Interleukin-13 receptor alpha2 is a novel therapeutic target for human adrenocortical carcinoma

Authors


  • This article is US Government work and, as such, is in the public domain in the United States of America.

Abstract

BACKGROUND.

Adrenocortical carcinoma (ACC) is a relatively rare but aggressive malignancy with limited therapeutic options. Previous genome-wide expression studies have demonstrated the overexpression of interleukin-13 receptor alpha2 (IL13Rα2) in some human malignancies.

METHODS.

The authors evaluated IL13Rα2 mRNA and protein expression in 21 normal samples, 78 benign samples, 10 primary malignant samples, and 25 metastatic/recurrent samples and performed functional analyses with IL13 ligand and IL13 Rα2 knockdown in vitro. The sensitivity of 2 ACC cell lines (NCI-H295R [high IL13Rα2 expression] and SW13 [low IL13Rα2 expression]) to a highly specific IL-13 conjugated with Pseudomonas exotoxin (IL-13-PE) also was evaluated in both in vitro and in vivo models.

RESULTS.

IL13Rα2 was overexpressed in malignant tumors compared with benign and normal samples (15-fold higher; P < .05). Immunohistochemistry also confirmed higher protein expression in malignant and benign tumors than in normal adrenocortical tissues (P < .05). The half-maximal inhibitory concentration for IL-13-PE was 1.3 ng/mL in the NCI-H295R cell line and 1000 ng/mL in the SW13 cell line. Mice that received intratumoral or intraperitoneal IL-13-PE injection had a significant reduction in tumor size and increased tumor necrosis compared with control groups (P < .05) and also had prolonged survival (P < .05). IL13Rα2 protein expression increased in cells that were treated with IL-13 ligand along with cell invasion (P < .05). Direct IL13Rα2 knockdown decreased cellular proliferation and invasion (P < .05).

CONCLUSIONS.

The current results indicated that IL13Rα2 is overexpressed in ACC and regulates cell invasion and proliferation. IL13Rα2 is a novel therapeutic target for the treatment of human ACC. Cancer 2012. © 2012 American Cancer Society.

INTRODUCTION

With an annual incidence of approximately 0.72 cases per million, adrenocortical carcinoma (ACC) is a rare malignancy of the adrenal cortex with a poorly understood mechanism of development.1-4 Currently, surgical resection is the only possible curative therapy for patients with ACC; however, because the majority of patients present with metastatic disease, the 5-year survival rate is <10%.1, 5 Therefore, there is a significant need for new therapeutic options that may be effective in patients with ACC.

Because the molecular mechanism of adrenocortical tumorigenesis is not clearly defined, genome-wide gene expression profiling analysis of adrenocortical tumors has been used to determine dysregulated gene expression associated with ACC.6-11 Interleukin-13 (IL-13) receptor alpha2 (IL13Rα2) has been identified as 1 gene that is overexpressed in ACC.10 IL13Rα2 is a high-affinity receptor of the T-helper 2 cell-derived cytokine IL-13 and is overexpressed in several tumors compared with low or absent expression in normal cells and tissues.12-18 Initial studies suggested that IL13Rα2 binds IL-13 ligand with high affinity but without activating any signal transduction pathways.19 Therefore, it was hypothesized that the function of the extracellular domain of IL13Rα2 was to serve as a decoy receptor to regulate the signaling of type II IL-13R complex (IL-13, IL13Rα1, and IL-4α) through the Janus kinase-signal transducer and activator of transcription 6 (JAK-STAT6) pathway.20, 21 However, recently, it was demonstrated that high-affinity binding of IL-13 ligand to IL13Rα2 does signal through a STAT6-independent activator protein 1 (AP-1) pathway, which leads to increased transforming growth factor-β activity.22

Targeting cell-surface receptors or antigens with small molecules or immunotoxin therapies is an attractive strategy for developing effective cancer therapy.23, 24 It has been demonstrated that a chimeric fusion protein that consists of IL-13 and a mutated form of Pseudomonas exotoxin (IL-13-PE) is highly cytotoxic to IL13Rα2-positive cancer cells in both in vitro and vivo models of several malignancies.15, 25, 26

In the current study, we demonstrate that IL13Rα2 is overexpressed in ACC. Furthermore, IL13Rα2 influences ACC cell invasion and is a good therapeutic target in ACC cells in both in vitro and in vivo models using IL-13-PE immunotoxin.

