtained from all patients and/or parents. The clinical and histopathological features of children and adults with adrenocortical tumors are summarized in supplemental Tables 1 and 2, respectively, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org. Samples of sporadic adrenocortical tumors were obtained from 23 children (16 girls and 7 boys; 0.9–15 yr) and 34 adult patients (29 women and 5 men; 18–66 yr). The mean follow-up period was 57.8 ± 6.2 months. In the pediatric group, we observed that seven of 13 tumors with Weiss score of at least 4 had benign evolution, confirming that this isolated criterion is not reliable for children’s tumor classification (9). Consequently, the diagnosis of malignancy in this group was established in six of 23 pediatric adrenocortical tumors by an advanced tumor stage (III or IV) and/or poor clinical outcome. Adult adrenocortical tumors were classified according to Weiss criteria: 20 adrenocortical adenomas (Weiss score ≤3) and 14 carcinomas (Weiss score ≥4) (26). The p53 tumor suppressor gene was previously studied in 22 children and 26 adults, and the known Arg337His mutation was identified in 77 and 12% of them, respectively (7, 27).

Quantitative real-time PCR

After surgical resection, tumor fragments were immediately frozen in liquid nitrogen and stored at –80 C until total RNA extraction using the Trizol reagent (Invitrogen, Carlsbad, CA). cDNA was generated from 1 µg of total RNA using the high capacity kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed in the ABI Prism 7700 sequence detector using TaqMan gene expression assays for the gene quantification according to the manufacturer’s instructions (Applied Biosystems). The assay IDs were: IGF-II, Hs01005963_m1; IGF-IR, Hs00181385_m1; β-actin, 43263; 3-β-hydroxysteroid dehydrogenase type II (HSD3B2) Hs00605123_m1; 11-β-hydroxylase (CYP11B1) Hs01596404_m1; 21-hydroxylase (CYP21A2) Hs00416901_g1. A cycle threshold (CT) value in the linear range of amplification was selected for each sample in triplicate and normalized to β-actin expression levels. The relative expression levels were analyzed using the 2-{Delta}{Delta}CT method, where the {Delta}{Delta}CT is the difference between the selected {Delta}CT value of a particular sample and the {Delta}CT of a pool of 61 normal adrenals from autopsies (CLONTECH, Palo Alto, CA) (28). The mean expression of the target genes in the pool of normal adrenals was assigned an expression value of 1.0, and fold increase in the expression levels was determined for each tumor sample and adrenocortical tumors cell lines.

Adrenocortical cell lines

The NCI H295 cell line, previously established from an invasive primary adrenocortical carcinoma, was kindly provided by Dr. Walter L. Miller (University of California, San Francisco, CA) (29). A new pediatric transitory cell line was obtained from a functioning adrenocortical adenoma (weight 10 g; stage I) diagnosed in a 1.1-yr-old girl with mixed syndrome (virilization and Cushing syndrome) (patient 15; supplemental Table 1). The tumor fragments (0.67 g), obtained from viable nonhemorrhagic areas, were digested by sequential 4 mg/ml collagenase plus 1 µg/ml deoxyribonuclease I (Life Technologies, Inc., Paisley, UK), 30 min digestion and mechanical disaggregated with gentile movements in a volumetric pipette. The digested material was then filtered in a 100-µm nylon filter to retain nondigested material and thereafter pelleted at 700 rpm for 10 min. Two media were used: DMEM and reduced serum medium modification of MEM (Opti-MEM I) supplemented with, respectively, 10 and 2% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Initial growth, at a slow rate, occurs in both medium, and irrespective of medium used, the cells were adherent and spindle shaped. Cells were cultured in DMEM supplemented with 10% FBS for growth and steroid secretion studies. The pediatric adrenocortical adenoma cell morphological features were examined under phase-contrast microscope and light microscope after stained with hematoxylin and eosin. The adrenocortical tumor cell lines were maintained at 37 C in a 95% air-5% CO2 fully humidified environment and cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin. All in vitro experiments and steroid analyses were performed in the fifth passage of the pediatric adrenocortical adenoma cell line.

Steroid hormone analysis

Steroid secretion was measured in 5 d clarified supernatant medium of pediatric tumor cell culture by commercial kits: cortisol and testosterone, fluorometric assay (AutoDELFIA; Wallac, Oy, Finland); androstenedione, chemiluminescent enzyme immunoassay (Immulite 2000; Siemens, Siemens Medical Solutions Diagnostics, Los Angeles, CA); 17-OH progesterone, RIA (Diagnostic Systems Laboratories, Webster, TX).

