Preclinical Targeting of the Type I Insulin-Like Growth Factor Receptor in Adrenocortical Carcinoma
Ferdous M. Barlaskar, Aaron C. Spalding, Joanne H. Heaton, Rork Kuick, Alex C. Kim, Dafydd G. Thomas, Thomas J. Giordano, Edgar Ben-Josef, and Gary D. Hammer
Department of Internal Medicine-Division of Metabolism, Endocrinology, and Diabetes (F.M.B., J.H.H., A.C.K., G.D.H.), Departments of Radiation Oncology (A.C.S., E.B .- J.) and Department of Pathology (D.G.T., T.J.G.) and Biostatistics Core, Comprehensive Cancer Center (R.K.), University of Michigan Medical School, Ann Arbor, Michigan 48109
Context: Drug therapy for adrenocortical carcinoma (ACC), a rare and lethal malignancy, is largely empirical and ineffective. New treatments directed at molecular targets critical to the pathophys- iology of ACC may prove more efficacious.
Objective: The objective of the study was to profile human adrenal tumors and ACC cell lines to assess activated IGF signaling and determine the efficacy of two IGF receptor (IGF-1R) antagonists alone and in combination with mitotane.
Experimental Design: ACC cell lines that display or lack activated IGF signaling are used to assess the effects of two IGF-1R antagonists in cultured cells and ACC xenograft tumors.
Results: Transcriptional profiling data derived from DNA microarray analysis of human adrenal tumors implicate /GF2 as the single highest up-regulated transcript in the vast majority of carci- nomas. We show that the majority of ACC cell lines tested display constitutive IGF ligand production and activation of downstream effector pathways. Both IGF-1R antagonists cause significant dose- dependent growth inhibition in ACC cell lines. Furthermore, we observe that mitotane, the first- line adrenolytic drug used in patients with ACC, results in enhanced growth inhibition when used in combination with the IGF-1R antagonists. We next examined the activity of IGF-1R antagonists against ACC xenografts in athymic nude mice. IGF inhibition markedly reduced tumor growth greater than that observed with mitotane treatment, and combination therapy with mitotane significantly enhanced tumor growth suppression.
Conclusion: These findings establish a critical role of IGF signaling in ACC pathophysiology and provide rationale for use of targeted IGF-1R antagonists to treat adrenocortical carcinoma in future clinical trials. (J Clin Endocrinol Metab 94: 204-212, 2009)
A drenocortical carcinoma (ACC) is a rare endocrine malig- nancy characterized by a limited understanding of its de- velopment and pathophysiology, dismal clinical prognosis, and lack of efficacious therapeutic regimens. The annual incidence of ACC ranges from 0.5 to 2 cases per million (1). Whereas com- plete operative resection remains the only potentially curative option for ACC, approximately half of all patients present with metastatic disease (1, 2). This results in a 5-yr survival rate of less than 10% (1, 3). A better understanding of the etiology and pathogenesis of this devastating disease could lead to more ef-
fective drug designs and the development of molecularly targeted treatments.
ACC’s association with a select number of genetic syndromes such as Beckwith-Wiedemann syndrome (BWS) has provided insights into its pathophysiology. BWS arises from a loss of het- erozygosity and/or a loss of imprinting of the 11p15.5 chromo- somal region. This locus includes the mitogenic hormone, IGF-2 gene (IGF2), and locus dysregulation results in significant over- expression of this gene. Transcriptional profiling of sporadic ACC tissues provides additional support for this hormone’s
pathogenic role. We and others have shown IGF2 as the single most up-regulated transcript in 80-90% of ACCs (4-6). IGF-II mainly elicits its cellular effects through the ubiquitously ex- pressed type 1 IGF receptor (IGF-1R). Importantly, human ACCs also exhibit elevated levels of IGF-1R mRNA and protein (7). Taken together, these observations suggest that activation of the IGF pathway is a common pathological mechanism used by tumor cells during adrenocortical tumorigenesis.
In this study, we analyzed a large series of benign and malig- nant human adrenal tumors and a panel of ACC cell lines to confirm enhanced IGF signaling in ACCs. We used a small mol- ecule inhibitor (NVP-AEW541) and a fully human monoclonal antibody (IMC-A12), both targeting IGF-1R, to demonstrate specific abrogation of IGF-mediated signaling and concomitant inhibition of proliferation. Only ACC lines with increased IGF signaling responded to both agents. Synergistic antiproliferative effects were observed when IGF-1R inhibition was combined with mitotane in culture. In vivo, both IGF-1R antagonists mark- edly attenuated human ACC xenograft growth in athymic nude mice. Moreover, IGF inhibition combined with mitotane signif- icantly enhanced single agent tumor growth inhibition. Our re- sults validate IGF-1R as an important target in ACC and provide rationale for the testing of IGF-1R antagonists as a promising therapeutic agent in clinical trials.
