1,594

Novel Signaling Pathways in Osteosarcoma

Alan Nguyen, Vi Nguyen, Dalton Pham, Marco Mravic, Michelle A Scott, Aaron W James

Alan Nguyen, Vi Nguyen, Dalton Pham, School of Dentistry, University of California, Los Angeles, CA, the United States
Alan Nguyen, Marco Mravic, Aaron W James, Department of Pathology & Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA, the United States
Michelle A Scott, Children’s Hospital Los Angeles, Los Angeles, CA, the United States

Correspondence to: Aaron W James, MD, Department of Pathology & Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, 10833 Le Conte Ave, CHS A3-251, Los Angeles, CA 90077, the United States.
Email: awjames@mednet.ucla.edu
Received: June 25, 2014
Revised: July 25, 2014
Accepted: July 30, 2014
Published online: October 3, 2014

ABSTRACT

Osteosarcomas are the most common primary malignant bone tumors. Major advances in the treatment of osteosarcoma (OS) have revolutionized their clinical and surgical care. Despite these improvements, no specific targeted therapy has yet been employed for OS treatment. The present review will outline significant cytokine signaling pathways important in OS tumor biology, including the Bone morphogenetic protein (BMP), Hedgehog, and Wnt signaling pathways. Also discussed are the inherent challenges in studying cytokine signaling in OS, including the diversity in tumor location, grade, and lines of differentiation. Multiple BMP ligands and receptors are expressed across most OS cell lines and OS subtypes. Available data suggest that BMP signaling has pro-migratory effects in OS cells, as in the case with other sarcomas. Activation of Hedgehog and Wnt signaling has been observed in OS cell lines and/or primary human OS specimens. Emerging data suggests that Wnt signaling inhibition may prevent osteosarcomagenesis and/or sensitize OS cells to traditional chemotherapeutic agents. Likewise, interference with Hedgehog signaling transduction may reduce OS cell proliferation and in vivo tumor growth. In summary, multiple signaling factors show preclinical promise for cell targeted therapy in osteosarcoma.

© 2014 The Authors. Published by ACT Publishing Group Ltd.

Key Words: Osteosarcoma; BMP; Hedgehog; Wnt

Nguyen A, Nguyen V, Pham D, Mravic M, Scott MA, James AW. Novel Signaling Pathways in Osteosarcoma. International Journal of Orthopaedics 2014; 1(3): 73-84 Available from: URL: http://www.ghrnet.org/index.php/ijo/article/view/890

Introduction

Osteosarcoma is the most common, non-hemopoietic, primary malignancy of bone. When untreated, osteosarcoma (OS) is essentially a fatal disease characterized by early hematogenous dissemination. With a combination of surgical resection and chemotherapy, long term disease-free survival is achievable in the majority of patients. Despite these vast improvements in patient outcome, targeted therapies for OS have not yet been realized.

The characterization of cell signaling pathways in OS presents multiple practical challenges, principally derived from the significant variation from tumor to tumor. For example, OS tumors vary by line of differentiation, histologic grade, tumor location, and treatment history. While all OS show some element of osteogenic differentiation, this can be highly variable. By convention, OS can be classified into osteoblastic, chondroblastic, and fibroblastic differentiation. Accordingly, the extent of bone matrix formation has been observed to correlate with various osteoinductive cytokine expression. Meanwhile, tumor grading predicts biological behavior based on histological appearance, with higher grade tumors showing increased cellularity, nuclear atypia, and mitotic figures. Considering the signaling cascade activity of BMP alone, for example, there exists a spectrum of activity and expression between low-grade and high-grade tumors. Perhaps the simplest distinguishing feature of OS is the anatomic location. Intramedullary tumors tend to be more common (conventional OS), while superficial (periosteal) lesions are more infrequent. Finally, OS specimens vary in their treatment history and responsiveness to preoperative chemotherapy. Tumor necrosis with or without inflammation shows dramatic changes to OS cytokine signaling. With this variation of OS in mind, this review will focus on cell signaling pathways important in OS tumor biology.

Focus of review

The development of new, targeted therapies for OS hinges on improved understanding of cell signaling pathways important in osteosarcomagenesis, tumor growth, invasion, and metastasis. Surprisingly, few studies have specifically examined key signaling cascades in the context of OS tumor biology. For example, BMP, Hedgehog, and Wnt signaling cascades are relatively infrequently studied in the context of OS. Further, the majority of studies treat OS as a single disease entity, rather than taking into account the variety inherent in OS tumors. Conversely, studies focusing on only a single OS subtype may be limited in scope and conclusions. With these limitations in mind, this review will specifically focus on the role of BMP, Hedgehog, and Wnt signaling in OS and other malignant skeletal tumors. This review will highlight the recent advances in understanding the role of each signaling pathway in OS biology, with an emphasis on the divergent findings by various researchers.

BMP Signaling in OS

Introduction to BMP signaling

Members of the transforming growth factor-β (TGF-β) superfamily, Bone Morphogenetic Proteins (BMPs) are extracellular cytokines originally isolated from bone extracts[1]. While responsible for numerous cell regulatory processes, BMPs are most studied for their ectopic induction of bone and cartilage[2]. To date, 31 different types of BMP ligands have been identified[3], of which BMP-2, -4, -7, and -9 are most commonly studied in the context of osteogenesis[4,5]. As expected from a key regulator of osteogenesis, investigators have observed BMP expression in both benign and malignant skeletal tumors, including benign bone-forming lesions[6], as well as benign non-bone forming lesions[7]. BMP-like protein was first identified in murine OS in 1974[8], confirmed soon after in human OS[9]. Since then, the significance of BMP signaling in OS has been studied by multiple investigators. Despite these studies, however, conflicting conclusions have been drawn in regards to the role of BMPs in OS[10].

Overview of BMP signaling

The BMP signaling cascade initiates through extracellular ligand interaction with transmembrane type I and type II BMP receptors (BMPRs), both being serine-threonine kinases (Figure 1). Type II BMPRs handle initial BMP ligand interaction, binding via a high affinity mechanism which results in recruitment and activation of type I BMPRs, which are classified as low-affinity receptors[2,11,12]. While seven known variants of type I BMPRs exist, few actually contribute to osteogenic differentiation, predominantly BMPR-IA and BMPR-IB[13]. BMP signaling propagation then proceeds downstream to activate several pathways via phosphorylation, including the Smad1/5/8, MAP Kinase, and c-Jun N-terminal kinase (JNK) signaling pathways[13,14]. It is principally through Smad1/5/8 signaling that BMPs induce osteogenic programming (see[15] for a more detailed review of BMP signaling transduction).



Significance of BMP signaling in bone development

In regards to its role in bone development, the BMP signaling pathway plays a crucial role in the induction and regulation of osteogenic differentiation through both autocrine and paracrine pathways[16,17]. Through genetic modification of BMP receptors, inhibitors, and ligands in murine models, investigators have shown a role for BMP in signaling bone formation[18-21]. For instance, modification of BMPR-IA receptors in transgenic mice results in reduced bone mass as well as irregular patterns of calcification[18]. Conversely, overexpression of BMP signaling inhibitors, such as Noggin and Gremlin, has led to impaired bone formation[22-24]. Of the 31 known BMP ligands, only several are known to induce osteogenic commitment and terminal differentiation in mesenchymal stem cells (MSC); these include BMP-2, -4, -6, -7, and -9[5,25]. Of these, BMP-2 is most well- studied, exhibiting induction of osteogenesis in MSC both in vitro and in vivo[26-33]. As BMP signaling exerts significant pro-osteogenic effects across multiple cell types and bone injury models, interest in the expression and manipulation of BMP signaling in OS tumor biology has been significant.

BMP Signaling in OS

BMP signaling elements and activity has been identified in several OS cell lines and primary cells, most notably in murine cell lines[8,34,35] and human OS cells lines[36-38]. Independent of their osteoinductive properties in vivo, expression of BMP-2, -4, and -6 have been observed in murine OS cell lines[39]. Via polymerase chain reaction, Gobbi et al observed BMP-2 through -8, along with BMP receptors, across five OS cell lines[40]. With regards to human OS specimens, BMP-2/4 expression has been observed in both immunohistochemical and in situ hybridization[41,42]. In fact, high-grade OS was shown to have the highest and most consistent expression of BMP-2/4[41]. In comparison, however, high-grade OS specimens exhibited reduced levels of BMP signaling activity in comparison to the benign osteoblastoma, as indicated by reduced phospho-Smad1 expression[43]. In a similar comparison, gene microarray compared osteoblastic to non-osteoblastic OS specimens[44]. BMP-7 expression was significantly increased in osteoblastic OS as compared to non-osteoblastic OS specimens. BMP-6, in contrast, was most highly expressed in chondroblastic OS[45]. Thus, the majority of OS specimens exhibit BMP signaling activity. Studies suggest that specific BMP ligand expression is influenced by the histological subtype and tumor grade, although this is incompletely defined.

