Abstract SY42-01: Translational control and tumor stem cell radioresponse
Authors: Tofilon Philip J. 
Published: May 2015

Analogous to prokaryotic cells, radiation-induced gene expression in eukaryotic cells has long been assumed to comprise an adaptive or protective response against radiation-induced death. Thus, defining the inducible and putatively protective genes may not only provide insight into the fundamental mechanisms regulating radioresponse, but may also lead to the identification of targets for radiation modifiers. Along these lines, the radiation-induced changes in total cellular mRNA (i.e., the transcriptome) have been extensively cataloged using single gene and whole genome approaches. However, analyses reported for a variety of irradiated tumor and normal cell lines have revealed few commonly affected genes, even among cell lines originating from the same histology. Moreover, the vast majority of mRNA changes detected after irradiation do not correlate with changes in the corresponding protein, a situation that has made it difficult to assign functional significance to radiation-induced gene expression. In contrast to prokaryotes, eukaryotic cell gene expression is heavily dependent on post-transcriptional processes, i.e. translational control, which is not accounted for by profiling the cellular transcriptome. Using the microarray analysis of polysome-bound RNA, we have shown in established cell lines that radiation selectively regulates gene translation, a process that operates independently from changes in the transcriptome. In contrast to the radiation-induced transcriptome, characteristics of the radiation-induced translational control of gene expression include a correlation between the genes whose translational activity is affected by radiation and the expression of their corresponding protein. These initial results indicate that radiation does modify gene expression but does so primarily via post-transcriptional processes. Moreover, these results suggested that defining the mRNAs whose translation is affected by radiation and the molecules that mediate this form of translational control should not only contribute to the fundamental understanding of cellular radioresponse but also suggest novel strategies for tumor radiosensitization.

Along these lines, we have begun to extend our studies on the radiation-induced translational control of gene expression to glioblastoma cells. Radiotherapy remains a primary treatment modality for glioblastomas (GBMs) significantly contributing to the prolongation of patient survival. However, whereas many GBMs initially respond, they essentially all recur; even in combination with surgery and chemotherapy, the median survival of patients with GBM continues to be dismal with the vast majority succumbing to disease within 2 years of diagnosis. Defining the processes and molecules responsible for this radioresistance should provide a rational basis for designing target based strategies that enhance GBM radiosensitivity and therapeutic response. Investigations aimed at delineating the determinants of GBM radioresponse have traditionally focused on in vitro cultures of long established glioma cell lines. The biology of these glioma cell lines as reflected by genetic abnormalities, gene expression and orthotopic growth patterns, however, has little in common with GBM in situ. With respect to a more biologically accurate model system, data now suggest that GBMs are driven and maintained by a subpopulation of clonogenic cells referred to as glioblastoma stem-like cells (GSCs). GSCs have a number of in vitro properties in common with normal neural stem cells and, in contrast to the traditional glioma cell lines, when implanted into the brains of immuno-compromised mice grow as invasive neoplasms.

To investigate the radiation-induced translational control of gene expression in GSCs, initial analyses were performed on the CD133+ cell line NSC11, which was isolated from neurospheres generated from a human GBM surgical specimen. Whereas previous studies on traditional cell lines were limited to analysis at 6h after irradiation, in the current experiment GSCs were collected at 1 and 6h after exposure to 2 Gy. For each treatment group total cellular RNA (transcriptome) and polysome-bound RNA (translatome) were isolated and subjected to microarray analysis using U133A 2.0 Affymetrix GeneChip. Differential gene expression was defined according to p ≤ 0.05 and a fold change of greater or less than 1.5 in irradiated versus untreated cells. In total RNA preparations the number of genes whose mRNA levels were modified at 1h after exposure to 2Gy was approximately 25; at 6h after irradiation the number increased to approximately 114 genes. There were no commonly affected genes in the radiation-induced transcriptomes determined at 1 and 6h after irradiation. Analysis of the translatome revealed that the polysome association of greater than 3000 genes was modified at 1h after irradiation, which was reduced to approximately 700 at 6h. Importantly, greater than 90% of the genes whose translational activity was affected at 6h were also affected at 1h. These data indicate that the GSC translatome is considerably more susceptible to radiation-induced changes than is the transcriptome. Moreover, in contrast to the transcriptome, modifications in the translatome are a relatively early event after irradiation as illustrated by the substantial reduction in gene changes from 1 to 6h. Finally, there was essentially no overlap between the genes comprising the radiation-induced changes in the translatome and transcriptome suggesting that the 2 processes are mediated through different mechanisms.

As an alternative method for comparing the radiation-induced transcriptome and translatome in NSC11 cells RNA-seq. was performed. In this study cells were exposed to 2 Gy and collected 1h later. Total cellular RNA and polysome-bound RNA were then subjected to RNA-seq. Differential gene expression was defined according to p ≤ 0.05 and a fold change of greater or less than 1.5 in irradiated versus untreated cells. As expected, RNA-seq. was more sensitive in detecting changes in gene expression after irradiation with greater than 5000 genes in the polysome fraction and approximately 80 in total RNA. Comparison of the radiation-induced translatomes defined by microarray analysis and RNA-seq. showed a significant overlap in the genes detected by both methods. These results are thus consistent with translational control serving as the primary mechanism mediating radiation-induced changes in gene expression in GSCs.

As an initial investigation into whether radiation-induced translational control influences radiosensitivity our studies have focused on mTOR kinase, which is a critical regulator of cap-dependent translation. To inhibit mTOR we used INK128, a clinically applicable, ATP competitive mTOR inhibitor that targets both mTORC1 and mTORC2 activity. Treatment of GSCs with INK128 effectively inhibited mTOR activity as indicated by the reductions in phosphorylation of 4E-BP1, S6 kinase and Akt. As determined by the microarray analysis of polysome-bound RNA, INK128 exposure also significantly reduced the number of genes whose translational activity was modified by radiation. Finally, the radiosensitivity of GSCs as defined by clonogenic analysis was enhanced by INK128 added to cultures after irradiation. These data suggest that radiation-induced translational control is a determinant of GSC radiosensitivity and that competitive mTOR inhibitors may enhance GBM radiosensitivity.

Citation Format: Philip J. Tofilon. Translational control and tumor stem cell radioresponse. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY42-01. doi:10.1158/1538-7445.AM2014-SY42-01