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IGF2 and IGF1R in pediatric adrenocortical tumors: roles in metastasis and
2 steroidogenesis
3 4 Régia Caroline Peixoto Lira1, Paola Fernanda Fedatto1, David Santos Marco Antonio2,
5 Letícia Ferro Leal1, Carlos Eduardo Martinelli Jr1, Margaret de Castro3, Silvio Tucci Jr4,
6 Luciano Neder5, Leandra Ramalho5, Ana Luiza Seidinger6, Izilda Cardinalli6, Maria José
7 Mastellaroº, José Andres Yunes6,7, Silvia Regina Brandalise6,7, Luiz Gonzaga Tone1, Sonir 8 Roberto Rauber Antonini1, Carlos Alberto Scrideli1
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9 1Department of Pediatrics, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
11 12 2 Research and Development Department, Fleury Group, Sao Paulo, SP, Brazil.
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13 3Department of Internal Medicine, 4Department of Surgery, 5Department of Pathology,
Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
14 15 16 Brazil.
‘Boldrini Children Center, 1State University of Campinas (UNICAMP), Campinas, SP,
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18 Correspondence should be addressed to Carlos Alberto Scrideli
19 Department of Pediatrics, Ribeirao Preto Medical School, University of Sao Paulo - USP.
20 Avenida dos Bandeirantes, 3900, 14049-900, Ribeirao Preto, SP, Brazil. Phone: +55 16
21 3602 2672. E-mail: scrideli@fmrp.usp.br
22 23 Short title: IGF1R in pediatric adrenocortical tumor
24 Keywords: IGF2, IGF1R, childhood, adrenocortical tumor, OSI-906
25 Word count: 4858 26
27 Abstract
28 29 30 Deregulation of the IGF system observed in human tumors suggests a role in malignant cell transformation and in tumor cell proliferation. Although overexpression of the IGF2 and IGF1R genes was described in adrenocortical tumors (ACTs), few studies reported 31 their profiles in pediatric ACTs. In this study, the IGF2 and IGF1R expression was 32 evaluated by RT-qPCR according to the patient’s clinical/pathological features in 60 33 34 35 36 pediatric ACT samples and the IGF1R protein was investigated in 45 samples by immunohistochemistry (IHC). Whole transcriptome and functional assays were conducted after IGF1R inhibition with OSI-906 in NCI-H295A cell line. It was found significant IGF2 overexpression in tumor samples when compared to non-neoplastic samples 37 38 (P<0.001), significantly higher levels of IGF1R in patients with relapse/metastasis (P=0.031) and moderate/strong IGF1R immunostaining in 62.2% of ACTs, but no other 39 relation with patient survival, clinical/pathological features were observed. OSI-906 40 treatment down-regulated genes associated with MAPK activity, induced limited reduction 41 of cell viability and increased the apoptosis rate. After 24 hours, the treatment also
42 decreased the expression of genes related to the steroid biosynthetic process, the protein 43 levels of the steroidogenic acute regulatory protein (StAR) and androgen secretion in cell medium, supporting the role of IGF1R in steroidogenesis of adrenocortical carcinoma 44
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cells. Our data showed that the IGF1R overexpression could be indicative of aggressive
46 ACTs in children. However, in vitro treatments with high concentrations of OSI-906 (>
47 1 µM) showed limited reduction of cell viability, suggesting that OSI-906 alone could not
48 be a suitable therapy to abolish carcinoma cell growth.
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Introduction
Pediatric adrenocortical carcinoma (ACC) is a rare neoplasia with a worldwide incidence estimated at 0.2-0.3 cases/million children under 15 years per year (Else et al. 2014). The incidence in Southern of Brazil is 10-15 higher than the worldwide rate, which is related to the inherited germline TP53 mutation (p.R337H) (Ribeiro & Figueiredo 2004). Some of prognostic factors include older age, mitotic rate, tumor weight, tumor size and presence of metastasis (Klein et al. 2011). Complete surgical resection remains the only treatment to achieve cure and long-term survival, which is less than 30% in patients with advanced stages of the disease (Fassnacht et al. 2011; Lorea et al. 2012). Adjuvant mitotane, chemotherapy and/or radiotherapy have been often recommended in order to reduce local recurrence. For palliative cases, the arterial chemoembolization, radiotherapy and radiofrequency ablation should be also considered (Berruti et al. 2012; Fassnacht et al. 2012; Else et al. 2014).
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Among molecular markers, the overexpression of Insulin Growth Factor 2 (IGF2) has been commonly found in pediatric tumors (Wilkin 2000; Lerario et al. 2013). Additionally, few studies have evaluated the Insulin Growth Factor 1 Receptor (IGF1R) gene expression (West et al. 2007; Almeida et al. 2008). The IGF signaling is activated by IGF1, IGF2 and/or insulin binding to the IGF1R, that autophosphorylates and begins downstream cascades such as PI3K and MAPK, promoting cell proliferation and differentiation, exerting anti-apoptosis effects and angiogenesis (Riedemann & Macaulay 2006; Pollak 2012). Although adrenal tumorigenesis involves several genetic abnormalities, the IGF2 overexpression seems to occur at an earlier stage of tumor formation, but it is unable to cause tumor formation alone (Assié et al. 2014; Pinto et al. 2015).
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77 78 79 80 81 Evidences of the IGF pathway role in malignant cell transformation and proliferation have conducted to the study of IGF1R target-drugs. Among them, OSI-906 (Linsitinib) is a potent, oral and selective inhibitor of the IGF1R and Insulin receptor (IR) autophosphorylation, reducing cell proliferation in different types of cancer cell lines (Mulvihill et al. 2009). Recently, a large phase III trial in adults with advanced ACC 82 demonstrated no differences in overall survival between patients treated with OSI-906 and the placebo group (Fassnacht et al. 2015; Kirschner 2015). Therefore, it remains to be established the real role of IGF1R in ACT and whether anti-IGF1R therapy alone or combined with other drugs is efficient as a preferred treatment.
