YM155 decreases radiation-induced invasion and reverses epithelial-mesenchymal transition by targeting STAT3 in glioblastoma

Abstract

Background: Radiotherapy constitutes a standard arm of therapy in the multimodal treatment of patients with glioblastoma (GBM). Ironically, studies have recently revealed that radiation can augment malignant progression, by promoting migration and invasion, which make the disease especially difficult to cure. Here, we investigated the anticancer effects of YM155, a purported radiosensitizer, in GBM cell lines.

Methods: GBM cell lines U251 and U87 were treated with YM155 to assess cytotoxicity and activity of the molecule in vitro. Nude mice were implanted with cells to generate orthotopic xenografts for in vivo studies. Response of cells to treatment was examined using cell viability, immunofluorescence, wound healing, and the Transwell invasion assay. Molecules potentially mediating response were examined through western blot analysis, phospho-kinase arrays, and qPCR. Cells were transfected with siRNA knockdown and gene expression constructs to identify molecular mediators of response.

Results: YM155 reduced viability of U251 and U87 cells and enhanced radiosensitivity through inhibition of homologous recombination. Besides, YM155 decreased invasion caused by radiation and led to expression changes in molecular markers associated with EMT. STAT3 was one of 10 molecules identified on a phosphokinase array exhibiting significant change in phosphorylation under YM155 treatment. Transfection with STAT3 siRNAs or expression constructs demonstrated that EMT changes were achieved by inhibiting the phosphorylation of STAT3 and were survivin-independent. Finally, combining YM155 and radiation in orthotopic xenografts reduced growth and prolonged overall survival of animals.

Conclusions: YM155 decreased radiation-induced invasion in GBM cell lines in vitro and in vivo through inhibition of STAT3.

Keywords: Epithelial–mesenchymal transition; Glioblastoma; Radiation; STAT3; YM155.

Fig. 1

YM155 decreases cell viability and inhibits proliferation of GBM cells in vitro. a Cell viability of U251 and U87 cells after treatment for 48 h with increasing concentrations of YM155 using the CCK-8 assay. b Representative images of EdU incorporation performed on U251 and U87 cells treated with YM155 for 48 h at the concentrations indicated. c Graphic representation of the quantitation of EdU incorporation following treatment of cells with YM155 at the concentration indicated. **P < 0.01; ***P < 0.001; size bars = 50 µm

Fig. 2

YM155 impairs HR. a Cell viability of U251 and U87 pretreated with DMSO or 5 nM YM155 after increased doses of radiation. b Bar graph representation of the number of colonies formed as related to control (DMSO). c Bar graph representation of the percentage of apoptosis as related to control (DMSO). Early apoptosis is measured using annexin V and late apoptosis is measured using PI. d Western blots to assess protein levels of γ-H2AX after treatment of indicated cells with DMSO, YM155, radiation and combined treatment (5 nM YM155 + radiation 4 Gy). e Quantitation of chemiluminescence of western blot analysis in (d). f Western blot analysis for protein levels of Rad51, BRCA1 and γ-H2AX in lysates prepared from cells treated with increasing concentrations of YM155. g HR efficiency of U251 and U87 cells after treatment with DMSO or 5 nM YM155 evaluated using qPCR to detect levels of a recombined fragment. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 3

YM155 decreases invasion induced by radiation and reverses EMT in GBM cells. a Representative images of Transwell Matrigel assays from U251 and U87 cells in DMSO, radiation, and combination treatment (YM155 5 nM + radiation 4 Gy) fixed and stained with crystal violet. b Quantitation of migrated cell numbers from (a). c Morphology of U251 and U87 cells under bright field microscopy after treatment with vehicle or YM155. d Representative fluorescence images of phalloidin staining of DMSO and YM155 (5 nM) treated U251 and U87 cells. e Western blot analysis of E-cadherin, N-cadherin, β-catenin, Zeb1, and slug in U251 and U87 cells incubated with increasing concentrations of YM155. **P < 0.01; ***P < 0.001; size bars = 50 µm

Fig. 4

STAT3 mediates EMT reversal induced by YM155 independently of survivin. a Morphology of U251 and U87 cells before and after knockdown with survivin siRNA, or knockdown with YM155 treatment. b Phospho-kinase array of 43 phosphorylated kinases in U251 cells treated with 5 nM YM155 relative to DMSO control. c Graphic representation of the quantitation of 10 molecules with the most significant difference in phosphorylation status between YM155 treatment and control. d Western blot analysis of pSTAT3 (Ser727), pSTAT3 (Tyr705), STAT3, Cyclin D1 and c-Myc of U251 and U87 cells incubated with increasing concentrations of YM155. e Western blot analysis of pSTAT3 (Ser727), pSTAT3 (Tyr705), STAT3, N-cadherin and E-cadherin in U251 and U87 cells treated with DMSO, radiation, and combination treatment (YM155 5 nM + radiation 4 Gy)

Fig. 5

STAT3 mediates EMT reversal induced by YM155. a Western blot analysis of pSTAT3 (Ser727), pSTAT3 (Tyr705), STAT3, E-cadherin, and N-cadherin in U251 and U87 cells before and after knockdown with STAT3 siRNA. b Morphology of U251 and U87 cells before and after STAT3 siRNA knockdown. c Crystal violet staining of Transwell Matrigel assay for U251 and U87 cells control or transfected with STAT3 siRNA after radiation treatment. d Quantitation of migrating cell number in experiments from (c). e Western blot analysis of pSTAT3 (Ser727), pSTAT3 (Tyr705) and STAT3 in lysates prepared from control cells and cells stably expressing STAT3. f Crystal violet staining of Transwell Matrigel assay from control or stable ectopic expression of STAT3 U251 cells after control, radiation (4 Gy), YM155 (5 nM) and combination treatment (5 nM YM155 + 4 Gy radiation). g Quantitation of migrating cell number in experiments from (f). h Crystal violet staining of Transwell Matrigel assay from YM155 treated U251 and U87 cells or stable ectopic expression of STAT3 U251 and U87 cells following YM155 treatment. i Quantitation of migrating cell number in experiments from (h). **P < 0.01; ***P < 0.001; size bars = 50 µm

Fig. 6

YM155 increases the antitumor effect of radiotherapy in vivo. a Survival curve of animals implanted with U251 cells in the four different treatment groups indicated. b Representative images of hematoxylin and eosin staining of brain sections from animals in each of the four different treatment groups indicated. c IHC for pSTAT3 (Ser727) and pSTAT3 (Tyr705) in xenograft sections from animals in each of the four different treatment groups. Quantitation of positive staining nuclei. Positively staining cells are presented as a percentage of total number of cells counted. **P < 0.01; size bars = 50 µm. d Representative images of subcutaneous tumors with U87 cells. Tumor volume and tumor weight were measured. **P < 0.01