Cancer stem cells (CSC) are essential for tumorigenesis. The transcription factor Sox2 is overexpressed in brain gliomas, and is essential to maintain CSC. In mouse high‐grade glioma pHGG cells in culture, Sox2 deletion causes cell proliferation arrest and inability to reform tumors after transplantation in vivo; in Sox2‐deleted cells, 134 genes are derepressed. To identify genes mediating Sox2 deletion effects, we overexpressed into pHGG cells nine among the most derepressed genes, and identified four genes, Ebf1, Hey2, Zfp423, and Cdkn2b, that strongly reduced cell proliferation in vitro and brain tumorigenesis in vivo. CRISPR/Cas9 mutagenesis of each gene, individually or in combination (Ebf1 + Cdkn2b), significantly antagonized the proliferation arrest caused by Sox2 deletion. The same genes also repressed clonogenicity in primary human glioblastoma‐derived CSC‐like lines. These experiments identify a network of critical tumor suppressive Sox2‐targets whose inhibition by Sox2 is involved in glioma CSC maintenance, defining new potential therapeutic targets.

Sizes of neuronal, astroglial and oligodendroglial complements forming the neonatal cerebral cortex largely depend on rates at which pallial stem cells give rise to lineage-committed progenitors and the latter ones progress to mature cell types. Here, we investigated the spatial articulation of pallial stem cells’ (SCs) commitment to astrogenesis as well as the progression of committed astroglial progenitors (APs) to differentiated astrocytes, by clonal and kinetic profiling of pallial precursors. We found that caudal-medial (CM) SCs are more prone to astrogenesis than rostro-lateral (RL) ones, while RL-committed APs are more keen to proliferate than CM ones. Next, we assessed the control of these phenomena by 2 key transcription factor genes mastering regionalization of the early cortical primordium, Emx2 and Foxg1, via lentiviral somatic transgenesis, epistasis assays, and ad hoc rescue assays. We demonstrated that preferential CM SCs progression to astrogenesis is promoted by Emx2, mainly via Couptf1, Nfia, and Sox9 upregulation, while Foxg1 antagonizes such progression to some extent, likely via repression of Zbtb20. Finally, we showed that Foxg1 and Emx2 may be implicated—asymmetrically and antithetically—in shaping distinctive proliferative/differentiative behaviors displayed by APs in hippocampus and neocortex.

The Sox2 transcription factor is expressed in different neural tumors. In particular, it is active within the "cancer stem cell" (CSC) subpopulation of tumor cells, able to reinitiate tumorigenesis after conventional chemotherapy (to which it is usually resistant). This led to hypothesize that Sox2 (and its downstream regulated genes) may qualify as promising targets for therapeutic strategies directed against CSC. However, the potential relevance of Sox2 in this regard depends on whether it is functionally important to maintain CSC. Here, we comparatively examine the effects of Sox2 genetic ablation within mouse models of different neural tumor types. Sox2 ablation in mouse glioma (and in human glioblastomaderived CSC) demonstrated a critical function for Sox2 in the maintenance of CSC. Surprisingly, however, Sox2 ablation in two different mouse models of melanoma (a neural crest-related tumor), and in a mouse model of medulloblastoma of the Sonic Hedgehog subgroup, showed that, in these contexts, Sox2 is dispensable for tumorigenesis. This heterogeneous situation has a parallel in the normal development of the nervous system, where generalized Sox2 ablation in neural stem/ progenitor cells selectively affects the development of some neural regions, but not other ones. Molecular mechanisms underlying these specificities may involve the regulation, by Sox2, of different sets of target genes in different tumors, but also a redundant regulation of the same targets by different Sox transcription factors, differentially coexpressed with Sox2 in different tumors. Collectively, these findings point to the need to experimentally address the requirement for Sox2, and its downstream targets, within different tumor types, as a prerequisite to fully exploit its potential as a target for novel therapeutic approaches.

Cancer stem cells (CSC) are essential for tumorigenesis. The transcription factor Sox2 is overexpressed in brain tumors. In gliomas, Sox2 is essential to maintain CSC. In mouse high-grade glioma pHGG, Sox2 deletion causes cell proliferation arrest and inability to reform tumors in vivo;134 genes are significantly derepressed. To identify genes mediating the effects of Sox2 deletion, we overexpressed into pHGG cells nine among the most derepressed genes, and identified four genes, Cdkn2b, Ebf1, Zfp423 and Hey2, that strongly reduced cell proliferation in vitro and brain tumorigenesis in vivo. CRISPR/Cas9 mutagenesis, or pharmacological inactivation, of each of these genes, individually, showed that their activity is essential for the proliferation arrest caused by Sox2 deletion. These Sox2-inhibited antioncogenes also inhibited clonogenicity in primary human glioblastoma-derived cancer stem-like cell lines. These experiments identify critical anti-oncogenic factors whose inhibition by Sox2 is involved in CSC maintenance, defining new potential therapeutic targets for gliomas. Main PointsSox2 maintains glioma tumorigenicity by repressing the antioncogenic activity of a regulatory network involving the Ebf1, Hey2, Cdkn2b and Zfp423 genes.Mutation of these genes prevents the cell proliferation arrest of Sox2-deleted glioma cells.

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