Accepted Manuscript

Insulin-Like Growth Factor and SLC12A7 Dysregulation: A Novel Signaling Hallmark of Non-Functional Adrenocortical Carcinomas

Journal of the American College of Surgeons

Taylor C. Brown, MD, MHS, Norman G. Nicolson, MD, Adam Stenman, MD, PHD, C Christofer Juhlin, MD, PHD, Courtney E. Gibson, MD, MS, FACS, Glenda G. Callender, MD, FACS, Reju Korah, PhD, Tobias Carling, MD, PHD, FACS

PII:	S1072-7515(19)30293-5
DOI:	https://doi.org/10.1016/j.jamcollsurg.2019.04.018
Reference:	ACS 9501
To appear in:	Journal of the American College of Surgeons
Received Date:	13 March 2019
Accepted Date: 11 April 2019

Please cite this article as: Brown TC, Nicolson NG, Stenman A, Juhlin CC, Gibson CE, Callender GG, Korah R, Carling T, Insulin-Like Growth Factor and SLC12A7 Dysregulation: A Novel Signaling Hallmark of Non-Functional Adrenocortical Carcinomas, Journal of the American College of Surgeons (2019), doi: https://doi.org/10.1016/j.jamcollsurg.2019.04.018.

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Insulin-Like Growth Factor and SLC1247 Dysregulation: A Novel Signaling Hallmark of Non-Functional Adrenocortical Carcinomas

Taylor C Brown, MD, MHS1, Norman G Nicolson, MD1, Adam Stenman, MD, PhD2, C Christofer Juhlin, MD, PhD2,3, Courtney E Gibson, MD, MS, FACS1, Glenda G Callender, MD, FACS1, Reju Korah, PhD1, Tobias Carling, MD, PHD, FACS1

1 Department of Surgery & Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT

2 Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden

3 Department of Pathology and Cytology, Karolinska University Hospital, Stockholm, Sweden

Disclosure Information: Nothing to disclose.

Support: This study was supported by an Ohse Research Grant.

Presented at the 99th Annual Meeting of the New England Surgical Society, Portland, ME, September 2018.

Correspondence:

Tobias Carling, MD, PhD Department of Surgery, Yale Endocrine Neoplasia Laboratory Yale University School of Medicine 333 Cedar Street, TMP FMB130A, P.O. Box 208062, New Haven, CT 06520, U.S.A. Tel: +1-203-737-2036 Fax: +1-203-737-4067 Email: tobias.carling@yale.edu

Reprint requests should be addressed to Tobias Carling (tobias.carling@yale.edu)

Brief Title: Insulin-Like Growth Factor and Adrenocortical Carcinoma

ACCEPTED NUMERO NUMERO FRE ERO

ABSTRACT

Background: Insulin-like growth factor (IGF) dysregulation and gene copy number variations (CNV) are hallmarks of adrenocortical carcinoma (ACC). The contribution of IGF CNVs in adrenal carcinogenesis has not been previously studied. Moreover, studies demonstrating an association between SLC1247 gene amplifications and enhanced metastatic behavior in ACC, as well as reported IGF-SLC12A7 signaling interactions in other cancers, suggests a potential IGF- SLC12A7 signaling circuitry in ACC. Here we investigate the potential complicity of IGF- SLC12A7 signaling in ACC.

Study Design: IGF CNVs were determined by whole-exome sequencing analysis in an exploratory cohort of ACC. Quantitative PCR methods determined IGF1 and IGF2 expression levels and were evaluated for correlation with SLC1247 expression and tumor characteristics. IGF CNVs and expression patterns were compared to The Cancer Genome Atlas (TCGA). In vitro studies determined the relationship of IGF and SLC1247 co-expression in two ACC cell lines, SW-13 and NCI-H295R. Immunohistochemistry assessed IGF1 receptor (IGFR) activation.