In the present study we analyzed IGF1R and IGF2 gene expression profiles according to the clinical and pathological features of adrenocortical tumors in a large series of pediatric patients samples and investigated the effect of IGF1R inhibition by OSI-906 in the ACC cell line NCI-H295A as well as the altered downstream signaling pathways.
Subjects, Material and methods
Patients
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A total of 60 pediatric adrenocortical tumor samples were obtained from the University Hospital, Ribeirao Preto Medical School, University of Sao Paulo and Boldrini Children Center, Campinas. All patients underwent clinical and hormonal evaluation by biochemical and imaging investigation. Abdominal and chest CT and bone scintigraphy were conducted for metastasis detection at diagnosis and during follow-up. The disease stage at diagnosis was based on modified Sandrini’s classification of childhood ACTs (Michalkiewicz et al. 2004). Ten non-neoplastic adrenal samples were used as control, which were collected during nephrectomy due to Wilms tumor, before chemotherapy and from children without Beckwith-Wiedemann syndrome. The study was approved by the
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local Ethics Committees (protocol number: 8380/2010) and a signed statement of informed consent was obtained from the children’s parents.
The group of patients diagnosed with ACT consisted of 46 girls and 14 boys with a mean age at diagnosis of 40.5 months (range 5-187 months). Three patients had non- secreting tumors, while 57 had hormone-secreting tumors (37 androgen, 18 mixed 107 cortisol/androgen and 2 cortisol). Thirty-five patients were classified as stage I, 10 as stage 108 109 110 II, 8 as stage III, and 7 as stage IV. The germline TP53 p.R337H mutation was evaluated by direct genomic DNA sequencing and was detected in 52/60 (86.6%) patients. In most tumors, tumor DNA was sequenced and the loss of heterozygosity (LOH) at the 17q locus was confirmed. The analysis of the entire coding and boundary regions of the TP53 gene revealed the absence of other mutations. Median follow-up was 68.3 months (range: 8-168 months). Twenty patients (33.3%) presented metastasis at diagnosis (n = 7) or relapsed (n = 13). The clinical and pathological features of these patients were described previously (Leal et al. 2011; Lorea et al. 2012; Gomes et al. 2014) .
Real time PCR (qPCR)
Tumor fragments were collected during surgical resection, frozen in liquid nitrogen, microdissected and revised by a pathologist. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. The cDNA was generated from 1 µg total RNA using the High Capacity Kit (Applied Biosystems, Foster City, CA, USA). The human genes IGF2 (Hs01005963_m1), IGF1R (Hs00609566_m1), MAPK1 (Hs01046830_m1), MAPK3 (Hs00385075_m1) and PIK3R5 (Hs01046353_m1) were amplified by qRT-PCR using TaqMan gene assays and the ABI 7500 Real Time PCR System (Applied Biosystems Inc., Foster City, CA, USA). All samples were analyzed in triplicate and normalized to the endogenous reference human genes ACTB and GUSB (Applied Biosystems, Foster City, CA, USA) as previously described (Leal et al. 2011;
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Leite et al. 2014). The relative expression was determined by the 2-44 method (Livak & Schmittgen 2001) and the median gene expression values of all non-neoplastic adrenal tissues was used as reference and defined as 1 for analysis of tumor samples. For in vitro assays, samples were normalized to the GUSB gene and untreated cells were used as reference samples.
Immunohistochemistry
Immunohistochemistry for IGF1R was performed by the avidin-biotin peroxidase complex (ABC) method (Novocastra, Newcastle-upon-Tyne, United Kingdom) in 45 137 tumor samples from the 60 patients evaluated in this study. Fifteen samples were excluded because they presented few or none tumor representative area anymore. A small group of 21 tumors was also evaluated for IGF2. The primary antibody was applied for overnight incubation (Mouse monoclonal anti-IGF-1R, 1:300; Biocare Medical - CM 414 A. C .; Concord, CA, USA; Mouse monoclonal anti-IGF2, 1:50; Santa Cruz Biotechnology Inc., sc-74119; Santa Cruz, CA, USA) and a biotinylated secondary antibody was incubated. The visualization was performed with streptavidin peroxidase followed by diaminobenzidine coloring (Gibco, Gaithersburg, Maryland, USA) and Harris’ hematoxylin counterstaining. The positive control was a breast carcinoma sample (IGF1R) and placenta (IGF2) and the negative control was obtained by replacing the primary antibody with PBS 1X.
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The immunohistochemistry analysis classified the samples as negative (no/weak staining) or positive (moderate/strong staining) according to the intensity of IGF1R/IGF2 staining. Regarding the localization, the samples were grouped into cytoplasmic, cytoplasmic/membrane or nuclear staining.
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Cell line and reagents
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The human adrenocortical carcinoma cell line NCI-H295A was cultivated in RPMI medium as previously described (Gomes et al. 2014). Cell line authentication was conducted by examining CSF1PO, D13S317, D16S539, D5S818, D7S820, THO1, TPOX, vWA, and AMEL polymorphic loci by short tandem repeat profiling (STR). To avoid genetic drift or selection of variant subclones, all experiments were performed under standard cell culture conditions in an incubator at 37°℃ in a humidified atmosphere of 5% CO2 and cells were used at low passages (<10).
The OSI-906 inhibitor was acquired from Selleck Chemicals (LLC, Houston, TX, USA), dissolved in dimethyl sulfoxide (DMSO) at stock concentration of 10 mM and stored at -20℃. Control groups were prepared for all experiments using cells grown in medium with DMSO only.