MATERIALS AND METHODS

Tissue Specimens

Our Institutional Review Board approved the protocol, and all materials were collected after written informed consent was obtained from patients. Adrenal tissues were snap frozen at the time of surgery and stored at −80°C. IL13Rα2 mRNA and protein expression were determined in 134 human adrenocortical tissue specimens (21 normal tissues, 78 benign adrenocortical tumors, 10 primary ACCs, 23 ACC metastases, and 2 ACC recurrences). The diagnosis of unequivocal ACC was confirmed in all cases by histologic examination, the presence of lymph nodes metastasis, distant metastasis, and/or the development of locoregional recurrent disease during follow-up.

Cell Culture and Reagents

The NCI-H295R and SW13 ACC cell lines (American Type Culture Collection, Rockville, Md) were grown and maintained in Dulbecco modified Eagle medium supplemented with 1% insulin transferrrin selenium (BD Biosciences, Bedford, Mass) and 2.5% Nu-Serum I (BD Biosciences) in a standard humidified incubator at 37°C in a 5% CO2 atmosphere. PM-RCC, a renal cell carcinoma cell line, was maintained in Dulbecco modified Eagle medium with 10% fetal bovine serum (Bio-Whittaker Inc., Walkersville, Md), 1 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mm nonessential amino acids, 100 μg/mL penicillin, and 100 μg/mL streptomycin (Bio-Whittaker Inc.). The recombinant IL-13-PE used in our studies has been described previously.25, 27 IL-13 was obtained from Sigma Chemical Company (St. Louis, Mo).

Immunohistochemical and Immunocytochemical Staining

Sections were deparaffinized, rehydrated, and incubated with the primary anti-IL13Rα2 goat polyclonal antibody (AF146; R&D Systems, Inc., Minneapolis, Minn) at 15 μg/mL dilution overnight at 4°C followed by biotinylated secondary antibody (1:200 dilution; Vector Laboratories, Burlingame, Calif) for 1 hour at room temperature. Sections were developed using 3,3′-diaminobenzidine DAB as the chromogen (ABC Elite Kit; Vector Laboratories) and counterstained with hematoxylin. The slides were scanned under an Olympus light microscope (Nikon, Tokyo, Japan), and images were acquired at ×20 and ×40 magnification. A semiquantitative scoring system was used to analyze IL13Rα2 expression; the expression level was classified as 0 (no staining), 1 (<30% stained cells), 2 (30%-50% stained cells), and 3 (>50% stained cells). Two observers, who were blinded to the tumor type, independently scored each sample.

Immunofluorescence staining was done using cultured NCI-H295R cells (1 × 105 cells in 1 mL) on chamber slides (Lab-Tek, Rochester, NY). The cells were allowed to adhere to slides for 48 hours and were incubated with IL-13 at 50 ng/mL for another 48 hours. Cells were washed with phosphate-buffered saline and fixed with 4% formaldehyde for 30 minutes. The cells were incubated with primary anti-IL13Rα2 goat polyclonal antibody at 1:100 dilution overnight (AF146; R&D Systems Inc.) at 4°C. The primary antibodies were detected with fluorophore conjugated with RedTX antigoat immunoglobulin G (IgG) (Invitrogen, Carlsbad, Calif). The slides were then rinsed and mounted with 4′, 6-diamidino-2-phenylindole mounting solution. Images were captured with a Zeiss Axioskop-2 microscope (Carl Zeiss, Inc., Oberkochen, Germany) at ×20 and ×40 magnification.