Results: The IGF1 gene was amplified in 9 of 19 ACC samples, similar to findings in the TCGA database. IGF1 overexpression was observed in 5 samples and was associated with SLC12A7 overexpression and non-functional, early stage tumors (p<0.05). In contrast, IGF2 overexpression was associated with larger tumors (p<0.05). In vitro IGF treatment of ACC cell lines did not stimulate SLC1247 expression, while endogenous overexpression and silencing of SLC12A7 significantly altered IGF1 and IGFIR expression without impacting other IGFs. IGF1R activation was associated with IGF1 overexpression in ACC tumor samples. Conclusion: These findings indicate that IGF1 overexpression caused, in part, by gene

amplifications, is correlated with SLC1247 overexpression in non-functional, early stage ACCs, suggesting a potentially targeted IGF1-SLC12A7 therapeutic opportunity for these tumors. Keywords: Adrenocortical Carcinoma, IGFs, IGF1R, and SLC12A7.

ACCEPTED MANUS CRIPT

ABBREVIATIONS

Adrenocortical carcinoma, ACC; American Type Cell Collection, ATCC; Copy number variations, CNV; Dulbecco’s modified Eagle’s medium, DMEM; Insulin like growth factor, IGF; Solute carrier family 12 member 7, SLC12A7; The Cancer Genome Atlas, TCGA; Whole-exome sequencing, WES.

ACCEPTED MANUS CRIP Wolever

INTRODUCTION

Adrenocortical carcinoma (ACC) is a rare cancer of the adrenal cortex, with a reported incidence of 0.7-2 cases per million people per year (1). Despite recent advancements in our understanding of the molecular causes of adrenocortical tumorigenesis, little improvement has been made in overall treatment outcomes, especially in those patients with advanced and/or metastatic diseases. The overall prognosis for ACC is poor and a recent analysis of the National Cancer Data Base demonstrated a 5-year survival for stage IV tumors to be less than 50% (2). Furthermore, recurrence rates can be very high after curative intent resections, indicating the need for improved adjuvant therapies after RO resection (3).

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The three most common molecular alterations observed in ACC include dysregulation of WNT, TP53 and IGF signaling pathways (4). A gain-of-function mutation of the beta-catenin gene (CTNNB1), a proto-oncogene, promotes its nuclear persistence by inhibiting its programmed cytoplasmic degradation by a destruction complex. This in turn enhances constitutive target gene transcription and tumor formation (5). Germline TP53 mutations are frequently found in childhood cases of ACC (6, 7), while somatic TP53 mutations occur commonly in aggressive adult tumors and are believed to represent a late event in adrenal carcinogenesis (8). However, the most frequent molecular event observed in ACC and an area of intense interest for developing targeted therapies, is the overexpression and activation of the insulin like growth factor (IGF) signaling system.

In normal adrenal tissue, insulin like growth factor 2 (IGF2) expression is highest during embryogenesis, while insulin like growth factor 1 (IGF1) expression primarily occurs after birth. Both IGFs bind the IGF1 receptor (IGF1R) and promote adrenal growth and cortisol production (9-12). In adrenal malignancy, IGF2 is the predominant IGF overexpressed, driven, in part, by

alterations in methylation patterns and is associated with more aggressive tumors. This is best exemplified by the development of pediatric ACCs in Beckwith-Wiedemann syndrome, a genetically heterogeneous condition due to epigenetic alterations on chromosome 11p15.5, which causes constitutive IGF2 up-regulation in subsets of patients. A few studies also report heightened expression of IGF1 in ACC, implicating a global role for the IGF system in adrenal tumorigenesis (13, 14). Interestingly, neither overexpression of IGF1 or IGF2 alone has been shown to cause malignant transformation in the adrenal gland (9). Apart from changes in promoter methylation of the IGF2 gene in a subset of ACC’s, the cause(s) of aberrant IGF signaling observed frequently in ACC is poorly understood. Gene copy number alterations, a common genome-wide phenomenon in ACC (15), have not been specifically analyzed at the IGF loci and are studied here.

Other than its known role in promoting cellular growth, the underlying mechanisms by which aberrant IGF signaling potentially modulates a more aggressive phenotype in ACC has not been fully clarified. Recent studies identified potassium chloride transporter, solute carrier family 12 member 7 (SLC1247) overexpression as one of the aberrant processes that promotes the aggressive behavior of ACC’s via alterations in membrane architecture and invasive properties (16, 17). Interestingly, co-expression of IGF1 and SLC12A7 has been observed in several gynecological tumor types, especially in metastatic lesions. Furthermore, tumor cell stimulation with IGF1 was associated with SLC12A7 membrane localization and increased tumor cell invasion kinetics (18). We hypothesize that a similar invasion-promoting role for SLC12A7 in ACC is potentially promoted, in part, by deregulated IGF-signaling and is investigated here.