Despite numerous studies examining BMPs in OS, relatively few have addressed the potential role for BMP signaling in tumor formation or disease progression. BMP-2 has been reported to exhibit an inhibitory effect on in vivo sarcomagenesis as seen in OS ‘cancer stem cells’ (OS99-1 cell line)[46]. Conversely, BMP-2 and -9 were found to exert pro-mitogenic effects in OS with ‘differentiation defects,’ tumors where overexpression of either BMP ligand failed to induce terminal osteogenesis[47]. Instead, tumor growth was increased, which suggests that some OS cells gain resistance to BMP induced osteogenesis and develop a concomitant shift towards sarcoma growth[47]. Lastly, in another study, OS cells were exposed to various extracellular matrix components in either the presence or absence of BMP-2; results showed that BMP-2 induces OS cell migration through regulating fibronectin-Integrinβ1 signaling[48]. In summary, few studies have examined the mechanisms via which BMP signaling may affect sarcomagenesis, sarcoma growth, and invasion – with somewhat conflicting results.

Lastly, studies have shown that the BMP signaling pathway is upregulated in conditions predisposing to OS, such as Paget’s disease and fibrous dysplasia. For example, Urist et al examined patients with Paget’s disease, finding significantly increased BMP levels by radioimmunoassay in serum as compared to the normal control group[49]. Other investigators have examined BMP signaling in fibrous dysplasia. For example, Khurana et al described BMP-2 and -4 expression fibrous dysplasia by RT-PCR, Northern blot, and immunohistochemistry[7]. Likewise, in fibroblast-like cells of fibrous dysplasia, Sakamoto et al found more frequent immunoexpression of TGF-β and fibroblast growth factor-2 (FGF-2), in addition to BMP-2[50]. Lastly, in bone tissue of women with fibrous dysplasia, a gene analysis using RT-PCR found nine upregulated genes and 18 downregulated genes in comparison to non-fibrous dysplasia bone tissue[51]. These altered genes corresponded to collagen molecules, adhesion molecules, and growth factors, to name a few. Taken together, these studies regarding predisposing conditions of osteosarcoma further reinforce the central role that BMP plays in bone formation, and why regulation of this signaling pathway holds potential in context of OS tumor biology.

Hedgehog Signaling in OS

Introduction to Hedgehog signaling

Hedgehog (HH) signaling pathway is essential for mesodermal tissue patterning and differentiation. Originally identified in Drosophila, Hedgehog was later identified across all vertebrate organisms. Three homologues exist for this protein: Sonic Hedgehog (SHH), Indian Hedgehog (IHH), and Desert Hedgehog (DHH)[52]. While DHH expression is limited and not of known importance in bone biology[53-56], both SHH and IHH are critical in embryonic development. Knockout of either SHH and IHH in murine models results in multiple congenital anomalies and neonatal lethality[57-59]. While IHH is more prominent in regulating chondrogenesis and endochondral formation[52], SHH has a role in vertebrate organogenesis, promoting adult stem cell proliferation in hematopoietic, mammary, and neural tissues[60]. Furthermore, SHH plays a role in skeletogenesis and development of facial, appendicular, and axial skeletal patterns[52]. With a significant role in cell differentiation and stimulation of osteogenesis, the HH pathway serves as an attractive potential target in OS tumor biology.

Overview of Hedgehog signaling

The Hedgehog signaling pathway mechanism remains conserved across the three different homologues. Following initial pathway activation via autocatalytic cleavage, the C-terminus is modified with cholesterol while the N-terminus is modified with palmitoyl acid, thus increasing membrane solubility in the process (Figure 2)[61]. Hedgehog acyltransferase (HHAT) has also been deemed necessary in the post-translational palmitoylation of HH; in the absence of HHAT, HH secretion is downregulated[62]. The resulting multimeric form of the activated ligand undergoes paracrine signaling; secretion via 12-pass transmembrane protein, Dispatched (DISP), allows for the activation of 12-pass transmembrane Patched (PTCH) receptor on the receiving cell[63]. In its unbound state, PTCH functions as an inhibitor of Smoothened (SMO). From interaction with the activated HH ligand, Smoothened is released from constitutive inhibition by PTCH. Once free, SMO shuttles through the cilia and activates downstream target genes through utilization of the transcription factor glioblastoma gene product family, Gli2/3 complex and Gli1[63]. At the same time, this action inhibits the Gli3 repressor form. HH-Gli activators have been shown to function in regulating lineage commitment during cell differentiation[64].

Hedgehog signaling in bone biology

The HH signaling pathway plays a critical role in differentiation, directing cells toward an osteogenic lineage. HH signaling (SHH, Gli1) regulates osteoprogenitor cell differentiation by enhancing osteogenic differentiation and inhibiting adipogenic differentiation in multiple mesenchymal cell types[65]. For example, SHH added to adipose derived stromal cells (ASC) simultaneously induced osteoblast differentiation while suppressing adipocyte formation, both in vitro and in vivo[66]. Likewise, the IHH ligand has also been shown to exhibit an inverse relationship between osteogenic and adipogenic mesenchymal stem cell differentiation[67]. Expectedly, ossification is greatly impaired in developing animals with improper SHH and IHH signaling, leading to a variety of mutations; SHH mutant mice develop a cleft palate associated with disrupted FGF signaling[68]. In mice with mutations for IHH, an endochondral ossification defect led to severely shortened limbs while irregular intramembranous ossification led to skull vault defects[69]. Furthermore, endogenous HH is involved in regulation of angiogenesis and bone vascularization. Similarly, exogenous HH induces vascular formation in vitro. Specifically, implantation of recombinant SHH catalyzes in vitro vascular structure formation and enhances in vivo perfusion of artificial tissue and formation of de novo mature bone tissue[70]. The critical role of HH in stimulation of calvarial and appendicular bone development and basic bone biology has led to interest in the manipulation of HH signaling in OS tumor biology.

Hedgehog signaling in the biology and treatment of OS

Dysregulation of the HH pathway has been associated with tumorigenesis, angiogenesis, and metastasis in a variety of cancers. For example, in the benign cartilage tumor osteochondroma, the lack of EXT expression and heparin sulfate production disrupts IHH signaling, resulting in excess proliferation[71]. To date, three key methods have been proposed to explain how HH signaling leads to tumor formation[72]. In the first, both increased expression of HH ligands and ectopic Gli or PTCH will lead to autocrine or juxtacrine HH activation. Second, activation of HH occurs independent of the ligand, where mutation of either transmembrane PTCH or SMO enables SMO to remain active on downstream Gli elements. By contrast, in the last approach, paracrine HH activation occurs in a ligand-dependent manner; ectopic expression or mutated elements can be passed from the tumor to the supporting stroma. This signaling has been shown to increase both tumor invasiveness and proliferation. Furthermore, activation of the HH pathway causes increase in Snail protein expression and decrease in E-cadherin and tight junctions[73], leading to a stimulation of angiogenesis and metastasis[74]. Lastly, it is worth noting that abnormal activation of HH has been shown to upregulate angiogenic factors angiopoietin-1 and angiopoietin-2[75], as well as antiapoptotic genes[76].

In comparison to regular osteoblasts, human OS cell lines show increased mRNA expression of HH ligands, receptors, and downstream transcriptional activators[77]. Accordingly, in keeping with the known roles of HH in tumorigenesis, pharmaceutical antagonists of HH signaling have been examined[78]. One recent example is the development of Vismodegib, the first FDA approved drug to manipulate the HH pathway to inhibit tumor growth in basal cell carcinoma[79]. Its mechanism involves SMO inhibition, which inactivates transcription factors Gli1, thus hindering expression of tumorigenic genes. Currently, Vismodegib is undergoing clinical trials by the National Cancer Institute to test its potency in treating extraskeletal osteosarcoma, metastatic osteosarcoma. Likewise, FDA-approved arsenic trioxide (ATO) used for the treatment of leukemias has shown preclinical success in OS both in vitro and in vivo. Specifically, ATO induces apoptosis of p53 deficient OS MG63 cells by inhibiting catalase[80]. Moreover, injection of ATO in vivo reduced tumor size in mice by 300% after an eight week period[81]. Finally, ATO also inhibits OS invasiveness and metastasis by reducing cell motility, migration, and adhesion[82].

The HH pathway has also been observed to be altered in patients with predisposing conditions of OS. For instance, Cohen noted that abnormal bone in fibrous dysplasia were caused by mutations in GNAS1, downstream of the SHH pathway[83]. Likewise, Regard et al had previously demonstrated that GNAS gain-of-function contributed to fibrous dysplasia in osteoblast progenitor cells, but via upregulated WNT signaling[84]. In a subsequent study, Regard et al found that Gαs, which is encoded by GNAS, restricts bone formation via inhibition of HH[85]. This suggests a crosstalk phenomenon between the WNT and HH signaling pathways, in which Gαs serves as a key regulator of osteoblast differentiation. Thus, the consistent findings in predisposing conditions of OS further reaffirm the central role of HH signaling in OS development.

