Sox2 is required for tumor development and cancer cell proliferation in osteosarcoma.

The stem cell transcription factor Sox2 is highly expressed in many cancers where it is thought to mark cancer stem cells (CSCs). In osteosarcomas, the most common bone malignancy, high Sox2 expression marks and maintains a fraction of tumor-initiating cells that show all the properties of CSC. Knockdown of Sox2 expression abolishes tumorigenicity and suppresses the CSC phenotype.

Myeloid Zinc Finger 1 and GA Binding Protein Co-Operate with Sox2 in Regulating the Expression of Yes-Associated Protein 1 in Cancer Cells.

The transcription factor (TF) yes-associated protein 1 (YAP1) is a major effector of the tumor suppressive Hippo signaling pathway and is also necessary to maintain pluripotency in embryonic stem cells. Elevated levels of YAP1 expression antagonize the tumor suppressive effects of the Hippo pathway that normally represses YAP1 function. High YAP1 expression is observed in several types of human cancers and is particularly prominent in cancer stem cells (CSCs).

Phosphoproteomics of Fibroblast Growth Factor 1 (FGF1) Signaling in Chondrocytes: Identifying the Signature of Inhibitory Response.

Fibroblast growth factor (FGF) signaling is vital for many biological processes, beginning with development. The importance of FGF signaling for skeleton formation was first discovered by the analysis of genetic FGFR mutations which cause several bone morphogenetic disorders, including achondroplasia, the most common form of human dwarfism. The formation of the long bones is mediated through proliferation and differentiation of highly specialized cells - chondrocytes.

PPARγ agonists promote differentiation of cancer stem cells by restraining YAP transcriptional activity.

Osteosarcoma (OS) is a highly aggressive pediatric bone cancer in which most tumor cells remain immature and fail to differentiate into bone-forming osteoblasts. However, OS cells readily respond to adipogenic stimuli suggesting they retain mesenchymal stem cell-like properties. Here we demonstrate that nuclear receptor PPARγ agonists such as the anti-diabetic, thiazolidinedione (TZD) drugs induce growth arrest and cause adipogenic differentiation in human, mouse and canine OS cells as well as in tumors in mice.

Sox2 antagonizes the Hippo pathway to maintain stemness in cancer cells.

The repressive Hippo pathway has a profound tumour suppressive role in cancer by restraining the growth-promoting function of the transcriptional coactivator, YAP. We previously showed that the stem cell transcription factor Sox2 maintains cancer stem cells (CSCs) in osteosarcomas. We now report that in these tumours, Sox2 antagonizes the Hippo pathway by direct repression of two Hippo activators, Nf2 (Merlin) and WWC1 (Kibra), leading to exaggerated YAP function.

FIREWACh: high-throughput functional detection of transcriptional regulatory modules in mammalian cells.

Promoters and enhancers establish precise gene transcription patterns. The development of functional approaches for their identification in mammalian cells has been complicated by the size of these genomes. Here we report a high-throughput functional assay for directly identifying active promoter and enhancer elements called FIREWACh (Functional Identification of Regulatory Elements Within Accessible Chromatin), which we used to simultaneously assess over 80,000 DNA fragments derived from nucleosome-free regions within the chromatin of embryonic stem cells (ESCs) and identify 6,364 active regulatory elements.

Morphological comparison of the craniofacial phenotypes of mouse models expressing the Apert FGFR2 S252W mutation in neural crest- or mesoderm-derived tissues.

Bones of the craniofacial skeleton are derived from two distinct cell lineages, cranial neural crest and mesoderm, and articulate at sutures and synchondroses which represent major bone growth sites. Premature fusion of cranial suture(s) is associated with craniofacial dysmorphogenesis caused in part by alteration in the growth potential at sutures and can occur as an isolated birth defect or as part of a syndrome, such as Apert syndrome. Conditional expression of the Apert FGFR2 S252W mutation in cells derived from mesoderm was previously shown to be necessary and sufficient to cause coronal craniosynostosis.

SOX2 regulates YAP1 to maintain stemness and determine cell fate in the osteo-adipo lineage.