Immunocytochemistry analysis

Approximately 1–2 x 104 cells were seeded onto coverslips in DMEM containing 10% FBS and fixed with formaldehyde 4% for 20 min. Immunocytochemistry stains were performed using antibodies against vimentin (mouse antihuman monoclonal, 1:100; Novocastra, Newcastle upon Tyne, UK) and melan A/mart 1 (prediluted mouse antihuman monoclonal clone A103; Chemicon, Temecula, CA). The immune complex was detected by immunoperoxidase staining using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine as previously described (30). Culture cells from human normal skin fibroblasts and human melanoma cell line LB373-MEL were used for positive controls for vimentin and melan-A, respectively. Cell cultures incubated in nonimmune primary antibody yielded negative results.

A selective IGF-IR kinase inhibitor (NVP-AEW541)

NVP-AEW541, a pyrrolo[2,3-d]pyrimidine derivate highly selective against IGF-IR, was kindly provided by Novartis Pharma (Basel, Switzerland) (24). Stock solution of this drug was prepared in dimethylsulfoxide and stored at –20 C.

Cell proliferation and caspase-3/7 activity assays

Adrenocortical tumor cell lines were plated in 96-well plates at a density of 20,000 cells/well. After starvation for 24 h in DMEM, cells were treated or not (control cells) with crescent concentrations of NVP-AEW541 (0.3–30 µM) with or without IGF-II (50 ng/ml; R&D Systems, Minneapolis, MN) stimulation. After 24 to 96 h, the CellTiter 96 AQueous One solution (Promega, Madison, WI) was added and cells incubated for 3 h. The OD was measured at 450 nm in an ELISA reader.

Apoptosis analysis was based on the caspase-3/7 activity after treatment with NVP-AEW541 (0.3–30 µM). After 3–9 h treatment, cells were incubated with the Caspase-Glo 3/7 Assay (Promega) for 1 h, and the luminescent signal was measured in a luminometer. All cell proliferation and apoptotic experiments were performed in triplicate.

Statistical analysis

All statistical analyses were performed with the SPSS software (SPSS 13.0; SPSS, Inc., Chicago, IL). Continuous data are expressed as mean ± SEM. Differences in expression levels between adenomas and carcinomas were analyzed by means of the two-tailed Mann-Whitney U test. Predictive factors of metastases were identified by means of Cox proportional hazards regression models, which was used to estimate hazard ratios (HR) and their 95% confidence intervals in univariate analysis. The time of event (metastases) was defined as the time between the diagnosis of primary tumor and the first metastases. P < 0.05 was considered significant. Repeated measures of absorbance and luminescence were compared by ANOVA, followed by Bonferroni’s post hoc test. The level of significance for the Bonferroni adjusted tests was set at 0.0024.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
IGF-II and IGF-IR expression

IGF-II transcripts were overexpressed in both pediatric adrenocortical carcinomas and adenomas (50.8 ± 18.5 vs. 31.2 ± 3.7, respectively; P = 0.23) (Fig. 1AGo). Otherwise, IGF-II was mainly overexpressed in adult adrenocortical carcinomas, compared with adenomas (270.5 ± 130.2 vs. 16.1 ± 13.3; P = 0.0001) (Fig. 1BGo) according to previous studies (14, 15, 22). IGF-IR mRNA levels were significantly higher in childhood adrenocortical carcinomas than adenomas (9.1 ± 3.1 vs. 2.6 ± 0.3; P = 0.0001), whereas similar IGF-IR expression levels were detected in adult adrenocortical carcinomas and adenomas (1.6 ± 0.3 vs. 1.8 ± 0.5, respectively; P = 0.75) (Fig. 1Go, C and D).


Figure 1
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  		FIG. 1. Expression of IGF-II (A and B) and IGF-IR (C and D) in 57 sporadic adrenocortical tumors (23 children and 34 adults) using quantitative real-time PCR. Y axis shows fold increase in IGF-II and IGF-IR expression of tumor samples relative to the mean IGF-II and IGF-IR expression levels of a pool of normal adrenals. The horizontal line within the box plot represents the median value, the box plot limits refer to 25th to 75th percentiles, and the box plot bars includes the 10th to 90th percentiles for mRNA levels.

