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Amplification of the Insulin-Like Growth Factor 1 Receptor Gene Is a Rare Event in Adrenocortical Adenocarcinomas: Searching for Potential Mechanisms of Overexpression

Context. IGF1R overexpression appears to be a prognostic biomarker of metastatic pediatric adrenocortical tumors. However, the molecular mechanisms that are implicated in its upregulation remain unknown. Aim. To investigate the potential mechanisms involved in IGF1R overexpression. Patients and Methods. We studied 64 adrenocortical tumors. IGF1R copy number variation was determined in all patients using MLPA and confirmed using real time PCR. In a subgroup of 32 patients, automatic sequencing was used to identify IGF1R allelic variants and the expression of microRNAs involved in IGF1R regulation by real time PCR. Results. IGF1R amplification was detected in an adrenocortical carcinoma that was diagnosed in a 46-year-old woman with Cushing's syndrome and virilization. IGF1R overexpression was demonstrated in this case. In addition, gene amplification of other loci was identified in this adrenocortical malignant tumor, but no IGF1R copy number variation was evidenced in the remaining cases. Automatic sequencing revealed three known polymorphisms but they did not correlate with its expression. Expression of miR-100, miR-145, miR-375, and miR-126 did not correlate with IGF1R expression. Conclusion. We demonstrated amplification and overexpression of IGF1R gene in only one adrenocortical carcinoma, suggesting that these combined events are uncommon. In addition, IGF1R polymorphisms and abnormal microRNA expression did not correlate with IGF1R upregulation in adrenocortical tumors.


Introduction
Adrenocortical tumors are rare endocrine malignancies [1]. However, the incidence of these tumors is remarkably high in Southern Brazil, where it is estimated to be 10-15 times greater than the worldwide incidence [2,3]. These tumors can occur in all age groups and have been characterized as having a bimodal age distribution, with the first peak occurring before 5 years of age and the second between the fourth and fifth decades [3].
The insulin-like growth factor (IGF) signaling system plays an important role in the growth and development of many tissues, including the adrenal gland [4]. Insulin-like growth factors are mitogens that regulate cell proliferation, differentiation, and apoptosis by interacting with the IGF1 receptor (IGF1R) [5]. This system has also been implicated in various pathophysiological conditions and is thought to play a particular role in tumorigenesis [6]. The IGF1R pathway is important in promoting oncogenic transformation, growth, and survival of cancer cells [7], and IGF1R overexpression has been demonstrated in many cancers [8,9].
We previously demonstrated IGF1R overexpression in 65% of children and 13% of adults with adrenocortical tumors  [10]. Interestingly, this IGF1R upregulation was a predictor of metastases in children with adrenocortical tumors. Additionally, a selective IGF1R kinase inhibitor exhibited antitumor effects in adult and pediatric adrenocortical tumor cell lines, suggesting that IGF1R inhibitors represent a promising therapy for human adrenocortical carcinoma [10].
The IGF1R upregulation was confirmed in another cohort of pediatric patients [11]. The molecular mechanisms that lead to increased IGF1R expression in these tumors remain unexplained. In this study, we investigated whether IGF1R gene amplification and allelic variations could be implicated in IGF1R upregulation. In addition, the expression of four distinct microRNAs (miRNA) that were involved in IGF1R regulation was evaluated.

Patients
The study included 64 Brazilian patients with sporadic adrenocortical tumors (Table 1) [10], and the overexpression of this receptor was observed in 17 children and 5 adults [10]. In 32 out of these 45 patients automatic sequencing was used to identify IGF1R allelic variants and the expression of microRNAs involved in IGF1R regulation was determined by real time PCR. Eight normal adrenal gland cortices, which were obtained from children and adults (range of age: 1 to 72 years) during renal surgery or autopsies, were used as controls. The fragments of adrenal cortices were properly selected by an experienced pathologist.

Molecular Analysis
3.1. DNA Extraction. Genomic DNA was extracted from frozen tumor samples using a Wizard Genomic DNA Purification Kit (Promega, WI, USA) and was stored at −35 ∘ C. The concentration and purity of the genomic DNA were measured, using spectrophotometer at an absorbance of 260 and 280 nm.

