The stem cell–determining transcription factor Sox2 is required for the maintenance of normal neural stem cells. In this study, we investigated the requirement for Sox2 in neural cancer stem-like cells using a conditional genetic deletion mutant in a mouse model of platelet-derived growth factor–induced malignant oligodendroglioma. Transplanting wild-type oligodendroglioma cells into the brain generated lethal tumors, but mice transplanted with Sox2-deleted cells remained free of tumors. Loss of the tumor-initiating ability of Sox2-deleted cells was reversed by lentiviral-mediated expression of Sox2. In cell culture, Sox2-deleted tumor cells were highly sensitive to differentiation stimuli, displaying impaired proliferation, increased cell death, and aberrant differentiation. Gene expression analysis revealed an early transcriptional response to Sox2 loss. The observed requirement of oligodendroglioma stem cells for Sox2 suggested its relevance as a target for therapy. In support of this possibility, an immunotherapeutic approach based on immunization of mice with SOX2 peptides delayed tumor development and prolonged survival. Taken together, our results showed that Sox2 is essential for tumor initiation by mouse oligodendroglioma cells, and they illustrated a Sox2-directed strategy of immunotherapy to eradicate tumor-initiating cells. Cancer Res; 74(6); 1833–44. ©2014 AACR.

Background: Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an increasingly popular technique to map genome-wide binding profiles of histone modifications, transcription factors, and co-factors. The ENCODE project and others have compiled blacklists for ChIP-seq which have been widely adopted: these lists contain regions of high and unstructured signal, regardless of cell type or protein target, indicating that these are false positives. While CUT&RUN obtains similar results to ChIP-seq, its biochemistry and subsequent data analyses are different. We found that this results in a CUT&RUN-specific set of undesired high-signal regions. Results: We compile suspect lists based on CUT&RUN data for the human and mouse genomes, identifying regions consistently called as peaks in negative controls. Using published CUT&RUN data from our and other labs, we show that the CUT&RUN suspect regions can persist even when peak calling is performed with SEACR or MACS2 against a negative control and after ENCODE blacklist removal. Moreover, we experimentally validate the CUT&RUN suspect lists by performing reiterative negative control experiments in which no specific protein is targeted, showing that they capture more than 80% of the peaks identified. Conclusions: We propose that removing these problematic regions can substantially improve peak calling in CUT&RUN experiments, resulting in more reliable datasets.

The transcription factor Sox2 has been shown to play essential roles during embryonic development as well as in cancer. To more precisely understand tumor biology and to identify potential therapeutical targets, we thoroughly investigated the expression and function of Sox2 in medulloblastoma, a malignant embryonic brain tumor that initiates in the posterior fossa and eventually spreads throughout the entire cerebrospinal axis. We examined a large series of tumor samples (n = 188) to show that SOX2 is specifically expressed in Sonic hedgehog (SHH)-associated medulloblastoma with an interesting preponderance in adolescent and adult cases. We further show that cerebellar granule neuron precursors (CGNP), which are believed to serve as the cell of origin for this medulloblastoma subgroup, express Sox2 in early stages. Also, Shh-associated medulloblastoma can be initiated from such Sox2-positive CGNPs in mice. Independent of their endogenous Sox2 expression, constitutive activation of Shh signaling in CGNPs resulted in significantly enhanced proliferation and ectopic expression of Sox2 in vitro and Sox2-positive medulloblastoma in vivo. Genetic ablation of Sox2 from murine medulloblastoma did not affect survival, most likely due to a compensatory overexpression of Sox3. However, acute deletion of Sox2 from primary cultures of CGNPs with constitutive Shh signaling significantly decreased proliferation, whereas overexpression of Sox2 enhanced proliferation of murine medulloblastoma cells. We conclude that Sox2 is a marker for Shh-dependent medulloblastomas where it is required and sufficient to drive tumor cell proliferation. Cancer Res; 73(12); 3796–807. ©2013 AACR.

Human teeth contain a variety of mesenchymal stem cell populations that could be used for cell-based regenerative therapies. However, the isolation and potential use of these cells in the clinics require the extraction of functional teeth, a process that may represent a significant barrier to such treatments. Fibroblasts are highly accessible and might represent a viable alternative to dental stem cells. We thus investigated and compared the in vitro differentiation potential of human dental pulp stem cells (hDPSCs), gingival fibroblasts (hGFs) and foreskin fibroblasts (hFFs). These cell populations were cultured in osteogenic and adipogenic differentiation media, followed by Alizarin Red S and Oil Red O staining to visualize cytodifferentiation. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was performed to assess the expression of markers specific for stem cells (NANOG, OCT-4), osteogenic (RUNX2, ALP, SP7/OSX) and adipogenic (PPAR-γ2, LPL) differentiation. While fibroblasts are more prone towards adipogenic differentiation, hDPSCs exhibit a higher osteogenic potential. These results indicate that although fibroblasts possess a certain mineralization capability, hDPSCs represent the most appropriate cell population for regenerative purposes involving bone and dental tissues.

Transcriptional cofactors that participate in Wnt/β-catenin signaling act in the cytoplasm to promote proper development of tooth enamel.