The osteoblastic and adipocytic lineages arise from mesenchymal stem cells (MSCs), but few regulators of self-renewal and early cell-fate decisions are known. Here, we show that the Hippo pathway effector YAP1 is a direct target of SOX2 and can compensate for the self-renewal defect caused by SOX2 inactivation in osteoprogenitors and MSCs. Osteogenesis is blocked by high SOX2 or YAP1, accelerated by depletion of either one, and the inhibition of osteogenesis by SOX2 requires YAP1.

The B55α regulatory subunit of protein phosphatase 2A mediates fibroblast growth factor-induced p107 dephosphorylation and growth arrest in chondrocytes.

Fibroblast growth factor (FGF)-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types and underlies several genetic skeletal disorders caused by activating FGF receptor (FGFR) mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the retinoblastoma protein (Rb) family member p107 by protein phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report, we show that the PP2A-B55α holoenzyme (PP2A containing the B55α subunit) is responsible for this phenomenon.

Isolation and analysis of DNA derived from nucleosome-free regions.

Precise regulation of the levels and timing of gene expression is fundamental to all biological processes and is largely determined by the activity of cis-regulatory modules, containing the binding sites for transcription factors, within promoters and enhancers. The global identification of these transcriptional regulatory elements within mammalian genomes, and understanding when and where they are active, is an important effort that will require the development and implementation of several different technologies. Here we detail a means for the identification of transcriptional regulatory elements using functional assays.

Overexpression of cyclin E/CDK2 complexes overcomes FGF-induced cell cycle arrest in the presence of hypophosphorylated Rb proteins.

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent.

Mesodermal expression of Fgfr2S252W is necessary and sufficient to induce craniosynostosis in a mouse model of Apert syndrome.

Coordinated growth of the skull and brain are vital to normal human development. Craniosynostosis, the premature fusion of the calvarial bones of the skull, is a relatively common pediatric disease, occurring in 1 in 2500 births, and requires significant surgical management, especially in syndromic cases. Syndromic craniosynostosis is caused by a variety of genetic lesions, most commonly by activating mutations of FGFRs 1-3, and inactivating mutations of TWIST1.

Perspectives on cancer stem cells in osteosarcoma.

Osteosarcoma is an aggressive pediatric tumor of growing bones that, despite surgery and chemotherapy, is prone to relapse. These mesenchymal tumors are derived from progenitor cells in the osteoblast lineage that have accumulated mutations to escape cell cycle checkpoints leading to excessive proliferation and defects in their ability to differentiate appropriately into mature bone-forming osteoblasts. Like other malignant tumors, osteosarcoma is often heterogeneous, consisting of phenotypically distinct cells with features of different stages of differentiation.

Regulation of cranial morphogenesis and cell fate at the neural crest-mesoderm boundary by engrailed 1.

The characterization of mesenchymal progenitors is central to understanding development, postnatal pathology and evolutionary adaptability. The precise identity of the mesenchymal precursors that generate the coronal suture, an important structural boundary in mammalian skull development, remains unclear. We show in mouse that coronal suture progenitors originate from hedgehog-responsive cephalic paraxial mesoderm (Mes) cells, which migrate rapidly to a supraorbital domain and establish a unidirectional lineage boundary with neural crest (NeuC) mesenchyme.

Distinct functions of Sox2 control self-renewal and differentiation in the osteoblast lineage.

The transcription factor Sox2 is a key player in the maintenance of pluripotency and "stemness." We have previously shown that Sox2 maintains self-renewal in the osteoblast lineage while inhibiting differentiation (U. Basu-Roy et al.

The Sox2 high mobility group transcription factor inhibits mature osteoblast function in transgenic mice.

We have previously shown that in osteoblasts Sox2 expression can be induced by Fgfs, and can inhibit Wnt signaling and differentiation. Furthermore, in mice in which Sox2 is conditionally deleted in the osteoblastic lineage, bones are osteopenic, and Sox2 inactivation in cultured osteoblasts leads to a loss of proliferative ability with a senescent phenotype. To help understand the role of Sox2 in osteoblast development we have specifically expressed Sox2 in bone from a Col1α1 promoter, which extended Sox2 expression into more mature osteoblasts.