Materials and Methods
Reagents
IMC-A12 was provided by ImClone Systems (New York, NY) (8). NVP-AEW541 was provided by Novartis (Basel, Switzerland) (9). Re- combinant human IGF-I and IGF-II ligands were from Peprotech (Rocky Hill, NJ). Mitotane, lectin-fluorescein isothiocyanate (FITC), and anti- B-actin were from Sigma (St. Louis, MO). Anti-IGF-1Rß was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), whereas anti-Akt and anti-phospho-AktSer473 were from Cell Signaling Technology (Danvers, MA). Anti-phospho-tyrosine (4G10) was from Millipore Bioscience (Billerica, MA).
Cell lines and cell culture
All standard cell culture reagents were purchased from Invitrogen Life Technologies (Carlsbad, CA). The cell lines NCI-H295 (10), Y1 (11), and SW13 (12) were obtained from American Type Culture Col- lection (Manassas, VA). The RL251 (13) cell line was previously de- scribed and generously provided by Dr. David Schteingart (University of Michigan, Ann Arbor, MI), whereas the ST5 (14) cell line was generated and provided by Dr. Synthia Mellon (University of California, San Fran- cisco, San Francisco, CA).
Cell proliferation assay
Cells were seeded on 96-well plates with increasing concentrations of the compounds added in triplicate wells. Incubation continued for 72 h (for NVP-AEW541 treatment) or 7 d (for IMC-A12 treatment) and then an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul- fophenyl)-2H-tetrazolium (MTS) assay (Promega Corp., Madison, WI) was performed according to the manufacturer’s protocol.
Immunoblotting, immunohistochemistry, and RT-PCR
Assays were performed as described previously (15) with primer se- quences located in supplementary Table 1, published as supplemental
data on The Endocrine Society’s Journals Online Web site at http:// jcem.endojournals.org.
Mice
Animal studies were performed in accordance to an institutionally approved protocol under the auspice of the University Committee on Use and Care of Animals at the University of Michigan. Tumor xenografts were established by sc injection of 1 × 106 RL251 or 2.5 × 106 H295 cells into flank regions of 4- to 6-wk-old female athymic mice. Animals were treated with IMC-A12 (8) or NVP-AEW541 (9). Mitotane was refor- mulated in 10% Tween 80/0.9% normal saline and administered by ip injection once daily at 300 mg/kg.
DNA microarray analysis
The full details of the DNA microarray analysis will be reported separately (manuscript in press). The Affymetrix array data have already been deposited into Gene Expression Omnibus as series GSE10927.
Human adrenal tissue microarray
An adrenal-specific tissue array was constructed using human for- malin-fixed, paraffin-embedded tissues that correspond to the tissues used for DNA microarray analysis and was stained with either phospho- AktSer473 (Invitrogen Life Technologies, Carlsbad, CA) or phospho-IGF- 1RTyr1158/62/63 (Millipore). Immunoreactivity was scored blindly by a four-tier [negative, low (1+), medium (2+), and high (3+) positive] grading scheme.
Statistical analysis
Tests between pairs of groups were performed with two-sided, two- sample t tests. For in vivo and in vitro data testing combinations of agents, two-way ANOVA models were used to test differences in cell viability or tumor size between difference combinations of agents and test for interactions. We also used Calcusyn software to determine com- bination indices with in vitro mitotane and NVP-AEW541 MTS assay.