METHODS

Study cohort. Following approval by the Yale University and Karolinska Institute institutional review boards, 33 cases (8 Yale, 25 Karolinska) of histologically confirmed ACCs were selected for biochemical and clinical analysis. Clinical characteristics of the patients are shown in Table 1. All fresh-frozen adrenal tissues samples were maintained in a prospectively maintained endocrine tumor repository and experienced endocrine pathologists reviewed tissue sections for confirmation of the diagnosis prior to investigation.

Tumor gene copy analysis. Genomic DNA from ACC samples was recently subjected to whole-exome sequencing (WES) in which chromosomal, arm-level copy number variations (CNV) were reported by Juhlin et al (19). A separate reanalysis was performed with an alternative algorithm to identify single-gene CNVs for the IGF loci on chromosome 11 and 12 in 19 samples by assessing coverage depth analysis of WES reads between tumor and adjacent normal adrenal DNA using the genomic identification of significant targets in cancer (i.e., GISTIC), version 2.0, algorithm output for gene-level data. A larger confirmatory cohort from The Cancer Genome Atlas (TCGA) database was analyzed (Xena Browser, UC Santa Cruz) to determine gene copy alterations of IGF1 and IGF2 as well (20).

Tumor gene expression analysis. RNA was isolated from fresh frozen samples using the AllPrep DNA/RNA/Protein Kit (Qiagen). Quantity and quality of isolated RNA was assessed by spectrophotometry (NanoDrop Technologies, Inc) and two hundred ng of RNA was used for cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad). Real-time quantitative PCR (RT-qPCR) was performed on a CFX96 Real-Time System thermo cycler (Bio-Rad) using TaqMan PCR master mix with primers and probes (Applied Biosystems) specific to IGF1 (Hs01547656_m1), IGF2 (Hs04188276_m1), SLC12A7 (Hs00986431_m1), and the housekeeping gene large ribosomal protein 0 (RPLP0; Hs00420895 gH). Relative expression

levels were calculated using the Livak method (21). The normal reference tissue analyzed in this study for comparison included 10 samples of histologically normal adrenal tissue surgically removed along with adjacent adrenal adenoma samples. A larger confirmatory cohort analyzing the TCGA database RNA sequence reads (Xena Browser, UC Santa Cruz) determined gene expression levels of IGF1 and IGF2. Assays were performed in triplicates.

Cell culture. The authenticated ACC cell lines SW-13 and NCI-H295R were purchased from the American Type Cell Collection (ATCC) and maintained per ATCC protocol, as previously described (17, 22). Briefly, SW-13 cells were grown under sterile conditions in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% certified fetal bovine serum and 1% of 100X penicillin/streptomycin (ThermoFisher Scientific) in a standard humidified incubator at 37.0 C and 5% CO2. NCI-H295R cells were grown under sterile conditions in DMEM/F12 supplemented with 5% NuSerum, 0.1% Insulin-Transferrin-Selenium, and 1% of 100X penicillin/streptomycin (ThermoFisher Scientific) in a standard humidified incubator at 37.0 ℃ and 5% CO2.

In vitro IGF stimulation and expression. Two hundred and fifty thousand cells of SW- 13 were seeded per well into a 6-well plate in 3 mL of growth medium. After overnight incubation to allow for cell adherence, cells were then treated with various concentrations of recombinant IGF1 or IGF2. After 6, 12, and 24 hours of incubation, mRNA was isolated using RNeasy Plus Mini Kit (Qiagen) and mRNA levels of SLC1247 were determined. Similarly, mRNA expression levels of IGF1, IGF2, and IGFIR were also measured in SW-13 cells, which have low endogenous expression of SLC1247, and in NCI-H295R cells, which have high endogenous expression of SLC1247 (17). Assays were performed in triplicates.