Multiplex Ligation-Dependent Probe Amplification
(MLPA). The IGF1R copy number of the adrenocortical tumors samples was measured using the SALSA MLPA kit P217 IGF1R (MRC-Holland, Amsterdam, Netherlands). This molecular assay was designed to detect deletions/duplications of one or more exons of the IGF1R (chromosome 15q26) and IGFBP3 (chromosome 7p13) genes. The P217 IGF1R probe mix contained 22 specific probes for IGF1R, 5 probes for IGFBP3, and 9 control probes. The FGFR4 and NSD1 genes analysis was performed using the SALSA MLPA kit P026-C1 Sotos (MRC-Holland, Amsterdam, The Netherlands), which contains two probes for FGFR4 gene and 24 probes for NSD1 gene (5q35.1). MLPA was performed with a total of 200 ng of genomic DNA for each sample, as previously described [12]. The PCR products were submitted to capillary electrophoresison an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems, The Perkin-Elmer Corporation, CA, USA), and the MLPA results were analyzed using the Genescan 3.7 software and further evaluated using the Excel spreadsheet software (Microsoft Corporation, Redmond, WA, USA). The tumor sample normalized peak height was then divided by the average normalized peak height of normal adrenals. Dosage quotient areas outside the range of 0.70-1.3 were considered abnormal. In addition, visual comparison of peak profiles was performed, three control samples were included in each MLPA experiment, and all results were confirmed by two independent tests. for mRNA sequences) was used as a house-keeping gene, and specific forward and reverse primers for IGF1R were used. The PCR was performed in 25 L containing 2.5 L of DNA (50 ng), 10 pmol of each primer, and the power SYBR Green PCR master mix (Applied Biosystems, Forster, CA). The thermal cycling conditions consisted of denaturation for 10 min at 95 ∘ C, followed by 40 cycles of denaturation for 15 sec at 95 ∘ C and annealing/extension for 1 min at 60 ∘ C. A cycle threshold (CT) value in the linear range of amplification was selected for each sample in triplicate and normalized to NPC1. The IGF1R dosage was determined using the 2 −ΔΔCT method [13]. The normalized value (ΔCT) for each tumor sample was then compared with the mean ΔCT of five normal adrenals to generate a fold change ratio (normal = 1) that was multiplied by 2 to obtain the copy number (normal = 2). 3.6. P53 Analysis. Genomic DNA was extracted from frozen tumor specimens using a QIAmp DNA mini Kit (QIA-GEN, Hilden, Germany). The entire exon 10 was amplified by PCR using the following intronic primers: 5 -GCTGTATAGGTACTTGAAGTGCAG-3 and 5 -GATGA-GAATGGAATCCTATG-3 . The amplification protocol consisted of denaturing at 94 ∘ C for 5 min, followed by 35 cycles consisting of annealing at 50 ∘ C for 30 sec, primer extension at 72 ∘ C for 30 sec, and denaturing at 94 ∘ C for 30 sec.

IGF1R Sequence
All amplified fragments were examined on 1% agarose gel electrophoresis. The PCR products were pretreated with an enzymatic combination of exonuclease I and shrimp alkaline phosphatase (United Stated Biochemical Corp., Cleveland, OH) and directly sequenced using the BigDye terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA) in an ABI PRISM 310 automatic sequencer (Perkin-Elmer Corp.).

IGF1R Copy Number. IGF1R
amplification was detected using MLPA and confirmed using real time PCR in an adrenocortical carcinoma that was diagnosed in a 46-yearold female patient with Cushing's syndrome associated with clinical virilization (Figure 1). The tumor's size was 10 × 8 × 6 cm, and it was classified as ENSAT stage III and presented a Weiss score of 7 upon histopathological analysis. IGF1R mRNA levels were 5 times higher in this tumor than in the normal total adrenal gland pool. Additionally, the amplification of the IGFBP3, FGFR4 (data not shown), and NSD1 (data not shown) genes was also detected in this carcinoma, but IGF1R amplification was not demonstrated in the remaining cases with adrenocortical tumors.  0.01 to 12.6)) had variable expression in the adrenocortical tumors that were evaluated; however, these values were not correlated with the expression values of IGF1R in adrenocortical adenomas and carcinomas. miR-126 (median 596.6 (from 30.8 to 2667.4)) was overexpressed in benign and malignant adrenocortical tumors and was not related to IGF1R expression (Figure 2).

Statistical Analysis.
Statistical analysis was performed using SPSS Software (PASW version 19.0; SPSS Inc., Chicago, IL). Continuous data are expressed as median values (from minimum to maximum). Differences in expression levels between two groups were analyzed by means of the two-tailed Mann-Whitney test.

