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. 2020 Aug;20(2):1989-1998.
doi: 10.3892/ol.2020.11699. Epub 2020 Jun 5.

STAT3 is a key molecule in the oncogenic behavior of diffuse intrinsic pontine glioma

Jinju Park  1 , Woochan Lee  2 , Sangil Yun  1 , Saet Pyoul Kim  1 , Kyung Hyun Kim  1   3 , Jong-Il Kim  2 , Seung-Ki Kim  3 , Kyu-Chang Wang  1   3 , Ji Yeoun Lee  1   3
Affiliations

STAT3 is a key molecule in the oncogenic behavior of diffuse intrinsic pontine glioma

Jinju Park et al. Oncol Lett. 2020 Aug.

Abstract

Diffuse intrinsic pontine glioma (DIPG) is one of the most lethal childhood brain tumors. This tumor is unique because it is detected exclusively in the ventral pons of patients aged between 6 and 7 years, which suggests a developmental nature of its formation. Signal transducer and activator of transcription 3 (STAT3) is a critical molecule for the differentiation of neural stem cells into astrocytes during neurodevelopment. Additionally, STAT3 is associated with oncogenesis and the epithelial-mesenchymal transition (EMT) in various types of tumor. In recent years, several studies have demonstrated the oncogenic role of STAT3 in high-grade gliomas. However, the role of STAT3 in DIPG at the cellular level remains unknown. To assess the possible association between gliogenesis and DIPG, the expression levels of various molecules participating in the differentiation of neural stem cells were compared between normal brain control tissues and DIPG tissues using open public data. All of the screened genes exhibited significantly increased expression in DIPG tissues compared with normal tissues. As STAT3 expression was the most increased, the effect of STAT3 inhibition in a DIPG cell line was assessed via STAT3 short hairpin (sh)RNA transfection and treatment with AG490, a STAT3 inhibitor. Changes in viability, apoptosis, EMT and radiation therapy efficiency were also evaluated. Downregulation of STAT3 resulted in decreased cyclin D1 expression and cell viability, migration and invasion. Additionally, treatment with STAT3 shRNA or AG490 suppressed the EMT phenotype. Finally, when radiation was administered in combination with STAT3 inhibition, the therapeutic efficiency, assessed by cell viability and DNA damage repair, was increased. The present results suggest that STAT3 is a potential therapeutic target in DIPG, especially when combined with radiation therapy.

Keywords: astrogliogenesis; diffuse intrinsic pontine glioma; epithelial-mesenchymal transition; signal transducer and activator of transcription 3.