Reverse Transcription and Real-Time Quantitative Polymerase Chain Reaction

RNA samples from tissue samples and cell lines were subjected to reverse transcriptase-polymerase chain reaction (PCR) analysis. Total RNA (200-500 ng) was reverse-transcribed using a High Capacity Reverse Transcription cDNA kit according to the manufacturer's instructions (Applied Biosystems, Foster City, Calif). Real-time quantitative PCR was used to measure mRNA expression levels, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA amplification from the same samples served as an internal control. The normalized gene expression level was represented as 2ˆ Ct of the gene of interest − Ct of GAPDH) × 100%, where Ct is the PCR cycle threshold. The PCR primer probes for IL13Rα2 (Hs_010180383_m1) and GAPDH (Hs99999905_m1) were obtained from Applied Biosystems. All reactions were performed in a final volume of 10 μL with 2 μL of cDNA template on a 7900HT Fast Real-Time PCR System (Applied Biosystems). The thermal cycler condition was 95°C for 12 minutes followed by 40 cycles at 95°C for 15 seconds and at 60°C for 1 minute.

Cell Culture and Transient Transfection

Cells were seeded onto 240-well and 6-well plates (4 × 104 cells in 0.5 mL and 1.6 × 105 cells in 2 mL, respectively). Twenty-four hours after plating, the cells were transfected either with a nonspecific negative control small interfering RNA (siRNA) (AM4613) or with a combination of IL13Rα2-specific siRNAs at a final concentration of 80 nM (s7376 and s7374; Applied Biosystems). The TransIT-siQuest reagent (Mirus Bio LLC, Madison, Wis) was used to deliver siRNA to the cells according to the manufacturer's instructions.

Cell Proliferation

Cells were plated at a concentration of 5 × 103 cells per 100 μL culture medium in a 96-well plate in 6 replicates. Cell numbers were determined by using the CyQUANT assay kit (Invitrogen) and a fluorometric microplate reader (Molecular Devices, Sunnyvale, Calif) at 480 nm/520 nm.

Flow Cytometry

Seven days after transfection, the cells (1 × 105) were incubated with 5 μg/mL of mouse monoclonal anti-IL13Rα2 (Diaclone; B-D13; Rockland, Gilbertsville, Pa). Positive staining or binding in the cells was detected using secondary goat-antimouse IgG conjugated with fluorescein isothiocyanate (FITC) (Sigma-Aldrich, St. Louis, Mo). The fluorescence associated with the live cells was measured using a FACS Calibur device (BD Biosciences). The cells were gated using secondary FITC-conjugated antibody control.

Cell Invasion Assay

The extent of cell invasion was assessed using the BD BioCoat Matrigel Invasion Chamber (BD Biosciences) according to the manufacturer's protocol. In total, 1 × 105 cells were seeded onto the inserts (8-μM pore sized polycarbonate membranes) coated with a thin layer of Matrigel Basement Membrane Matrix (BD Biosciences). The inserts were placed into a 24-well plate with 10% serum-containing culture medium as a chemoattractant. The plates were incubated for 48 hours at 37°C. Cells that invaded the Matrigel matrix to the lower surface of the membrane were fixed and stained with Diff-Quik (Dade Behring, Newark, NJ) and counted under a light microscope.

Protein Synthesis Inhibition Assay

In total, 1 × 104 cells were plated in 96-well plate in 100 μL of complete growth medium for 24 hours. After 24 hours, the plates were replaced with leucine-free medium (Biofluids, Rockville, Md) for 6 hours. IL-13-PE immunotoxin was added to the cells at 0.1 to 1000 ng/mL and incubated for 20 hours at 37°C. Then, 1 μCi of (3H)-labeled leucine (NEN Research Products, Boston, Mass) was added to each well and incubated for an additional 4 hours. The cells were harvested, and labeled leucine incorporation was measured by a β plate counter (Perkin Elmer, Waltham, Mass). The half-maximal inhibitory concentration (IC50) was defined as the concentration of a drug at which the proliferation was reduced by 50%.

A protein synthesis inhibition assay was performed on Day 3 after IL13Rα2 siRNA and negative control transfection. The transfection was carried out at a final concentration of 100 nM IL13Rα2 siRNA and negative control using 0.2 μL per well of TransIT-siQUEST transfection reagent (Mirus Bio LLC). After 48 hours, the cells were treated with different concentrations of IL-13-PE immunotoxin to assess the biologic activity of silenced IL13Rα2. The IC50 values of the silenced cells were compared with negative control and medium. The experiment was performed in quadruplicate with 2 independent biologic repeats.