SLC12A7 siRNA knock-down and IGF expression. RNAi gene silencing of NCI- H295R cells were carried out with three unique 27-mer siRNA duplexes targeting SLC1247 (Human) using the standard protocol as previously described (17). Universal scrambled siRNA was used as non-specific control (all from Origene). Lipofectamine 3000-mediated transfection was carried out in Opti-MEM medium according to the manufacturer’s recommendations (ThermoFisher Scientific) in 6-well plates with starting densities of 100,000 cells/well. Transfection medium was replaced with growth medium after 6 hours of transfection. Cells were lysed for RNA extraction and gene expression analysis at 24 hours post-transfection. IGF1 expression levels were determined in NCI-H295R cells undergoing siRNA knock-down of SLC12A7 expression, relative to the parental NCI-H295R cells that express high endogenous levels of SLC12A7. Confirmation of SLC12A7 overexpression or RNAi-knock-down were confirmed by RT-qPCR, as previously reported (17). Assays were performed in duplicates.

Western blot detection of IGF1 & IGF1R expression. Altered protein expression of IGF1 and IGF1R was confirmed via Western blot technique using goat anti-IGF1 and mouse monoclonal anti-IGF1R antibodies, followed by anti-goat-HRP and anti-mouse-HRP antibodies (all from Invitrogen), mini-PROTEAN TGX gel (BioRad), PVDF blotting membrane (BioRad), and enhanced chemiluminescence detection reagents (ThermoFisher Scientific) according to the manufacturer’s protocols. Equivalent protein loading was ensured via Western blot detection of beta-actin expression using anti-ß-actin mouse monoclonal antibody (Santa Cruz Biotech.) followed by anti-mouse-HRP secondary antibody (Invitrogen). Western blot analysis was performed in duplicate.

Immunohistochemistry. Representative sections of histologically confirmed ACCs and colon adenocarcinoma (positive control) from formalin fixed paraffin embedded tissue samples

were selected for study. Using immunohistochemistry methods per the manufacturer’s protocol (Abcam), target epitopes were detected using anti-IGF1R (phosphoY1161) antibody at 1;200 dilution followed by secondary antibody. 3,3’-diaminobenzidine tretrachydrochloride (DAB) was utilized for antigen detection. Sections were counterstained with hematoxylin. Photomicrographs were taken at 400x magnification.

Statistical Analysis. A 2-tailed t-test or Mann-Whitney test was used to assess difference in two groups with continuous distribution, for normal and non-normal variables, respectively. A one-sample t-test was used to determine the significance of gene copy alterations. For variables with greater than two dependent values, a one-way analysis of variance (ANOVA) was used. Pearson correlation was used to compare matched continuous variables. Survival data were assessed by Kaplan-Meier methods and differences were compared by the Mantel-Cox test. Statistical analyses were performed using GraphPad Prism 7. P-values < 0.05 were considered as statistically significant.

RESULTS

Nineteen ACC and matched normal samples that were previously subjected to WES (19) were analyzed for gene copy alterations at the IGF1 and IGF2 loci. The IGF1 gene was found significantly amplified, ranking in the top 10% of all the genes determined to be amplified, with nine samples demonstrating a gain of at least one gene copy (p<0.05, Figure 1A). In contrast, the IGF2 locus was not significantly amplified, with only two samples demonstrating gene copy gains. Analysis of the TCGA database also demonstrated the IGF1 gene to be significantly amplified (p<0.05, Figure 1B), with mostly a copy-neutral status observed for the IGF2 gene locus.

A total of 33 cases of ACC were analyzed for IGF1 and IGF2 gene expression using RT- qPCR methods. RNA from three samples did not yield reliable amounts of cDNA for either IGF1 or IGF2 determination and were excluded from further analysis wherever applicable. Ten samples demonstrated relative increase in expression of IGF1 compared to normal adrenal tissue, with 5 cases demonstrating greater than a two-fold increase in expression levels. Overall relative expression levels were 2.2 times higher compared to normal adrenal tissue (Figure 2A). This finding is in contrast with two previous reports that demonstrated unaltered IGF1 expression in ACC tumors (23, 24). IGF2 expression was in accordance with previous findings and showed an average overall relative higher expression greater than one hundred fold. Only five samples in this cohort demonstrated decreased IGF2 expression (Figure 2B). Analysis of TCGA RNA sequence data showed that the majority of IGF1 amplifications occurred in samples with IGF1 mRNA overexpression (p<0.05, Figure 3A), while similar results were not shown for IGF2 (Figure 3B).