Results
Expression profile of IGF2 and downstream signaling in human ACC tissues
Using DNA microarray technology, we analyzed human tis- sues derived from normal adrenal cortex, adrenocortical adeno- mas (ACAs), and ACCs to reveal gene expression profiles (manu- script in press). From these data, we specifically examined the 11p15.5 chromosomal region in which locus dysregulation has been associated with adrenocortical cancers (Fig. 1A). The vast majority of ACCs display overexpression of IGF2 gene tran- scripts, whereas the H19 [a micro-RNA negatively regulating IGF2 expression (16, 17)] and CDKN1C (encoding the cell cycle dependent kinase inhibitor, p57kip2) genes are down-regulated, suggesting an imprinting defect or loss of heterozygosity of this chromosomal region, similar to that commonly observed in BWS. To validate these microarray results, quantitative RT-PCR was performed on RNA isolated from three randomly selected ACAs and three ACCs (Fig. 1B). We found a greater than 60-fold increase of IGF2 transcripts in all three ACC samples when com- pared with IGF2 levels in ACA samples. Further analysis of ac- tive IGF signaling with these six human tumor samples was per- formed by immunoblotting for levels of total IGF-1R protein and phosphorylated AktSer473, a downstream mediator of active IGF signaling (Fig. 1C). Expression of IGF-1R was observed in all six
A
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ACA
ACC
H19_224646_x_at
H19_224997_x_at
IGF2_210881_s_at
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CDKN1C_213182_x_at
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Fold change from the median
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Relative IGF-II Expression
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IGF-IRß
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p-IGF-IR
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tissues, whereas two ACC samples possessed far greater levels of the receptor. Immunoblotting for phospho-AktSer473 suggested active IGF signaling in all three ACC samples and in only one ACA sample. To further validate the observation that IGF-me- diated signaling was specifically increased in ACC compared with normal and adenomatous samples, tissue microarray slides containing 24 ACC, 22 ACA, and four normal adrenals were stained for phospho-IGF-1R and phospho-AktSer473 (Fig. 1D). A marked increase in signal intensity of phospho-IGF-1R and phospho-AktSer473 was observed in ACC samples compared with ACA and normal adrenal tissue, represented here as a shift in the percentage of ACC samples with high intensity staining for these activated proteins. In summary, molecular profiling of hu- man adrenal tumors demonstrated overexpression of two critical components (IGF2 and IGF-1R) of the IGF signaling cascade and concomitant activation of the downstream effector, Akt. These results are consistent with the IGF pathway playing a critical role in ACC pathogenesis.
Expression of IGF pathway members and downstream signaling in ACC cell lines
To begin to determine the biologic relevance of IGF-1R in ACC, we first examined the endogenous expression profiles of IGF ligands, IGF-1R, and downstream effectors of IGF-mediated signaling in five ACC cell lines (Fig. 2). We chose two mouse lines (Y1 and ST5) and three human lines derived from invasive pri- mary adrenocortical carcinomas (NCI-H295, SW13, and RL251). Using RNA purified from cells maintained in serum-free or serum-containing media, RT-PCR was performed to detect IGF1 and IGF2 gene expression. The two mouse cell lines ex- pressed IGF1, whereas the H295 cells expressed IGF2 transcript, in agreement with a previous study describing an autocrine role of IGF-II in H295 cells (18). To assess protein expression, IGF- 1RB was immunoprecipitated from whole-cell lysates followed by immunoblot analysis. As shown in Fig. 2, we observed only minimal levels of IGF-1R protein in RL251 cells compared with the four other cell lines. However, there was also variable protein
Y1
ST5
H295
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RL251
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expression between the four IGF-1R-positive cell lines. Immu- noprecipitates were also probed for phospho-tyrosine and dem- onstrated active signaling in all cell lines except RL251. Pathway activation was additionally confirmed by immunoblotting for phospho-AktSer473. Immunoblots for total protein levels of Akt were used as a loading control. The H295 cell line emerged as the most efficient in vitro recapitulation of ACC, as assessed by its constitutive overexpression of IGF-II and active IGF signaling. Therefore, this was the cell line of choice for further experiments, although other ACC cell lines were used to confirm all initial results obtained from H295 experiments.
Inhibition of IGF signaling by IGF-1R antagonists
To assess the ability of both IGF antagonists (IMC-A12 and NVP-AEW541) to specifically inhibit IGF-II/IGF-1R-mediated signaling, log-phase H295 cells were subjected to increasing amounts of IMC-A12 or NVP-AEW541 in the presence of 10 nM IGF-I/ IGF-II (Fig. 3). Immunoblotting of cell lysates incubated with increasing concentrations of IMC-A12 resulted in a dose- dependent reduction of IGF-1R levels, with an approximately 80% receptor decrease achieved at 100 nM concentrations of antibody (Fig. 3A). This decrease may be attributed to antibody- mediated receptor internalization and degradation, a phenom- enon not seen when IGF ligands are incubated alone (8). IMC- A12 attenuated phospho-AktSer473 in a dose-dependent manner (100 nM treatment resulted in ~70% decrease in the ratio of p-Akt to Akt band intensities over controls), indicating targeted decrease of IGF signaling. We also found NVP-AEW541 inhib- ited IGF-1R signaling in H295 cells (Fig. 3B). At 5 µM NVP- AEW541 concentrations, receptor phospho-tyrosine levels were undetectable and phospho-AktSer473 levels were also completely abrogated. No change in total IGF-1R or Akt levels was observed with treatment. Taken together, these results indicate that both pharmacologic agents are able to specifically target and inhibit IGF-mediated signaling in ACC cell lines.
