Abstract A57: The mesenchymal phenotype in recurrent glioblastoma is due to irradiation induced Snail expression and resultant EMT

Background: The mesenchymal subgroup of glioblastoma is more prevalent in recurrent tumors, which are known to present a worst prognosis. Ionizing irradiation is a known cause of epithelial to mesenchymal transition (EMT) in various cell types, but yet is still a vital part of glioblastoma treatment. Here we show that this mesenchymal change is due to an EMT effect in response to irradiation, and reflects the expression of the transcription factor Snail. Purpose: In this study we investigated the effect of irradiation on the EMT process and relate it to the mesenchymal phenotype that is demonstrated clinically. [Materials and Methods] Using the LINAC III accelerator, malignant glioma cell lines KMG4 and T98 along with primary patient cultures cells were irradiated at single dose 10Gy and multi fractionated 20Gy. Using qRT-PCR and Western blot analysis, mRNA and proteins were quantified in both the apoptotic and repopulating cells after irradiation. The re-populating cells after 21 days were monitored for phenotypic changes by invasion and wound healing assays, stemness marker SOX2 was quantified by immunofluorescence and tumor reformation ability was assessed by colony formation and xenograft assays, mortality and morbidity was quantified by orthotopic transplant. siRNA was used to knockdown, Snail, and transient overexpression of Snail gene, was done to determine the exact role that it plays in the progression to more mesenchymal phenotypes. We examined the phenotypic changes that occur based on standard EMT findings, including motility, invasiveness, expression of stemness markers, xenograft formation and colony formation assay. Results: In the short term, 48 hours after irradiation there is an increase in the EMT marker Snail and a further increase of this marker in the repopulating cells that would make up the recurrent tumor. Knockdown of Snail, not only results in decreased mesenchymal markers, but also decreased motility and invasion, these results are consistent in both single dose 10Gy and multi fractionated 20Gy irradiation levels. We also showed that the phosphorylation of the MAPK pathway plays a vital role in the downstream activation of EMT whereas the SMAD pathway remains non-activated and the inactivation of the GSK3β molecule results in higher Snail levels with a greater EMT effect. In cases where TGFβ concentration is increased, by co-culturing with fibroblasts, and/or monocytes, we showed that fibroblasts are the main source of TGFβ where as it is not secreted by glioma cells. Conclusion: Snail may be a useful target for inhibition to result in a decrease of recurrent mesenchymal phenotypes in malignant glioma producing a higher cure rate. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A57. Citation Format: Roshan Mahabir, Mishie Tanino, Aiman Elmansuri, Masumi Tsuda, Taichi Kimura, Lei Wang, Hiroshi Nishihara, Shinya Tanaka. The mesenchymal phenotype in recurrent glioblastoma is due to irradiation induced Snail expression and resultant EMT. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A57.