IGF expression patterns were assessed for correlation with tumor characteristics including gender, age, tumor size, stage, hormonal status, and survival. Increased IGF1 expression was found to be associated with non-functional tumors (p<0.05, Table 1), with nearly an 8-fold increase in relative expression compared to hormonally active tumors. IGF1 expression was also 7 times higher in early stage (ENSAT I-II) tumors (p<0.05, Table 1). In contrast, IGF2 expression was not associated with either hormone status or stage, but did correlate with tumor weight (p<0.05, Table 1) and size (p=0.08, Table 1). Increased IGF1 expression was found also to be associated with SLC1247 overexpression (p<0.05, Figure 4A), but no similar correlation was observed with IGF2 (Figure 4B). It should be noted that previous studies have analyzed the

prognosis of IGF expression in ACC, but those findings were contradictory, most likely limited by their overall small cohort sizes (25, 26).

Previous studies have shown a role for IGF in promoting SLC12A7-mediated invasive activity in ovarian cancer cells, potentially through IGF1-induced SLC12A7 trafficking (27). However, a SLC12A7 transcriptional regulatory role for the IGF system or vice versa, has not been reported in ACC. To explore the possibility of an IGF signaling-mediated paracrine/autocrine loop for modulating SLC12A7 expression and thereby a more aggressive behavior in adrenal cancer, ACC cell line SW-13 with low endogenous SLC12A7 expression (17) was treated with recombinant IGF1 and IGF2 for varying time intervals. No significant effect was seen on SLC1247 mRNA expression (data not shown) by exogenous IGF1/2 treatments. On the other hand, SW-13 cells with low endogenous expression of SLC1247 (17), were shown to have low expression of IGF1 and IGFIR, while NCI-H295R cells, which have relatively high endogenous expression of SLC1247 (17), were shown to have a 93-fold and 13- fold increase in expression of IGF1 and IGFIR (Figure 5), respectively. Moreover, RNAi- silencing of SLC1247 in NCI-H295R cells demonstrated nearly a 50% decrease in expression of IGF1 (Figure 6A). Western blot analysis further demonstrated decreased protein expression of IGF1 and IGF1R (Figure 6B) with RNAi-silencing, suggesting an IGF1-SLC12A7 circuitry in ACC development.

Previous studies have demonstrated that IGF1 binding of IGF1R causes IGF1R autophosphorylation and downstream signal transduction, ultimately promoting cellular growth, transformation, and anti-apoptotic properties (28). To determine whether increased IGF1 expression in ACC promotes IGF1R activation, representative samples were tested for IGF1R phosphorylation using immunohistochemical techniques. As shown in Figures 7 and 8, samples

with low IGF1 expression (Figure 8A and 8B) demonstrated minimal expression of phosphorylated IGF1R, while samples with high IGF1 expression showed increased expression of phosphorylated IGF1R (Figure 8B and 8C). These results suggest that IGF1 overexpression observed in subsets of ACC tumors may promote tumor formation or progression, in part, through IGF1 specific IGF1R activation.

DISCUSSION

Multiple comprehensive next-generation sequencing studies underscored the diversity of genetic and signaling aberrations, including widely reported IGF dysregulation, in promoting adrenocortical carcinogenesis (19, 20, 29). While IGF2 overexpression has been previously associated with alterations in gene methylation levels, it has been unclear what changes, if any, affected the IGF1 locus. Here we show that the IGF1 gene copy number is significantly affected by gene amplifications in the Yale/Karolinska study cohort, supported further by another independent cohort (TCGA) with similar levels of gene copy alterations measured. These findings again highlight that gene copy number variations may play a significant role in ACC tumorigenesis and that additional studies are warranted to investigate the potential contributions of CNVs in the global signaling dysregulations generally noted in ACCs.

