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. 2021 Apr 8;11(4):284.
doi: 10.3390/jpm11040284.

IL13Rα2 Is Involved in the Progress of Renal Cell Carcinoma through the JAK2/FOXO3 Pathway

Mi-Ae Kang  1 , Jongsung Lee  2 , Chang Min Lee  3 , Ho Sung Park  4   5   6 , Kyu Yun Jang  4   5   6 , See-Hyoung Park  3
Affiliations

IL13Rα2 Is Involved in the Progress of Renal Cell Carcinoma through the JAK2/FOXO3 Pathway

Mi-Ae Kang et al. J Pers Med. .

Abstract

Previously, we reported a close relationship between type II IL4Rα and IL13Rα1 complex and poor outcomes in renal cell carcinoma (RCC). In this study, we investigated the clinicopathologically significant oncogenic role of IL13Rα2, a kind of the independent receptor for IL13, in 229 RCC patients. The high expression of IL13Rα2 was closely related to relapse-free survival in specific cancers in univariate and multivariate analysis. Then, the oncogenic role of IL13Rα2 was evaluated by performing in vitro assays for cell proliferation, cell cycle arrest, and apoptosis in A498, ACHN, Caki1, and Caki2, four kinds of RCC cells after transfection of siRNA against IL13Rα2. Cell proliferation was suppressed, and apoptosis was induced in A498, ACHN, Caki1, and Caki2 cells by knockdown of IL13Rα2. Interestingly, the knockdown of IL13Rα2 decreased the phosphorylation of JAK2 and increased the expression of FOXO3. Furthermore, the knockdown of IL13Rα2 reduced the protein interaction among IL13Rα2, phosphorylated JAK2, and FOXO3. Since phosphorylation of JAK2 was regulated by IL13Rα2, we tried to screen a novel JAK2 inhibitor from the FDA-approved drug library and selected telmisartan, a clinically used medicine against hypertension, as one of the strongest candidates. Telmisartan treatment decreased the cell proliferation rate and increased apoptosis in A498, ACHN, Caki1, and Caki2 cells. Mechanistically, telmisartan treatment decreased the phosphorylation of JAK2 and increased the expression of FOXO3. Taken together, these results suggest that IL13Rα2 regulates the progression of RCC via the JAK2/FOXO3-signaling path pathway, which might be targeted as the novel therapeutic option for RCC patients.

Keywords: FOXO3; IL13Rα2; JAK2; renal cell carcinoma; telmisartan.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Immunohistochemical expression and survival analysis for the expression of IL13Rα2 in renal cell carcinomas. (A) Immunohistochemical expression of IL13Rα2 in clear cell renal cell carcinoma, chromophobe renal cell carcinoma, and papillary renal cell carcinoma tissue. Original magnification, ×400. (B) Receiver operator characteristic curve analysis to determine the cutoff point of IL13Rα2 immunostaining. The cutoff point is determined to predict cancer-specific survival of renal cell carcinoma patients. The cutoff point has the highest area under the curve (AUC). Arrow indicates a cutoff point for the IL13Rα2 immunostaining. (C) Kaplan–Meier survival analysis for cancer-specific survival and relapse-free survival according to the immunohistochemical positivity for IL13Rα2 in 229 cell renal cell carcinomas.
Figure 2
Figure 2
Kaplan–Meier survival analysis in histologic subtypes of renal cell carcinomas. Kaplan–Meier survival curves for cancer-specific survival (CSS) and relapse-free survival (RFS) according to the expression of IL13Rα2 in clear cell renal cell carcinoma (A), chromophobe renal cell carcinoma (B), and papillary renal cell carcinoma (C).
Figure 3
Figure 3
Antiproliferative effect by transfection of siRNA against IL13Rα2 in A498, ACHN, Caki1, and Caki2 cells. Cell viability and proliferation rate were determined by WST-1 (A), cell counting assay (B) for 24, 48, and 72 h, and Colony formation assay for 14 days (C). This result is representative data of at least three independent experiments, and the error bar indicates mean ± standard error (STE). * stands for the P-value < 0.05. Cell cycle arrest for 48 h after transfection was determined by cell cycle analysis (D). Apoptosis for 48 h after transfection was determined by Annexin V staining analysis (E) and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (F). This result represents at least three independent experiments (G) Western blotting analysis of proteins related to cell cycle arrest and apoptosis for 48 h after transfection. β-actin was used for a gel-loading control.
Figure 4
Figure 4
Protein interaction between IL13Rα2 and JAK2. Knock-down of IL4Rα2 in A498, ACHN, Caki1, and Caki2 cells reduced the interaction between IL13Rα2 and JAK2. Cells were transfected with siRNA against IL4Rα2 or control siRNA. Then cell lysates were immunoprecipitated with antibodies against IL4Rα2 (A), JAK2 (B), or FOXO3 (C). The immunoprecipitated proteins were immunoblotted by IL4Rα2, pJAK2, JAK2, and FOXO3 antibodies. Light chain of IgG was used for the loading control. (D) 293T cells were co-transfected with Myc-IL4Rα2 and HA-JAK2 (O.E.) or a control plasmid DNA (pCMV6-C-Myc-Flag and pCMV3-C-HA, Con.) as indicated. Then cell lysates were immunoprecipitated with antibodies against Myc or HA. The immunoprecipitated proteins were immunoblotted by Myc, HA, IL4Rα2, JAK2 antibodies. Light chain of IgG and Coomassie Blue staining of SDS–PAGE were used for the loading control.
Figure 5
Figure 5
Antiproliferative effect by telmisartan treatment in A498, ACHN, Caki1, and Caki2 cells. Cell viability and proliferation rate were determined by WST-1 (A), cell counting assay (B) for 24, 48, and 72 h, and Colony formation assay for 14 days (C) after treatment of telmisartan (0, 20, and 40 μM). This result is representative data of at least three independent experiments, and the error bar indicates mean ± standard error (STE). * stands for the P-value < 0.05. Cell cycle arrest for 48 h after treatment of telmisartan (40 μM) was determined by cell cycle analysis (D). Apoptosis for 48 h after treatment of telmisartan (40 μM) was determined by Annexin V staining analysis (E) and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (F). This result is representative data of at least three independent experiments. (G) Western blotting analysis of proteins related to cell cycle arrest and apoptosis for 48 h after treatment of telmisartan (40 μM). β-actin was used for a gel-loading control.
Figure 6
Figure 6
A diagram for the possible oncogenic role of IL13Rα2 in renal cell carcinoma (RCC) by activation of JAK2 and inhibition of FOXO3.
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