Identification of a novel role of IL-13Rα2 in human Glioblastoma multiforme: interleukin-13 mediates signal transduction through AP-1 pathway

Abstract

Background: Previously, we have demonstrated that Interleukin 13 receptor alpha 2 (IL-13Rα2) is overexpressed in approximate 78% Glioblastoma multiforme (GBM) samples. We have also demonstrated that IL-13Rα2 can serve as a target for cancer immunotherapy in several pre-clinical and clinical studies. However, the significance of overexpression of IL-13Rα2 in GBM and astrocytoma and signaling through these receptors is not known. IL-13 can signal through IL-13R via JAK/STAT and AP-1 pathways in certain cell lines including some tumor cell lines. Herein, we have investigated a role of IL-13/IL-13Rα2 axis in signaling through AP-1 transcription factors in human glioma samples in situ.

Methods: We examined the activation of AP-1 family of transcription factors (c-Jun, Fra-1, Jun-D, c-Fos, and Jun-B) after treating U251, A172 (IL-13Rα2 +ve) and T98G (IL-13Rα2 -ve) glioma cell lines with IL-13 by RT-qPCR, and immunocytochemistry (ICC). We also performed colorimetric ELISA based assay to determine AP-1 transcription factor activation in glioma cell lines. Furthermore, we examined the expression of AP-1 transcription factors in situ in GBM and astrocytoma specimens by multiplex-immunohistochemistry (IHC). Student t test and ANOVA were used for statistical analysis of the results.

Results: We have demonstrated up-regulation of two AP-1 transcription factors (c-Jun and Fra-1) at mRNA and protein levels upon treatment with IL-13 in IL-13Rα2 positive but not in IL-13Rα2 negative glioma cell lines. Both transcription factors were also overexpressed in patient derived GBM specimens, however, in contrast to GBM cell lines, c-Fos is also overexpressed in patient derived specimens. Astrocytoma specimens showed lesser extent of immunostaining for IL-13Rα2 and three AP-1 factors compared to GBM specimens. By transcription factor activation assay, we demonstrated that AP-1 transcription factors (C-Jun and Fra-1) were activated upon treatment of IL-13Rα2 + GBM cell lines but not IL-13Rα2 - GBM cell line with IL-13. Our results demonstrate functional activity of AP-1 transcription factor in GBM cell lines in response to IL-13.

Conclusions: These results indicate that IL-13/IL-13Rα2 axis can mediate signal transduction in situ via AP-1 pathway in GBM and astrocytoma and may serve as a new target for GBM immunotherapy.

Keywords: AP-1; Glioblastoma; IL-13; IL-13Rα2; Transcription factors.

Fig. 1

Evaluation of AP-1 transcription factors in IL-13 treated U251 and T98G cell lines. a IL13Rα2 staining with Streptavidin 594 (red) and DAPI used for nuclear staining (blue). b c Jun staining with Streptavidin 594 (red) and DAPI used for nuclear staining (blue). c Fra-1 staining with Streptavidin 594 (red) and DAPI used for nuclear staining (blue). d Jun B staining with Streptavidin 594 (red) and DAPI used for nuclear staining (blue). All images were captured at ×1000 magnification

Fig. 2

AP-1 transcription factors expression in GBM cell lines. mRNA was analyzed by RT-qPCR for AP-1 transcription factors expression in three GBM cell lines. Values are mean of three independent experiments each done in quadruplicate determinations. Results are expressed as % relative fluorescence units (RFU) normalized to β-actin expression

Fig. 3

AP-1 transcription factor activation assay by ELISA. Nuclear extracts from two IL-13Rα2 positive GMB cell lines, U251 and A172 and IL-13Rα2 negative GBM cell line T98G were analyzed for AP-1 transcription factors after stimulation with IL-13. The assay was run in quadruplicate and results are expressed as arbitrary units after measuring the absorbance at 450 nm

Fig. 4

Analysis of AP-1 transcription factors in Human GBM samples and normal human brain sample. a IL13Rα2 staining with Streptavidin 594 (red) and DAPI used for nuclear staining (blue). b c Jun staining with Streptavidin 594 (red) and DAPI used for nuclear staining (blue). c c Fos staining with Streptavidin 594 (red) and DAPI used for nuclear staining (blue). d Jun B staining with Streptavidin 594 (red) and DAPI used for nuclear staining (blue). Immunostained sections were photographed at ×1000 magnification. Extent of immunostaining and percent positive field were counted and analyzed

Fig. 5

Evaluation of AP-1 transcription factors in human Glioblastoma, astrocytoma and normal brain specimens. Expression of AP-1 family members was analyzed in situ by IFA. The specimens were evaluated in a blinded manner by 3 investigators and the results are shown as mean ± SD. Extent of immunostaining for each phenotype corresponds to ≤ 1+, negative; 2 + , positive; 3 + , strongly positive; 4 + , more strongly positive

Fig. 6

Analysis of AP-1 transcription factors in situ. Schematic representation of the results in human brain tissue specimen outlining the signaling mechanism of AP-1 in GBM, Astrocytoma and Normal brain tissues