Recent studies also showed a clear role for dysregulated potassium-chloride channel protein SLC12A7 expression in conferring an aggressive phenotype to ACC (16, 17). However, a potential role for dysregulated IGF signaling in modulating SLC12A7-promoted tumor behavior seen in other cancer types, has not been reported in ACC. This potential relationship is investigated in this study using a large cohort of ACC patient samples and two widely studied established ACC cell lines. Here we report increased expression of either IGF1 (16% of samples) or IGF2 (83% of samples). Although two previous studies reported absence of overexpression

of IGF1 mRNA transcripts (23, 24) in ACC, we found relatively higher IGF1 expression in 5 of 33 samples of ACC, similar to an early 1991 study that showed IGF1 overexpression in ACC using immunohistochemical techniques (14). The disagreement in IGF1 expression profiles reported by these studies could be related to the quasi-quantitative methods used and/or the relatively low numbers of samples analyzed in those studies. Furthermore, it remains unclear whether uncharacterized post-transcriptional and/or post-translational mechanisms are also involved in modulating the expression patterns of IGFs in ACC.

The significant association between IGF1 overexpression and the non-functional status of the tumors found in this study could be associated with SLC12A7 overexpression, a potassium and chloride ion channel. Related ion channel dysregulation has recently been shown to alter hormone secretion in benign adrenal tumors (30, 31), however further investigation would be needed to determine the potential causal relationship observed in malignant ACCs. Moreover, analogous relationships between IGF1 signaling and SLC12A7 has been previously reported in multiple cancer types (18, 32).

Previous studies have also demonstrated that IGF1 stimulates recruitment of SLC12A7 from the cytosolic pool to the cell membrane with associated increase in activity of KCC related ion channels including SLC12A7 (32). IGF stimulation alone may not be the consummate cause of SLC12A7 overexpression in ACC, but may support its function by facilitating its transportation at the cell membrane. Indeed, it has been previously shown that ACC cells overexpressing SLC12A7 localize preferentially at the leading edge of cell extensions (17).

Similar to previous reports, we have shown overexpression of IGF2 in nearly all tumors tested, which has been consistently shown to be a molecular hallmark of ACC (9). Comprehensive genetic analyses also have shown IGF2 overexpression to be present in all

genetic subtypes of ACC (20), possibly indicating that IGF2 signally does not represent a unique molecular signature that applies to a subset of adrenal tumors, but rather a global signaling event complementing to a variety of driving events in adrenal tumorigenesis, such as CTNNB1 and TP53 mutations. Although we observed higher IGF2 expression in larger tumors, it did not portend any effect on survival. This could be due, in part, to the limited number of stage 1 tumors in our cohort, or the mere bulk of the tumor tissue synchronously producing IGFs.

Despite a plethora of in vitro and in vivo data indicating the IGF system to be a promising target in ACC treatment, a recent phase 3 clinical trial inhibiting IGF1R signaling with Linsitinib (OSI-906) failed to show a survival benefit compared to the placebo control and was stopped early because of a lack of response (33). However, an earlier study targeting IGF1R and mammalian target of rapamycin (mTOR) simultaneously demonstrated stable disease in 11 of 26 patients (34). These findings suggest that targeting IFG signaling alone may not be sufficient in improving treatment outcomes, but could prove to be helpful in combination with other targets such as dysregulated ion channels as suggested in this study.

CONCLUSION

Here we show that increased IGF1 stimulation is correlated with SLC12A7 overexpression in non-functional, early stage ACCs, suggesting a targeted SLC12A7/IGF1 therapeutic opportunity for the subset of ACCs profiled for their co-expression.

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Table 1. Association of Insulin-Like Growth Factor Expression Levels and Patient Characteristics
Characteristic	Data (n=33)	IGF1 expression p value	IGF2 expression p value
Sex, n		0.43	0.20
Male	12
Female	21
Age, y, mean±SD	57.6±13.8	0.56	0.28
Tumor size
Diameter, cm, mean±SD	13.0±4.4	0.40	0.08
Weight, g, mean±SD	755 ±667.1	0.95	0.01*
ENSAT stage, n		0.01*	0.71
I-II	17
III-IV	16
Hormone status, n		0.01*	0.78
Aldosterone	1
Cortisol	8
Androgen	3
Multi-secretingt	5
Non-functional	12
Unknown	4
Outcome, n		0.26	0.74
Alive, no recurrence	10
Alive, recurrent	3
Death from disease	16
Death other causes	4

*Statistically significant

* Tumors secreting two or more of the following hormones: aldosterone, cortisol, testosterone, or dehydroepiandrosterone.