 
Metastases were documented in six of 23 children and seven of 34 adults with adrenocortical tumors. In pediatric adrenocortical tumors, tumor weight [HR 16.6, 95% confidence interval (CI) 1.54–178.9; P = 0.02], histopathological features of malignancy (Weiss score) (HR 2.11; 95% CI 1.2–3.72; P = 0.009), and IGF-IR mRNA levels (HR 1.84; 95% CI 1.28–2.66; P = 0.01) were associated with a higher risk of metastatic disease in univariate analysis (Table 1Go). In adult adrenocortical tumors, male sex (HR 9.96; 95% CI 1.93–51.29; P = 0.006), tumor weight (HR 6.39; 95% CI 1.44–28.36; P = 0.01), and Weiss score (HR 1.74; 95% CI 1.24–2.44; P = 0.015) were associated with a higher risk of metastatic disease in univariate analysis. IGF-II expression levels were not a significant predictor of metastases (Table 1Go).


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  		TABLE 1. Predictive factors of metastases in pediatric and adult patients with adrenocortical tumors according to univariate analyses

 
Characterization of the new pediatric adrenocortical tumor cell line

To dissect the cellular consequences of IGF-IR inhibition in pediatric and adult adrenocortical tumors, two adrenocortical cell lines were studied. The NCI H295 cell line was previously obtained from an invasive primary adrenocortical carcinoma in a 48-yr-old woman (29). To date, cell lines derived from pediatric adrenocortical tumors are lacking. Here we obtained a new pediatric adrenocortical tumor cell culture from a functioning adrenocortical adenoma. This adrenocortical adenoma cell line has continuously been growing after eight passages. Steroid secretion was detected in 5 d supernatant medium of the fifth subcultured pediatric cell culture (cortisol 238 µg/dl, testosterone 1098 ng/dl, androstenedione >8.5 ng/ml, and 17-hydroxyprogesterone >20 ng/ml). In addition, the expression of several enzymes involved in steroid biosynthesis (3-β-hydroxysteroid dehydrogenase type II, 11-β-hydroxylase, 21-hydroxylase) was demonstrated by quantitative real-time PCR (data not shown).

The pediatric adrenocortical adenoma cell line had a fibroblastoid and spindle-shaped appearance at phase-contrast microscopy and hematoxylin and eosin staining, respectively (Fig. 2Go, A and B). Pediatric adrenocortical adenoma cell culture showed cytoplasmic immunoreactivity for melan-A in 100% of the culture cells (Fig. 2Go, C and D). The melan-A is a melanocytic differentiation marker, which has the useful property of staining steroid hormone-producing tumors, such as adrenocortical adenomas and carcinomas (31). A strong cytoplasmic expression of vimentin, the major intermediate filament protein of mesenchymal cells, was also detected in 100% of these adrenocortical cells (Fig. 2EGo and F). This homogeneity pattern of this cell culture suggests one cellular type isolated from the tumor fragments.


Figure 2
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  		FIG. 2. The pediatric adrenocortical adenoma cells had a fibroblastoid and spindle-shaped appearance at phase contrast microscopy (A, x10 magnification) and hematoxylin and eosin staining (B). A positive immunoreactivity for melan-A, a melanocytic differentiation marker, was detected by immunocytochemistry in 100% of the pediatric tumor cells (C, melan-A immunoreactivity; D, negative control cells). A strong cytoplasmic expression of vimentin, an intermediate filament of mesenchymal cells, was also evidenced in 100% of these adrenocortical cells (E, vimentin immunoreactivity; F, negative control cells).

 
IGF-IR was expressed in both NCI H295 (1.5 ± 0.3) and pediatric adrenocortical adenoma (0.4 ± 0.06) cell lines, whereas IGF-II was mainly overexpressed in NCI H295 cells (42.4 ± 12.5), compared with pediatric adrenocortical adenoma cells (4.5 ± 1.1).

NVP-AEW541 effects on adrenocortical tumor cell lines

An additional implication of our findings concerns their potential exploitation for the identification of novel therapeutic targets. To achieve this goal, we evaluated the effects of NVP-AEW541 on blocking IGF-II stimulated proliferation of human NCI H295 and pediatric adrenocortical adenoma cells. IGF-II significantly increased proliferation of NCI H295 and pediatric adrenocortical adenoma control cells after 72 and 48 h, respectively (P = 0.0001). The NVP-AEW541 treatment had a significant effect on proliferation reduction of NCI H295 cells at increasing concentrations of 10 µM (70 ± 10%) and 30 µM (33.3 ± 6.7%), compared with untreated cells (100 ± 6.7%) at 24 h (P = 0.0001). The treatment with this IGF-IR inhibitor significantly decreased NCI H295 cell proliferation at 0.3 µM (70.3 ± 2.7%), 1.0 µM (51.4 ± 5.4%), 3.0 µM (35.1 ± 2.7%), 10 µM (18.9 ± 0.5%), and 30 µM (2.7 ± 0.27%), compared with untreated cells (100 ± 2.7%) at 48 h (P = 0.0001). NVP-AEW541 treatment promoted a near-total reduction in NCI H295 cell proliferation at 10 and 30 µM after 96 h (Fig. 3AGo).