In summary, with mutation and dysregulation of the HH pathway correlated to tumor progression, targeted inhibition of HH signaling has enabled new strategies in the development of novel OS treatments. Inhibition of the cell surface receptors PTCH and SMO downregulates Gli1 and Gli2 transcriptional activity, thereby impeding OS progression through apoptosis. Collectively, these findings warrant additional research, as the possibility of using Hedgehog pathway inhibitors holds therapeutic promise for the treatment of osteosarcoma.

Wnt Signaling in OS

Introduction to Wnt signaling

The Wnt signaling pathway has been shown to play a variety of crucial roles in regards to cell fate, development, self-renewal, and tissue morphogenesis[86]. In bone development, Wnt signaling is required for limb bud initiation, early limb patterning, and late limb morphogenesis events[87]. Dysregulation of Wnt signaling can lead to osteoporosis[88], but also is associated with numerous and diverse diseases such as Parkinson’s disease[89], retinopathy[90], colon cancer[91], and melanoma[92]. Over 19 known Wnt ligands have been identified to date, with 15 receptors and co-receptors across seven protein families[86,92]. Furthermore, Wnt ligands activate distinct intracellular signaling cascades, including the Wnt/β-catenin pathway, the Wnt/Ca2+ pathway, and the Wnt/planar cell polarity (Wnt/PCP) pathway[93]. Due to its myriad of cellular functions, tremendous attention has been focused particularly on the key role of the Wnt/β-catenin signaling pathway in the regulation of bone mass[94,95] and on characterizing the ligands, receptors, and effectors of the Wnt pathway.

Overview of Wnt signaling

The Wnt signaling pathways can essentially be separated into two main categories: the β-catenin-dependent pathway (canonical Wnt pathway) and the β-catenin-independent pathway (non-canonical Wnt pathway). The canonical Wnt pathway has been studied more extensively, and functions by regulating the amount of the transcriptional coactivator β-catenin, which controls key developmental gene expression programs[96]. This β-catenin dependent mechanism initiates with Wnt ligand binding to the cysteine-rich portion of the seven-pass transmembrane receptors of the Frizzled family (Frz), as well as co-receptors such as lipoprotein receptor-related protein (LRP)-5/6 (Figure 3)[96-98]. The Wnt-Frz-LRP-5/6 complex then pairs with the intracellular phosphoprotein Disheveled (Dsh), thereby phosphorylating LRP-5/6 and recruiting Axin. As a result, the cytoplasmic Axin complex—consisting of the scaffolding protein, Axin, the tumor suppressor adenomatous polyposis coli gene product (APC), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3)—is unable to phosphorylate cytoplasmic β-catenin. Thus, the E3 ubiquitin ligase β-Trcp fails to recognize the β-catenin for proteosomal degradation[96,99]. This stabilization and accumulation of cytoplasmic β-catenin allows β-catenin to enter the nucleus, combine with transcription factors of the lymphoid enhancer-binding factor/T cell factor (LEF/TCF) family, and regulate gene expression[100]. Moreover, the canonical Wnt pathway regulates mesenchymal lineage specification via this mechanism[101].

Several similarities exist between the canonical and non-canonical pathway mechanisms, namely the binding of Wnt ligand to Frz receptors to activate Dsh. However, the downstream pathways are activated by binding of a non-canonical Wnt ligand which no longer require β-catenin; hence, the non-canonical Wnt pathway is also known as the β-catenin-independent pathway. The non-canonical Wnt pathway contains two main pathways: the planar cell polarity (PCP) pathway and the Ca2+ pathway. In the Ca2+ pathway, the binding of a non-canonical Wnt ligand to Frz and a co-receptor, increases intracellular calcium levels, decreases cyclin GMP (cGMP) levels, and activates protein kinase C (PKC)[102].



The binding of Frz receptors in the PCP pathway, on the other hand, activates Dsh, which then signals modulation of cytoskeletal elements including actin and microtubules via small GTPases[102,103]. By inducing modifications to the actin cytoskeleton, the PCP pathway mediates asymmetric cytoskeletal organization and the polarization of cells[103]. Both non-canonical pathways play a role in development, cell polarization, motility, and homeostasis[104]. Furthermore, non-canonical Wnt ligands can stimulate osteogenic differentiation through the activation of both PCP and Ca2+ pathways[105,106].

Significance of Wnt signaling in bone development

Wnt signaling plays vital roles in the development, growth, and homeostasis of various organs including the skeletal system[107-110]. Extensive investigation has adequately characterized the role of canonical Wnt signaling in maintaining bone homeostasis. Riddle et al. demonstrated that expression of both LRP-5 and LRP-6 are required within mature osteoblasts for normal postnatal bone development[111]. When LRP-5 was mutated upstream of β-catenin, loss-of-function LRP-5 mutations resulted in osteoporosis pseudoglioma (OPPG) syndrome and a low bone mass phenotype, whereas gain-of-function mutations in LRP-5 led to an osteosclerotic, high bone mass phenotype[112-115]. Furthermore, mice selectively lacking either LRP-5 or LRP-6 in mature osteoblasts demonstrated significant reductions in whole-body bone mineral density. LRP-6 mutant mice failed to accumulate normal amounts of trabecular bone, while LRP-5 mutant mice exhibited normal trabecular bone volume but later showed trabecular bone loss as they matured. Both mutants exhibited significant alterations in cortical bone structure[111]. Mice lacking both LRP-5 and LRP-6 developed severe osteopenia, and calvarial mesenchymal cells deficient for both LRP-5 and -6 fail to form osteoblasts when cultured in osteogenic media, instead attaining a chondrocyte-like phenotype[111]. Additionally, the various roles of β-catenin through canonical Wnt signaling have been verified as a regulator of both osteoblast and osteoclast function in mature cells as well as in the early stages of osteogenesis and postnatal development[116]. Reduced levels of β-catenin in mesenchymal progenitor cells arrests osteoblast development at an early stage, resulting in fetal skeletal defects[116-119]. Furthermore, genetically induced β-catenin deficiencies in terminally differentiated osteoblasts can result in impaired maturation and mineralization along with upregulation of an osteoclast differentiation factor, leading to significant bone resorption[120,121].

Inhibiting Wnt antagonists have also been studied as a method of stimulating canonical Wnt pathway activity to induce formation of new bone along with limiting bone resorption. In particular, Wnt antagonists Sclerostin and Dickkopf-1 (DKK-1) inhibit canonical Wnt signaling by bind to co-receptors LRP-5/6 in order to downregulate the accumulation of β-catenin, thus inhibiting β-catenin’s influence on gene expression[122,123]. Morvan et al demonstrated that mice lacking a single allele of DKK-1 showed a markedly increased trabecular bone volume and elevated trabecular bone formation rate[124]. Yaccoby et al used antibodies raised against DKK-1 in the treatment of a mouse model of multiple myeloma revealed increased numbers of osteoblasts, reduced number of osteoclasts, and reduced myeloma burden in the antibody-treated mice[125]. Inhibiting the production or action of sclerostin resulting in enhanced canonical Wnt signaling led to increased bone mass in preclinical studies, where monoclonal antibody against sclerostin promoted trabecular bone formation rapidly in monkeys and rats without increases in resorption parameters[126]. Moreover, introducing inhibitors of antagonists Sclerostin and DKK-1, anti-Sclerostin and anti-DKK-1 respectively, has demonstrated the ability to stimulate bone formation and increase bone mineral density, with successful Phase I and II clinical trials[127,128]. Overall, the Wnt signaling pathway has been shown to have numerous and diverse effects in bone biology, including skeletal development, regulation of osteoblast/osteoclast function, and maintenance of bone homeostasis. Thus, research regarding this multifaceted signaling pathway in context of OS tumor biology has been significant.