IGF-1R antagonists inhibit proliferation of ACC cell lines
IGF signaling regulates several aspects of cellular function including the enhancement of proliferation. To test the func- tional consequence of IGF-1R inhibition, MTS proliferation as- says were performed on H295 cells treated with IMC-A12 or NVP-AEW541 (Fig.4). We used the RL251 cell line as a negative control in these assays because it lacks expression of IGF-1R
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IMC-A12 [nM]
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Proliferation (%)
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Proliferation (%)
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IMC-A12 Concentration [nM]
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protein in comparison with other ACC lines. As predicted, in- creasing doses of IMC-A12 had no substantial effect on the RL251 cells, whereas high concentrations of the antibody ex- hibited antiproliferative effects on H295 cells (Fig. 4, left panel). The IMC-A12 IC50 value for the H295 cells was 90 nM, but 50% inhibition was not achieved with RL251 cells, even at the highest IMC-A12 dose (P < 0.001 comparing RL251 vs. H295 at 100 nM A12). The NVP-AEW541 IC50 value for H295 cells was 2.7 UM, whereas for RL251 cells, the IC50 was 36 µM (P < 0.001 com- paring RL251 vs. H295 at 3 µM NVP-AEW541), again demon- strating targeted suppression of the H295 cell line’s ability to proliferate due to the presence of IGF-1R.
Targeting of IGF-1R delays tumor xenograft growth
Stemming from our data revealing IGF-1R inhibition results in a significant decrease of downstream signaling and proliferation in vitro, we assessed its in vivo activity using human ACC cell lines. Using the rationale discussed above, we used the RL251 cell line as an additional control in these assays. H295 or RL251 cells were inoculated sc into athymic nude mice, and the mice were subse- quently randomized into three treatment groups (n = 8 tumors per group for H295 and n = 10 for RL251): placebo, IMC-A12 treatment, and NVP-AEW541 treatment. Tumor volume mea- surements were taken three times a week for 21 d. The data were plotted as the log ratios of tumor size over initial tumor size to more accurately reflect the percent reduction in tumor size and, at the same time, account for the variable initial tumor sizes in each mouse (Fig. 5A). RL251 xenografts had a higher tumor engraftment rate than H295 xenografts (94 vs. 75%, data not shown). IMC-A12 was well tolerated in treated mice with no substantial adverse effects or weight changes observed between groups (data not shown). The treatment of H295 tumors with IMC-A12 over a 21-d span (Fig. 5A, left panel) resulted in a significant reduction in tumor size over untreated controls (P < 0.001 from day 7 onwards). Treatment in RL251 xenografts (Fig. 5A, right panel) resulted in a statistically insignificant re- duction in tumor volume at each time point, underscoring the specificity of IMC-A12. Tests comparing the tumor growth in both cell lines showed that IMC-A12 reduced H295 tumor vol- umes (compared with tumors receiving no treatment) signifi-
cantly more than for RL251 xenografts (P = 0.017). Whereas H295 xenografts treated with NVP-AEW541 were similar in histological appearance to those treated with IMC-A12 and ex- hibited a 34% reduction in tumor burden over controls, this decrease fell short of being statistically significant (P = 0.086) (supplementary Fig. 2, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem. endojournals.org). NVP-AEW541 treatment of mice harboring RL251 xenografts also had an insignificant reduction in tumor volume (P = 0.57). NVP-AEW541 was well tolerated in treated mice with no substantial adverse effects or weight changes ob- served during treatment (data not shown).
To determine whether blocking IGF-1R resulted in decreased IGF signaling in vivo, H295 xenografts were subjected to im- munoblot analysis (Fig. 5B). Lysates prepared from two separate tumors from control and IMC-A12-treated mice were immuno- blotted for Akt and phospho-AktSer473 levels. Although total Akt expression levels were similar in both treatment arms, phospho- AktSer473 levels were decreased in the tumors of mice treated with IMC-A12, thus revealing a targeted decrease in IGF signaling. Taken together, these results indicate in vivo IGF-1R inhibition caused significant and targeted tumor growth delay of human ACC xenografts and underscores the importance of IGF signal- ing in ACC pathophysiology.