ENSAT, European Network for the Study of Adrenal Tumors

Figure legend

Figure 1: Insulin-like growth factor (IGF) gene copy analysis. (A) Nineteen samples that previously underwent whole exome sequencing (19), were analyzed for gene copy alterations at the IGF1 and IGF2 loci. The IGF1 gene was significantly amplified, ranking in the top 10% of all genes amplified using GISTIC2 analysis (*, p<0.05; 2N, diploid). Average value (base-2 logarithmic scale) is noted above each gene analyzed. CNV; copy number variation. GISTIC2; genomic identification of significant targets in cancer version 2.0. IGF; insulin-like growth factor. Horizontal bar, mean; Error bars, standard deviation. (B) Analysis of the TCGA ACC cohort database also demonstrated the IGF1 gene to be significantly amplified, with minimal alterations observed in the IGF2 gene (*, p<0.05; 2N, diploid). Horizontal bar, mean; Error bars, standard deviation.

Figure 2: Insulin-like growth factor (IGF) gene expression analysis. (A) Relative messenger RNA expression levels of IGF1 in adrenocortical carcinoma (ACC) samples (n=31) were measured by real-time quantitative polymerase chain reaction (RT-qPCR) and compared with expression levels in normal adrenal tissues (n=10). Five samples demonstrated a > 2-fold increase in expression levels. Horizontal bar, mean; Error bars, standard deviation. (B) Relative messenger RNA expression levels of IGF2 in ACC samples (n=30) were measured by RT-qPCR and compared with expression levels in normal adrenal tissues (n=10). Twenty-five of 30 samples demonstrated a > 2-fold increase in expression levels. Horizontal bar, mean; Error bars, standard deviation.

Figure 3: Insulin-like growth factor gene expression analysis of The Cancer Genome Atlas (TCGA) adrenocortical carcinoma cohort. (A) IGF1 expression levels were determined in the TCGA database and compared to TCGA IGF1 gene copy alterations. Overall, higher gene

expression was observed in samples with IGF1 gene amplifications (*, p<0.05). IGF; insulin-like growth factor. Horizontal bar, mean; Error bars, standard deviation. (B) IGF2 expression levels were also determined and compared to IGF2 gene copy alterations. No difference in expression levels was observed between IGF2-non-amplified and amplified genes. IGF; insulin-like growth factor. Horizontal bar, mean; Error bars, standard deviation.

Figure 4: Insulin-like growth factor 1 and SLC12A7 Expression in adrenocortical carcinoma. (A) IGF1 expression levels were assessed for possible correlation with SLC1247 expression, which was previously determined (16). Normal adrenal tissue served as reference to calculate relative expression levels. Overexpression of IGF1 and SLC1247 overexpression was correlated (*, p<0.05). IGF; insulin-like growth factor. SLC12A7; Solute Carrier Family 12 Member 7. Horizontal bar, mean; Error bars, range. (B) A similar association was not observed with IGF2 and SLC1247 expression levels. Horizontal bar, mean; Error bars, range.

Figure 5: Insulin-like growth factor 1 (IGF1) and IGF1 receptor and SLC12A7 expression in vitro. (A) IGF1 expression levels were measured in adrenocortical carcinoma (ACC) lines SW- 13 and NCI-H295R. SW-13 cells, with low endogenous expression of SLC1247 (17), were shown to have low expression of IGF1 as well, while NCI-H295R cells with high endogenous expression of SLC1247 (17) were shown to have a 93-fold increase in expression of IGF1. IGFIR expression was also found to be higher in NCI-H295R cells compared to SW-13 cells. SW-13 cells served as reference tissue to calculate relative expression levels. IGF; insulin-like growth factor. SLC12A7; Solute Carrier Family 12 Member 7. Horizontal bar, mean; Error bars, standard deviation.