Wnt signaling in OS

Dysregulation of the Wnt/β-catenin pathway is closely associated with a variety of human cancers, including OS. β-catenin is required for differentiation and proliferation of cells in a wide variety of tissues, including the skeletal, neuromuscular, and cardiovascular system among many others. The critical involvement of aberrant activation of the Wnt/β-catenin pathway in oncogenesis is relatively well documented[129,130]; studies have demonstrated that overexpression of numerous Wnt components, including Wnt ligands and Frizzled and LRP receptors, highlighted the implications of aberrant Wnt/β-catenin signaled in the development and progression of OS. For example, Chen et al identified expression of multiple Wnt ligands (Wnt-2b, Wnt-3, Wnt-5a, Wnt-5b, and Wnt-14) and receptors (Frz-1, Frz-2, Frz-3, Frz-6, Frz-7, LRP-5, and LRP-6) across two human OS cell lines: MG63 and HOS[131]. Furthermore, two murine OS cell lines (K7M2 and K12) also demonstrated Wnt ligand and receptor expression[131]. Similarly, Hoang et al examined Wnts, Frzs, and LRP-5 expression using reverse transcription polymerase chain reaction (RT-PCR) in four OS cell lines: U2OS, HOS, 143B, and Saos-2[132]; all cell lines were found to express Frz-1, Frz-2, Frz-4, Frz-5, Frz-9, and LRP-5. Furthermore, out of 44 high-grade OS patient biopsies surveyed for mRNA, Hoang et al detected LRP-5 expression in 50% of samples, and found a significant correlation between LRP-5 expression and nuclear β-catenin accumulation with tumor metastasis[132]. Additional studies suggest a role for LRP-5 in OS; Guo et al demonstrated that Saos-2 cells transfected with dominant-negative, soluble LRP-5 resulted in an increase in E-cadherin and decrease in N-cadherin, leading to the reversal of the epithelial-to-mesenchymal transition (EMT), a hallmark of OS[133]. The increase in E-cadherin also caused a corresponding decrease in Wnt-responsive transcriptional repressors SLUG and TWIST, thereby suggesting that LRP-5 promotes EMT and OS invasiveness[133]. Using real-time quantitative PCR analysis, Ma et al found that all major elements in the Wnt/β-catenin pathway—including Wnt-3a, β-catenin, and Lef-1—were consistently upregulated in human OS cell lines Saos-2 cells compared to human fetal osteoblasts (hFOB)[134]. Ma et al also demonstrated that expression level of β-catenin consistently correlated with the invasiveness of OS. Western blotting analysis further confirmed that the protein levels of both total and active β-catenin were increased in Saos-2 cells compared to hFOBs[134]. Moreover, knocking down β-catenin in Saos-2 cells, Ma et al observed increased Saos-2 sensitivity to methotrexate (MTX) induced cell death, sensitizing the cells to chemotherapy[134].

In addition to the overexpression of numerous Wnt components in OS, the epigenetic silencing of genes encoding endogenous Wnt pathway inhibitors, such as Wnt inhibitor factor 1 (WIF-1), underscore the implications of aberrant Wnt signaling in the development and progression of OS. WIF-1 mRNA expression was significantly decreased in numerous OS cell lines in comparison to normal human osteoblasts, attributed to WIF-1 promoter hypermethylation; it was found that WIF-1 expression downregulates the expression of MMP-9 and 14, thereby preventing the invasion and mobility of OS cells[135]. Kansara et al further confirmed that WIF-1 is epigenetically silenced in human OS, and target disruption of WIF-1 accelerates OS formation in mice[136]. Expression of other Wnt/β-catenin inhibitors, such as FrzB/sFRP3, has been found to be consistently suppressed in OS[137]. Likewise, LEF/TCF transcription factors have been shown to be negative regulators of the Wnt pathway[138]. Another suspected negative regulator of the canonical Wnt pathway is TWIST, a basic helix-loop-helix transcription factor that is typically expressed in low levels in OS[139]. Inducing overexpression of TWIST in vitro in Saos-2 and MG63 lines led to a decrease in β-catenin, while knockout of TWIST with RNA interference (RNAi) led to increased levels of β-catenin[139]. Further studies have demonstrated that increased TWIST expression in Saos-2 OS cells causes an increase in sensitivity to cisplatin-induced apoptosis, whereas TWIST RNAi will actually increase resistance to apoptosis[139]. Taken together, the majority of the current literature supports the notion that aberrant activation of the Wnt pathway contributes to OS development.

Following the notion that aberrant activation of the Wnt pathway contributes to OS, investigated treatment methods commonly target the Wnt pathway and regulators to inhibit and downregulate Wnt signaling activity[140]. Cadigan et al conducted a review showing that in the absence of Wnt binding to Frz and LRP-5/6 cell-surface receptors, a cytoplasmic complex consisting of axin, adenomatosis polyposis coli (APC), and glycogen synthase kinase 3 (GSK3) phosphorylates β-catenin, thereby promoting degradation of β-catenin, thus contributing to the downregulation of Wnt signaling activity[141]. To reduce β-catenin-dependent Wnt pathway activity, Brun et al silenced FHL2 (four and a half LIM domains protein 2) using shRNA in OS cells, which reduced the expression of Wnt-responsive genes as well as Wnt-5a and Wnt-10a, inhibiting OS cell proliferation, invasion, and migration in vitro. Moreover, the study demonstrated that FHL2 silencing markedly reduced tumor growth and lung metastasis occurrence in mice, associated with the decreased Wnt signaling in the tumors[142].

Several studies have suggested that Wnt pathway inhibition has anti-proliferative, pro-apoptotic effects in OS cells[143,144]. Activating the Wnt pathway can inhibit apoptosis, but when combined with chemotherapeutic drugs, doing so can be counterproductive[142,145]. Whether or not Wnt inhibition can directly induce apoptosis is not well documented, and the precise mechanisms remain unknown.

Finally, aberrant Wnt signaling is also observed in predisposing conditions of OS, such as fibrous dysplasia and Paget’s disease. For example, Regard et al showed that activation of the Wnt/ β-catenin pathway in osteoblast progenitors led to the fibrous dysplasia phenotype. Specifically, they analyzed the interaction between the Gα protein families and the Wnt/β-catenin pathway, and identified abnormally increased signaling levels as a cause of fibrous dysplasia[146]. Furthermore, the removal of the Gα protein mutations decreased Wnt/β-catenin signaling and correspondingly decreased bone formation. In patients with Paget’s disease of bone, an inhibitor of Wnt signaling, Dickkoph-1 (Dkk-1), was found to have elevated levels in the serum[147]. Overexpression of Dkk-1 is implicated in osteolytic disease as it is expected to inhibit bone formation, as well as induce bone breakdown. Previous studies have also demonstrated increased Dkk-1 expression and protein in osteoblastic and stromal cell cultures from Paget’s lesions[148]. Thus, studies in predisposing conditions of OS reaffirm the importance of the Wnt signaling mechanism in OS development.

Other Signaling Pathways in OS

Numerous other signaling pathways play a role in osteogenic differentiation, and likewise are suspected to play a role in OS tumor biology. This review will briefly discuss Notch, Insulin-like Growth Factor (IGF), Transforming growth factor beta (TGF- β), and Fibroblast Growth Factor (FGF) in relation to OS.

The Notch signaling pathway plays a central role in the commitment of mesenchymal cells to osteoblastic lineage. For example, Ramasamy et al demonstrated that disruption of Notch signaling in mice led to impaired growth, reduced osteogenesis, shortening of long bones, chondrocyte defects, loss of trabeculae, and decreased bone mass[149]. Hughes et al showed that the Notch pathway is a significant contributor to the metastatic ability of osteosarcoma cells. In comparison to normal human osteoblasts and non-metastatic osteosarcoma cell lines, metastatic osteosarcoma cells exhibited higher levels of Notch1, Notch2, the Notch ligand DLL1, Hes1, a Notch-induced gene[150]. Thus, Notch is believed to hold an essential role in osteosarcoma metastasis, and regulation of this pathway can be crucial to the development of new therapies (please see[151] for a more detailed review).

IGF plays a critical role in skeletal maturation during fetal growth and development. Specifically, IGF is heavily involved in limb morphogenesis. Agrogiannis et al observed growth retardation and delayed skeletal development in mice lacking the Igf gene[152]. In regards to osteosarcoma, the IGF-1 receptor has been an area of focus due to its role in pathogenesis; compared to normal osteoblasts, osteosarcoma cells are characterized by overexpression of the IGF-1R signaling pathways. Thus, IGF-1R has served as a target of regulation in development of new therapeutic drugs. In one study, R1507, a monoclonal antibody to IGF-1R, is currently undergoing phase II clinical trials in patients diagnosed with OS[153]. Kuijjer et al also showed an inhibitor of IGF-1R, OSI-906, decreased osteosarcoma cell line proliferation via inhibition of phosphorylation of IRS-1, a downstream target of IGF-1R signaling[154]. Thus, regulation of the IGF signaling pathway holds potential in the development of novel OS therapies (please see[155] for a more detailed review).

TGF-β is another signaling pathway involved in numerous aspects of bone biology, including osteoprogenitor proliferation, differentiation, and commitment to the osteoblastic lineage. Lee et al demonstrated that TGF-β-2 can lead to more rapid calvarial bone expansion through activation of Erk-MAPK, which stimulates differentiation of osteoprogenitor cells into osteoblasts[156]. In the context of OS, Franchi et al showed that 17 of 23 different osteosarcoma cell lines demonstrated cytoplasmic reactivity for TGF-β1, -2, and -3. Furthermore, high-grade osteosarcomas showed higher expression of TGF-β1 in comparison to low-grade osteosarcomas[157]. Overall, due to its implication in cell differentiation and commitment to the osteoblastic lineage, TGF-β is a target pathway that warrants further research in the development of new OS therapies (please see[158] for a more detailed review).

Lastly, the FGF cascade is an important signaling pathway involved in skeletal development and bone regeneration. Through activation of FGF receptors (FGFR), this signaling pathway controls osteoprogenitor cell replication and differentiation into osteoblasts[159], aberrations in which can lead to various congenital skeletal diseases[160]. In context of OS tumor biology, Shimizu et al showed that Fgf2 enhanced proliferation, migration, and motility of osteosarcoma AX cells in mice[161]. With a significant role in osteosarcoma pathogenic progression, inhibition of the FGF pathway is a potential target area of research that can lead to new OS therapies (please see[162] for a more detailed review).