Tumor Spheroids

In total, 1 × 105 NCI-H295R cells per well (in 0.5 mL) were plated into 24-well Ultra Low Cluster Plates (Costar, Corning, NY) to generate tumor spheroids. The plates were cultured at 37°C in 5% CO2 for 1 week, and the medium was changed every 3 days. After 1 week of culture, tumor spheroids were photographed under a dissecting microscope and were treated with different concentrations of IL-13-PE (0.13-6.5 ng/mL) or vehicle (0.2% human serum albumin in phosphate-buffered saline) in duplicate. The tumor spheroids were treated for 2 weeks (and were dosed every first and third day).

Adrenocortical Carcinoma Xenografts in Athymic Nude Mice

The institutional Animal Care and Use Committee approved the ACC xenograft animal study protocol. Five-week to 6-weeks old (body weight, 20-22 g) female athymic NCr nu/nu mice were obtained from the Frederick Cancer Center Animal Facilities (National Cancer Institute, Frederick, Md). The mice were maintained according to the guidelines of the institute's Animal Advisory Committee. NCI-H295R cells were implanted by subcutaneous injection into the left flank with 4 × 106 cells in 100 μL Dulbecco modified Eagle medium and Matrigel (1:1 dilution). Tumor sizes were measured every 7 days and recorded in mm3 (length × width2/2). For tumor therapy experiments, the mice were grouped into 5 groups with 6 mice per group. Group I and II mice received intratumor injections (using Hamilton syringes; Hamilton-80400-22s,2 inch) with 25 μL IL-13-PE (100 μg/kg) every other day for 1 week and 2 weeks, respectively. Group III mice received intraperitoneal injection (using 27-gauge needles) of 50 μL IL-13-PE (50 μg/kg) every other day for 1 week. Group IV and V mice received 0.2% human serum albumin (vehicle) every other day for 2 week by intratumor and intraperitoneal injection, respectively. The mice were followed until the protocol endpoint (when the tumor size reached ≥2 cm) and were sacrificed for further investigations. Tumors and vital organs, such as liver, kidney, spleen, and lung, were removed and fixed in 10% formalin for histopathology examinations.

Statistical Analyses

Continuous data are reported as mean ± standard deviation values. Two-tailed analysis of variance multicomparison t tests were used to assess differences between normal, benign, and malignant samples. A P value < .05 was considered significant. Statistical analyses were done using Stat View software (version 5.0; SAS Institute, Inc., Cary, NC) and SPSS (version 16.0; SPSS Inc., Chicago, Ill).

RESULTS

Interleukin-13 Receptor Alpha2 Is Overexpressed in Adrenocortical Carcinoma

Expression levels of IL13Rα2 mRNA were significantly higher in ACC compared with levels in normal adrenocortical tissues and benign adrenocortical tumors (P < .001) (Fig. 1A). In addition, IL13Rα2 mRNA expression levels were higher in metastatic and recurrent tumors compared with levels in normal adrenocortical tissue but lower than levels in primary ACC (P < .001). Consistent with the mRNA expression data (Fig. 1A), IL13Rα2 protein expression also was higher in ACC and benign adrenocortical tumor samples than in normal adrenocortical tissue (Fig. 1B). Semiquantitative scoring indicated a significant difference between normal versus benign or malignant tumors (P < .5) (Fig. 1C). IL13Rα2 was specifically and uniformly overexpressed in malignant cells but not in adjacent normal tissues.

Figure 1.

Interleukin-13 receptor alpha2 (IL13Rα2) mRNA expression is illustrated in adrenocortical carcinoma (ACC). (A) IL13Rα2 mRNA expression in normal adrenal cortex (n = 21), benign adrenocortical tumors (n = 78), primary ACC (n = 10), and metastatic and recurrent ACC (n = 25). Asterisks indicates a statistically significant difference (P < .05; 1-way analysis of variance post hoc tests) A single asterisk indicates primary ACC versus metastases (P < .001); double asterisks, primary ACC versus benign and normal adrenal tissue (P < .001). GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase. (B) Immunohistochemistry for IL13Rα2 protein expression is viewed in (1,2) normal adrenocortical tissue, (3,4) benign tumors, and (5,6) ACC. Photomicrographs 7 and 8 show corresponding hematoxylin and eosin-stained images of malignant samples with invasive features. Representative images are provided for each category (original magnification, ×10 and ×20, as indicated). Arrows indicate positive cytoplasmic and membrane staining for IL13Rα2. (C) Semiquantitative IL13Rα2 immunohistochemistry scores are indicated in samples of normal tissue (n = 7), benign tumor (n = 8), and ACC (n = 6). Columns represent the average score (obtained from scoring) ±standard deviation. A black asterisk indicates a significant difference between benign and normal tissue samples, red asterisk, the difference between malignant and normal tissue samples.