Whole-transcriptome analysis
After NCI-H295A treatment with OSI-906 (2 µM) for 6 and 24 hours, total cellular RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) and stored in DEPC-treated water at -80℃ and the quantity and quality of samples was evaluated with an ND-1000 NanoDrop spectrophotometer (NanoDrop Products, Wilmington, DE, USA). The mRNA library was constructed using 200 ng of total RNA and investigated with the Whole Human Genome Microarray Kit, 4x44K (Agilent Technology, Santa Clara, CA, USA) to determine the gene expression profiles. Spot images were processed by Feature Extraction Software v10.7.3.1 (Agilent technology, Santa Clara, CA, USA). All steps of
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quality evaluation, normalization, background correction and statistical analysis were performed using R statistical language (Gentleman et al. 2004; R Core Team 2014) and the Bioconductor package Agi4x44PreProcess: PreProcessing of Agilent 4x44 array data (Lopez-romero 2012). After background correction the samples were quantile normalized and the expression of the genes was obtained using a linear model fitted to each gene so
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180 that the fold change between conditions and its associated errors could be estimated. 181 Differentially expressed genes were obtained from those with lowest P-value and extreme values of fold-change. To annotate the differentially expressed genes (DEG), EnricheR tool was used for Gene Ontology (GO) and KEGG pathway analysis with criterion of p- value < 0.05 and at least three genes per process (Chen et al. 2013). 182 183 184 185
Cell viability assay
The resazurin reduction method was used to investigate cellular metabolic activity after treatment with OSI-906 (O’Brien et al. 2000). For the assay, the cells were treated with OSI-906 inhibitor at different concentrations (0.125 uM to 3 uM) for 24, 48 or 72 hours. After the treatment period, resazurin (Sigma-Aldrich Co., Saint Louis, MO, EUA) was added and the plates were incubated for 4 hours at 37℃, 5% CO2. Absorbance at 570 nm wavelength was then read with a reference wavelength of 595 nm using an iMark Microplate Absorbance Reader (BioRad Laboratories, Inc, CA, EUA). The effects of OSI- 906 in the cell viability were reported as mean ± standard deviation (SD) of at least three independent experiments performed in triplicate. The EC50 values were calculated using Calcusyn software (Biosoft, Ferguson, Missouri, USA) (Chou & Talalay 1984).
Apoptosis assay
Apoptotic cell death was determined by labeling with annexin V fluorescein isothiocyanate (BD Biosciences Pharmigen, San Jose, California, USA) and propidium iodide (PI) staining. Briefly, after 72 hours of OSI-906 treatment, 1.5 x 105 cells were trypsinized and centrifuged at 1000 rpm for 5 min at 4℃, washed with ice-cold PBS 1X, and then resuspended in 350 ul of annexin V binding buffer (BD Biosciences Pharmigen, San Jose, California, USA). Cells were stained with 5 ul of annexin V and 50 ul of PI 50 umol/l and incubated at room temperature in the dark. The samples were analyzed using a
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BD FACSCalibur flow cytometer (BD Biosciences Pharmigen, San Jose, California, USA).
Cell lysis and Western blot analysis
After treatment with OSI-906 (2 uM) for 3, 6 or 24 hours, cells were lysed in RIPA buffer (Sigma-Aldrich Co., Saint Louis, MO, USA) in the presence of protease and phosphatase inhibitors. Equal amounts of whole-cell lysates were resolved by 12% SDS- polyacrylamide gel electrophoresis (PAGE) followed by transfer to nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA), which were incubated in Tris-buffered saline - 0.1% Tween-20 containing 5% (w/v) dried nonfat milk for 1 hour at room temperature and probed with appropriately diluted primary antibodies overnight at 4℃. Blots were incubated with biotin-labeled horseradish peroxidase-conjugated species- specific secondary antibodies (1:10000; GE Healthcare, Piscataway, NJ, USA) followed by chemiluminescence detection using the ECLTM Western blotting Analysis System Kit (Amersham GE Healthcare, Buckinghamshire, UK) and ChemiDoc System (BioRad Laboratories, CA, USA).
Rabbit monoclonal antibodies against phospho- and total-ERK1/2 were obtained from Cell Signaling (respectively, #9101 and #9102; both 1:800; Danvers, MA, USA). A rabbit polyclonal antibody against StaR was purchased from Santa Cruz Biotechnology (1:200; sc-25806; Dallas, TX, USA) and a mouse monoclonal antibody against GAPDH from Santa Cruz Biotechnology was used as loading control (1:1000; sc-47724).
Hormone measurements
After 24 hours of OSI-906 treatment (2 uM), the supernatant medium was collected from each well prior to cell lysis for RNA or protein extraction and stored at -80 ℃ until hormone quantification. Dehydroepiandrosterone sulfate (DHEAS), D4-androstenedione
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(D4) and Testosterone concentrations were determined by radioimmunoassay (RIA) as previously described (Moreira & Elias 1992). All measurement of hormones from the NCI-
H295A medium were conducted in triplicate and the mean was normalized to the cell viability effects in the same treatment conditions.
Evaluable data from circulating hormone concentrations (Cortisol, DHEAS, D4, 237 Testosterone) of patient prior tumor treatment was used for correlations analysis.
Statistical Analyses
The expression of IGF2 and IGF1R genes according to following variables: age (<versus ≥4 years), tumor weight (< versus ≥ 100 g), tumor size (< versus ≥ 200 cm3), TP53 p.R377H mutation (positive versus negative) and disease stage was compared by the 243 Mann-Whitney test. Differences between the expression values are reported as fold change (FC) by dividing the median expression values for each variable analyzed. Event-free survival (EFS) analysis (with relapse and/or death due to any cause being considered as unfavorable events) was carried out based on Kaplan-Meier curves, using the gene expression median values from ACTs as the cut-off point for IGF2 and IGF1R. The curves for different groups were compared by the log-rank test. Correlations were determined using the Spearman correlation coefficient (rho).