Figure 6: Insulin-like growth factor 1 expression in adrenocortical carcinoma (ACC) cells silenced for SLC12A7 expression. (A) NCI-H295R cells were subjected to SLC1247 RNAi gene

silencing, and IGF1 gene expression levels were measured. IGF1 expression levels were lower in NCI-H295R cells compared to non-transfected cells (lipofectamine only, p<0.05) and cells transfected with scrambled siRNA (p=0.09). IGF; insulin-like growth factor. Horizontal bar, mean; Error bars, standard deviation. (B) NCI-H295R subjected to SLC12A7 RNAi gene silencing also demonstrated decrease protein expression of IGF1 (upper panel) and IGF1R (lower panel) at 36 and 48 hours post-transfection respectively, as determined by Western immunobloting. Beta-actin expression is used as protein loading control. SCRN; scrambled RNA.

Figure 7: Insulin-like growth factor 1 (IGF1) expression of selected adrenocortical carcinoma (ACC) samples. Samples 10 and 11 have relatively low expression of IGF1 compared to normal adrenal tissue, while samples 12 and 33 both have a greater than 10-fold increase in expression. Figure 8. Insulin-like growth factor 1 receptor (phosphoY1161) immunoreactivity in adrenocortical carcinoma (ACC) samples. All photomicrographs are magnified x400. (A-B) ACC cases 10 and 11 respectively displaying negative immunoreactivity. (C) ACC case 12 displaying regional nuclear and cytoplasmic immunoreactivity, whereas other areas of the same tumor displayed only weak expression (data not shown). (D) ACC case 33 displaying cytoplasmic immunoreactivity and absence of nuclear staining. This pattern was heterogenous, with adjacent areas displaying negative staining (data not shown). (E) Positive control (colon mucosa). (F) Omission of the primary antibody for each tissue (as represented here for ACC case 1559) served as negative control.

Precis

This study analyzes the role of insulin-like growth factor (IGF) signaling in adrenocortical carcinoma. Our results demonstrate that IGF1 overexpression caused, in part, by gene copy amplification, is associated with non-functional, early stage tumors as well as aberrant expression of the metastasis promoting gene SL12AC7.

ACCEPTED MANUS CRIPTS

0.26

0.63

0.08

1.5-

GISTIC2 Copy Number

1.0-

0.5-

0.0

-0.5

AVG CNV

IGF1 CNV

IGF2 CNV

GISTIC2 copy number (mean +/- SD)

0.72

-0.1

›

-1

IGF1

IGF2

ACCEPTED MAN

20-

Relative IGF1 Gene Expression

Normal (n=10)

ACC (n=31)

600-

Relative IGF2 Gene Expression

400-

200-

Normal (n=10)

ACC (n=30)

ACCEPTED MAN

log2(counts +1) [mean +/- SD]

IGF1 copy gain

no IGF1 copy gain

log2(counts +1) [mean +/- SD]

10.

IGF2 copy gain

no IGF2 copy gain

ACCEPTED MANUS

ACCEPTED MANUSCRIPT

40- 35-

30- 25

20 5

Relative IGF1 Expression Compared to Normal Tissue

Normal Tumor SLC12A7 Exp (n=16)

High Tumor SLC12A7 Exp (n=14)

600-

Relative IGF2 Expresion Compared to Normal Tissue

400-

200-

Normal Tumor SLC12A7 Exp (n=16)

High Tumor SLC12A7 Exp (n=13)

ACCEPTED MANU

150

100

Relative Gene Expression Compared to Normal Tissue

SW-13 IGF1

NCI-H295R IGF1

SW-13 IGF1R

NCI-H295R IGF1R

Relative IGF1 Expression

1.0-

0.5-

0.0

Lipofectamine

Scramble SİRNA

SLC12A7 SİRNA

IGF1

B-Actin

IGF1R

B-Actin

LIPO

SCRN

Si-SLC12A7

ACCEPTED MAN

Normal

Sample 10

Sample 11

Sample 12

sample 33

0.00

0.05

0.10

Relative IGF1 Gene Expression

ACCEPTED MANUSC