Discussion

In summary, much remains unknown regarding those cytokine signaling pathways that drive osteosarcomagenesis, as well as sarcoma proliferation, invasion, and metastasis. Various signaling cascades with importance in basic bone biology have been studied in OS tumor biology, including BMP, Hedgehog, and Wnt signaling. Other signaling cascades not discussed here include Notch[163], Insulin-like Growth Factor (IGF)[153,164], Transforming growth factor beta (TGF-β)[165,166], and Fibroblast Growth Factor (FGF)[167,168], to name a few. Although not directly discussed, crosstalk between signaling cascades is a rich area of investigation. Therefore, it is vital to continue investigating inhibition of these signaling pathways as a strategy to combat diseases such as OS. Development of targeted therapies for osteosarcoma hinges on improved understanding of those signaling pathways important in OS tumor biology.

Acknowledgements

Funding was provided by the UCLA Department of Pathology and Laboratory Medicine and the Translational Research Fund. The authors have no conflicts of interest. The authors thank AS James for his excellent technical assistance.

CONFLICT OF INTEREST

There are no conflicts of interest with regard to the present study.

REFERENCES

1 Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA. Novel regulators of bone formation: molecular clones and activities. Science 1988; 242: 1528-1534

2 Chen D, Zhao M, Mundy GR. Bone morphogenetic proteins. Growth factors 2004; 22: 233-241

3 Ducy P, Karsenty G. The family of bone morphogenetic proteins. Kidney international 2000; 57: 2207-2214

4 Bragdon B, Moseychuk O, Saldanha S, King D, Julian J, Nohe A. Bone morphogenetic proteins: a critical review. Cell Signal 2011; 23: 609-620

5 Kang Q, Song WX, Luo Q, Tang N, Luo J, Luo X, Chen J, Bi Y, He BC, Park JK, Jiang W, Tang Y, Huang J, Su Y, Zhu GH, He Y, Yin H, Hu Z, Wang Z, Chen L, Zuo GW, Pan X, Shen J, Vokes T, Reid RR, Haydon RC, Luu HH, He TC. A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells. Stem Cells Dev 2009; 18: 545-559

6 Okuda S, Myoui A, Nakase T, Wada E, Yonenobu K, Yoshikawa H. Ossification of the ligamentum flavum associated with osteoblastoma: a report of three cases. Skeletal radiology 2001; 30: 402-406

7 Khurana JS, Ogino S, Shen T, Parekh H, Scherbel U, DeLong W, Feldman MD, Zhang PJ, Wolfe HJ, Alman BA. Bone morphogenetic proteins are expressed by both bone-forming and non-bone-forming lesions. Archives of pathology & laboratory medicine 2004; 128: 1267-1269

8 Amitani K, Nakata Y, Stevens J. Bone induction by lyophilized osteosarcoma in mice. Calcified tissue research 1974; 16: 305-313

9 Urist MR, Grant TT, Lindholm TS, Mirra JM, Hirano H, Finerman GA. Induction of new-bone formation in the host bed by human bone-tumor transplants in athymic nude mice. The Journal of bone and joint surgery American volume 1979; 61: 1207-1216

10 Yoshikawa H, Nakase T, Myoui A, Ueda T. Bone morphogenetic proteins in bone tumors. Journal of orthopaedic science: official journal of the Japanese Orthopaedic Association 2004; 9: 334-340

11 Miyazono K, Maeda S, Imamura T. BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk. Cytokine & growth factor reviews 2005; 16: 251-263

12 Nohe A, Keating E, Knaus P, Petersen NO. Signal transduction of bone morphogenetic protein receptors. Cell Signal 2004; 16: 291-299

13 Chen D, Ji X, Harris MA, Feng JQ, Karsenty G, Celeste AJ, Rosen V, Mundy GR, Harris SE. Differential roles for bone morphogenetic protein (BMP) receptor type IB and IA in differentiation and specification of mesenchymal precursor cells to osteoblast and adipocyte lineages. The Journal of cell biology 1998; 142: 295-305

14 Nishimura R, Hata K, Matsubara T, Wakabayashi M, Yoneda T. Regulation of bone and cartilage development by network between BMP signalling and transcription factors. J Biochem; 151: 247-254

15 Li X, Cao X. BMP signaling and skeletogenesis. Annals of the New York Academy of Sciences 2006; 1068: 26-40

16 Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, Luu HH, An N, Breyer B, Vanichakarn P, Szatkowski JP, Park JY, He TC. Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). The Journal of bone and joint surgery American volume 2003; 85-A: 1544-1552.

17 Suzawa M, Takeuchi Y, Fukumoto S, Kato S, Ueno N, Miyazono K, Matsumoto T, Fujita T. Extracellular matrix-associated bone morphogenetic proteins are essential for differentiation of murine osteoblastic cells in vitro. Endocrinology 1999; 140: 2125-2133

18 Mishina Y, Starbuck MW, Gentile MA, Fukuda T, Kasparcova V, Seedor JG, Hanks MC, Amling M, Pinero GJ, Harada S, Behringer RR. Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast function and bone remodeling. The Journal of biological chemistry 2004; 279: 27560-27566

19 Okamoto M, Murai J, Yoshikawa H, Tsumaki N. Bone morphogenetic proteins in bone stimulate osteoclasts and osteoblasts during bone development. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 2006; 21: 1022-1033

20 Gazzerro E, Smerdel-Ramoya A, Zanotti S, Stadmeyer L, Durant D, Economides AN, Canalis E. Conditional deletion of gremlin causes a transient increase in bone formation and bone mass. The Journal of biological chemistry 2007; 282: 31549-31557

21 Gazzerro E, Pereira RC, Jorgetti V, Olson S, Economides AN, Canalis E. Skeletal overexpression of gremlin impairs bone formation and causes osteopenia. Endocrinology 2005; 146: 655-665

22 Qian SW, Tang Y, Li X, Liu Y, Zhang YY, Huang HY, Xue RD, Yu HY, Guo L, Gao HD, Liu Y, Sun X, Li YM, Jia WP, Tang QQ. BMP4-mediated brown fat-like changes in white adipose tissue alter glucose and energy homeostasis. Proc Natl Acad Sci U S A 2013; 110: E798-807

23 Davis SW, Camper SA. Noggin regulates Bmp4 activity during pituitary induction. Developmental biology 2007; 305: 145-160

24 Zhu W, Kim J, Cheng C, Rawlins BA, Boachie-Adjei O, Crystal RG, Hidaka C. Noggin regulation of bone morphogenetic protein (BMP) 2/7 heterodimer activity in vitro. Bone 2006; 39: 61-71

25 Dorman LJ, Tucci M, Benghuzzi H. In vitro effects of bmp-2, bmp-7, and bmp-13 on proliferation and differentation of mouse mesenchymal stem cells. Biomed Sci Instrum, 48:81-87

26 Reid J, Gilmour HM, Holt S. Primary non-specific ulcer of the small intestine. Journal of the Royal College of Surgeons of Edinburgh 1982; 27: 228-232

27 Varkey M, Kucharski C, Haque T, Sebald W, Uludag H. In vitro osteogenic response of rat bone marrow cells to bFGF and BMP-2 treatments. Clinical orthopaedics and related research 2006; 443: 113-123

28 Partridge K, Yang X, Clarke NM, Okubo Y, Bessho K, Sebald W, Howdle SM, Shakesheff KM, Oreffo RO. Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds. Biochemical and biophysical research communications 2002; 292: 144-152

29 Wegman F, Bijenhof A, Schuijff L, Oner FC, Dhert WJ, Alblas J. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo. Eur Cell Mater 2011; 21: 230-242; discussion 242

30 Park KH, Kim H, Moon S, Na K. Bone morphogenic protein-2 (BMP-2) loaded nanoparticles mixed with human mesenchymal stem cell in fibrin hydrogel for bone tissue engineering. Journal of bioscience and bioengineering 2009; 108: 530-537

31 Tang Y, Tang W, Lin Y, Long J, Wang H, Liu L, Tian W. Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats. Cell biology international 2008; 32: 1150-1157

32 Kempen DH, Lu L, Hefferan TE, Creemers LB, Maran A, Classic KL, Dhert WJ, Yaszemski MJ. Retention of in vitro and in vivo BMP-2 bioactivities in sustained delivery vehicles for bone tissue engineering. Biomaterials 2008; 29: 3245-3252

33 Cheng SL, Lou J, Wright NM, Lai CF, Avioli LV, Riew KD. In vitro and in vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene. Calcified tissue international 2001; 68: 87-94

34 Hanamura H, Urist MR. Osteogenesis and chondrogenesis in transplants of Dunn and Ridgway osteosarcoma cell cultures. The American journal of pathology 1978; 91: 277-298