Data of the functional assays such as cell viability and apoptosis, as well as the gene expression after OSI-906 treatment were evaluated by one-way ANOVA with the Bonferroni pos-test. Moreover, differences of hormone concentrations in the cell medium were analyzed by the t-test for equality of means. All analyses were carried out using the IBM® SPSS® Statistics software version 20 (SPSS Inc, IL, USA), with the level of significance set at 0.05.
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Results
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IGF2 gene is overexpressed in adrenocortical tumors
A significant overexpression of IGF2 was detected in ACT samples when compared to non-neoplastic adrenal tissue, (15.9-fold, P<0.001) (Figure 1A), however IGF2 overexpression was not related to relapse or metastasis in the patients (P=0.243), neither with clinical-pathological features nor with 5-year EFS (P=0.753).
IGF1R gene expression is higher in patients with relapse and metastasis
Although IGF1R gene expression was very similar for non-neoplastic adrenal samples and ACTs (P=0.822), we observed significant overexpression of IGF1R in patients with tumor relapse or metastasis when compared with the group of patients with complete remission (2.7-fold, P=0.031) (Figure 1B). However, IGF1R overexpression (1x or 2x > median) was not indicative of significant differences in the EFS of patients with ACT (58,7% ± 13,0% versus 80,6% ± 6,2%, 2x > median; P=0,103). Interestingly, we found significant positive correlation between IGF1R gene expression and DHEAS concentrations in patient’s serum (rho=0.663; P=0.02).
Transcripts do not correlate with protein expression
The IGF1R protein expression evaluated by IHC did not agree with the gene expression profiles observed by qRT-PCR. Positive immunoreactivity was found in 32/45 (71.1%) of the ACT samples, with 16 cases (35.5%) classified as strong, 12 (26.7%) as moderate and 4 (8.9%) as weak. The remaining 13 samples (28.9%) as well as the non- neoplastic tissue adjacent to the tumor presented no IGF1R staining (Figure 2). Moderate or strong immunostaining were considered IGF1R positive, while samples with weak or absent staining were classified as IGF1R negative. The median of IGF1R mRNA expression was 0.58 for IHC-positive samples (mean: 1.46, range: 0.10-8.26) and 0.55 for IHC-negative samples (mean: 1.23, range: 0.17-4.88) (P=0.933). Cytoplasmic staining was
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observed in 26 tumor samples, cytoplasmic and focal membrane staining was present in 4 samples and focal nuclear staining was detected in 2 cases. There was no significant difference in 5-year EFS between patients with moderate/strong and negative/weak IGF1R immunoreactivity (P=0.613) or with pure cytoplasmic versus membranous/nuclear staining (P=0.726).
Additionally, the IHC for IGF2 showed no correlation with transcripts, being 43% of negative cases, 43% with weak staining, 10% with moderate and 5% with strong staining. The positive samples presented predominantly cytoplasmatic staining (data not shown) and no significant differences were found according to the clinical and pathological features.
Identification of differentially expressed genes after treatment of adrenocortical carcinoma cells with OSI-906
In order to investigate the role of IGF1R in adrenocortical carcinoma cells (NCI- H295A), the global gene expression by microarray analysis was performed after functional blockage of IGF1R with OSI-906. After normalization, the 2000 most differentially expressed genes (DEGs) at two time points during treatment (6 and 24 hours) were selected for further analysis. Biological processes (Gene Ontology - GO) and pathway mappings (KEGG) were evaluated for the 200 most up-regulated and the 200 most down- regulated genes for each treatment time. The results demonstrated that up-regulated DEGs were significantly enriched in the GO factors involved in biological processes such as cytoskeleton organization and NFKB cascade, while the down-regulated DEGs were enriched in hormone metabolic/biosynthetic processes and MAPK activity.
We next evaluated whether treatment with OSI-906 could also impair time- dependent changes in the biological processes. At both times the up-regulated DEGs were enriched in GO terms such as activation/positive regulation of caspases and cell cycle
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310 311 312 313 314 regulation/arrest, while one KEGG pathway was identified only at 6 hours of treatment (HSA04115 - P53 signaling pathway; P=0.03). On the other hand, the group of down- regulated DEGs were associated with positive regulation/activation of the MAPK pathway and cell motility at 6 hours (Figure 3A) and significantly enriched in lipid and hormone synthesis/metabolism, response to external stimuli and lipid metabolic processes (Figure 315 3B) at 24 hours. The down-regulated DEGs were also enriched in KEGG pathways related to hormone metabolism (Table 1), supporting the findings of the biological processes enrichment. Moreover, differential expression of specific genes such as higher expression of DEPTOR (FC=1.4) and IRS2 (FC=1.4) at 24 hours, as well as reduction of PPARGC1B (FC =- 1.6) at 24 hours and PIK3C2G (FC =- 1.4) at 6 hours suggests inactivation of mTOR signaling downstream IGF1R.
Confirming the microarray analysis by qRT-PCR, the cells treated with OSI-906 showed significant reduction of MAPK1 at 6 hours and PI3K at both times. MAPK3 expression was slightly lower at 6 hours and higher at 24 hours (not significant) (Figures 4A-4C). Moreover the treated cells showed reduction of phosphorylated and total Erk1/2 proteins when compared to untreated cells (Figures 4D).
