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

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.

Copyright © 2012 American Cancer Society.

Figures

Figure 1

IL13Rα2 mRNA expression in 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). Statistical significant difference was indicated by an asterisk (*) (p<0.05, One-Way ANOVA post-hoc tests). **Primary ACC or benign vs. normal (p < 0.001) and *primary ACC vs. metastases (p < 0.001). B) Immunohistochemistry for IL13Rα2 protein expression in normal adrenocortical tissue (1–2), benign tumors (3–4) and malignant ACC (5–6). (7–8) The corresponding H&E image of the malignant sample representing invasive features. Representative images are shown for each category at 10X and 20X magnifications. Arrows indicate the positive cytoplasmic and membrane staining for IL13Rα2. C) Semiquantitative IL13Rα2 IHC scores are shown in the normal (n=7), benign (n=8) and ACC (n=6) samples. Columns represent the average score (obtained from two individual investigators) ± standard deviation. Black asterisk indicates significant difference between benign and normal while red asterisk indicate the difference between malignant and normal tissue samples.

Figure 1

IL13Rα2 mRNA expression in 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). Statistical significant difference was indicated by an asterisk (*) (p<0.05, One-Way ANOVA post-hoc tests). **Primary ACC or benign vs. normal (p < 0.001) and *primary ACC vs. metastases (p < 0.001). B) Immunohistochemistry for IL13Rα2 protein expression in normal adrenocortical tissue (1–2), benign tumors (3–4) and malignant ACC (5–6). (7–8) The corresponding H&E image of the malignant sample representing invasive features. Representative images are shown for each category at 10X and 20X magnifications. Arrows indicate the positive cytoplasmic and membrane staining for IL13Rα2. C) Semiquantitative IL13Rα2 IHC scores are shown in the normal (n=7), benign (n=8) and ACC (n=6) samples. Columns represent the average score (obtained from two individual investigators) ± standard deviation. Black asterisk indicates significant difference between benign and normal while red asterisk indicate the difference between malignant and normal tissue samples.

Figure 2

IL-13 increases IL13Rα2 expression in H295R cells. A) Exposure of IL-13 at 50 ng/ml increased IL13Rα2 expression after 48 hours as analyzed by immunoflouroscence. Nuclei were stained by DAPI (blue), red color indicates IL13Rα2 expression. The upper panel image represents untreated cells and the lower panel represents treated cells at 10X and 40X magnification. B)IL13Rα2 siRNA knockdown in the NCI-H295R adrenocortical carcinoma cell line. Transient transfection was done in H295R cells using IL13Rα2 siRNA and negative control and cells were analyzed for mRNA expression after day 1, 3, 5 and 7 of treatment. Columns represent remaining IL13Rα2 mRNA expression percentage relative to negative control ± standard deviation of four experiments.

Figure 2

IL-13 increases IL13Rα2 expression in H295R cells. A) Exposure of IL-13 at 50 ng/ml increased IL13Rα2 expression after 48 hours as analyzed by immunoflouroscence. Nuclei were stained by DAPI (blue), red color indicates IL13Rα2 expression. The upper panel image represents untreated cells and the lower panel represents treated cells at 10X and 40X magnification. B)IL13Rα2 siRNA knockdown in the NCI-H295R adrenocortical carcinoma cell line. Transient transfection was done in H295R cells using IL13Rα2 siRNA and negative control and cells were analyzed for mRNA expression after day 1, 3, 5 and 7 of treatment. Columns represent remaining IL13Rα2 mRNA expression percentage relative to negative control ± standard deviation of four experiments.

Figure 3

A) IL13Rα2 protein expression at the cell surface in siRNA transfected cells was assessed by flow cytometry. Representative histograms demonstrating cell surface expression of IL13Rα2 after 7 days of IL13Rα2 siRNA or negative control treatment. The x-axis indicates FITC fluorescence intensity and Y-axis indicates cell count. The red color represents the isotype control, and the black and blue lines represent IL13Rα2 siRNA knockdown and negative control cells, respectively. B) IL13Rα2 siRNA knockdown reduced invasion in NCI-H295R cell line. Representative images are shown at 20X. C) Quantitative analysis of the number of cells invading with and without IL13Rα2 siRNA as compared to siRNA negative control group. Columns represent the mean ± standard deviation (SD) of three independent experiments performed in triplicate. *p<0.05 IL13Rα2 siRNA vs. siRNA negative control. D) IL13Rα2 siRNA knockdown and cell proliferation in the NCI-H295R cell line. The distribution of number of cells for the IL13Rα2 siRNA treated and negative control treated groups are shown at indicated time points (significant values are indicated by asterisk (*) (*** p<0.001; ** p<0.01; relative to negative control). Error bars represent ± standard error of mean and is representative of four experiments.

