In vivo radiation response of proneural glioma characterized by protective p53 transcriptional program and proneural-mesenchymal shift

Abstract

Glioblastoma is the most common adult primary brain tumor and has a dismal median survival. Radiation is a mainstay of treatment and significantly improves survival, yet recurrence is nearly inevitable. Better understanding the radiation response of glioblastoma will help improve strategies to treat this devastating disease. Here, we present a comprehensive study of the in vivo radiation response of glioma cells in a mouse model of proneural glioblastoma. These tumors are a heterogeneous mix of cell types with differing radiation sensitivities. To explicitly study the gene expression changes comprising the radiation response of the Olig2(+) tumor bulk cells, we used translating ribosome affinity purification (TRAP) from Olig2-TRAP transgenic mice. Comparing both ribosome-associated and total pools of mRNA isolated from Olig2(+) cells indicated that the in vivo gene expression response to radiation occurs primarily at the total transcript level. Genes related to apoptosis and cell growth were significantly altered. p53 and E2F were implicated as major regulators of the radiation response, with p53 activity needed for the largest gene expression changes after radiation. Additionally, radiation induced a marked shift away from a proneural expression pattern toward a mesenchymal one. This shift occurs in Olig2(+) cells within hours and in multiple genetic backgrounds. Targets for Stat3 and CEBPB, which have been suggested to be master regulators of a mesenchymal shift, were also up-regulated by radiation. These data provide a systematic description of the events following radiation and may be of use in identifying biological processes that promote glioma radioresistance.

Fig. 1.

Ten gray of IR induces transient cell cycle arrest, asynchronous apoptosis, and depletion of Olig2⁺ cells in mouse glioma. Staining for phospho-histone H3 (A), TUNEL (B), Olig2 (C), and Nestin (D). (E) TUNEL⁺ cells predominantly in tumor bulk rather than Nestin⁺ perivascular region 24 h after IR. (F) Quantification of Olig2⁺ nuclei in glioma following IR. Error bars are SD. *P < 0.05, **P < 0.005.

Fig. 2.

In vivo gene expression changes induced by IR in RCAS-PDGF Ink4a/Arf^−/− gliomas collected 6 h after 10-Gy IR. (A) Log₂ change in total mRNA vs. log₂ change in ribosome-bound translating mRNA 6 h after 10-Gy IR. R, Pearson correlation. (B) All probes altered more than twofold in translating mRNA, and corresponding changes in total mRNA and translational efficiency. (C) Volcano plot of expression changes 6 h after 10-Gy IR. Genesets most significantly enriched (D) and depleted (E) among translating mRNAs after IR, with selected enrichment plots. FDR q-val, calculated false discovery rate; NES, Normalized Enrichment Score.

Fig. 3.

p53 and E2F are major drivers of in vivo radiation response. (A) Transcription factors whose targets are most significantly enriched and depleted among translating mRNAs after IR. (B) Enrichment plots for targets of p53 and E2F. (C) Unsupervised hierarchical clustering of genes significantly altered by IR in PDGF and PDGF/p53shRNA gliomas. (D) Kaplan–Meier survival curves of unirradiated and irradiated PDGF/p53shRNA gliomas in Ink4a/Arf^−/− mice after postnatal day 18 randomization and single-dose 10-Gy IR. (E) Fold changes of the 50 most induced transcripts 6 h after 10-Gy IR in total RNA from PDGF-driven gliomas, and the corresponding fold change in total RNA from PDGF/p53shRNA tumors.

Fig. 4.

IR induces proneural-to-mesenchymal shift in PDGF gliomas. (A) Enrichment plots of CEBP and STAT3 targets in translating mRNA 6 h after 10-Gy IR in PDGF Ink4a/Arf^−/− gliomas. (B) Time course of CEBP and STAT3 target up-regulation in PDGF Ink4a/Arf^+/− gliomas. Average of total and translating mRNA pools is shown; dotted lines are SE. (C) Enrichment plots of mesenchymal and proneural signature genes in translating mRNA after 10-Gy IR in PDGF-driven Ink4a/Arf^−/−, Ink4a/Arf^−/−/PTEN^−/−, and Ink4a/Arf^−/−/p53shRNA gliomas. (D) Enrichment plots of mesenchymal and proneural signature genes in the relative changes of translating mRNA after IR of PDGF Ink4a/Arf^−/− tumors vs. PDGF Ink4a/Arf^−/−/p53shRNA tumors.