OSI-906 reduces tumor cell viability and induces apoptosis
Treatments with 1, 2 and 3 µM of OSI-906 for 24 hours decreased cell viability by 13%, 15% and 18% respectively, while at 48 and 72 hours, the reduction was significant with all doses of treatment (0.125 uM-3 uM) (P<0.001). However, limited reductions were observed at concentrations beyond 0.5 uM, which were characterized by a plateau in the graph of cell viability (Figure 4E). The most effective reduction was observed at 72 hours with 1 µM (40%). The time-dependent effect was confirmed with distinct EC50 (>20 uM±15.2 for 24 hours; 2.64 µM±0.23 for 48 hours; 1.99 uM+0.78 for 72 hours). We also
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observed a significant dose-dependent increase of cell apoptosis rate at 72 hours, reaching 31% of apoptotic cells with 1 µM of OSI-906 (CI=62.4-79.3; P<0.001) (Figure 4F).
Treatment with OSI-906 reduces hormone biosynthesis in adrenocortical carcinoma cells
To validate the microarray findings concerning the reduction of genes associated with hormone synthesis and lipid metabolism, we investigated the effects of OSI-906 on cellular hormone production after 24 hours. All steroid hormones were reduced, but it was significant only to testosterone (P=0.02) (Figure 5A-C). Interestingly, StaR protein expression decreased 55% at 24 hours (Figure 5D).
Discussion
The characteristics of our samples regarding clinical data such as age, sex, tumor stage, clinical symptoms, survival and prognostic factors were similar to those described by the International Pediatric Adrenocortical Tumor Registry (IPACTR) (Michalkiewicz et al. 2004).
One hallmark of pediatric ACT in Brazil is the TP53 p.R337H germline mutation, which disrupts protein tetramer formation and reduces the p53 activity in higher pH levels (Wasserman et al. 2012). Interestingly, in response to cellular stress such as oncogene activation, the p53 can shutdown the IGF pathway, reducing IGF2 and IGF1R expression and the receptor tyrosine phosphorylation (Sampaoli et al. 2012). Similar to other Brazilian childhood ACT series (Sandrini et al. 2005; Custódio et al. 2013), we observed high frequency of TP53 p.R337H mutation in tumor samples, but none significant differences in IGF2 or IGF1R expression profiles between samples with or without the mutation.
In vitro studies have shown that apoptosis and inhibition of ACC cells growth after ionizing radiation is dependent of p53 protein stabilization (Sampaoli et al. 2012) and
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TP53 somatic mutations confer resistance for anti-IGF1R therapy in colorectal carcinoma cells (Wang et al. 2013). Interestingly, NCI-H295 lacks TP53 p.R337H, but presents other mutations affecting p53 functions (Sampaoli et al. 2012; Leal et al. 2015), which also could be related to reduced effectiveness of therapies, such as anti-IGF1R.
The role of the IGF system in the development and growth of adrenal cortex is well known, with high levels of IGF2 detected in the adrenal glands and serum during the fetal stage, followed by a strong decline during the postnatal period. However, overexpression of IGF2 has been associated with a higher risk of ACT recurrence in adults (Boulle et al. 1998; Fottner et al. 2004). In children, we observed that IGF2 overexpression was not associated with tumor recurrence or disease poor outcome, which is in agreement with a few studies reporting IGF2 higher expression in pediatric ACT (Wilkin 2000; West et al. 2007; Almeida et al. 2008).
Among the four receptors of the IGF pathway described in mammals, the tyrosine kinase receptor type 1 (IGF1R) originates signals that facilitate cell transformation by other agents in different types of tumors (Wang & Sun 2002). In agreement with its pivotal role in cell growth and homeostasis, the overexpression of IGF1R in several human cancers is not surprising and suggests the involvement of IGF1R in tumor growth and progression (Maki 2010). In the present study, IGF1R expression was quite similar for non-neoplastic and tumor samples, but patients with metastasis or relapse showed significant higher IGF1R expression levels. Similar to our findings, Almeida et al. (2008) observed IGF1R overexpression in adrenocortical carcinomas and significant association with higher risk of metastasis in a study with 23 pediatric ACT samples. Herein, all control samples consisted of non-neoplastic adrenal tissue with both cortical and medullary cells, which somehow could be a potential bias for the analysis.
The correlation between correspondent IGF1R and IGF2 protein expression and their transcripts were not significant in the evaluated ACT samples. Other studies on
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387 388 389 390 cancer have revealed that the lack of correlation between mRNA and protein levels can be attributed to mRNA translational silencing, cleavage or alternative splicing (Dziadziuszko et al. 2010; Mountzios et al. 2013). In the IGF1R context, alternative mRNA splicing can lead to distinct protein degradation rates in cancer cells (Mountzios et al. 2013) while the 391 frequently expressed isoform (alpha) of IGF1R is not directly associated with global IGF1R gene expression (Pollak 2012). Interestingly, reports have demonstrated the impact of IGF1R protein localization on the biological behavior and prognosis of human breast cancer and in clear renal cancer cells (Aleksic et al. 2010; Tamimi et al. 2011). The ACT samples presented more IGF1R pure cytoplasmic than mixed
cytoplasmic/membranous/nucleus staining, but no significant differences were observed regarding patients’ survival, suggesting that IGF1R localization has no prognostic relevance for pediatric ACTs.
Evidence of IGF signaling in malignant cell transformation and tumor cell proliferation has encouraged the development of many IGF1R target-drugs. Among them, OSI-906 is known to reduce tumor cells proliferation because of its selective effect on both IGF1R and IR (Mulvihill et al. 2009). According to the global gene expression analysis of NCI-H295A cells, the inhibition of IGF1R with OSI-906 seems to up-regulate caspases activity and to induce cell cycle arrest. Gene array analysis conducted on a broad panel of colorectal cancer cell lines revealed that OSI-906-sensitive cells present up-regulation of the P53 pathway, while resistant cells present MAPK pathway up-regulation (Pitts et al. 2010). At six hours of OSI-906 treatment, we observed up-regulation of P53 signaling pathway genes and reduction of genes associated with MAPK pathway activation, suggesting a sensitive profile of the NCI-H295A cell line. In fact, it was obtained a significant dose-dependent increasing of apoptosis rate (31%), but cell viability was limited to 40% even after high doses of OSI-906. Since resistant colorectal cancer cells present up-regulation of the WNT pathway (Pitts et al. 2010), the mild decrease of cell
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viability in NCI-H295A cells could be related to the constitutive activation of Wnt/beta- catenin signaling due to S45P mutation in this cell line (Tadjine et al. 2008).
