Comprehensive analysis of the proximity-dependent nuclear interactome for the oncoprotein NOTCH1 in live cells.

Notch signaling plays a critical role in cell fate decisions in all cell types. Furthermore, gain-of-function mutations in NOTCH1 have been uncovered in many human cancers. Disruption of Notch signaling has recently emerged as an attractive disease treatment strategy. However, the nuclear interaction landscape of the oncoprotein NOTCH1 remains largely unexplored. We therefore employed here a proximity-dependent biotin identification approach to identify in vivo protein associations with the nuclear Notch1 intracellular domain in live cells. We identified a large set of previously reported and unreported proteins that associate with NOTCH1, including general transcription and elongation factors, DNA repair and replication factors, coactivators, corepressors, and components of the NuRD and SWI/SNF chromatin remodeling complexes. We also found that Notch1 intracellular domain associates with protein modifiers and components of other signaling pathways that may influence Notch signal transduction and protein stability such as USP7. We further validated the interaction of NOTCH1 with histone deacetylase 1 or GATAD2B using protein network analysis, proximity-based ligation, in vivo cross-linking and coimmunoprecipitation assays in several Notch-addicted cancer cell lines. Through data mining, we also revealed potential drug targets for the inhibition of Notch signaling. Collectively, these results provide a valuable resource to uncover the mechanisms that fine-tune Notch signaling in tumorigenesis and inform therapeutic targets for Notch-addicted tumors.

Selective Targeting of Class I Histone Deacetylases in a Model of Human Osteosarcoma.

Dysregulation of histone deacetylases (HDACs) is associated with the pathogenesis of human osteosarcoma, which may present an epigenetic vulnerability as well as a therapeutic target. Domatinostat (4SC-202) is a next-generation class I HDAC inhibitor that is currently being used in clinical research for certain cancers, but its impact on human osteosarcoma has yet to be explored. In this study, we report that 4SC-202 inhibits osteosarcoma cell growth in vitro and in vivo. By analyzing cell function in vitro, we show that the anti-tumor effect of 4SC-202 involves the combined induction of cell-cycle arrest at the G2/M phase and apoptotic program, as well as a reduction in cell invasion and migration capabilities. We also found that 4SC-202 has little capacity to promote osteogenic differentiation. Remarkably, 4SC-202 revised the global transcriptome and induced distinct signatures of gene expression in vitro. Moreover, 4SC-202 decreased tumor growth of established human tumor xenografts in immunodeficient mice in vivo. We further reveal key targets regulated by 4SC-202 that contribute to tumor cell growth and survival, and canonical signaling pathways associated with progression and metastasis of osteosarcoma. Our study suggests that 4SC-202 may be exploited as a valuable drug to promote more effective treatment of patients with osteosarcoma and provide molecular insights into the mechanism of action of class I HDAC inhibitors.

Development and characterization of the novel human osteosarcoma cell line COS-33 with sustained activation of the mTOR pathway.

Outcomes have not improved for metastatic osteosarcoma for several decades. In part, this failure to develop better therapies stems from a lack of understanding of osteosarcoma biology, given the rarity of the disease and the high genetic heterogeneity at the time of diagnosis. We report here the successful establishment of a new human osteosarcoma cell line, COS-33, from a patient-derived xenograft and demonstrate retention of the biological features of the original tumor. We found high mTOR signaling activity in the cultured cells, which were sensitive to a small molecule inhibitor, rapamycin, a suppressor of the mTOR pathway. Suppressed mTOR signaling after treatment with rapamycin was confirmed by decreased phosphorylation of the S6 ribosomal protein. Increasing concentrations of rapamycin progressively inhibited cell proliferation in vitro. We observed significant inhibitory effects of the drug on cell migration, invasion, and colony formation in the cultured cells. Furthermore, we found that only a strong osteogenic inducer, bone morphogenetic protein-2, promoted the cells to differentiate into mature mineralizing osteoblasts, indicating that the COS-33 cell line may have impaired osteoblast differentiation. Grafted COS-33 cells exhibited features typical of osteosarcoma, such as production of osteoid and tumorigenicity in vivo. In addition, we revealed that the COS-33 cell line retained a complex karyotype, a homozygous deletion of the TP53 gene, and typical histological features from its original tumor. Our novel cellular model may provide a valuable platform for studying the etiology and molecular pathogenesis of osteosarcoma as well as for testing novel drugs for future genome-informed targeted therapy.

Targeting the Wnt/ß-catenin pathway in human osteosarcoma cells.

Aberrant activation of Wnt signaling has been implicated in human osteosarcoma, which may provide a genetic vulnerability that can be targeted in osteosarcoma treatment. To test whether Wnt activation is necessary for osteosarcoma growth, colony formation, invasion, and metastasis, we treated human osteosarcoma cells with a small molecule inhibitor of Wnt/ß-catenin, PRI-724, which suppresses Wnt/ß-catenin-mediated transcription. We found increased protein levels of endogenous active-ß-catenin in five human osteosarcoma cell lines. Treatment with PRI-724 was sufficient to inhibit human osteosarcoma 143B and SJSA-1 cell proliferation. Suppressed Wnt signaling was confirmed by decreased protein levels of the Wnt target Cyclin D1. Furthermore, we revealed significant inhibitory effects on cell migration, invasion, and colony formation in the human osteosarcoma cells. Using deposited data from next generation sequencing studies, we analyzed somatic mutations and gene expression of components in the Wnt/ß-catenin pathway. We found somatic mutations and upregulated gene expression of many components in the Wnt/ ß-catenin pathway, indicating activated Wnt signaling. Taken together, our results illustrate the critical role of Wnt/ß-catenin signaling in human osteosarcoma pathogenesis and growth, as well as the therapeutic potential of Wnt inhibitors in the treatment of human osteosarcoma.

Molecular genetics of osteosarcoma.

Osteosarcoma is the predominant form of bone cancer, affecting mostly adolescents. Recent progress made in molecular genetic studies of osteosarcoma has changed our view on the cause of the disease and ongoing therapeutic approaches for patients. As we draw closer to gaining more complete catalogs of candidate cancer driver genes in common forms of cancer, the landscape of somatic mutations in osteosarcoma is emerging from its first phase. In this review, we summarize recent whole genome and/or whole exome genomic studies, and then put these findings in the context of genetic hallmarks of somatic mutations and mutational processes in human osteosarcoma. One of the lessons learned here is that the extent of somatic mutations and complexity of the osteosarcoma genome are similar to that of common forms of adult cancer. Thus, a much higher number of samples than those currently obtained are needed to complete the catalog of driver mutations in human osteosarcoma. In parallel, genetic studies in other species have revealed candidate driver genes and their roles in the genesis of osteosarcoma. This review also summarizes newly identified drivers in genetically engineered mouse models (GEMMs) and discusses our understanding of the impact of nature and number of drivers on tumor latency, subtypes, and metastatic potentials of osteosarcoma. It is becoming apparent that a synergistic team composed of three drivers (one 'first driver' and two 'synergistic drivers') may be required to generate an animal model that recapitulates aggressive osteosarcoma with a short latency. Finally, new cancer therapies are urgently needed to improve survival rate and quality of life for osteosarcoma patients. Several vulnerabilities in osteosarcoma are illustrated in this review to exemplify the opportunities for next generation molecularly targeted therapies. However, much work remains in order to complete our understanding of the somatic mutation basis of osteosarcoma, to develop reliable animal models of human disease, and to apply this information to guide new therapeutic approaches for reducing morbidity and mortality of this rare disease.

Hypoxia-Mimicking Nanofibrous Scaffolds Promote Endogenous Bone Regeneration.

Utilizing biomimetic materials to potentiate endogenous cell growth or signaling is superior to relying on exogenous cells or signals for bone formation. Desferoxamine (DFO), which is a hypoxia-mimetic agent that chelates iron (Fe3+), mimics hypoxia to encourage bone healing. However, high cytotoxicity, off-target effects, and the short half-life of DFO have significantly impeded its further applications. We mitigated these side effects by locally immobilizing DFO onto a gelatin nanofibrous (GF) scaffold that retained DFO's ability to chelate Fe3+. Moreover, DFO-functionalized GF (GF-DFO) scaffolds, which have similar micro/macrostructures to GF scaffolds, not only demonstrated decreased cytotoxicity on both human umbilical vein endothelial cells and human mesenchymal stem cells but also significantly increased vascular endothelial growth factor (VEGF) expression in vitro. Most importantly, in our in vivo experiments on a critical-sized cranial bone defect mouse model, a significant amount of bone was formed in most of the GF-DFO scaffolds after six weeks, while very little new bone was observed in the GF scaffolds. These data suggest that use of a hypoxia-mimicking nanofibrous scaffold is a promising strategy for promoting endogenous bone formation.

