Faculty Bibliography

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.

Macrophages adopt an alternatively activated phenotype (AAM) when activated by the IL-4Ralpha. AAM can be derived either from proliferation of tissue resident macrophages, or recruited inflammatory monocytes, but it is not known whether these different sources generate AAM that are phenotypically and functionally distinct. By transcriptional profiling analysis, we show here that while both monocyte and tissue-derived AAM expressed high levels of Arg1, Chi3l3 and Retnla, only monocyte-derived AAM upregulated Raldh2 and PD-L2. Monocyte-derived AAM were also CX3CR1-GFPhigh and expressed CD206, whereas tissue-derived AAM were CX3CR1-GFP and CD206 negative. Monocyte-derived AAM had high levels of aldehyde dehydrogenase (ALDH) activity and promoted the differentiation of FoxP3+ cells from naive CD4+ cells via production of retinoic acid. In contrast, tissue-derived AAM expressed high levels of UCP1. Hence monocyte-derived AAM have properties associated with immune regulation and the different physiological properties associated with AAM function may depend on the distinct lineage of these cells.

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 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. SOX2 favors adipogenesis and induces PPARgamma, but adipogenesis can only occur with moderate levels of YAP1. YAP1 induction by SOX2 is restrained in adipogenesis, and both YAP1 overexpression and depletion inhibit the process. YAP1 binds beta-catenin and directly induces the Wnt antagonist Dkk1 to dampen pro-osteogenic Wnt signals. We demonstrate a Hippo-independent regulation of YAP1 by SOX2 that cooperatively antagonizes Wnt/beta-catenin signals and regulates PPARgamma to determine osteogenic or adipocytic fates.

Background: Supernumerary teeth are often observed in patients suffering from cleidocranial dysplasia due to a mutation in Runx2 that results in haploinsufficiency. However, the underlying molecular mechanisms are poorly defined. In this study, we assessed the roles of Runx2 and its functional antagonist Twist1 in regulating fibroblast growth factor (FGF) signaling using in vitro biochemical approaches. Results: We showed that Twist1 stimulated Fgfr2 and Fgf10 expression in a mesenchymal cell line and that it formed heterodimers with ubiquitously expressed E12 (together with E47 encoded by E2A gene) and upregulated Fgfr2 and Fgf10 promoter activities in a dental mesenchyme-derived cell line. We further demonstrated that the bHLH domain of Twist1 was essential for its synergistic activation of Fgfr2 promoter with E12 and that the binding of E12 stabilized Twist1 by preventing it from undergoing lysosomal degradation. Although Runx2 had no apparent effects on Fgfr2 and Fgf10 promoter activities, it inhibited the stimulatory activity of Twist1 on Fgfr2 promoter. Conclusions: These findings suggest that Runx2 haploinsufficiency might result in excessive unbound Twist1 that can freely bind to E12 and enhance FGF signaling, thereby promoting the formation of extra teeth. Developmental Dynamics 241:1708-1715, 2012. (c) 2012 Wiley Periodicals, Inc.

Tumors are thought to be sustained by a reservoir of self-renewing cells, termed tumor-initiating cells or cancer stem cells. Osteosarcomas are high-grade sarcomas derived from osteoblast progenitor cells and are the most common pediatric bone malignancy. In this report we show that the stem cell transcription factor Sox2 is highly expressed in human and murine osteosarcoma (mOS) cell lines as well as in the tumor samples. Osteosarcoma cells have increased ability to grow in suspension as osteospheres, that are greatly enriched in expression of Sox2 and the stem cell marker, Sca-1. Depletion of Sox2 by short-hairpin RNAs in independent mOS-derived cells drastically reduces their transformed properties in vitro and their ability to form tumors. Sox2-depleted osteosarcoma cells can no longer form osteospheres and differentiate into mature osteoblasts. Concomitantly, they exhibit decreased Sca-1 expression and upregulation of the Wnt signaling pathway. Thus, despite other mutations, these cells maintain a requirement for Sox2 for tumorigenicity. Our data indicate that Sox2 is required for osteosarcoma cell self renewal, and that Sox2 antagonizes the pro-differentiation Wnt pathway that can in turn reduce Sox2 expression. These studies define Sox2 as a survival factor and a novel biomarker of self renewal in osteosarcomas, and support a tumor suppressive role for the Wnt pathway in tumors of mesenchymal origin. Our findings could provide the basis for novel therapeutic strategies based on inhibiting Sox2 or enhancing Wnt signaling for the treatment of osteosarcomas.Oncogene advance online publication, 19 September 2011; doi:10.1038/onc.2011.405

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. Sox2 also interferes with Wnt signaling by binding beta-catenin, a central mediator of the Wnt pathway. Here we show that these multiple functions of Sox2 are encoded in distinct domains. The self-renewal function of Sox2 is dependent on its transcriptional activity, and requires both its DNA-binding and C-terminal activation regions, while only the third C-terminal-transactivation region is required for binding beta-catenin and interfering with Wnt-induced transcription. Gene expression analysis upon Sox2 deletion strongly supports the notion that Sox2 maintains stemness. We also show that Sox2 suppresses differentiation by attenuating Wnt signaling by posttranscriptional and transcriptional mechanisms and that negative regulators of the Wnt pathway, APC and GSK3beta are direct Sox2 targets in osteoblasts. Several genes associated with stemness such as FoxP1 and BMI-1 are downregulated upon Sox2 inactivation. Constitutive expression of the polycomb complex member, BMI-1, can bypass the Sox2 requirement for self-renewal, but does not affect differentiation. Our results establish a connection between Sox2 and BMI-1 in maintaining self-renewal and identify BMI-1 as a key mediator of Sox2 function

The development and maintenance of most tissues and organs require the presence of multipotent and unipotent stem cells that have the ability of self-renewal as well as of generating committed, further differentiated cell types. The transcription factor Sox2 is essential for embryonic development and maintains pluripotency and self-renewal in embryonic stem cells. It is expressed in immature osteoblasts/osteoprogenitors in vitro and in vivo and is induced by fibroblast growth factor signaling, which stimulates osteoblast proliferation and inhibits differentiation. Sox2 overexpression can by itself inhibit osteoblast differentiation. To elucidate its function in the osteoblastic lineage, we generated mice with an osteoblast-specific, Cre-mediated knockout of Sox2. These mice are small and osteopenic, and mosaic for Sox2 inactivation. However, culturing calvarial osteoblasts from the mutant mice for 2-3 passages failed to yield any Sox2-null cells. Inactivation of the Sox2 gene by Cre-mediated excision in cultured osteoblasts showed that Sox2-null cells could not survive repeated passage in culture, could not form colonies, and arrested their growth with a senescent phenotype. In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability. Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression. These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage