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. Here we show that, in a mouse model of osteosarcoma, osteoblast-specific Sox2 conditional knockout (CKO) causes a drastic reduction in the frequency and onset of tumors. The rare tumors detected in the Sox2 CKO animals were all Sox2 positive, indicating that they arose from cells that had escaped Sox2 deletion. Furthermore, Sox2 inactivation in cultured osteosarcoma cells by CRISPR/CAS technology leads to a loss of viability and proliferation of the entire cell population. Inactivation of the YAP gene, a major Hippo pathway effector which is a direct Sox2 target, causes similar results and YAP overexpression rescues cells from the lethality caused by Sox2 inactivation. These effects were osteosarcoma-specific, suggesting a mechanism of cell "addiction" to Sox2-initiated pathways. The requirement of Sox2 for osteosarcoma formation as well as for the survival of the tumor cells suggests that disruption of Sox2-initiated pathways could be an effective strategy for the treatment of osteosarcoma.
MZF1 and GABP Cooperate with Sox2 in Regulating the Expression of YAP1 in Cancer Cells
The transcription factor 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. The stem cell transcription factor Sox2, which marks and maintains cancer stem cells in osteosarcomas, promotes YAP1 expression by binding to an intronic enhancer element and YAP1 expression is also crucial for the maintainance of osteosarcoma stem cells. To further understand the regulation of YAP1 expression in osteosarcomas we subjected the YAP1 intronic enhancer to scanning mutagenesis to identify all DNA cis-elements critical for enhancer function. Through this approach we identified two novel transcription factors, GABP and MZF1, which are essential for basal YAP1 transcription. These factors are highly expressed in osteosarcomas and bind to distinct sites in the YAP1 enhancer. Depletion of either factor leads to drastically reduced YAP1 expression and thus a reversal of stem cell properties. We also found that YAP1 can regulate the expression of Sox2 by binding to two distinct DNA binding sites upstream and downstream of the Sox2 gene. Thus Sox2 and YAP1 reinforce each other expression to maintain stemness and tumorigenicity in osteosarcomas, but the activity of MZF1 and GABP is essential for YAP1 transcription.
Phosphoproteomics of 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. Chondrocytes respond to FGF with growth inhibition, a unique response which differs from the proliferative response of the majority of cell types; however its molecular determinants are still unclear. Quantitative phosphoproteomic analysis was utilized to catalogue the proteins whose phosphorylation status is changed upon FGF1 treatment. The generated dataset consists of 756 proteins. We were able to localize the divergence between proliferative (canonical) and inhibitory (chondrocyte specific) FGF transduction pathways immediately upstream of AKT kinase. Gene Ontology (GO) analysis of the FGF1 regulated peptides revealed that many of the identified phosphorylated proteins are assigned to negative regulation clusters, in accordance with the observed inhibitory growth response. This is the first time a comprehensive subset of proteins involved in FGF inhibitory response is defined. We were able to identify a number of targets and specifically discover glycogen synthase kinase3beta (GSK3beta) as a novel key mediator of FGF inhibitory response in chondrocytes.
PPARgamma 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 PPARgamma 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. Gene expression analysis reveals that TZDs induce lipid metabolism pathways while suppressing targets of the Hippo-YAP pathway, Wnt signaling and cancer-related proliferation pathways. Significantly, TZD action appears to be restricted to the high Sox2 expressing cancer stem cell population and is dependent on PPARgamma expression. TZDs also affect growth and cell fate by causing the cytoplasmic sequestration of the transcription factors SOX2 and YAP that are required for tumorigenicity. Finally, we identify a TZD-regulated gene signature based on Wnt/Hippo target genes and PPARgamma that predicts patient outcomes. Together, this work highlights a novel connection between PPARgamma agonist in inducing adipogenesis and mimicking the tumor suppressive hippo pathway. It also illustrates the potential of drug repurposing for TZD-based differentiation therapy for osteosarcoma.
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. Repression of Nf2, WWC1 and high YAP expression marks the CSC fraction of the tumor population, while the more differentiated fraction has high Nf2, high WWC1 and reduced YAP expression. YAP depletion sharply reduces CSCs and tumorigenicity of osteosarcomas. Thus, Sox2 interferes with the tumour-suppressive Hippo pathway to maintain CSCs in osteosarcomas. This Sox2-Hippo axis is conserved in other Sox2-dependent cancers such as glioblastomas. Disruption of YAP transcriptional activity could be a therapeutic strategy for Sox2-dependent tumours.
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. Here we used micro computed tomography images of mice expressing the Apert mutation constitutively in either mesoderm- or neural crest-derived cells to quantify craniofacial shape variation and suture fusion patterns, and to identify shape changes in craniofacial bones derived from the lineage not expressing the mutation, referred to here as secondary shape changes. Our results show that at postnatal day 0: (i) conditional expression of the FGFR2 S252W mutation in neural crest-derived tissues causes a more severe craniofacial phenotype than when expressed in mesoderm-derived tissues; and (ii) both mesoderm- and neural crest-specific mouse models display secondary shape changes. We also show that premature suture fusion is not necessarily dependent on the expression of the FGFR2 S252W mutation in the sutural mesenchyme. More specifically, it appears that suture fusion patterns in both mouse models are suture-specific resulting from a complex combination of the influence of primary abnormalities of biogenesis or signaling within the sutures, and timing.
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. Many of these represent newly discovered ESC-specific enhancers, showing enriched binding-site motifs for ESC-specific transcription factors including SOX2, POU5F1 (OCT4) and KLF4. The application of FIREWACh to additional cultured cell types will facilitate functional annotation of the genome and expand our view of transcriptional network dynamics.
The role of FGF/FGFR signaling in cranial integration: Implications for primate evolution [Meeting Abstract]
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. The cancer stem cell hypothesis posits that tumors are maintained by stem cells and it is the incomplete eradication of a refractory population of tumor-initiating stem cells that accounts for drug resistance and tumor relapse. In this review we present our current knowledge about the biology of osteosarcoma stem cells from mouse and human tumors, highlighting new insights and unresolved issues in the identification of this elusive population. We focus on factors and pathways that are implicated in maintaining such cells, and differences from paradigms of epithelial cancers. Targeting of the cancer stem cells in osteosarcoma is a promising avenue to explore to develop new therapies for this devastating childhood cancer.
The B55alpha 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-B55alpha holoenzyme (PP2A containing the B55alpha subunit) is responsible for this phenomenon. Only the B55alpha (55-kDa regulatory subunit, alpha isoform) regulatory subunit of PP2A was able to bind p107, and this interaction was induced by FGF in chondrocytes but not in other cell types. Small interfering RNA (siRNA)-mediated knockdown of B55alpha prevented p107 dephosphorylation and FGF-induced growth arrest of RCS (rat chondrosarcoma) chondrocytes. Importantly, the B55alpha subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55alpha is also the site of cyclin-dependent kinase (CDK) binding, B55alpha association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55alpha subunit itself on several serine residues that drastically increases the affinity of B55alpha for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55alpha dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals.