Co-operative roles of IL-4Rα and IL-13Rα1 in the progression of ovarian carcinomas and the survival of ovarian carcinoma patients.

Dear Editor, Recently, there has been a rise in interest in the roles of cytokines and cytokine receptors in malignant human tumors. Cytokine receptors, especially type II interleukin-4 receptor (IL-4R), have recently been examined as novel therapeutic targets of human cancers [1-3]. The type II IL-4R complex is composed of IL-4Rα and interleukin-13 receptor α1 (IL-13Rα1), and the expression of IL-4Rα and IL-13Rα1 was higher in cancer tissues compared with normal counterpart tissues [3, 4]. Furthermore, higher expression of IL-4Rα or IL-13Rα1 was associated with shorter survival of cancer patients [5, 6]. This expression of IL-4Rα and IL-13Rα1 in human cancer is related to their roles in regulating signaling mechanisms involved in cancer progression, including the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway [3, 5]. Therefore, the IL-4R-related pathway is targeted in anti-cancer therapeutic strategies [3, 5, 6]. However, despite extensive investigations into the role of IL-4Rα and IL-13Rα1 in the regulation of the biological mechanism of immune cells and tumor cells [2-4], there have been limited reports focused on the role of IL-4Rα/IL-13Rα1 in ovarian carcinomas. Recently, cytokine-related activation of the JAK2/STAT3 pathway was implicated in the progression of ovarian cancers through the induction of epithelial-to-mesenchymal transition (EMT) [7]. In addition, it was shown that EMT is a vital mechanism in the progression of human cancer and resistance of cancers to conventional anti-cancer therapies [8, 9]. Therefore, when considering the roles of IL-4Rα and IL-13Rα1 in JAK-related cancer progression [2, 3, 5], IL-4Rα and IL-13Rα1 might be involved in the progression of ovarian cancers. Therefore, this study investigated the expression of IL-4Rα and IL-13Rα1 in human ovarian cancer tissues and further evaluated the roles of IL-4Rα and IL-13Rα1 in the progression of ovarian carcinomas in conjunction with the EMT phenotype. Detailed study methods are described in the Supplementary Materials and Methods. In human ovarian carcinomas, IL-4Rα and IL-13Rα1 were primarily expressed in the cytoplasm and nuclei of tumor cells (Figure 1A). Clear expression of IL-4Rα or IL-13Rα1 in the cytoplasmic membrane was not seen. The positivity of immunohistochemical expression of IL-4Rα and IL-13Rα1 was determined with receiver operating characteristic curve analysis (Supplementary Figure S1). IL-4Rα positivity was significantly associated with older age, increased serum level of cancer antigen 125, higher TNM stage, higher histologic grade, and IL-13Rα1 positivity, whereas IL-13Rα1 positivity was significantly associated with higher tumor stage and bilateral tumor (Supplementary Table S2). In univariate survival analysis, IL-4Rαand IL-13Rα1 expression were significantly associated with both overall survival (OS) and relapse-free survival (RFS) (all P < 0.01; Figure 1B, Supplementary Table S3). In addition, the expression of IL-4Rα and IL-13Rα1 were significantly associated with OS and RFS in 97 serous carcinomas (Supplementary Figure S2A), but limited significance in low-grade serous carcinomas, high-grade serous carcinomas, endometrioid carcinomas, and mucinous carcinomas (Supplementary Figure S2B-E). In multivariate analysis, IL-13Rα1 positivity (OS, P = 0.010) and IL-4Rα positivity (RFS, P = 0.029) were independent indicators of patient survival (Supplementary Table S3). Furthermore, the combined expression patterns of IL-4Rα and IL-13Rα1 were significantly associated with the survival of overall ovarian carcinomas (Figure 1C-D, Supplementary Tables S4-S5), serous carcinomas, and mucinous carcinomas (Supplementary Figure S3A-B), but limited significance in low-grade serous carcinomas, high-grade serous carcinomas, and endometrioid carcinomas (Supplementary Figure S3C-E). Multivariate analysis indicated the combined expression patterns of IL-4Rα and IL-13Rα1 to be an independent prognostic indicator of OS (P = 0.005) and RFS (P = 0.025) (Supplementary Table S5). Furthermore, in OVCAR3 and SKOV3 cells, knockdown of either IL-4Rα or IL-13Rα1 significantly inhibited the proliferation of cells (Figure 1E-F, Supplementary Figure S4). In contrast, overexpression of either IL-4Rα or IL-13Rα1 significantly increased the proliferation of ovarian cancer cells (Figure 1E-F, Supplementary Figure S4). In addition, the knockdown of either IL-4Rα or IL-13Rα1 significantly decreased the migration and invasion activities of OVCAR3 and SKOV3 cells, but overexpression of either IL-4Rα or IL-13Rα1 significantly increased these activities (Figure 1G-H, Supplementary Figure S5). Furthermore, IL-4Rα- or IL-13Rα-mediated invasiveness of ovarian cancer cells was associated with the expression of the molecules related to EMT. The knockdown of IL-4Rα or IL-13Rα1 increased the expression of E-cadherin, an important indicator of EMT, and decreased the expression of N-cadherin, Snail, transforming growth factor β1 (TGF-β1), β-catenin, and Cyclin D1 (Figure 1I-J, Supplementary Figure S6). In contrast, overexpression of IL-4Rα or IL-13Rα1 decreased the expression of E-cadherin and increased the expression of N-cadherin, Snail, TGF-β1, β-catenin, and Cyclin D1 (Figure 1I-J, Supplementary Figure S6). The knockdown or overexpression of IL-4Rα influenced the expression of IL-13Rα1 protein and vice versa (Figure 1I). However, the overexpression of IL-4Rα or IL-13Rα1 did not influence the expression of mRNA of IL-4Rα or IL-13Rα1 (Figure 1J). Therefore, these data suggest that IL-4Rα and IL-13Rα1 might be involved in the stabilization of each other. As expected, immunoprecipitation of IL-4Rα or IL-13Rα1 showed direct binding of IL-4Rα and IL-13Rα1 proteins (Supplementary Figure S7). In addition, cycloheximide-mediated degradation of IL-4Rα was accelerated with the knockdown of IL-13Rα1 and vice versa (Figure 1K). Furthermore, poly-ubiquitination of IL-4Rα increased with the knockdown of IL-13Rα1, and poly-ubiquitination of IL-13Rα1 increased with the knockdown of IL-4Rα (Figure 1L). This result suggests that both IL-4Rα and IL-13Rα1 are involved in the post-translational stabilization of each other. Thereafter, we evaluated the co-operative effects of IL-4Rα and IL-13Rα1 in tumor growth after co-transfection (Supplementary Figure S8A). Overexpression of both IL-4Rα and IL-13Rα1 significantly increased proliferation and co-knockdown of IL-4Rα and IL-13Rα1 significantly decreased proliferation of SKOV3 cells in a CCK8 assay and a colony-forming assay (Supplementary Figure S8B-C). In a xenograft animal model, overexpression of either IL-4Rα or IL-13Rα1 significantly increased proliferation compared with controls (Figure 1M-O). Furthermore, tumors overexpressing both IL-4Rα and IL-13Rα1 were significantly larger than tumors overexpressing IL-4Rα or IL-13Rα1 (Figure 1M-O). Knockdown of IL-4Rα, IL-13Rα1, and both IL-4Rα and IL-13Rα1 significantly decreased tumor size compared with controls (Figure 1M-O). Therefore, when considering the results of the expression of IL-4Rα and IL-13Rα1 in human ovarian cancers and ovarian cancer cells, our results suggest that IL-4Rα and IL-13Rα1 are co-operatively involved in the progression of ovarian carcinomas by stabilizing each other. In addition, based on our finding that the knockdown of IL-4Rα and/or IL-13Rα1 inhibits the proliferation and invasiveness of ovarian cancer cells, blocking IL-4, IL-13, IL-4Rα, IL-13Rα1, or molecules downstream of IL-4Rα and IL-13Rα1 signaling might be effective therapeutic targets for ovarian cancers. In conclusion, this study showed high expression of IL-4Rα and IL-13Rα1 to be an indicator of poor prognosis of ovarian carcinoma patients, and both IL-4Rα and IL-13Rα1 are co-operatively involved in the progression of ovarian carcinomas by stabilizing each other from proteasomal degradation. In addition, blocking IL-4Rα and IL-13Rα1 inhibited proliferation, invasiveness, and the EMT phenotype in ovarian cancer cells. Therefore, this study suggests that a therapeutic modality targeting the IL-4, IL-13, IL-4Rα, or IL-13Rα1 pathway might be a novel therapeutic approach for ovarian carcinoma patients with tumors expressing high levels of IL-4Rα and/or IL-13Rα1. WKC, UKH, AGA, JZ, KMK, ARA, HSP, SHP, DHC, and KYJ participated in the study design. WKC, UKH, AGA, JZ, KMK, ARA, HSP, SHP, DHC, and KYJ performed the experiment. WKC, HSP, DHC, and KYJ were involved in data collection and data interpretation. WKC, UKH, AGA, JZ, SHP, DHC, and KYJ participated in the statistical analyses. WKC, UKH, AGA, JZ, KMK, ARA, HSP, SHP, DHC, and KYJ wrote the manuscript. All authors read and approved the final manuscript. We thank DB Leveson-Gower for providing medical writing services. The authors declare that they have no competing interests. This work was supported by grants from the Medical Research Center Program (2017R1A5A2015061) through the National Research Foundation (NRF), which is funded by the Korean government, the Ministry of Science, ICT and Future Planning (MSIP). Not applicable This study obtained institutional review board approval from Jeonbuk National University Hospital (IRB number, CUH 2021-04-047-001) and was performed according to the Declaration of Helsinki. Based on the retrospective and anonymous character of the study, the approval contained a waiver for written informed consent. All animal experiments were performed with the approval of the institutional animal care and use committee of Jeonbuk National University (approval number: JBNU 2021-0164). The datasets used in the current study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.