Discussion
IGF1R has potent mitogenic, antiapoptotic, and transforming activities [17]. Normal growth and differentiation are result of a fine balance between the process of cell proliferation and death, and the disruption of one or more factors involved in these actions can result in pathologic phenotypes, including malignancy [17]. Indeed, IGF1R is overexpressed in several primary tumors and cancer-derived cells [18,19]. It has been demonstrated that IGF1R expression is a fundamental prerequisite for cellular transformation because enhanced IGF1R levels and IGF1 signaling are considered key factors for the cell to adopt the proliferative and oncogenic pathways [20]. Increased expression of the human IGF1 receptor promotes ligand-dependent neoplastic transformation in different cells [6,21]. On the other hand, the absence or decrease of levels of the IGF1 receptor prevents malignant growth and transformation [22].
The IGF1R copy number in 64 adrenal tumor samples was analyzed using MLPA in this study. Amplification of IGF1R was detected in only one adrenocortical carcinoma (Weiss score 7) that was diagnosed in a woman who had an endocrine hyperfunction that was characterized by hypercortisolism and hyperandrogenism. This patient was treated with mitotane and she had a favorable evolution over the previous 10 years. As expected, the adrenocortical tumor with IGF1R amplification diagnosed in this patient had higher IGF1R mRNA levels compared to normal adrenal samples. MLPA analysis also demonstrated a multiplegene amplification, suggesting a potential aneuploidy. IGF1R gene amplification was not demonstrated in the remaining adrenocortical tumors, which suggested that other mechanisms could be implicated in IGF1R upregulation. Additionally only the previously known exomic and intronic polymorphisms were observed in tumors with or without IGF1R overexpression.
Stimulatory and inhibitory transcription factors determine the level of IGF1R expression, and, consequently, the proliferative cell status. The IGF1R gene is controlled by a number of tumor suppressors, including p53, which is the most frequently mutated molecule in human cancer. P53 was able to suppress IGF1R promoter activity by approximately 90%, whereas its mutant forms were associated with IGF1R upregulation [31]. A specific germinative mutation (p.R337H) that affected the tetramerization domain of p53 has been identified in high frequency in Brazilian children with adrenocortical tumors [32,33]. This unique p53 defect was detected in 22 of the 64 (34.3%) patients of the Brazilian cohort that was studied here (data not shown). Nevertheless, IGF1R overexpression and gene amplification were not associated with the presence of this p53 mutation. In addition, the adrenocortical carcinoma with IGF1R amplification did not harbor the p.R337H mutation in blood or tumor tissue DNA (data not shown).
MicroRNA abnormalities are potential mechanisms that may be associated with IGF1R overexpression. Calin et al. [34] demonstrated that more than 50% of microRNA genes were located in cancer-associated genomic regions. In the present work, we demonstrated that the expression of miR-100 and 375 was decreased in benign and malignant adrenocortical tumors; however, no correlation was found between the selected microRNAs and IGF1R expression. miR-145 also did not correlate with IGF1R expression values. Our data corroborate with those from previous studies that showed that miR-100 and 375 expression was decreased in adrenocortical tumors and could be tumor suppressor microRNAs [11,35]. In addition, Doghman et al. [11] showed that miR-100 regulated the expression of IGF1R and mTOR signaling in adrenocortical cells by modulating the expression of key proteins involved in these pathways [11]. miR-375 is also downregulated in gastric cancers and hepatocellular carcinomas [15]. Previous studies in esophagus carcinoma cells showed that miR-375 has a tumor suppressor effect by inhibiting IGF1R [15]. Shi et al. [14] identified that IGF1R and its docking protein, the insulin receptor substrate-I, are targets of miR-145. miR-126 was overexpressed in benign and malignant adrenocortical tumors. This microRNA suppresses endothelial migration by targeting a set of genes in cancer cells that drive endothelial migration, by activating IGF1R, which is a promoter of endothelial migration, and inhibiting the endothelial MERTK receptor, which is a suppressor of endothelial migration [16].
In conclusion, we detected concurrent IGF1R gene copy number variation and IGF1R overexpression in a single functioning malignant adrenocortical tumor. This data suggests that the IGF1R overexpression, which is common in pediatric adrenocortical tumors, is not driven by increased gene copy number. In addition, IGF1R polymorphisms and the abnormal expression of the microRNAs miR-100, miR-145, miR-375, and miR-126 did not correlate with IGF1R overexpression in these tumors.