Figure 3

A) IL13Rα2 protein expression at the cell surface in siRNA transfected cells was assessed by flow cytometry. Representative histograms demonstrating cell surface expression of IL13Rα2 after 7 days of IL13Rα2 siRNA or negative control treatment. The x-axis indicates FITC fluorescence intensity and Y-axis indicates cell count. The red color represents the isotype control, and the black and blue lines represent IL13Rα2 siRNA knockdown and negative control cells, respectively. B) IL13Rα2 siRNA knockdown reduced invasion in NCI-H295R cell line. Representative images are shown at 20X. C) Quantitative analysis of the number of cells invading with and without IL13Rα2 siRNA as compared to siRNA negative control group. Columns represent the mean ± standard deviation (SD) of three independent experiments performed in triplicate. *p<0.05 IL13Rα2 siRNA vs. siRNA negative control. D) IL13Rα2 siRNA knockdown and cell proliferation in the NCI-H295R cell line. The distribution of number of cells for the IL13Rα2 siRNA treated and negative control treated groups are shown at indicated time points (significant values are indicated by asterisk (*) (*** p<0.001; ** p<0.01; relative to negative control). Error bars represent ± standard error of mean and is representative of four experiments.

Figure 3

A) IL13Rα2 protein expression at the cell surface in siRNA transfected cells was assessed by flow cytometry. Representative histograms demonstrating cell surface expression of IL13Rα2 after 7 days of IL13Rα2 siRNA or negative control treatment. The x-axis indicates FITC fluorescence intensity and Y-axis indicates cell count. The red color represents the isotype control, and the black and blue lines represent IL13Rα2 siRNA knockdown and negative control cells, respectively. B) IL13Rα2 siRNA knockdown reduced invasion in NCI-H295R cell line. Representative images are shown at 20X. C) Quantitative analysis of the number of cells invading with and without IL13Rα2 siRNA as compared to siRNA negative control group. Columns represent the mean ± standard deviation (SD) of three independent experiments performed in triplicate. *p<0.05 IL13Rα2 siRNA vs. siRNA negative control. D) IL13Rα2 siRNA knockdown and cell proliferation in the NCI-H295R cell line. The distribution of number of cells for the IL13Rα2 siRNA treated and negative control treated groups are shown at indicated time points (significant values are indicated by asterisk (*) (*** p<0.001; ** p<0.01; relative to negative control). Error bars represent ± standard error of mean and is representative of four experiments.

Figure 4

IL13Rα2 positive H295R cells are sensitive to IL-13-PE. A) IL13Rα2 mRNA expression in ACC cell lines (H295R and SW13). Y axis represents % IL13Rα2 mRNA expression relative to GAPDH. Protein synthesis inhibition assay was done to assess the cytotoxicity of IL-13-PE to H295R (B) and SW13 (C) cells. PM-RCC cells (renal carcinoma cell line) were used as positive control. Mean ± SD of quadruplicate determination. D) IL-13-PE was administered at concentration of 1.3 ng/ml to cells with IL13Rα2 knockdown and negative control. Upper panel represents negative control and siRNA knockdown cells treated with vehicle (0.2% HSA) and lower panel represents the cells treated with IL-13-PE. All images are at 10X magnification and representative of two experiments done in duplicates. E) Protein synthesis inhibition assay was done to assess the cytotoxicity of IL-13-PE to H295R transfected cells with IL13Rα2 siRNA and negative control used at 80 nM concentration. F) Effect of IL-13-PE in H295R spheroids. Spheroids were treated with IL-13-PE and vehicle at different concentrations of 0.13 ng/ml to 6.5 ng/ml. Upper panel represents IL-13PE treatment at 12.5X magnification and lower panel represents vehicle treatment at 50X magnification.

Figure 4

IL13Rα2 positive H295R cells are sensitive to IL-13-PE. A) IL13Rα2 mRNA expression in ACC cell lines (H295R and SW13). Y axis represents % IL13Rα2 mRNA expression relative to GAPDH. Protein synthesis inhibition assay was done to assess the cytotoxicity of IL-13-PE to H295R (B) and SW13 (C) cells. PM-RCC cells (renal carcinoma cell line) were used as positive control. Mean ± SD of quadruplicate determination. D) IL-13-PE was administered at concentration of 1.3 ng/ml to cells with IL13Rα2 knockdown and negative control. Upper panel represents negative control and siRNA knockdown cells treated with vehicle (0.2% HSA) and lower panel represents the cells treated with IL-13-PE. All images are at 10X magnification and representative of two experiments done in duplicates. E) Protein synthesis inhibition assay was done to assess the cytotoxicity of IL-13-PE to H295R transfected cells with IL13Rα2 siRNA and negative control used at 80 nM concentration. F) Effect of IL-13-PE in H295R spheroids. Spheroids were treated with IL-13-PE and vehicle at different concentrations of 0.13 ng/ml to 6.5 ng/ml. Upper panel represents IL-13PE treatment at 12.5X magnification and lower panel represents vehicle treatment at 50X magnification.

Figure 4

IL13Rα2 positive H295R cells are sensitive to IL-13-PE. A) IL13Rα2 mRNA expression in ACC cell lines (H295R and SW13). Y axis represents % IL13Rα2 mRNA expression relative to GAPDH. Protein synthesis inhibition assay was done to assess the cytotoxicity of IL-13-PE to H295R (B) and SW13 (C) cells. PM-RCC cells (renal carcinoma cell line) were used as positive control. Mean ± SD of quadruplicate determination. D) IL-13-PE was administered at concentration of 1.3 ng/ml to cells with IL13Rα2 knockdown and negative control. Upper panel represents negative control and siRNA knockdown cells treated with vehicle (0.2% HSA) and lower panel represents the cells treated with IL-13-PE. All images are at 10X magnification and representative of two experiments done in duplicates. E) Protein synthesis inhibition assay was done to assess the cytotoxicity of IL-13-PE to H295R transfected cells with IL13Rα2 siRNA and negative control used at 80 nM concentration. F) Effect of IL-13-PE in H295R spheroids. Spheroids were treated with IL-13-PE and vehicle at different concentrations of 0.13 ng/ml to 6.5 ng/ml. Upper panel represents IL-13PE treatment at 12.5X magnification and lower panel represents vehicle treatment at 50X magnification.

Figure 4

IL13Rα2 positive H295R cells are sensitive to IL-13-PE. A) IL13Rα2 mRNA expression in ACC cell lines (H295R and SW13). Y axis represents % IL13Rα2 mRNA expression relative to GAPDH. Protein synthesis inhibition assay was done to assess the cytotoxicity of IL-13-PE to H295R (B) and SW13 (C) cells. PM-RCC cells (renal carcinoma cell line) were used as positive control. Mean ± SD of quadruplicate determination. D) IL-13-PE was administered at concentration of 1.3 ng/ml to cells with IL13Rα2 knockdown and negative control. Upper panel represents negative control and siRNA knockdown cells treated with vehicle (0.2% HSA) and lower panel represents the cells treated with IL-13-PE. All images are at 10X magnification and representative of two experiments done in duplicates. E) Protein synthesis inhibition assay was done to assess the cytotoxicity of IL-13-PE to H295R transfected cells with IL13Rα2 siRNA and negative control used at 80 nM concentration. F) Effect of IL-13-PE in H295R spheroids. Spheroids were treated with IL-13-PE and vehicle at different concentrations of 0.13 ng/ml to 6.5 ng/ml. Upper panel represents IL-13PE treatment at 12.5X magnification and lower panel represents vehicle treatment at 50X magnification.

Figure 5

Intratumoral (IT) and intraperitoneal (IP) IL-13-PE treatment of ACC xenografts significantly reduced tumor growth. A) Graph representing the comparison of tumor growth between vehicle and IL-13-PE IT treatment groups (100 µg/kg once a day for one and two weeks indicated by vertical arrows). B) Graph representing the comparison of tumor growth between vehicle group and IL-13-PE intraperitoneal treatment group (50 µg/kg; twice a day for a week). Each point represents the mean ± standard error of mean (SEM) from 6 mice in two independent experiments. Statistically significant values are indicated by an asterisk (*p<0.05; **p<0.01). C) Tumor regression in mice with ACC xenograft after treatment with IL-13-PE. (1–4) Representative H&E images from IT (1–2) and IP (3–4) vehicle group (0.2% human serum albumin) with presence of viable tumor cells. (4–8) Images represent H&E stained sections from IT (100 µg/kg; 5–6) and IP (50 µg/kg; 7–8) treated mice with IL-13-PE and demonstrates presence of few viable cells with necrotic mass. Images are represented at 2.5X and 20X magnifications.

Figure 5

Intratumoral (IT) and intraperitoneal (IP) IL-13-PE treatment of ACC xenografts significantly reduced tumor growth. A) Graph representing the comparison of tumor growth between vehicle and IL-13-PE IT treatment groups (100 µg/kg once a day for one and two weeks indicated by vertical arrows). B) Graph representing the comparison of tumor growth between vehicle group and IL-13-PE intraperitoneal treatment group (50 µg/kg; twice a day for a week). Each point represents the mean ± standard error of mean (SEM) from 6 mice in two independent experiments. Statistically significant values are indicated by an asterisk (*p<0.05; **p<0.01). C) Tumor regression in mice with ACC xenograft after treatment with IL-13-PE. (1–4) Representative H&E images from IT (1–2) and IP (3–4) vehicle group (0.2% human serum albumin) with presence of viable tumor cells. (4–8) Images represent H&E stained sections from IT (100 µg/kg; 5–6) and IP (50 µg/kg; 7–8) treated mice with IL-13-PE and demonstrates presence of few viable cells with necrotic mass. Images are represented at 2.5X and 20X magnifications.