415 419 420 Reduction of cell viability after treatment with OSI-906 has been frequently 416 reported in different types of cancer cells. In this study, we used doses between 0.125 uM and 3 uM, which are comparable with other preclinical studies and are also within the 418 range of human maximal plasma concentrations (Cmax from 1.705 to 3.110 uM) after oral administration of OSI-906 (150 mg of OSI-906 twice daily) (Fassnacht et al. 2015; Puzanov et al. 2015). Sensitive cells, including the ACC cell line NCI-H295R, usually 421 present EC50 < 1 uM at 72 hours (Buck et al. 2010; Zhao et al. 2012; Janku et al. 2013). 422 423 424 425 426 427 428 429 430 Surprisingly we found higher EC50 values (>20 uM at 24 hours, 2.6 uM at 48 hours and 2.0 µM at 72 hours) which, together with restoration of ERK-1/2 expression after 24 hours of treatment, suggest a resistant profile for NCI-H295A after long periods of treatment (Zinn et al. 2013). Since OSI-906 inhibits kinase activities from both IR and IGF1R, we ruled out a resistance mechanism through compensatory IR signaling (Buck et al. 2010). However, new functions for IGF1R have been described (Boucher et al. 2010; Janku et al. 2013), which can explain the significant increase in apoptosis with only a mild decrease in NCI-H295A viability observed after OSI-906 treatment. Janku et al. (2013) showed that IGF1R is able to keep intracellular glucose levels, supporting tumor cell survival 431 432 433 434 independent of their kinase activity. Moreover, IGF1R interacts with other receptor tyrosine kinases such as epidermal growth factor receptors (EGFRs), vascular endothelial growth factor receptor (VEGFR), mesenchymal-epithelial transition factor (MET), platelet- derived growth factor receptor (PDGFR), estrogen receptors (ER) and others, which are 435 436 437 438 frequently found upregulated in cancer cells and may play a role in resistance to therapies anti-IGF1R. The activation of common downstream effectors through other receptors has provided new approaches regarding co-targeting strategies for anticancer therapies (Singh et al. 2014; Brahmkhatri et al. 2015).
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In addition, OSI-906 reduced PIK3C2G (Catalytic Subunit Type 2 Gamma of PI3K) gene expression in the microarray analysis and the PI3K gene by qPCR. The PI3 kinases family transduces signals from various growth factors such as IGF-IGF1R, which activates downstream mTOR (mammalian target of rapamycin) signals (Liu et al. 2009). Activation of mTOR is induced by two complexes (TSC1 and TSC2), driving cancer cells growth and proliferation (Advani 2010). After OSI-906 treatment, the cells also expressed higher levels of DEPTOR gene, a negative regulator of mTORC1 and mTORC2, as well as lower expression of PPARGC1B and increased expression of IRS2, which together with PI3K reduction, suggest inactivation of mTOR pathway (Laplante & Sabatini 2009; Brouwer-Visser & Huang 2015) .
Adrenocortical tumors are frequently characterized by hormonal secretion, inducing clinical symptoms of the disease or revealing tumor recurrence during follow-up (Gönç et al. 2014). As expected, most of children evaluated presented pure androgen or mixed (androgen and cortisol) secreting tumors, while only two patients presented cortisol secretion alone. In vitro studies have shown that IGF1/IGF1R stimulates hormone synthesis in different steroidogenic cells by inducing expression of steroidogenic genes and the steroidogenic acute regulatory (StaR) protein through MAPK/ERK signaling (Ramanjaneya et al. 2011). In agreement, we found significant positive correlation between IGF1R gene expression and DHEAS levels in diagnosis patient’s serum. Moreover, cells treated with OSI-906 presented lower expression of genes related to hormone synthesis/metabolism and StaR protein impairment of 55%, which is considered the first key mediator of steroidogenesis (Samandari et al. 2007).
To a lesser extent, only testosterone concentration was significantly reduced after OSI-906 treatment, suggesting that steroidogenesis impairment started with lower StaR protein expression could not be sufficient to exerts downstream effects in the synthesis of all steroid hormones at 24 hours.
439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464
465 466 467 468 469 In summary, IGF2 gene overexpression in a relatively large series of children diagnosed with ACT was not related to any clinical or biological features analyzed here while IGF1R gene expression was significant higher in children who presented tumor relapse and metastasis, which was not true for the IGF1R protein expression analyzed by IHC. In vitro, blockage of IGF1R signaling down-regulated MAPK activity, causing 470 reduction of cellular viability and increase of apoptosis rate in a dose-dependent manner. In 471 addition, OSI-906 decreased the expression of genes related to the steroid biosynthetic 472 process and impaired the expression of StAR, a key steroidogenic enzyme, events that 173 were followed by reduction of testosterone production. These findings suggest that IGF1R could have a role in adrenocortical cancer, however its inhibition by OSI-906 in adrenocortical tumor cells seems to promote only a mild anti-tumoral effect in a similar way as observed in clinical trials. Thus, new studies which elucidate the mechanisms of resistance to OSI-906 as well as new therapy schedules, such as drugs combination, would be important for a clinical application of IGF1R as a therapeutic target in childhood adrenocortical cancer.