35 Asai T, Ueda T, Itoh K, Yoshioka K, Aoki Y, Mori S, Yoshikawa H. Establishment and characterization of a murine osteosarcoma cell line (LM8) with high metastatic potential to the lung. International journal of cancer Journal international du cancer 1998; 76: 418-422

36 Anderson HC, Hsu HH, Raval P, Hunt TR, Schwappach JR, Morris DC, Schneider DJ. The mechanism of bone induction and bone healing by human osteosarcoma cell extracts. Clinical orthopaedics and related research 1995; 313: 129-134

37 Ogose A, Motoyama T, Hotta T, Watanabe H, Takahashi HE. Bone formation in vitro and in nude mice by human osteosarcoma cells. Virchows Archiv: an international journal of pathology 1995; 426: 117-125

38 Hara A, Ikeda T, Nomura S, Yagita H, Okumura K, Yamauchi Y. In vivo implantation of human osteosarcoma cells in nude mice induces bones with human-derived osteoblasts and mouse-derived osteocytes. Laboratory investigation; a journal of technical methods and pathology 1996; 75: 707-717

39 Raval P, Hsu HH, Schneider DJ, Sarras MP, Jr., Masuhara K, Bonewald LF, Anderson HC. Expression of bone morphogenetic proteins by osteoinductive and non-osteoinductive human osteosarcoma cells. Journal of dental research 1996; 75: 1518-1523

40 Gobbi G, Sangiorgi L, Lenzi L, Casadei R, Canaider S, Strippoli P, Lucarelli E, Ghedini I, Donati D, Fabbri N, Warzecha J, Yeoung C, Helman LJ, Picci P, Carinci P. Seven BMPs and all their receptors are simultaneously expressed in osteosarcoma cells. International journal of oncology 2002; 20: 143-147

41 Yoshikawa H, Rettig WJ, Lane JM, Takaoka K, Alderman E, Rup B, Rosen V, Healey JH, Huvos AG, Garin-Chesa P. Immunohistochemical detection of bone morphogenetic proteins in bone and soft-tissue sarcomas. Cancer 1994; 74: 842-847

42 Yoshikawa H, Rettig WJ, Takaoka K, Alderman E, Rup B, Rosen V, Wozney JM, Lane JM, Huvos AG, Garin-Chesa P. Expression of bone morphogenetic proteins in human osteosarcoma. Immunohistochemical detection with monoclonal antibody. Cancer 1994; 73: 85-91

43 Mohseny AB, Cai Y, Kuijjer M, Xiao W, van den Akker B, de Andrea CE, Jacobs R, ten Dijke P, Hogendoorn PC, Cleton-Jansen AM. The activities of Smad and Gli mediated signalling pathways in high-grade conventional osteosarcoma. European journal of cancer 2012; 48: 3429-3438

44 Kubista B, Klinglmueller F, Bilban M, Pfeiffer M, Lass R, Giurea A, Funovics PT, Toma C, Dominkus M, Kotz R, Thalhammer T, Trieb K, Zettl T, Singer CF. Microarray analysis identifies distinct gene expression profiles associated with histological subtype in human osteosarcoma. International orthopaedics 2011; 35: 401-411

45 Sulzbacher I, Birner P, Trieb K, Pichlbauer E, Lang S. The expression of bone morphogenetic proteins in osteosarcoma and its relevance as a prognostic parameter. Journal of clinical pathology 2002; 55: 381-385

46 Wang L, Park P, Zhang H, La Marca F, Claeson A, Valdivia J, Lin CY. BMP-2 inhibits the tumorigenicity of cancer stem cells in human osteosarcoma OS99-1 cell line. Cancer biology & therapy 2011; 11: 457-463

47 Luo X, Chen J, Song WX, Tang N, Luo J, Deng ZL, Sharff KA, He G, Bi Y, He BC, Bennett E, Huang J, Kang Q, Jiang W, Su Y, Zhu GH, Yin H, He Y, Wang Y, Souris JS, Chen L, Zuo GW, Montag AG, Reid RR, Haydon RC, Luu HH, He TC. Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects. Laboratory investigation; a journal of technical methods and pathology 2008; 88: 1264-1277

48 Sotobori T, Ueda T, Myoui A, Yoshioka K, Nakasaki M, Yoshikawa H, Itoh K. Bone morphogenetic protein-2 promotes the haptotactic migration of murine osteoblastic and osteosarcoma cells by enhancing incorporation of integrin beta1 into lipid rafts. Experimental cell research 2006; 312: 3927-3938

49 Bitgood MJ, McMahon AP. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Developmental biology 1995; 172: 126-138.

50 Yao HH, Whoriskey W, Capel B. Desert Hedgehog/Patched 1 signaling specifies fetal Leydig cell fate in testis organogenesis. Genes & development 2002; 16: 1433-1440

51 Parmantier E, Lynn B, Lawson D, Turmaine M, Namini SS, Chakrabarti L, McMahon AP, Jessen KR, Mirsky R. Schwann cell-derived Desert hedgehog controls the development of peripheral nerve sheaths. Neuron 1999; 23: 713-724

52 Cohen MM, Jr.. The hedgehog signaling network. 2003; 123A: 5-28

53 Kawai Y, Noguchi J, Akiyama K, Takeno Y, Fujiwara Y, Kajita S, Tsuji T, Kikuchi K, Kaneko H, Kunieda T. A missense mutation of the Dhh gene is associated with male pseudohermaphroditic rats showing impaired Leydig cell development. Reproduction 2011; 141: 217-225

54 Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H, Beachy PA. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 1996; 383: 407-413

55 St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes & development 1999; 13: 2072-2086

56 Hayhurst M, McConnell SK. Mouse models of holoprosencephaly. Current opinion in neurology 2003; 16: 135-141

57 Ruat M, Roudaut H, Ferent J, Traiffort E. Hedgehog trafficking, cilia and brain functions. Differentiation; research in biological diversity 2012; 83: S97-104

58 Chamoun Z, Mann RK, Nellen D, von Kessler DP, Bellotto M, Beachy PA, Basler K. Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal. Science 2001; 293: 2080-2084

59 Dennis JF, Kurosaka H, Iulianella A, Pace J, Thomas N, Beckham S, Williams T, Trainor PA. Mutations in Hedgehog acyltransferase (Hhat) perturb Hedgehog signaling, resulting in severe acrania-holoprosencephaly-agnathia craniofacial defects. PLoS genetics 2012; 8: e1002927

60 Jiang Z, Cushing L, Ai X, Lu J. miR-326 Is Downstream of Sonic Hedgehog Signaling and Regulates the Expression of Gli2 and Smoothened. American journal of respiratory cell and molecular biology 2014.

61 Hojo H, Ohba S, Taniguchi K, Shirai M, Yano F, Saito T, Ikeda T, Nakajima K, Komiyama Y, Nakagata N, Suzuki K, Mishina Y, Yamada M, Konno T, Takato T, Kawaguchi H, Kambara H, Chung UI. Hedgehog-Gli activators direct osteo-chondrogenic function of bone morphogenetic protein toward osteogenesis in the perichondrium. The Journal of biological chemistry 2013; 288: 9924-9932

62 James AW, Leucht P, Levi B, Carre AL, Xu Y, Helms JA, Longaker MT. Sonic Hedgehog influences the balance of osteogenesis and adipogenesis in mouse adipose-derived stromal cells. Tissue engineering Part A 2010; 16: 2605-2616

63 James AW, Pang S, Askarinam A, Corselli M, Zara JN, Goyal R, Chang L, Pan A, Shen J, Yuan W, Stoker D, Zhang X, Adams JS, Ting K, Soo C. Additive effects of sonic hedgehog and Nell-1 signaling in osteogenic versus adipogenic differentiation of human adipose-derived stromal cells. Stem Cells Dev 2012; 21: 2170-2178

64 Day TF, Yang Y. Wnt and hedgehog signaling pathways in bone development. The Journal of bone and joint surgery American volume 2008; 90: 19-24

65 Rice R, Spencer-Dene B, Connor EC, Gritli-Linde A, McMahon AP, Dickson C, Thesleff I, Rice DP. Disruption of Fgf10/Fgfr2b-coordinated epithelial-mesenchymal interactions causes cleft palate. The Journal of clinical investigation 2004; 113: 1692-1700.