Interleukin-13 Signals Through Interleukin-13 Receptor Alpha2 and Influences Adrenocortical Carcinoma Cell Invasion

Given the high levels of IL13Rα2 expression in ACC, next, we determined whether the receptor was biologically active. Specifically, because it is believed that IL-13 signals through IL13Rα2, we attempted to evaluate the responsiveness of the IL13Rα2-overexpressing ACC cell line, NCI-H295R, to IL-13 ligand. We observed significant induction of IL13Rα2 in cells that were treated with 50 ng/mL of IL-13 ligand (Fig. 2A). In addition, IL13Rα2 immunofluorescence indicated that the receptor was expressed predominantly in cell membrane and cytoplasm (Fig. 2A).

Figure 2.

Interleukin-13 (IL-13) increases IL-13 receptor alpha2 (IL13Rα2) expression in NCI-H295R (H295R) cells. (A) Immunofluorescence analysis indicated that exposure of IL-13 at 50 ng/mL increased IL13Rα2 expression after 48 hours. Nuclei were stained with 4′6-diamidino-2-phenylindole (blue). Red indicates IL13Rα2 expression. Images in A represent (Top) untreated cells and (Bottom) treated cells (original magnification, ×10 [Left] and ×40 [Right]). (B) IL13Rα2 small interfering RNA (siRNA) knockdown is observed in the H295R adrenocortical carcinoma cell line. Transient transfection was done in H295R cells using IL13Rα2 siRNA, and negative controls and cells were analyzed for mRNA expression after days 1, 3, 5, 7, and 14 of treatment. Columns represent the remaining percentage of IL13Rα2 mRNA expression relative to negative controls (±standard deviation) from 4 experiments.

To better understand the potential biologic effects of IL-13 and IL13Rα2 signaling in ACC cells, we also evaluated invasion as a measure of metastatic potential in NCI-H295R cells in the presence or absence of IL-13. It was demonstrated previously in pancreatic cancer cell lines that IL-13 signals through IL13Rα2 to promote invasion.28 To assess this response in ACC, we used siRNA to knockdown IL13Rα2 expression in NCI-H295R cells. This approach resulted in a 84% to 88% reduction in mRNA expression within 24 hours compared with a negative control group, and the reduction lasted up to 7 days (Fig. 2B). Furthermore, we observed a 75% decrease in cell surface protein expression of IL13Rα2 compared with negative controls after 7 days of transfection (Fig. 3A). NCI-H295R cells were incubated with IL-13 to assess invasion. We observed that the number of invading cells increased by 15% with IL-13 treatment (Fig. 3B). In addition, after quantification, the number of invaded cells decreased by 44.7% in an IL13Rα2-silenced group compared with an siRNA-negative control in the presence of IL-13 (P = .007) and decreased by 30% in the absence of IL-13 (P = .006) (Fig. 3C). These results suggest that IL-13 signals through IL13Rα2 and promotes ACC cell invasion. In addition, there was a 28.5% to 33% decrease in cell proliferation after days 11 and 14 of IL13Rα2 knockdown compared with negative controls (P < .001) (Fig. 3D).

Figure 3.