RECQL4 Regulates p53 Function In Vivo During Skeletogenesis.

RECQ DNA helicases play critical roles in maintaining genomic stability, but their role in development has been less well studied. Rothmund-Thomson syndrome, RAPADILINO, and Baller-Gerold syndrome are rare genetic disorders caused by mutations in the RECQL4 gene. These patients have significant skeletal developmental abnormalities including radial ray, limb and craniofacial defects. To investigate the role of Recql4 in the developing skeletal system, we generated Recql4 conditional knockout mice targeting the skeletal lineage. Inactivation of Recql4 using the Prx1-Cre transgene led to limb abnormalities and craniosynostosis mimicking the major bone findings in human RECQL4 patients. These Prx1-Cre(+) ;Recql4(fl/fl) mice as well as Col2a1-Cre(+) ;Recql4(fl/fl) mice exhibited growth plate defects and an increased p53 response in affected tissues. Inactivation of Trp53 in these Recql4 mutants resulted in genetic rescue of the skeletal phenotypes, indicating an in vivo interaction between Recql4 and Trp53, and p53 activation as an underlying mechanism for the developmental bone abnormalities in RECQL4 disorders. Our findings show that RECQL4 is critical for skeletal development by modulating p53 activity in vivo.

The osteogenic niche promotes early-stage bone colonization of disseminated breast cancer cells.

Breast cancer bone micrometastases can remain asymptomatic for years before progressing into overt lesions. The biology of this process, including the microenvironment niche and supporting pathways, is unclear. We find that bone micrometastases predominantly reside in a niche that exhibits features of osteogenesis. Niche interactions are mediated by heterotypic adherens junctions (hAJs) involving cancer-derived E-cadherin and osteogenic N-cadherin, the disruption of which abolishes niche-conferred advantages. We elucidate that hAJ activates the mTOR pathway in cancer cells, which drives the progression from single cells to micrometastases. Human data set analyses support the roles of AJ and the mTOR pathway in bone colonization. Our study illuminates the initiation of bone colonization, and provides potential therapeutic targets to block progression toward osteolytic metastases.

Notch activation as a driver of osteogenic sarcoma.

Osteogenic sarcoma (OS) is a deadly skeletal malignancy whose cause is unknown. We report here a mouse model of OS based on conditional expression of the intracellular domain of Notch1 (NICD). Expression of the NICD in immature osteoblasts was sufficient to drive the formation of bone tumors, including OS, with complete penetrance. These tumors display features of human OS; namely, histopathology, cytogenetic complexity, and metastatic potential. We show that Notch activation combined with loss of p53 synergistically accelerates OS development in mice, although p53-driven OS is not Rbpj dependent, which demonstrates a dual dominance of the Notch oncogene and p53 mutation in the development of OS. Using this model, we also reveal the osteoblasts as the potential sources of OS.

miRNA-34c regulates Notch signaling during bone development.

During bone homeostasis, osteoblast and osteoclast differentiation is coupled and regulated by multiple signaling pathways and their downstream transcription factors. Here, we show that microRNA 34 (miR-34) is significantly induced by BMP2 during osteoblast differentiation. In vivo, osteoblast-specific gain of miR-34c in mice leads to an age-dependent osteoporosis due to the defective mineralization and proliferation of osteoblasts and increased osteoclastogenesis. In osteoblasts, miR-34c targets multiple components of the Notch signaling pathway, including Notch1, Notch2 and Jag1 in a direct manner, and influences osteoclast differentiation in a non-cell-autonomous fashion. Taken together, our results demonstrate that miR-34c is critical during osteoblastogenesis in part by regulating Notch signaling in bone homeostasis. Furthermore, miR-34c-mediated post-transcriptional regulation of Notch signaling in osteoblasts is one possible mechanism to modulate the proliferative effect of Notch in the committed osteoblast progenitors which may be important in the pathogenesis of osteosarcomas. Therefore, understanding the functional interaction of miR-34 and Notch signaling in normal bone development and in bone cancer could potentially lead to therapies modulating miR-34 signaling.