66 Lenton K, James AW, Manu A, Brugmann SA, Birker D, Nelson ER, Leucht P, Helms JA, Longaker MT. Indian hedgehog positively regulates calvarial ossification and modulates bone morphogenetic protein signaling. Genesis 2011; 49: 784-796

67 Rivron NC, Raiss CC, Liu J, Nandakumar A, Sticht C, Gretz N, Truckenmuller R, Rouwkema J, van Blitterswijk CA. Sonic Hedgehog-activated engineered blood vessels enhance bone tissue formation. Proc Natl Acad Sci U S A 2012; 109: 4413-4418

68 Jones KB, Morcuende JA. Of hedgehogs and hereditary bone tumors: re-examination of the pathogenesis of osteochondromas. The Iowa orthopaedic journal 2003; 23: 87-95

69 Gupta S, Takebe N, Lorusso P. Targeting the Hedgehog pathway in cancer. Therapeutic advances in medical oncology 2010; 2: 237-250

70 Li X, Deng W, Nail CD, Bailey SK, Kraus MH, Ruppert JM, Lobo-Ruppert SM. Snail induction is an early response to Gli1 that determines the efficiency of epithelial transformation. Oncogene 2006; 25: 609-621

71 Velcheti V. Hedgehog signaling is a potent regulator of angiogenesis in small cell lung cancer. Medical hypotheses 2007; 69: 948-949

72 Lavine KJ, White AC, Park C, Smith CS, Choi K, Long F, Hui CC, Ornitz DM. Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development. Genes & development 2006; 20: 1651-1666

73 Mimeault M, Batra SK. Frequent deregulations in the hedgehog signaling network and cross-talks with the epidermal growth factor receptor pathway involved in cancer progression and targeted therapies. Pharmacological reviews 2010; 62: 497-524

74 Hirotsu M, Setoguchi T, Sasaki H, Matsunoshita Y, Gao H, Nagao H, Kunigou O, Komiya S. Smoothened as a new therapeutic target for human osteosarcoma. Molecular cancer 2010; 9: 5

75 Ando K, Heymann MF, Stresing V, Mori K, Redini F, Heymann D. Current therapeutic strategies and novel approaches in osteosarcoma. Cancers 2013; 5: 591-616

76 Cirrone F, Harris CS. Vismodegib and the hedgehog pathway: a new treatment for basal cell carcinoma. Clinical therapeutics 2012; 34: 2039-2050

77 Wu Y, Dai J, Zhang W, Yan R, Zhang Y, Ruan C, Dai K. Arsenic trioxide induces apoptosis in human platelets via C-Jun NH2-terminal kinase activation. PloS one 2014; 9: e86445

78 Beauchamp EM, Ringer L, Bulut G, Sajwan KP, Hall MD, Lee YC, Peaceman D, Ozdemirli M, Rodriguez O, Macdonald TJ, Albanese C, Toretsky JA, Uren A. Arsenic trioxide inhibits human cancer cell growth and tumor development in mice by blocking Hedgehog/GLI pathway. The Journal of clinical investigation 2011; 121: 148-160

79 Tingting R, Wei G, Changliang P, Xinchang L, Yi Y. Arsenic trioxide inhibits osteosarcoma cell invasiveness via MAPK signaling pathway. Cancer biology & therapy 2010, 10(3): 251-257

80 Rao TP, Kuhl M. An updated overview on Wnt signaling pathways: a prelude for more. Circulation research 2010; 106: 1798-1806

81 Hartmann C, Tabin CJ. Dual roles of Wnt signaling during chondrogenesis in the chicken limb. Development 2000; 127: 3141-3159

82 Maeda K, Takahashi N, Kobayashi Y. Roles of Wnt signals in bone resorption during physiological and pathological states. Journal of molecular medicine 2013; 91: 15-23.

83 Berwick DC, Harvey K. The importance of Wnt signalling for neurodegeneration in Parkinson’s disease. Biochemical Society transactions 2012; 40: 1123-1128

84 Chen J, Stahl A, Krah NM, Seaward MR, Dennison RJ, Sapieha P, Hua J, Hatton CJ, Juan AM, Aderman CM, Willett KL, Guerin KI, Mammoto A, Campbell M, Smith LE. Wnt signaling mediates pathological vascular growth in proliferative retinopathy. Circulation 2011; 124: 1871-1881

85 Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 1997; 275: 1787-1790

86 Niehrs C. The complex world of WNT receptor signalling. Nature reviews Molecular cell biology 2012; 13: 767-779

87 Nelson WJ, Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways. Science 2004; 303: 1483-1487

88 Kato M, Patel MS, Levasseur R, Lobov I, Chang BH, Glass DA 2nd, Hartmann C, Li L, Hwang TH, Brayton CF, Lang RA, Karsenty G, Chan L. Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. The Journal of cell biology 2002; 157: 303-314

89 Hartikka H, Makitie O, Mannikko M, Doria AS, Daneman A, Cole WG, Ala-Kokko L, Sochett EB. Heterozygous mutations in the LDL receptor-related protein 5 (LRP5) gene are associated with primary osteoporosis in children. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 2005; 20: 783-789

90 MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Developmental cell 2009; 17: 9-26

91 Kestler HA, Kuhl M. From individual Wnt pathways towards a Wnt signalling network. Philosophical transactions of the Royal Society of London Series B, Biological sciences 2008; 363: 1333-1347

92 Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annual review of cell and developmental biology 2004; 20: 781-810

93 He X, Semenov M, Tamai K, Zeng X. LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way. Development 2004; 131: 1663-1677

94 Pandur P, Maurus D, Kuhl M. Increasingly complex: new players enter the Wnt signaling network. BioEssays: news and reviews in molecular, cellular and developmental biology 2002; 24: 881-884

95 Etheridge SL, Spencer GJ, Heath DJ, Genever PG. Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells. Stem cells 2004, 22(5): 849-860

96 Montcouquiol M, Crenshaw EB, 3rd, Kelley MW. Noncanonical Wnt signaling and neural polarity. Annual review of neuroscience 2006; 29: 363-386

97 Cai Y, Cai T, Chen Y. Wnt pathway in osteosarcoma, from oncogenic to therapeutic. J Cell Biochem 2014; 115: 625-631

98 Davis LA, Zur Nieden NI. Mesodermal fate decisions of a stem cell: the Wnt switch. Cellular and molecular life sciences : CMLS 2008; 65: 2658-2674

99 Boland GM, Perkins G, Hall DJ, Tuan RS. Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells. J Cell Biochem 2004; 93: 1210-1230

100 Kobayashi Y. Roles of Wnt signaling in bone metabolism. Clinical calcium 2012; 22: 1701-1706

101 Baron R, Kneissel M. WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nature medicine 2013; 19: 179-192

102 Krishnan V, Bryant HU, Macdougald OA. Regulation of bone mass by Wnt signaling. The Journal of clinical investigation 2006; 116: 1202-1209

103 Lories RJ, Corr M, Lane NE. To Wnt or not to Wnt: the bone and joint health dilemma. Nature reviews Rheumatology 2013; 9: 328-339

104 Nusse R, Varmus HE. Wnt genes. Cell 1992; 69: 1073-1087

105 Riddle RC, Diegel CR, Leslie JM, Van Koevering KK, Faugere MC, Clemens TL, Williams BO. Lrp5 and Lrp6 exert overlapping functions in osteoblasts during postnatal bone acquisition. PloS one 2013; 8: e63323

106 Williams BO, Insogna KL. Where Wnts went: the exploding field of Lrp5 and Lrp6 signaling in bone. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 2009; 24: 171-178

107 Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick MA, Wu D, Insogna K, Lifton RP. High bone density due to a mutation in LDL-receptor-related protein 5. The New England journal of medicine 2002; 346: 1513-1521

108 Gong Y, Slee RB, Fukai N, Rawadi G, Roman-Roman S, Reginato AM, Wang H, Cundy T, Glorieux FH, Lev D, Zacharin M, Oexle K, Marcelino J, Suwairi W, Heeger S, Sabatakos G, Apte S, Adkins WN, Allgrove J, Arslan-Kirchner M, Batch JA, Beighton P, Black GC, Boles RG, Boon LM, Borrone C, Brunner HG, Carle GF, Dallapiccola B, De Paepe A, Floege B, Halfhide ML, Hall B, Hennekam RC, Hirose T, Jans A, Jüppner H, Kim CA, Keppler-Noreuil K, Kohlschuetter A, LaCombe D, Lambert M, Lemyre E, Letteboer T, Peltonen L, Ramesar RS, Romanengo M, Somer H, Steichen-Gersdorf E, Steinmann B, Sullivan B, Superti-Furga A, Swoboda W, van den Boogaard MJ, Van Hul W, Vikkula M, Votruba M, Zabel B, Garcia T, Baron R, Olsen BR, Warman ML, Osteoporosis-Pseudoglioma Syndrome Collaborative Group. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 2001; 107: 513-523

109 Little RD, Carulli JP, Del Mastro RG, Dupuis J, Osborne M, Folz C, Manning SP, Swain PM, Zhao SC, Eustace B, Lappe MM, Spitzer L, Zweier S, Braunschweiger K, Benchekroun Y, Hu Y, Adair R, Chee L, FitzGerald MG, Tulig C, Caruso A, Tzellas N, Bawa A, Franklin B, McGuire S, Nogues X, Gong G, Allen KM, Anisowicz A, Morales AJ, Lomedico PT, Recker SM, Van Eerdewegh P, Recker RR, Johnson ML. A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. American journal of human genetics 2002; 70: 11-19

110 Chen J, Long F. beta-catenin promotes bone formation and suppresses bone resorption in postnatal growing mice. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 2013; 28: 1160-1169.