Figure 3
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  		FIG. 3. The NVP-AEW541 treatment had a significant effect on proliferation reduction of NCI H295 cells at increasing concentrations of 0.3–30 µM after treatment for 24–96 h (A). This IGF-IR inhibitor also led to a significant decrease of the human pediatric adrenocortical adenoma cell proliferation after treatment for 24–96 h (B). Tumor cells treated with NVP-AEW541 were compared with untreated cells stimulated by IGF-II. Data are expressed as mean ± SEM of triplicates.*, P = 0.0001.

 
NVP-AEW541 also promoted a significant decrease of pediatric adrenocortical adenoma cell proliferation at 0.3 µM (81.2 ± 3%), 1 µM (72.3 ± 6%), 3 µM (63.4 ± 6%), 10 µM (2 ± 0.3%), and 30 µM (2 ± 0.3%), compared with untreated cells (100 ± 5%) at 48 h (P = 0.0001). This IGF-IR inhibitor led to a progressive reduction on proliferation of pediatric adrenocortical adenoma cells at 0.3 µM (78.2 ± 5.6%), 1.0 µM (61.3 ± 1.6%), 3.0 µM (43.5 ± 2.4% OD), 10 µM (1.6 ± 0.4%), and 30 µM (1.6 ± 0.2%), compared with untreated cells (100 ± 1.6%) at 96 h (P = 0.0001) (Fig. 3BGo). NVP-AEW541 treatment of both adrenocortical tumor cell lines without exogenous IGF-II stimulation also promoted a significant reduction in cell proliferation (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org).

The IC50 values of NVP-AEW541 were 0.2 ± 0.02 and 2.2 ± 0.06 µM for NCI H295 and for pediatric adrenocortical adenoma cells after 96 h of treatment, respectively. The NCI H295 cells were more sensitive to NVP-AEW541, showing IC50 value at a submicromolar concentration.

We also investigated whether cells exposed to NVP-AEW541 underwent apoptosis. NCI H295 cells treated with NVP-AEW541 showed a significant increase in caspase-3/7 activity at 1.0 µM [3714 ± 248 relative light units (RLU)], 3.0 µM (5257 ± 311 RLU), 10 µM (7069 ± 801 RLU), and 30 µM (9060 ± 733 RLU), compared with untreated cells (2019 ± 329 RLU) at 3 h (P = 0.0001) (Fig. 4AGo). The IGF-IR inhibition in pediatric adrenocortical adenoma cells significantly increased caspase-3/7 activity at 3.0 µM (3220.4 ± 56.2 RLU), 10 µM (4056 ± 277.9 RLU), and 30 µM (6324.6 ± 198.3 RLU), compared with untreated cells (1732.9 ± 61.4 RLU) at 9 h (P = 0.0001) (Fig. 4BGo).


Figure 4
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  		FIG. 4. NVP-AEW541 induced apoptosis by a significant increase of caspase-3/7 activity in NCI H295 cells at concentrations of 0.3–30 µM after 3 and 6 h (A). NVP-AEW541 also increased caspase-3/7 activity in pediatric adrenocortical adenoma cells after treatment for 6 and 9 h (B). Tumor cells treated with NVP-AEW541 were compared with untreated cells. Data are expressed as mean ± SEM of triplicates.*, P = 0.0001.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The IGF signaling pathway plays an important role in the regulation of adrenal growth and differentiation (17). Binding of ligands to the IGF-IR or its own overexpression initiates a cascade of events, leading to stimulation of proliferation, angiogenesis, apoptosis, and inhibition of metastases (32). Indeed, increased expression of IGF-II and/or IGF-IR have been documented in various malignant tumors (33, 34). A strong overexpression of IGF-II is a dominant finding in adult adrenocortical cancer, occurring in approximately 83% of malignant adrenocortical tumors (14). Nonetheless, very few studies about IGF-II expression in pediatric adrenocortical tumors were reported (35, 36). We demonstrated that IGF-II expression was deregulated in a similar manner in both pediatric adrenocortical adenomas and carcinomas.