(A) Interleukin-13 receptor alpha2 (IL13Rα2) protein expression at the cell surface in small interfering RNA (siRNA)-transfected cells was assessed by flow cytometry. Representative histograms demonstrate cell surface expression of IL13Rα2 after 7 days of IL13Rα2 siRNA or negative control treatment. The x-axis indicates fluorescein isothiocyanate fluorescence intensity, and the y-axis indicates the cell count. The red line represents the isotype control, and black and blue lines represent IL13Rα2 siRNA knockdown and negative control cells, respectively. (B) IL13Rα2 siRNA knockdown reduced invasion in the NCI-H295R cell line (NC). Representative images are shown (original magnification, ×20). (C) This graph illustrates a quantitative analysis of the number of invasive cells with and without IL13Rα2 siRNA treatment compared with the siRNA-negative control group. Columns represent the mean ± standard deviation from 3 independent experiments performed in triplicate. A single asterisk indicates P < .05 (IL13Rα2 siRNA vs siRNA-negative control). (D) IL13Rα2 siRNA knockdown and cell proliferation are illustrated in the NCI-H295R cell line. The distribution of cells in the IL13Rα2 siRNA-treated group and the negative control-treated group is illustrated at the indicated time points (asterisks indicate significant values). Triple asterisks indicate P < .001; double asterisks, P < .01 (relative to negative controls). Error bars represent the standard error of the mean and are representative of 4 experiments.

Inteleukin-13 Receptor Alpha2-Positive Adrenocortical Carcinoma Cell Lines Are Sensitive to Interleukin-13-Pseudomonas Exotoxin Cytotoxin

Because IL13Rα2 is overexpressed in ACC, we evaluated its potential as a therapeutic target for ACC in vitro and in vivo. The sensitivity of 2 ACC cell lines (NCI-H295R with high IL13Rα2 expression and SW13 with low IL13Rα2 expression) to a highly specific immunotoxin (IL-13-PE) was determined (Fig. 4A). In addition, the renal cell carcinoma cell line PM-RCC, which has high IL13Rα2 mRNA expression, was used as a positive control. The IC50 for IL-13-PE was significantly lower (1.3 ng/mL and 0.2 ng/mL) in NCI-H295R and PM-RCC cells, respectively, with high IL13Rα2 expression (Fig. 4B) than the IC50 of 600 ng/mL in SW13 cells, which have lower IL13Rα2 expression (P < .05) (Fig. 4C). The IC50 of IL-13-PE also was administered in negative control and siRNA-transfected cells to evaluate the specificity of IL-13-PE to IL13Rα2. The treatment resulted in loss of sensitivity in siRNA-transfected cells compared with negative controls (Fig. 4D). The IC50 was determined using a protein synthesis inhibition assay and was significantly lower in the negative control and untreated groups (0.9 ng/mL, 2 ng/mL) compared with the siRNA-treated group (≥1000 ng/mL; P < .05) (Fig. 4E).

Figure 4.

Interleukin-13 (IL-13) receptor alpha2 (IL13Rα2)-positive NCI-H295R cells (H295R) are sensitive to IL-13-Pseudomonas exotoxin (IL-13-PE). (A) IL13Rα2 mRNA expression is illustrated in 2 adrenocortical carcinoma (ACC) cell lines (H295R and SW13). The y-axis indicates the percentage of IL13Rα2 mRNA expression relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. (B,C) A protein synthesis inhibition assay was used to assess the cytotoxicity of IL-13-PE (B) to H295R cells and (C) to SW13 cells. PM-RCC cells (a renal carcinoma cell line) were used as positive controls. Mean ± standard deviation values from 5 quadruplicate determinations are indicated. (D) IL-13-PE was administered at a concentration of 1.3 ng/mL to cells with IL13Rα2 knockdown and to negative controls (vehicle). Photomicrographs show (Top) negative control and small interfering RNA (siRNA) knockdown cells treated with vehicle (0.2% human serum albumin) and (Bottom) cells that were treated with IL-13-PE. All images are representative of 2 duplicate experiments (original magnification, ×10). (E) A protein synthesis inhibition assay was used to assess the cytotoxicity of IL-13-PE to H295R-transfected cells with IL13Rα2 siRNA and negative controls at 80 nM concentration. IC50 indicates half-maximal concentration. (F) The effects of IL-13-PE are observed in H295R spheroids. Spheroids were treated with IL-13-PE and vehicle at different concentrations from 0.13 ng/mL to 6.5 ng/mL, as indicated. The top 2 rows are images of IL-13-PE treatment, and the bottom 2 rows are images of vehicle treatment (original magnification, ×12.5 in top 2 rows, × 50 in bottom 2 rows).