Inhibition of IGF-1R enhances the inhibitory effects of mitotane
Mitotane is the standard of care for treatment of ACC because of its specific cytotoxic effects in cortical cells. To evaluate whether this response could be enhanced by additional IGF inhibition, we investigated the effect of combining mitotane with IGF antagonists in culture and in vivo tumor growth. First, MTS proliferation assays were performed on H295 and RL251 cells with increasing concen- trations of NVP-AEW541 and mitotane (Fig. 6A). Whereas both H295 and RL251 cells exhibited a dose-dependent reduction in proliferation when incubated with mitotane (no NVP-AEW541), only H295 cells demonstrated a further decrease in growth when incubated with mitotane and NVP-AEW541. Individually, 30 AM mitotane and 3 AM NVP-AEW541 reduced cell viability to 85 and 77% of control levels, respectively. An additive model for the com-
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binatorial effects of the two agents would predict about 65% cell viability compared with untreated cells. However, we observed just 9% cell viability for this combination compared with no treatment, which was significantly less than expected under the additive model (P = 4 × 10-15), indicating significant synergy (interaction) be- tween the two drugs. Analysis of these data using Calcusyn software to graphically quantitate combined drug effects also confirmed a synergistic effect of mitotane and NVP-AEW541 in vitro (supple- mentary Fig. 3).
To evaluate this effect in vivo, H295 xenografted nude mice were treated with mitotane or IMC-A12 either as single agents or in combination (n = 20 tumors per treatment group) (Fig. 6B). Treatment with antibody alone resulted in a 51% reduction in tumor size (P < 0.001), comparable with the 55% reduction observed in the monotherapy experiment detailed in Fig. 5A. Mitotane treatment as a single agent was not as effective as IMC- A12 but resulted in a 25% decrease in tumor volume (P = 0.035). The combination of IMC-A12 and mitotane resulted in a 70% decrease in tumor burden over untreated controls (P < 0.001). Although the combined treatment induced more than an additive effect predicted with mitotane and IMC-A12 (as calculated at d 21 of treatment), this difference was not sufficient to demon- strate synergy between these two compounds (P = 0.22, two sided F test). IMC-A12, mitotane, and their combination were well tolerated in treated mice with no substantial adverse effects or weight changes observed between groups (data not shown). After 21 d, xenograft specimens were collected for histochemical
analysis (Fig. 6C). Interestingly, hematoxylin and eosin staining revealed treatment results in a clear decrease in vascularity com- pared with control sections.
We investigated this further by immunohistochemically eval- uating microvessel density using a FITC-conjugated lectin mol- ecule that binds specifically to endothelial cells. Dense areas of lectin-positive endothelial cells are observed throughout control xenograft sections, but IMC-A12-treated xenograft sections dis- play a moderate decrease in endothelial cell density. Moreover, the greatest histologic decrease was observed in xenografts treated with the combination of IMC-A12 and mitotane. We hypothesized IGF inhibition may reduce the angiogenic potential imparted by the expression of IGF-II via induction of vascular endothelial growth factor (VEGF) expression, seen in other sys- tems (19, 20). Therefore, we generated cDNA from xenografts for quantitative RT-PCR to assess human VEGF expression us- ing PCR primers designed to recognize all four isoforms of VEGF transcripts (21) (Fig. 6D). Results demonstrated a 1.6-fold de- crease in human VEGF expression in IMC-A12-treated samples (P = 0.044) and a 2.2-fold decrease when antibody was combined with mitotane (P = 0.008). Mitotane treatment alone resulted in a small and statistically insignificant decrease (P = 0.46). Thus, inhibition of IGF signaling is the major factor causing the de- creased VEGF transcript levels in these tumors. These results, taken together, demonstrate that IGF signaling inhibition with IMC-A12 leads to a larger suppression of ACC tumor growth in comparison with mitotane and that combining both agents can
A
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RL251 Proliferation (%)
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specifically decrease ACC xenograft growth greater than either agent alone.