Declaration of interest
All authors declare that they had no conflict of interest that could be perceived to impair the impartiality of the research reported.
Funding
This work was supported by the Public Research Agencies: Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP), Grant number: 2010/07020-9; Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínica - FMRP/USP (FAEPA), Brazil.
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Acknowledgements
We thank Prof. Dr. Aguinaldo Luiz Simões for providing the authentication of the cell line.
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Figure legends Figure 1
(A) IGF2 mRNA overexpression in adrenocortical tumor samples when compared with non- neoplastic adrenal samples. (B) Significant IGF1R overexpression in samples from patients with relapse or metastasis compared to patients with complete remission. Real-time PCR analysis was performed with samples in triplicate, which were normalized against the ACTB and GUSB genes. Statistics: Mann-Whitney test; FC = Fold-change value.
Figure 2 Representative images of IGF1R protein expression in adrenocortical tumor tissues, detected by immunohistochemistry. (A) Non-neoplastic adrenal tissue adjacent to the tumor with negative staining for IGF1R; (B) Adrenocortical tumor cells with intense cytoplasmic staining surrounded by negative areas; (C) Tumor cells with both cytoplasmic and membranous staining; (D) Focal area of tumor cells with intense nuclear staining. Original magnification x400.
Figure 3 Main biological processes associated with up- and down-regulated differentially expressed genes (DEGs) after inhibition of IGF1R by treatment with OSI-906 in NCI-H295A cells. (A) Down-regulated DEGs after 6 hours of treatment were associated with positive regulation/activation of MAPK pathway and cell motility; (B) Down-regulated DEGs after 24 hours of treatment enriched in lipid and hormone synthesis/metabolism, response to external stimuli and lipid metabolic process. (A and B) Up-regulated DEGs were enriched in activation/positive regulation of caspases and cell cycle regulation/arrest at both treatment times.
Figure 4 Effects of OSI-906 in NCI-H295A cells. (A) Reduced MAPKI gene expression after 6 hours of treatment with 2 uM and 4 uM OSI-906, reinforcing the enrichment of the down-regulated DEGs at 6 hours of treatment; (B) Significant reduction of PI3K gene expression 24 hours after treatment; (C) Trend of reduction and increasing of MAPK3 gene expression after 6 and 24 hours of treatment respectively (n.s. = non-significant); (D) The western blotting analysis reinforced the qRT-PCR, showing reduced expression of phosphorylated and total ERK1/2, which reached the lowest level after 6 hours of treatment; (E) Significant and stable reduction of cell viability after treatment with OSI-906. At 24 hours the reduction was significant with doses 1 - 3 AM while after 48 and 72 hours all doses induced significant decrease of cell viability. Time-dependent reduction was observed since at the lowest dose. (* symbol means significant difference compared to the control DMSO; # symbol means time-dependent significant difference); (F) Significant increase of apoptotic cells after 72 hours of treatment with OSI-906. Bars represent the means of three independent experiments with three replicates per experiment. Error bars indicate the Standard Deviation. Statistics: One Way ANOVA with Bonferroni pos-test.
Figure 5 Reduction of hormone secretion by NCI-H295A cells treated with OSI-906 for 24 hours. (A) Dehydroepiandrosterone sulfate (DHEAS); (B) Testosterone; (C) D4-androstenedione (D4); (D) Reduction of StaR protein levels after 24 hours of treatment with OSI-906, confirming the inhibition of the general pathway of hormone synthesis. Statistics: T-test for equality of means; n.s. = non-significant.
| Term | Gene count | P-value | Z-score | Combined Score | Genes |
|---|---|---|---|---|---|
| HSA00140 - C21 steroid hormone metabolism | 5 | 9.65E-07 | -1.62 | 15.58 | HSD3B2; HSD3B1; CYP21A2; CYP11B1; CYP11B2 |
| HSA00330 - Arginine and proline metabolism | 4 | 0.001 | -1.88 | 5.84 | CKB; CKMT2; EPRS; OAT |
| HSA00150 - Androgen and estrogen metabolism | 4 | 0.006 | -2.00 | 4.10 | CYP11B1; CYP11B2; HSD3B2; HSD3B1 |
| HSA03320 - PPAR signaling pathway | 4 | 0.013 | -1.72 | 2.92 | APOA1; ACSL6; FADS2; FABP6 |
| HSA01510 - Neurodegenerative diseases | 3 | 0.014 | -1.57 | 2.66 | UCHL1; NEFH; PRNP |
| HSA01040 - Polyunsaturated fatty acid biosynthesis | 2 | 0.016 | -0.50 | 0.86 | FADS2; ELOVL5 |
| HSA00642 - Ethylbenzene degradation | 2 | 0.018 | -0.61 | 1.04 | ESCO2; DHRS2 |
| HSA00624 - 1 and 2 methylnaphthalene degradation | 2 | 0.044 | -1.00 | 0.96 | ESCO2; DHRS2 |
A
B
10,0-
mRNA relative expression (Log 10)
FC = 15
mRNA relative expression (Log 10)
FC = 2.7 (P= 0.031)
100,0-
(P < 0.001)
IGF2
IGF1R
10,0-
1,0-
1,0
,0-
.