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Figures

Figure 1.
Figure 1.
mRNA expression levels of astrogliogenesis-associated genes are high in DIPG. (A) In silico analysis of astrogliogenesis-associated gene mRNA expression in normal brain and DIPG tissues. (B) Relative STAT3 mRNA expression in normal brain and DIPG tissues. Each circle represents a tissue sample. DIPG, diffuse intrinsic pontine glioma; NOTCH1, Notch receptor 1; ID1, inhibitor of DNA binding 1; ACVR1, activin A receptor type I; HES1, Hes family bHLH transcription factor 1; SMAD1, SMAD family member 1; EP300, E1A binding protein p300; LIFR, LIF receptor subunit α; STAT3, signal transducer and activator of transcription 3.
Figure 2.
Figure 2.
STAT3 inhibition suppresses human diffuse intrinsic pontine glioma cell viability. SF8628 cells were treated with vehicle control (DMSO) or AG490. SF8628 cells were transfected shCtrl or shSTAT3. (A) mRNA STAT3 expression was determined via reverse transcription-semi-quantitative PCR. β-actin mRNA was used as the loading control. (B) Cell viability data of AG490-treated cells. Cells were treated with various concentrations of AG490. Cell viability was analyzed using a CCK-8 assay, and absorbance was measured at 420 nm. *P<0.05 vs. DMSO-treated cells using one-way ANOVA. Data are presented as the mean ± SD. (C and D) CCK-8 assay of control cells and STAT3-inhibited cells. Cell viability was analyzed using a CCK-8 assay, and absorbance was measured at 420 nm. *P<0.05 vs. DMSO-treated cells or shCtrl-transfected cells at 48 h using a two-tailed unpaired Student's t-test. Data are presented as the mean ± SD. (E) Western blot analysis of pSTAT3, total STAT3 and cyclin D1. Protein samples were extracted from SF8628 control cells and STAT3-inhibited cells; β-actin was used as the loading control. Exposure time was 3 min for pSTAT3, 1 min for STAT3, 30 sec for cyclin D1 and 15 sec for β-actin. STAT3, signal transducer and activator of transcription 3; pSTAT3, phosphorylated STAT3; shCtrl, control short hairpin RNA; shSTAT3, STAT3 short hairpin RNA; CCK-8, Cell Counting Kit-8.
Figure 3.
Figure 3.
STAT3 inhibition suppresses human diffuse intrinsic pontine glioma cell migration and invasion by regulating EMT. SF8628 cells were treated with vehicle control (DMSO) or AG490. SF8628 cells were transfected with shCtrl or shSTAT3. (A) Western blot analysis of EMT markers. Protein samples were extracted from SF8628 control cells and STAT3-inhibited cells. Protein samples were tested for E-cadherin, N-cadherin, Vimentin, Twist, Snail, MMP-9 and β-actin (loading control) expression. Exposure time was 5 min for E-cadherin, 1 min for N-cadherin, Twist and Snail, 10 sec for Vimentin, 3 min for MMP-9 and 15 sec for β-actin. (B) Organization of the actin cytoskeleton was determined by immunofluorescence staining. Alexa Fluor 633-conjugated phalloidin was used to visualize F-actin (red), and DAPI staining (blue) was used for visualization of cell nuclei. Original magnification, ×400. Scale bar, 2 µm. (C) Cells were seeded in the upper chamber of Transwell inserts, and the cell migration ability was assessed 48 h after cell plating. Magnification, ×40. Scale bar, 100 µm. (D) Cells were seeded in the upper chamber of Transwell inserts coated with Matrigel, and the cell invasion ability was measured 48 h after cell plating. Magnification, ×40. Scale bar, 100 µm. The results were calculated as percentages relative to control cells. Representative images of invasive cells are shown next to each bar graph. Data are presented as the mean ± SD. *P<0.05 using a two-tailed unpaired Student's t-test. STAT3, signal transducer and activator of transcription 3; shCtrl, control short hairpin RNA; shSTAT3, STAT3 short hairpin RNA; EMT, epithelial-mesenchymal transition; MMP-9, matrix metallopeptidase-9; DAPI, 4′6′-diamidio-2-phenoylindole.
Figure 4.
Figure 4.
STAT3 inhibition sensitizes human diffuse intrinsic pontine glioma cells to radiation by interfering with DNA damage repair. SF8628 cells were treated with control vehicle (DMSO) or AG490. SF8628 cells were transfected with shCtrl or shSTAT3. (A and B) CCK-8 assays of control cells and STAT3-inhibited cells at 0 and 24 h after radiation treatment. Cell viability was analyzed using a CCK-8 assay, and absorbance was measured at 420 nm. *P<0.05 using one-way ANOVA. Data are presented as the mean ± SD. (C and D) Analysis of DNA damage repair after treatment with 4 Gy radiation by visualizing the double-strand marker γH2AX. The panel shows representative images of γH2AX (green), DAPI-stained nuclei (blue) and merged images of SF8628 cells without irradiation and at 1, 4 and 24 h after radiation. Scale bar, 1 µm. STAT3, signal transducer and activator of transcription 3; shCtrl, control short hairpin RNA; shSTAT3, STAT3 short hairpin RNA; CCK-8, Cell Counting Kit-8; γH2AX, phosphorylated H2A X variant histone; DAPI, 4′6′-diamidio-2-phenoylindole.
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