To further confirm the cytotoxic effect of IL-13-PE in a 3-dimensional model that mimics solid tumors better, it also was administered to NCI-H295R tumor spheroids. NCI-H295R cells were cultured for 1 week to form spheroids; then, the spheroids were treated with different concentrations of IL-13-PE for 2 weeks. We observed a significant reduction in the spheroid size and disaggregation of cells during treatment within 1 week of treatment (Fig. 4F).

Treatment With Interleukin-13-Pseudomonas Exotoxin Causes Tumor Regression and Prolonged Survival in a Xenograft Model of Adrenocortical Carcinoma

On the basis of the profound effect of IL-13-PE on ACC cell line growth in vitro, the efficacy of IL-13-PE in ACC xenografts was determined in an athymic nude mice NCr-nu/nu model. Mice were injected with NCI-H295R cells, and treatment was initiated after the tumors reached a size of approximately 50 to 60 mm3. After 14 days, the mice were randomized into 5 groups. The routes of IL-13-PE administration were all well tolerated with no observed toxicity on histologic examination of vital organs (liver, kidney, spleen, and lung). In addition, there was no significant difference in weight between the groups. Nineteen days after treatment, mice with 1 week and 2 weeks of intratumoral treatment (Groups I and II) had a 70% reduction in tumor size compared with the vehicle control group (Group 1V; P < .05) (Fig. 5A), whereas the intraperitoneal group (Group III) had a 47% reduction by Day 25 compared with the vehicle control group (Group V; P < .05) (Fig. 5B). We also observed significantly longer survival in Groups I, II, and III (108 days, 106 days, and 111 days, respectively) compared with Groups IV and V (69 days and 84 days, respectively; P < .05). In addition, histologic analysis in the intraperitoneal and intratumoral control group demonstrated the presence of viable cells (Fig. 5C), whereas tumors treated with IL-13-PE had no evidence of necrosis (Fig. 5C). We observed no significant difference in IL13Rα mRNA or protein expression levels between IL-13-PE-treated and vehicle control tumor samples from the mice.

Figure 5.

Intratumoral (IT) and intraperitoneal (IP) treatment with interleukin-13 (IL-13) conjugated with Pseudomonas exotoxin (IL-13-PE) in adrenocortical carcinoma (ACC) xenografts significantly reduced tumor growth. (A) Tumor growth is compared between the vehicle-treated and IL-13-PE IT-treated groups (100 μg/kg once daily for 1 week and 2 weeks, as indicated by vertical arrows). (B) Tumor growth is compared between the vehicle treatment group and the IL-13-PE IP treatment group (50 μg/kg twice daily for 1 week). Each point represents the mean ± standard error of the mean for 6 mice from 2 independent experiments. Asterisks indicate statistically significant values (single asterisk, P < .05; double asterisks, P < .01). (C) Tumor histology is illustrated in mice that had ACC xenografts after treatment with IL-13-PE. These representative hematoxylin and eosin images from (1,2) the IT group and (3,4) and the IP vehicle group (0.2% human serum albumin) indicate the presence of viable tumor cells; and images of hematoxylin and eosin-stained sections from mice that received IL-13-PE in (5,6) the IT group (100 μ/kg) and (7,8) the IP group (50 μg/kg;) reveal the presence of few viable cells with necrotic mass (original magnification, ×2.5 and ×20, as indicated).

DISCUSSION

In the current study, for the first time, we analyzed IL13Rα2 expression and its biologic significance as a therapeutic target in ACC. Our results demonstrate that IL13Rα2 is overexpressed in ACC and is an excellent therapeutic target. IL13Rα2 knockdown in ACC cells resulted in the suppression of invasion, suggesting that it may influence cell-invasive phenotype in ACC. Our data also demonstrate that IL13Rα2 is a novel therapeutic target for the treatment of ACC with IL-13-PE.

Our study demonstrates IL13Rα2 mRNA and protein overexpression in ACC, as has been observed in other malignancies.12, 14, 16, 27, 29, 30 It is noteworthy that IL13Rα2 mRNA expression was lower in tumors from a cohort of patients with metastatic and recurrent ACC who received prior systemic chemotherapy before undergoing surgical resection, but expression levels still were well over 5-fold higher than levels in normal adrenocortical tissue. These results suggest that IL13Rα2 expression may vary because of treatment. Thus, because IL13Rα2 expression is low or absent in normal organs, it would be reasonable to consider IL-13-PE therapy for patients with metastatic or locally advanced ACC.