Discussion
Current therapy for adrenocortical cancer consists of surgical resection and adjuvant mitotane treatment or, in nonresectable
disease, mitotane in combination with other cytotoxic chemo- therapies (22). A recent retrospective study evaluating the effi- cacy of mitotane after radical resection in adrenocortical cancer revealed that mitotane may indeed prolong recurrence-free sur- vival to 2-3 times that of untreated patients (23, 24). However, mitotane, a structural isomer of the pesticide dichlorodiphenyl- trichloroethane, is associated with significant toxicity due to the high doses required for its adrenolytic effects. Unfortunately, a
large number of ACC patients present with metastatic disease, which typically precludes surgery and carries a dismal prognosis (25). These outcomes emphasize the need for new treatment strategies for this fatal disease with therapies predicated on iden- tification of specific pathways critical for ACC pathogenesis.
The adrenal gland requires dense vascularization to allow access of hormonal control from higher-order structures and for secretion of its hormone products to systemic blood. ACTH has been shown to increase the production and secretion of VEGF in the fetal adrenal, indicating coordinated growth and vascular programming (26). Patients with ACC have significantly greater serum VEGF levels than patients with benign adrenal tumors, and the highest levels are observed in ACC patients with cortisol hypersecretion (27). Additionally, our adrenal tumor gene ex- pression profiling also confirms increased VEGF transcript ex- pression in ACCs (data not shown). Therefore, it would be ra- tional to assess the effectiveness of angiogenesis inhibitors as single agents or in combination with mitotane and/or IGF inhi- bition in future preclinical and clinical studies.
Imprinting is an epigenetic mechanism by which cells regulate gene expression in a parent-of-origin-specific manner, whether maternally or paternally inherited, so that one allele is expressed, whereas the other is stably and efficiently silenced through DNA methylation. In pathologic situations, as in the case in the ma- jority of patients with BWS, this process goes awry and results in aberrant expression of imprinted genes (28). Our gene expres- sion profiling of sporadic human adrenal tumors revealed dys- regulation of the 11p15.5 expression profile that is reminiscent of what is commonly observed in BWS, suggesting that similar imprinting defects may underlie the pathogenesis of nonhered- itary ACC. Interestingly, during organ development in the hu- man fetus, the adrenal gland has one of the highest levels of IGF2 transcript expression, which subsequently declines after birth (29). The reactivation of IGF-II expression in sporadic ACCs lends credence to the hypothesis that this cancer may represent an aberrant epigenetic reprogramming in the repopulating cells (tissue specific stem/progenitor cells) of the adult adrenal. In the normal colon epithelium, loss of imprinting of IGF2 is frequently observed in patients with a higher risk of developing colorectal cancer. Transgenic mouse models overexpressing IGF through loss of imprinting expands the progenitor cell population of the colon (30). These proposed cancer stem cells may represent a bona fide malignant population of cells that both initiate tumor- igenesis and evade conventional chemotherapies. Whether ini- tiation of ACC is mediated through similar mechanisms is an area of active investigation.
In this report, we have demonstrated overexpression of IGF-1R and its ligand and activated IGF signaling in human ACC samples and ACC cell lines. Antagonizing this pathway with two pharmacological agents resulted in inhibition of growth in vitro and in vivo. Importantly, this targeted inhibition was more potent than mitotane treatment in decreasing xenograft growth and the combination of IGF inhibition and mitotane resulted in greater an- tiproliferative effects in vitro and greater xenograft growth inhibi- tion in vivo over single agent treatment. These data raise the pros- pect of using targeted disruption of IGF-1R signaling to attain a
therapeutic advantage when used as an adjuvant in mitotane ther- apy or possibly other chemotherapeutics in ACC patients.
Note Added in Proof
During the review of this manuscript, Almeida et al. reported similar findings in JCEM that IGF inhibition in ACC cell lines results in significant reduction in proliferation and concomitant activation of adoptosis, in vitro (31).
Acknowledgments
We thank ImClone Systems and Novartis for generously providing their respective targeted reagents. We thank Dr. David E. Schteingart for help- ful advice and Julie Pepera for technical support.
Address all correspondence and requests for reprints to: Dr. Gary D. Hammer, Department of Internal Medicine-Division of Metabolism, En- docrinology, and Diabetes, University of Michigan Medical School, 1502 BSRB, 109 Zina Pitcher Place, Ann Arbor, Michigan 48109-2200. E-mail: ghammer@umich.edu.
This work was supported by National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Grant DK 062027 (to G.D.H.) and the American Cancer Society Grant RSG-04- 236 (to G.D.H.). F.M.B. is supported through a predoctoral fellowship from National Institutes of Health/Training Program for Organogenesis and Grant T-32-HD007505 and is a fellow in the Medical Scientist Training Program. A.C.S. is supported by a Resident Seed Grant from ASTRO.
Disclosure Statement: The authors have nothing to disclose.
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