Regulation of non-classical FGF1 release and FGF-dependent cell transformation by CBF1-mediated notch signaling.

FGF1, a widely expressed proangiogenic factor involved in tissue repair and carcinogenesis, is released from cells through a non-classical pathway independent of endoplasmic reticulum and Golgi. Although several proteins participating in FGF1 export were identified, genetic mechanisms regulating this process remained obscure. We found that FGF1 export and expression are regulated through Notch signaling mediated by transcription factor CBF1 and its partner MAML.

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G₂arrest in RCS chondrocytes.

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G₁ phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G₂ phase of the cycle for the first 4-6 hours of the treatment.

Early onset of craniosynostosis in an Apert mouse model reveals critical features of this pathology.

Activating mutations of FGFRs1-3 cause craniosynostosis (CS), the premature fusion of cranial bones, in man and mouse. The mechanisms by which such mutations lead to CS have been variously ascribed to increased osteoblast proliferation, differentiation, and apoptosis, but it is not always clear how these disturbances relate to the process of suture fusion. We have reassessed coronal suture fusion in an Apert Fgfr2 (S252W) mouse model.

PP2A-mediated dephosphorylation of p107 plays a critical role in chondrocyte cell cycle arrest by FGF.

FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations. This phenomenon requires the function of the p107 and p130 members of the Rb protein family, and p107 dephosphorylation is one of the earliest distinguishing events in FGF-induced growth arrest. To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex.

Fibroblast growth factor signaling uses multiple mechanisms to inhibit Wnt-induced transcription in osteoblasts.

Fibroblast growth factor (FGF) and Wnt signals are both critical for proper bone development. We previously reported that the expression of activating FGF receptor mutations in osteoblasts downregulated the expression of many genes reported as targets of Wnt signaling, suggesting an antagonistic effect between Wnt signaling, which promotes osteoblast differentiation and function, and FGF signaling, which inhibits these processes. To analyze the effect of FGF on Wnt signaling in osteoblasts, we created reporter cell lines where a Wnt-responsive promoter drives luciferase expression and showed that Wnt3a-induced luciferase expression was specifically inhibited by FGF treatment.

Identification of active transcriptional regulatory modules by the functional assay of DNA from nucleosome-free regions.

The identification of transcriptional regulatory modules within mammalian genomes is a prerequisite to understanding the mechanisms controlling regulated gene expression. While high-throughput microarray- and sequencing-based approaches have been used to map the genomic locations of sites of nuclease hypersensitivity or target DNA sequences bound by specific protein factors, the identification of regulatory elements using functional assays, which would provide important complementary data, has been relatively rare. Here we present a method that permits the functional identification of active transcriptional regulatory modules using a simple procedure for the isolation and analysis of DNA derived from nucleosome-free regions (NFRs), the 2% of the cellular genome that contains these elements.

Osteoblast proliferation or differentiation is regulated by relative strengths of opposing signaling pathways.

Skeletal development requires the correct balance of osteoblast proliferation, survival, and differentiation which is modulated by a network of signaling pathways and transcription factors. We have examined the role of the AKT (PKB), and ERK1/2 signaling pathways in the osteoblast response to FGFs, which inhibit differentiation, and to IGF-1 and Wnt signaling, which promote it. Using osteoblastic cell lines as well as primary calvarial osteoblasts, we show that ERK1/2 and AKT have distinct effects in FGF-induced osteoblast proliferation and differentiation.

Downregulation of Akt activity contributes to the growth arrest induced by FGF in chondrocytes.

Unregulated FGF signaling produced by activating FGFR3 mutations causes several forms of dwarfism-associated chondrodysplasias in humans and mice. FGF signaling inhibits chondrocyte proliferation by activating multiple signal transduction pathways that all contribute to chondrocyte growth arrest and induction of some aspects of differentiation. Previous studies had identified the Stat1 pathway, dephosphorylation of the Rb family proteins p107 and p130, induction of p21 expression and sustained activation of MAP kinases as playing a role in the FGF response of chondrocytes.