,0-
Non-neoplastic
Tumors
Complete remission
Relapse or metastasis
291×119mm (96 x96DPI)
A
B
C
D
228x171mm (200 x 200 DPI)
A
GO: Biological Process - 6 hours
B
GO: Biological Process - 24 hours
Nucleobase, nucleoside and nucleic acid metabolic process (GO:0006139)
Organelle organization (GO:0006996)
Regulation of transcription from RNA polymerase II promoter (GO:0006357)
Protein catabolic process (GO:0030163)
Anatomical structure development (GO:0048856)
Proteolysis (GO:0006508]
Chromosome organization (GO:0051276)
Positive regulation of transcription, DNA-dependent (GO:0045893)
Establishment or maintenance of chromatin architecture (GO:0006325)
Positive regulation of hydrolase activity (GO:0051345)
Cell death (GO:0008219)
Skeletal system development (GO:0001501)
Positive regulation of hydrolase activity (GO:0051345)
Cell cycle arrest (GO:0007050]
Regulation of mitotic cell cycle (GO:0007346)
Regulation of peptidase activity (GO:0052547]
UP
Nucleotide metabolic process (GO:0009117)
UP
Intracellular receptor-mediated signaling pathway [GO:0030522]
Regulation of peptidase activity (GO:0052547)
Regulation of endopeptidase activity (GO:0052548)
Response to organic substance (GO:0010033)
Steroid hormone receptor signaling pathway [GO:0030518]
Regulation of endopeptidase activity [GO:0052548)
Positive regulation of caspase activity (GO:0043280)
Transmembrane receptor protein serine/threonine kinase pathway (GO:0007178)
Activation of caspase activity (00:UUU6919)
Phosphoinositide-mediated signaling (GO:0048015)
Microtubule cytoskeleton organization (GO:0000226)
Cellular amino acid derivative metabolic process (GO:0006575)
Positive regulation of caspase activity (GO:0043280)
Response to protein stimulus (GO:0051789]
Activation of caspase activity (GO:0006919)
Response to unfolded protein (GO:0006986)
BMP signaling pathway (GO:0030509)
Steroid biosynthetic process (GO:0006694)
Regulation of blood pressure (GO:0008217)
Positive regulation of MAP kinase activity (GO:0043406)
Keratinocyte differentiation (GO:0030216)
Activation of MAPK activity (GO:0000187)
Hormone biosynthetic process (00:0042446]
Cell motility [GO:0048870)
DOWN
C21-steroid hormone metabolic process (GO:0008207)
DOWN
RNA catabolic process (GO:0006401]
Regulation of MAP kinase activity (GO:0043405)
Cellular amino acid and derivative metabolic process (GO:0006519]
Negative regulation of transcription, DNA-dependent (GO:0045892)
Lipid biosynthetic process (GO:0008610]
Protein folding (GO:0006457)
Steroid metabolic process (GO:0008202)
Cell motion (GO:0006928)
Hormone metabolic process (GO:0042445)
Regulation of RNA metabolic process (GO:0051252)
Cell differentiation (GO:0030154)
Regulation of transcription, DNA-dependent (GO:0006355)
Response to external stimulus (GO:0009605]
Nucleobase, nucleoside and nucleic acid metabolic process (GO:0006139)
Catabolic process (GO:0009056)
Transport (GO:0006810)
Regulation of catalytic activity (GO:0050790)
Lipid metabolic process (GO:0006629)
0
2
4
6
8
10 12 14 16 18 20
0
2
4
6
8
10 1
14
Gene Count
Gene Count
Figure 3 Main biological processes associated with up- and down-regulated differentially expressed genes (DEGs) after inhibition of IGF1R by treatment with OSI-906 in NCI-H295A cells. (A) Down-regulated DEGs after 6 hours of treatment were associated with positive regulation/activation of MAPK pathway and cell motility; (B) Down-regulated DEGs after 24 hours of treatment enriched in lipid and hormone synthesis/metabolism, response to external stimuli and lipid metabolic process. (A and B) Up-regulated DEGs were enriched in activation/positive regulation of caspases and cell cycle regulation/arrest at both treatment times.
311x152mm (200 x 200 DPI)
A
MAPK1
B
PI3K
mRNA relative expression
1.5-
mRNA relative expression
1.5
P = 0.009
n. s.
P = 0.02
P = 0.078
P < 0.001
1.0-
1.0
T
T
T
T
T
0.5
0.5
T
I
T
0.0
6 hours
24 hours
0.0
6 hours
24 hours
DMSO
1.0 μΜ
2.0 μΜ
4.0 µM
DMSO
1.0 μΜ
2.0 μΜ
4.0 µM
C
MAPK3
D
mRNA relative expression
1.5
n. s.
n. s.
T
T
DMSO OSI-906 (2 μM)
1.0
T
Time (hours)
3
6
24
T
p-ERK 1/2
0.5
Total ERK 1/2
0.0
GAPDH
6 hours
24 hours
DMSO
1.0 µM
2.0 μΜ
4.0 µM
E
F
120-
#
#
#
#
#
#
*
*
Relative cell viability [%]
100
T
T
#
*
#
*
*
80
P < 0.001
T
*
*
T
*
T
80
T
*
*
T
*
T
Cell apoptosis rate (%)
*
*
*
*
60-
P = 0.022
60
T
T
T
40-
40
20
20-
0
DMSO
0.125
0.25
0.50
1.00
2.00
3.00
0
DMSO
0.125
0.25
0.50
1.00
Concentration (LM)
Concentration (uM)
24 hours
48 hours
72 hours
with Bonferroni pos-test. 261x336mm (96 x 96 DPI)
A
B
Dehydroepiandrosterone Sulfate (DHEAS) (ng/dL)
1700-
42-
P = 0.02
1650-
40-
Testosterone (ng/dl)
1600-
38-
36-
1550
34
1500-
32-
1450-
30
1400
n.s.
28-
DMSO
2 μΜ
DMSO
2 µM
C
D
DMSO OSI-906 (2 µM)
600-
Time (hours)
3
6
24
D4-androstenedione (ng/dl)
550
StaR
GAPDH
50C
1.0
45C
StaR / GAPDH Arbitrary units
0.8
40C-
0.6
0.4
350-
0.2
30C-
n.s.
DMSO
2 μΜ
0.0
DMSO
3
6
24
Time (hours)
264x291mm (96 x 96 DPI)