SOX2 Controls Fate of Brain Tumor Cells cells following 48 and 96 hours exposure 6 Dox (1 mg/mL). (D) Cell cycle analysis of i-SOX2-DAOY cultured in the absence or presence of Dox (1 mg/ mL) for 48 hours. Data for cells without Dox was collected as independent duplicates; whereas, samples with Dox were collected in triplicate. Error bars represent standard deviation. The student’s t-test was used to determine p-values. doi:10.1371/journal.pone.0044087.g006 in the level of endogenous transcripts containing the SOX2 39UTR. Additionally, transcript levels of CD133 were elevated significantly and exhibited an increase of more than three cycle- threshold values (Ct Values). Thus, exogenous expression of SOX2 induces the expression of the cell surface marker CD133 at both the RNA level (Figure 8B) and at the cell surface expression level (Figure 8A). To gain deeper insight into the changes in gene expression induced by elevation of SOX2, RNA isolated from i-SOX2- DAOY cells, cultured in the absence or presence of Dox for 24 hours, was subjected to Affymetrix microarray analysis (Table S2, Accession No. GSE36947, available at http://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc = GSE36947). Scatter plot analy- sis of the microarray data demonstrated significant changes in the gene expression profile of DAOY cells following the induction of SOX2 (Figure 8C). Of 8,518 transcripts represented on the microarray, 1.67% (142) were upregulated and 5.61% (478) were downregulated $2-fold. Gene ontology classification and analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID) to characterize the transcripts and biological processes changed $2-fold by elevation of SOX2 [32,33]. Genes associated with cell-adhesion, cell death and cell proliferation, were largely downregulated; whereas, genes associ- ated with neural development, metabolism and transcription were upregulated (Figure 8D). To confirm our microarray results, transcript levels of genes used as markers for neural development were examined more closely using RT-qPCR. As expected, PAX6 and MAP2, markers of neuronal differentiation [34,35], rose (Figure 8B); whereas, transcript levels of Nestin (NES), a neural progenitor cell marker and a marker of general central nervous system development, decreased upon exogenous SOX2 expression (Figure 8B). The changes observed in markers associated with neural development align with our observations above demonstrating that iSox-DAOY accumulate in the G1-cell cycle compartment and become quiescent (Figure 6D). Moreover, our RT-qPCR analysis closely aligned with the changes in gene expression determined by microarray analysis. Together, our findings argue that aberrant expression of SOX2 in i-SOX2-DAOY cells limits their pro- liferation and induces the DAOY cells to exhibit markers of neural differentiation. Discussion Improving our understanding of the fundamental mechanisms that govern cell proliferation and self-renewal is vital for the future development of therapeutic approaches for treating devastating cancers, such as glioblastoma and medulloblastoma. In this regard, studies to understand the basic biology of ESC have revealed that the transcription factor SOX2 is a central node in the vast gene regulatory networks necessary for maintaining the self-renewal and pluripotency of ESC. Moreover, studies conducted with ESC and NSC demonstrated that small changes in the expression of SOX2 dramatically alter their fate. In an important connection to these findings, SOX2 overexpression has been reported recently in many aggressive tumors, including tumors of the breast, lung, prostate and brain. In this study, we demonstrate that elevation of SOX2 levels ,2- fold in U87 and U118 glioblastoma cells leads to significant reductions in cellular proliferation by ,65% (Figure 1) and ,55% (Figure S1), respectively. Likewise, a 3-fold increase in SOX2 levels dramatically reduces DAOY medulloblastoma cell pro- liferation by ,90% (Figure 2). Importantly, neurosphere studies conducted with freshly isolated CD1332 DAOY cells, as well as putative tumor-initiating, CD133+ DAOY cells, support the conclusion that SOX2 levels must be carefully regulated in order to maintain the proliferation of DAOY medulloblastoma cells (Figure 7). In an effort to understand how elevating SOX2 retards the growth of DAOY cells, we probed for changes in cell-cycle distribution, gene expression and cell surface markers. In the case of the cell cycle, we observed significant changes 2 days after SOX2 levels were elevated (Figure 6D). Notably, there was a significant enrichment in the G1 compartment. Subsequent analysis demonstrated that this accumulation appears to reflect an increase in the proportion of quiescent cells in the population, rather than an increase in senescence as evidenced by a lack of b- galactosidase staining. Interestingly, RNA analysis indicated that elevation of SOX2 in i-SOX2-DAOY cells led to increases in the expression of gene markers associated with neural differentiation and reduced expression of markers associated with multipotent neural stem cells (Figure 8). Thus, our data suggest that ectopically elevating SOX2 in DAOY medulloblastoma cells causes them to undergo physiological changes that resemble a more differentiated phenotype. In addition to significant changes in cell fate, cell cycle and gene expression, elevating SOX2 alters the proportion of the DAOY cell population that expresses the cell surface markers CD133 and SSEA1. Induction of exogenous SOX2 shifts the distribution of DAOY cells towards CD133 and/or SSEA1 expressing cells. Importantly, this finding suggests that CD133/SSEA1 low and high populations are not static, but that these populations are fluid and can interconvert. Moreover, our studies suggest that over- expression of SOX2 imparts a significant stress upon brain tumor cells. Interestingly, other studies have shown that brain tumor cells can dynamically alter the distribution of cells that express CD133 in response to changes in their culture environment, specifically changes in oxygen tension [36]. Together, our findings with SOX2 and those observed at low oxygen levels raise the possibility that CD133 plays an important role in the stress response of brain tumor cells in particular, and in cellular physiology in general. Previous reports provide insight into regulatory mechanisms that may be essential for the careful regulation of SOX2 in brain tumors. The SOX2 gene promoter has been reported to be hypomethylated in clinical glioblastoma tumor samples, thereby enabling the expression of the SOX2 gene [12]. Thus, it is likely that regulatory mechanisms, which suppress the aberrant expres- sion of SOX2 in other neural tissues, must be modified to support the long-term expression of SOX2 in brain tumor cells. Brain tumor cells expressing SOX2 also have regulatory mechanisms in place to restrain its aberrant expression. In this regard, miR-145 has been shown to block the translation of SOX2 transcripts in glioblastoma cells [37]. Additionally, SOX2 has been shown to be modified by a number of post-translational modifications, in- cluding phosphorylation [38], sumoylation [39], methylation [40], acetylation and ubiquitination [41], that may affect the localiza- tion, stability and function of SOX2. Understanding which mechanisms are used to control the expression and function of PLOS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e44087