IGF-IR overexpression was previously demonstrated in adult adrenocortical carcinomas but not adrenocortical hyperplasias and adenomas (37, 38). In this study, we identified that IGF-IR expression was similar in benign and malignant adult adrenocortical tumors. Otherwise, a strong increase in IGF-IR expression was identified only in pediatric adrenocortical carcinomas. In our cohort, IGF-IR expression was a predictor of metastases in children with adrenocortical tumors. The mechanisms responsible for enhanced IGF-IR expression in pediatric adrenocortical tumors are still unclear. Changes in IGF system expression in cancerous cells may occur as a result of loss or altered expression of tumor suppressor genes (17). In normal cells, expression of wild-type P53 was shown to inhibit IGF-IR expression, whereas mutant P53 up-regulates IGF-IR expression in different tumors (39). In our cohort, 17 of 22 children with adrenocortical tumors (77%) harbored the Arg337His P53 mutation. Nevertheless, IGF-IR expression levels were not associated with the presence of this mutation (data not shown). Therefore, other molecular events, like IGF-IR gene amplification, may be involved in IGF-IR overexpression in pediatric adrenocortical tumors.

Adrenocortical carcinoma remains a disease of poor prognosis, with little expectation of long-term survival if complete surgical removal is not achieved (3). Therefore, the development of new inhibitory drugs that can target signaling pathways involved in adrenocortical tumorigenesis is strongly necessary. Although kinase inhibitors hold much promise for cancer therapy, their successful application requires preclinical strategies to identify molecular markers that define susceptible tumor subtypes. Analysis of tumor-derived cell lines provides an effective system for establishing the link between specific tumor molecular aspects and the response to molecular target drugs (40). We established a new transitory cell culture derived from a human pediatric adrenocortical adenoma, thus permitting the study of a specific signaling pathway that may interfere with adrenocortical tumor growth in children. We demonstrated that NVP-AEW541, a selective IGF-IR kinase inhibitor, was able to block cell proliferation in a dose- and time-dependent manner in two distinct human adrenocortical tumor cell lines. The inhibitory effects of the NVP-AEW541 were induced by a significant induction of apoptotic rate. In addition to antiproliferative and proapoptotic effects, the IGF-IR inhibition could also increase the efficacy of other therapeutic modalities, such as radiotherapy, in breast cancer cells (41).

The ability of the NVP-AEW541 to potently induce apoptosis was previously demonstrated in several cell lines by determining functional and morphological changes and caspase activation as well as fragmentation of nuclear DNA (25, 42, 43). Furthermore, NVP-AEW541 also inhibits cell cycle progression, inducing specific G1 arrest (25, 42). Regarding NVP-AEW541 sensitivity, NCI H295 cells showed an IC50 value comparable with that of the most sensitive cells, such as Ewing’s sarcoma and neuroblastoma cell lines (25, 44). The level of sensitivity of the pediatric adrenocortical tumor cells was similar to that of hepatocellular carcinoma and gastrointestinal tumor cells (42, 43).

In conclusion, IGF-IR overexpression was a biomarker of pediatric adrenocortical carcinomas. In addition, we demonstrated that a selective IGF-IR kinase inhibitor had antitumor effects in adult and pediatric adrenocortical tumor cell lines, suggesting that IGF-IR inhibitors represent a promising therapy for human adrenocortical carcinoma.


   Acknowledgments
 
We are in debt to Lourdes C. Martins (Department of Preventive Medicine, University of Sao Paulo) for the statistical review. We thank Valeria S. Lando and the staff of Laboratorio de Hormonios e Genetica Molecular LIM-42 for the steroid hormone analysis.


   Footnotes
 
This work was supported by Grants 05/04726–0; 06/00244–3 from the Fundação de Amparo à Pesquisa do Estado de São Paulo (to M.Q.A.) and by Grants 300469/2005–5 (to A.C.L.) and 300828/2005–5 (to B.B.M.) from the Conselho Nacional de Desenvolvimento Científico e Tecnológico.

Disclosure Statement: We declare no duality of financial interest or direct or indirect conflict of interest on the part of any author of this manuscript.

First Published Online July 8, 2008

Abbreviations: CI, Confidence interval; CT, cycle threshold; FBS, fetal bovine serum; HR, hazard ratio; IGF-IR, IGF-I receptor; RLU, relative light unit.

Received January 10, 2008.

Accepted June 27, 2008.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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