Given the overexpression of IL13Rα2 in ACC, we were interested in evaluating whether the receptor was functional in ACC. By using siRNA knockdown, the number of invading cells was reduced significantly compared with negative controls. In addition, incubation of cells with IL-13 ligand increased invasion compared with the negative control group, and the effect was lost in IL13Rα2-silenced cells. Therefore, these results suggest that IL13Rα2 is involved in ACC invasion and that IL13Rα2 mediates IL-13 signaling in NCI-H295R cells. In addition, an invasive feature (capsular invasion) was observed in the histology of malignant tumors that had higher IL13Rα2 overexpression. Previously, it was demonstrated that IL-13 activates AP-1 transcription factor, which, in turn, activates matrix metalloproteinases (MMPs) and thereby may influence metastatic potential in pancreatic cancer cells.28 MMPs are associated with tumor invasion and metastasis of malignant tumors with different histogenetic origins.31, 32 The current findings also are supported by other studies that reported elevated IL13Rα2 levels in breast cancer metastasis and glioma cells with mutant epidermal growth factor receptor.28, 33, 34 Our findings indicate that IL-13 mediates signaling through IL13Rα2 and influences cellular invasion and proliferation in ACC cells.

Because of the specific overexpression of IL13Rα2 in ACC and the lack of IL13Rα2 expression in the vast majority of normal tissues, we were interested in evaluating its role as a therapeutic target. It has been demonstrated that IL-13-PE inhibits the growth of different cancer cells in vitro and in vivo.26, 27 We tested the sensitivity and specificity of ACC cell lines to IL-13-PE in monolayer culture and observed that the IC50 was low in the IL13Rα2-positive NCI-H295R cell line compared with SW13, a receptor-negative cell line. In addition, IL-13-PE treatment led to a significant decrease in sensitivity and IC50 in siRNA-transfected cells (≥1000 ng/mL) compared with a negative control group (0.9 ng/mL). These results suggest that IL-13-PE is highly specific to IL13Rα2. With these encouraging results, we also evaluated sensitivity in 3-dimensional in vitro culture of spheroids to confirm the efficacy of IL-13-PE in ACC. The spheroid model resembles a solid tumor and is used for studying therapeutic drug testing, tumor growth, and cellular differentiation and invasion.35, 36 Our results revealed a dramatic effect on tumor spheroids at very low concentrations of IL-13-PE, confirming the efficacy and specificity of IL-13-PE to IL13Rα2-positive ACC cells.

On the basis of the positive cytotoxicity results in monolayer and spheroid culture, IL-13-PE efficacy also was determined in an in vivo model of subcutaneous xenografts. ACC xenografts were developed using NCI-H295R cells, and administration of IL-13-PE through intratumoral and intraperitoneal routes of injection resulted in significant reductions (70% and 47%, respectively) in established tumors within 3 weeks with no observable signs of toxicity. Intratumoral injections probably were more effective because of the direct route of administration, because longer treatment (higher cumulative dose) with this route of administration resulted in no significant difference tumor reduction. This result is in concordance with the previous studies in other IL13Rα2-positive tumor types, such as glioma, renal cell carcinoma, and pancreatic adenocarcinoma.15, 17, 27, 37 In addition, histologic analysis of the xenograft tumors revealed significant necrosis in tumors that were treated with IL-13-PE, whereas vehicle-treated tumors had evidence of viable cells. Thus, as demonstrated in previous studies using IL-13-PE–based immunotoxin, our results indicate that this agent causes cell death and tumor necrosis.38-40

In summary, to our knowledge, this is the first study to demonstrate that IL13Rα2 is overexpressed in ACC and is a novel therapeutic target for ACC. IL-13-PE immunotoxin is specific to IL13Rα2 and significantly inhibits tumor growth in vitro and in a human xenograft ACC animal model. Therefore, our results suggest that IL13Rα2 may be used as novel target and support the testing of IL-13-PE immunotoxin in a clinical trial among patients with advanced and metastatic ACC.

FUNDING SOURCES

This study was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health (grants 1ZIABC011286-01 and 1ZIABC011275-01).

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

Ancillary