Elevating SOX2 Levels Deleteriously Affects the Growth of Medulloblastoma and Glioblastoma Cells Jesse L. Cox, Phillip J. Wilder, Michelle Desler, Angie Rizzino* Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, United States of America Abstract Medulloblastomas and glioblastomas are devastating tumors that respond poorly to treatment. These tumors have been shown to express SOX2 and overexpression of SOX2 has been correlated with poor prognosis. Although knockdown of SOX2 impairs the growth and tumorigenicity of brain tumor cells, it was unclear how elevating SOX2 levels would affect their fate. Interestingly, studies conducted with neural stem cells have shown that small increases or decreases in the level of this transcription factor significantly alter their fate. Here, we report that elevating SOX2 3-fold above endogenous levels in U87 and U118 glioblastoma, and DAOY medulloblastoma cells significantly impairs their ability to proliferate. We extended these findings and determined that elevating SOX2 in DAOY cells remodels their cell-cycle profile by increasing the proportion of cells in the G1-compartment, and induces the expression of genes associated with differentiation. Furthermore, we show that elevating SOX2 leads to a dramatic induction of CD133 expression in DAOY cells, yet inhibits the ability of both CD133+ and CD1332 cells to form neurospheres. Together, these findings argue that SOX2 levels must be carefully controlled in glioblastomas and medulloblastomas to maintain their fate. Equally important, our data suggests that increases in the expression of SOX2 during brain tumor progression are likely to be linked closely with changes in other critical genes that work in concert with SOX2 to enhance the tumorigenicity of brain tumors. Importantly, we demonstrate that this is also likely to be true for other cancers that express SOX2. Moreover, these studies demonstrate the advantage of using inducible promoters to study the effects of SOX2 elevation, as compared to gene expression systems that rely on constitutive expression. Citation: Cox JL, Wilder PJ, Desler M, Rizzino A (2012) Elevating SOX2 Levels Deleteriously Affects the Growth of Medulloblastoma and Glioblastoma Cells. PLoS ONE 7(8): e44087. doi:10.1371/journal.pone.0044087 Editor: Xin-yuan Guan, The University of Hong Kong, China Received June 12, 2012; Accepted August 1, 2012; Published August 28, 2012 Copyright: 2012 Cox et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Nebraska Department of Health; Contract grant number: (2011–29; 2012–33). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: arizzino@unmc.edu Introduction Highly undifferentiated and aggressive brain tumors, including medulloblastoma and glioblastoma, are devastating diseases, which are difficult to treat. Glioblastoma multiforme, the most common and aggressive primary brain tumor, has a median survival time of 14 months despite considerable efforts to improve treatment [1]. Pediatric central nervous system tumors are the second most common malignancy in children (second only to leukemia), of which, medulloblastoma is the most common type. While the 5-year survival of medulloblastoma patients is nearly 80%, a treatment regime involving resection, radiotherapy and chemotherapeutics is associated with significant co-morbidities, including growth and endocrine abnormalities, as well as impairment of cognitive function [2,3]. Moreover, the use of chemotherapeutics and radiation treatments predisposes young patients to future treatment-induced neoplasms and malignancy. Thus, new approaches are needed to improve the treatment of patients with medulloblastomas and glioblastomas. Increased expression of the transcription factor SOX2 has been reported in a growing list of tumors, including breast, prostate, lung and in a number of highly aggressive central nervous system neoplasms, including both glioblastoma and medulloblastoma [4– 8]. The expression and requirement of SOX2 in central nervous system tumors is not surprising, given that SOX2 is expressed in neural progenitor cells and their progeny [9]. In brain tumor initiating cells [10,11], hypomethylation of the SOX2 promoter has been directly correlated with SOX2 expression, and the SOX2 promoter is hypomethylated in aggressive glioblastoma patient samples [12]. Additionally, it has recently been reported that the SOX2 gene is amplified in nearly 10% of glioblastomas and overexpressed in over 85% of these tumors [12]. Moreover, the knockdown of SOX2 has been shown to decrease the proliferation and tumorigenicity of glioblastomas [13]. SOX2 levels must be tightly controlled for proper development of the nervous system. In particular, perturbation of SOX2 levels in chick neural stem cells (NSC) has been shown to disrupt their fate. Aberrant elevation of SOX2 in chick NSC prevents their differentiation; whereas SOX2 knockdown induces the expression of gene markers of neural differentiation and causes the commitment of NSC to a differentiated fate [14,15]. The careful regulation of SOX2 levels is also required to support the self- renewal of pluripotent, embryonic stem cells (ESC). In this regard, a 2-fold increase [16] or the knockdown of Sox2 [17] in mouse ESC disrupts their cell fate, causing ESC to lose their capacity for self-renewal and pluripotency. Although NSC and ESC are adversely affected by increases in the levels of SOX2, other studies have reported that breast [18], prostate [19] and lung [20] cancer cells engineered to constitutively express elevated levels of SOX2 (,3- to 4-fold) PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e44087