One NOTCH Further: Jagged 1 in Bone Metastasis.

The outgrowth of metastatic cells to bone depends on the interaction between multiple intrinsic and host factors. In this issue of Cancer Cell, Sethi and colleagues report Notch signaling in bone cells as responsible for promoting this outgrowth and provide evidence for a beneficial treatment effect of NOTCH inhibitors.

E-selectin ligand-1 regulates growth plate homeostasis in mice by inhibiting the intracellular processing and secretion of mature TGF-beta.

The majority of human skeletal dysplasias are caused by dysregulation of growth plate homeostasis. As TGF-beta signaling is a critical determinant of growth plate homeostasis, skeletal dysplasias are often associated with dysregulation of this pathway. The context-dependent action of TFG-beta signaling is tightly controlled by numerous mechanisms at the extracellular level and downstream of ligand-receptor interactions. However, TGF-beta is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus, and the regulation of this posttranslational intracellular processing and trafficking is much less defined. Here, we report that a cysteine-rich protein, E-selectin ligand-1 (ESL-1), acts as a negative regulator of TGF-beta production by binding TGF-beta precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-beta by a furin-like protease, leading to reduced secretion of mature TGF-beta by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-beta/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation. Gain-of-function and rescue studies of the Xenopus ESL-1 ortholog in the context of early embryogenesis showed that this regulation of TGF-beta/Nodal signaling was evolutionarily conserved. This study identifies what we believe to be a novel intracellular mechanism for regulating TGF-beta during skeletal development and homeostasis.

Osteosclerosis owing to Notch gain of function is solely Rbpj-dependent.

Osteosclerosis is a pathologic bone disease characterized by an increase in bone formation over bone resorption. Genetic factors that contribute to the pathogenesis of this disease are poorly understood. Dysregulation or mutation in many components of the Notch signaling pathway results in a wide range of human developmental disorders and cancers, including bone diseases. Our previous study found that activation of the Notch signaling in osteoblasts promotes cell proliferation and inhibits differentiation, leading to an osteosclerotic phenotype in transgenic mice. In this study we report a longer-lived mouse model that also develops osteosclerosis and a genetic manipulation that completely rescues the phenotype. Conditionally cre-activated expression of Notch1 intracellular domain (NICD) in vivo exclusively in committed osteoblasts caused massive osteosclerosis with growth retardation and abnormal vertebrae. Importantly, selective deletion of a Notch nuclear effector--Rbpj--in osteoblasts completely suppressed the osteosclerotic and growth-retardation phenotypes. Furthermore, cellular and molecular analyses of bones from the rescued mice confirmed that NICD-dependent molecular alterations in osteoblasts were completely reversed by removal of the Rbpj pathway. Together, our observations show that the osteosclerosis owing to activation of Notch signaling in osteoblasts is canonical in nature because it depends solely on Rbpj signaling. As such, it identifies Rbpj as a specific target for manipulating Notch signaling in a cell-autonomous fashion in osteoblasts in bone diseases where Notch may be dysregulated.

Alteration of Notch signaling in skeletal development and disease.

Notch signaling is an evolutionarily conserved mechanism for specifying and regulating organogenesis and tissue renewal. Human and mouse genetic studies have demonstrated mutations in many components of the Notch signaling pathway that cause skeletal patterning defects. More recently, the in vivo effects of Notch signaling on osteoblast specification, proliferation, and differentiation have been demonstrated in addition to its regulation of osteoclast activity. However, while our understanding of canonical Notch signaling in skeletal biology is rapidly evolving, the role of noncanonical Notch signaling is still poorly understood. In a pathologic context, aberration of Notch signaling is also associated with osteosarcoma. These studies raise the question of how Notch may interact with other signaling pathways, such as Wnt. Finally, manipulation of Notch signaling for bone-related diseases remains complex because of the temporal and context-dependent nature of Notch signaling during mesenchymal stem cell and osteoblast differentiation.