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. 2019 Feb 4;9(1):1281.
doi: 10.1038/s41598-019-39018-3.

Interleukin-13 receptor α2 is a novel marker and potential therapeutic target for human melanoma

Hayato Okamoto  1 , Yasuhiro Yoshimatsu  1   2 , Taishi Tomizawa  1 , Akiko Kunita  3 , Rina Takayama  2 , Teppei Morikawa  3 , Daisuke Komura  4 , Kazuki Takahashi  2 , Tsukasa Oshima  5 , Moegi Sato  1 , Mao Komai  1 , Katarzyna A Podyma-Inoue  2 , Hiroaki Uchida  1   6 , Hirofumi Hamada  1 , Katsuhito Fujiu  5   7 , Shumpei Ishikawa  4 , Masashi Fukayama  3 , Takeshi Fukuhara  1   8 , Tetsuro Watabe  9   10
Affiliations

Interleukin-13 receptor α2 is a novel marker and potential therapeutic target for human melanoma

Hayato Okamoto et al. Sci Rep. .

Abstract

Malignant melanoma is one of the untreatable cancers in which conventional therapeutic strategies, including chemotherapy, are hardly effective. Therefore, identification of novel therapeutic targets involved in melanoma progression is urgently needed for developing effective therapeutic methods. Overexpression of interleukin-13 receptor α2 (IL13Rα2) is observed in several cancer types including glioma and pancreatic cancer. Although IL13Rα2 is implicated in the progression of various types of cancer, its expression and roles in the malignant melanoma have not yet been elucidated. In the present study, we showed that IL13Rα2 was expressed in approximately 7.5% melanoma patients. While IL13Rα2 expression in human melanoma cells decreased their proliferation in vitro, it promoted in vivo tumour growth and angiogenesis in melanoma xenograft mouse model. We also found that the expression of amphiregulin, a member of the epidermal growth factor (EGF) family, was correlated with IL13Rα2 expression in cultured melanoma cells, xenograft tumour tissues and melanoma clinical samples. Furthermore, expression of amphiregulin promoted tumour growth, implicating causal relationship between the expression of IL13Rα2 and amphiregulin. These results suggest that IL13Rα2 enhances tumorigenicity by inducing angiogenesis in malignant melanoma, and serves as a potential therapeutic target of malignant melanoma.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Tissue microarray analyses for IL13Rα2 expression. Multiple series of tissue microarrays were subjected to immunohistochemical analysis by using anti-IL13Rα2 antibody (KH7B9). Expression of IL13Rα2 was detected in the cytoplasm or membrane of melanoma cells (arrows). Red arrowheads indicate melanin pigment. (A) Benign naevus of the right face. (B) Metastatic malignant melanoma from the armpit (lymph node). (C) Malignant melanoma of the thigh. (D) Malignant melanoma of the cunnus. (E) Malignant melanoma of the skin. IL13Rα2 was expressed by melanoma cells (arrows) but not by stromal cells (S). (F) Malignant melanoma of the right sole. Scale bar: 50 μm (A), 20 μm (B–F).
Figure 2
Figure 2
Effects of IL13Rα2 expression on the in vitro proliferation of SK-MEL-28 melanoma cells. The SK-MEL-28 cells were transfected with the expression plasmid encoding IL13Rα2 to establish SK-IL13Rα2 to study the effect of IL13Rα2 on cell proliferation. (A) The expression of IL13Rα2 in the SK-MEL-28, SK-IL13Rα2 and A375 (IL13Rα2-positive) cells was determined by qRT-PCR. (B) 2 × 104 of SK-MEL-28 and SK-IL13Rα2 cells were seeded into 6-well plates and were allowed to grow for 5 days. The cells were harvested and were counted on the indicated day. All values are shown as the ratios to the number of the seeded cells on day 0 and are mean ± SD. *p < 0.05; Student’s t-test.
Figure 3
Figure 3
Effects of IL13Rα2 expression on the in vivo tumour formation of SK-MEL-28 melanoma cells. The SK-MEL-28 (n = 6) and SK-IL13Rα2 (n = 6) cells were subcutaneously transplanted into the immunodeficient mice. (A) Tumour growth was assessed for 102 days after transplantation by callipers and was calculated from minor axis and major radius. All values are mean ± SE. *p < 0.05; Student’s t-test. (B) Images of representative tumours at 102 days post-transplantation. Scale bar: 10 mm.
Figure 4
Figure 4
Effects of IL13Rα2 expression on tumour angiogenesis. (A) Sections of the tumours derived from the SK-MEL-28 (n = 5) and SK-IL13Rα2 (n = 6) cells were subjected to immunofluorescence staining with the anti-PECAM-1 antibodies. Scale bar: 50 μm. (B) PECAM-1 positive area represented as a fraction of the total image area. All values are mean ± SE. *p < 0.05; Student’s t-test.
Figure 5
Figure 5
Roles of IL13Rα2 in the in vivo tumour formation and angiogenesis in A375 xenograft model. IL13RA2 gene was knocked out in A375 melanoma cells. (A) The expression of IL13Rα2 in A375-Control and A375-IL13RA2 KO cells was determined by immunoblotting analysis with the anti-IL13Rα2 (KH7B9) and anti-β-actin antibodies. Cropped images from the same blots are shown. Full-length blots are presented in Supplementary Fig. S7. (B) In all, 3 × 103 of A375-Control (n = 6) and A375-IL13RA2 KO (n = 6) cells were seeded into 12-well plates and allowed to grow for 3 days. Cells were harvested and were counted at the indicated period. (C) The A375-Control and A375-IL13RA2 KO cells were subcutaneously transplanted into immunodeficient mice. Tumour growth was measured using callipers and was calculated from minor axis and major radius. (D) Images of representative tumours. Scale bar: 10 mm. (E) Sections of tumours derived from A375 Control (n = 5) and A375-IL13RA2 KO (n = 6) cells were subjected to immunofluorescence staining with the anti-PECAM-1 antibodies. Scale bar: 100 μm. (F) PECAM-1 positive area represented as the fraction of total image area. All values are mean ± SE. *p < 0.05; Student’s t-test.
Figure 6
Figure 6
Effects of IL13Rα2 on amphiregulin expression in various types of melanoma cells. (A) Total RNAs were prepared from tumour tissues derived from the SK-MEL-28 (#1, #2) and SK-IL13Rα2 (#7, #8) cells and were subjected to quantitative RT-PCR analyses for the expression of IL13Rα2 (top) and amphiregulin (bottom). (B) The A2058 and SK-MEL-28 melanoma cells were transfected with IL13Rα2 expression vector, followed by qRT-PCR analysis for the expression of IL13Rα2 (top) and amphiregulin (bottom). (C) IL13RA2 was knocked out in the A375 melanoma cells, followed by qRT-PCR analysis for the expression of IL13Rα2. (D) The A375 melanoma cells were transfected with negative control (NC) siRNAs or siRNAs for IL13Rα2 (#1 and 2), followed by qRT-PCR analysis for the expression of IL13Rα2 (top) and amphiregulin (bottom). All values are mean ± SD. *p < 0.05; Student’s t-test.
Figure 7
Figure 7
Effects of amphiregulin expression on the in vivo tumour formation and angiogenesis in the SK-MEL-28 xenograft model. The SK-MEL-28 cells were transfected with the expression plasmid encoding amphiregulin to establish SK-amphiregulin cells. The SK-amphiregulin and SK-GFP cells, were then subcutaneously transplanted into the immunodeficient mice, followed by the measurement of tumour size (A). (B) Sections of the tumours derived from the SK-GFP (n = 6) and SK-amphiregulin (n = 6) cells were examined by performing immunofluorescence staining with the anti-PECAM-1 antibodies. Scale bars, 200 μm. (C) PECAM-1 positive area represented as the fraction of the total image area. All values are mean ± SE. *p < 0.05; Student’s t-test.
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