111 Day TF, Guo X, Garrett-Beal L, Yang Y. Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Developmental cell 2005; 8: 739-750

112 Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C. Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Developmental cell 2005; 8: 727-738

113 Hu H, Hilton MJ, Tu X, Yu K, Ornitz DM, Long F. Sequential roles of Hedgehog and Wnt signaling in osteoblast development. Development 2005; 132: 49-60

114 Glass DA 2nd, Bialek P, Ahn JD, Starbuck M, Patel MS, Clevers H, Taketo MM, Long F, McMahon AP, Lang RA, Karsenty G. Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation. Developmental cell 2005; 8: 751-764

115 Holmen SL, Zylstra CR, Mukherjee A, Sigler RE, Faugere MC, Bouxsein ML, Deng L, Clemens TL, Williams BO. Essential role of beta-catenin in postnatal bone acquisition. The Journal of biological chemistry 2005; 280: 21162-21168

116 Niehrs C. Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene 2006; 25: 7469-7481.

117 Gatti D, Viapiana O, Fracassi E, Idolazzi L, Dartizio C, Povino MR, Adami S, Rossini M. Sclerostin and DKK1 in postmenopausal osteoporosis treated with denosumab. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 2012; 27: 2259-2263

118 Morvan F, Boulukos K, Clement-Lacroix P, Roman Roman S, Suc-Royer I, Vayssiere B, Ammann P, Martin P, Pinho S, Pognonec P, Mollat P, Niehrs C, Baron R, Rawadi G. Deletion of a single allele of the Dkk1 gene leads to an increase in bone formation and bone mass. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 2006; 21: 934-945

119 Yaccoby S, Ling W, Zhan F, Walker R, Barlogie B, Shaughnessy JD, Jr.. Antibody-based inhibition of DKK1 suppresses tumor-induced bone resorption and multiple myeloma growth in vivo. Blood 2007; 109: 2106-2111

120 Li X, Warmington KS, Niu QT, Asuncion FJ, Barrero M, Grisanti M, Dwyer D, Stouch B, Thway TM, Stolina M, Ominsky MS, Kostenuik PJ, Simonet WS, Paszty C, Ke HZ. Inhibition of sclerostin by monoclonal antibody increases bone formation, bone mass, and bone strength in aged male rats. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 2010; 25: 2647-2656.

121 Lim V, Clarke BL. New therapeutic targets for osteoporosis: beyond denosumab. Maturitas 2012; 73: 269-272

122 Padhi D, Jang G, Stouch B, Fang L, Posvar E. Single-dose, placebo-controlled, randomized study of AMG 785, a sclerostin monoclonal antibody. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 2011; 26: 19-26

123 Peifer M, Polakis P: Wnt signaling in oncogenesis and embryogenesis--a look outside the nucleus. Science 2000; 287: 1606-1609

124 Chen Y, Shi HY, Stock SR, Stern PH, Zhang M. Regulation of breast cancer-induced bone lesions by beta-catenin protein signaling. The Journal of biological chemistry 2011; 286: 42575-42584

125 Chen K, Fallen S, Abaan HO, Hayran M, Gonzalez C, Wodajo F, MacDonald T, Toretsky JA, Uren A. Wnt10b induces chemotaxis of osteosarcoma and correlates with reduced survival. Pediatric blood & cancer 2008; 51: 349-355

126 Hoang BH, Kubo T, Healey JH, Sowers R, Mazza B, Yang R, Huvos AG, Meyers PA, Gorlick R. Expression of LDL receptor-related protein 5 (LRP5) as a novel marker for disease progression in high-grade osteosarcoma. International journal of cancer Journal international du cancer 2004; 109: 106-111

127 Guo Y, Zi X, Koontz Z, Kim A, Xie J, Gorlick R, Holcombe RF, Hoang BH. Blocking Wnt/LRP5 signaling by a soluble receptor modulates the epithelial to mesenchymal transition and suppresses met and metalloproteinases in osteosarcoma Saos-2 cells. Journal of orthopaedic research: official publication of the Orthopaedic Research Society 2007; 25: 964-971

128 Ma Y, Ren Y, Han EQ, Li H, Chen D, Jacobs JJ, Gitelis S, O’Keefe RJ, Konttinen YT, Yin G, Li TF. Inhibition of the Wnt-beta-catenin and Notch signaling pathways sensitizes osteosarcoma cells to chemotherapy. Biochemical and biophysical research communications 2013; 431: 274-279

129 Rubin EM, Guo Y, Tu K, Xie J, Zi X, Hoang BH. Wnt inhibitory factor 1 decreases tumorigenesis and metastasis in osteosarcoma. Molecular cancer therapeutics 2010; 9: 731-741

130 Kansara M, Tsang M, Kodjabachian L, Sims NA, Trivett MK, Ehrich M, Dobrovic A, Slavin J, Choong PF, Simmons PJ, Dawid IB, Thomas DM. Wnt inhibitory factor 1 is epigenetically silenced in human osteosarcoma, and targeted disruption accelerates osteosarcomagenesis in mice. The Journal of clinical investigation 2009; 119: 837-851

131 Mandal D, Srivastava A, Mahlum E, Desai D, Maran A, Yaszemski M, Jalal SM, Gitelis S, Bertoni F, Damron T, Irwin R, O’connor M, Schwartz H, Bolander ME, Sarkar G. Severe suppression of Frzb/sFRP3 transcription in osteogenic sarcoma. Gene 2007; 386: 131-138

132 Dieudonne FX, Marion A, Hay E, Marie PJ, Modrowski D. High Wnt signaling represses the proapoptotic proteoglycan syndecan-2 in osteosarcoma cells. Cancer research 2010; 70: 5399-5408

133 Wu J, Liao Q, He H, Zhong D, Yin K. TWIST interacts with beta-catenin signaling on osteosarcoma cell survival against cisplatin. Molecular carcinogenesis 2014; 53: 440-446

134 Yang J, Zhang W. New molecular insights into osteosarcoma targeted therapy. Current opinion in oncology 2013; 25: 398-406.

135 Cadigan KM, Liu YI. Wnt signaling: complexity at the surface. Journal of cell science 2006; 119: 395-402

136 Brun J, Dieudonne FX, Marty C, Muller J, Schule R, Patino-Garcia A, Lecanda F, Fromigue O, Marie PJ. FHL2 silencing reduces Wnt signaling and osteosarcoma tumorigenesis in vitro and in vivo. PloS one 2013; 8: e55034

137 Lu W, Lin C, Roberts MJ, Waud WR, Piazza GA, Li Y. Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/beta-catenin pathway. PloS one 2011; 6: e29290

138 Dieudonne FX, Marion A, Marie PJ, Modrowski D. Targeted inhibition of T-cell factor activity promotes syndecan-2 expression and sensitization to doxorubicin in osteosarcoma cells and bone tumors in mice. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 2012; 27: 2118-2129

139 Pecina-Slaus N. Wnt signal transduction pathway and apoptosis: a review. Cancer cell international 2010; 10: 22

140 Yavropoulou MP, Yovos JG. The role of Notch signaling in bone development and disease. Hormones 2014; 13: 24-37

141 Kuchimaru T, Hoshino T, Aikawa T, Yasuda H, Kobayashi T, Kadonosono T, Kizaka-Kondoh S. Bone resorption facilitates osteoblastic bone metastasis by insulin-like growth factor and hypoxia. Von Kossa staining showing aberrant bone formation due to bone metastasis of murine osteosarcoma LM8 cells. Cancer science 2014; 105: Maycover

142 Pappo AS, Vassal G, Crowley JJ, Bolejack V, Hogendoorn PC, Chugh R, Ladanyi M, Grippo JF, Dall G, Staddon AP, Chawla SP, Maki RG, Araujo DM, Geoerger B, Ganjoo K, Marina N, Blay JY, Schuetze SM, Chow WA, Helman LJ. A phase 2 trial of R1507, a monoclonal antibody to the insulin-like growth factor-1 receptor (IGF-1R), in patients with recurrent or refractory rhabdomyosarcoma, osteosarcoma, synovial sarcoma, and other soft tissue sarcomas: Results of a Sarcoma Alliance for Research Through Collaboration study. Cancer 2014.

143 Cervini-Silva J, Antonio Nieto C, Gomez-Vidales V, Ramirez-Apan MT, Palacios E, Montoya A, Kaufhold S, Abidin Z, Theng BK. Lipid peroxidation and cytotoxicity induced by respirable volcanic ash. Journal of hazardous materials 2014; 274C: 237-246

144 Brescia AC, Simonds MM, McCahan SM, Fawcett PT, Rose CD. The Role of Transforming Growth Factor beta Signaling in Fibroblast-like Synoviocytes From Patients With Oligoarticular Juvenile Idiopathic Arthritis: Dysregulation of Transforming Growth Factor beta Signaling, Including Overexpression of Bone Morphogenetic Protein 4, May Lead to a Chondrocyte Phenotype and May Contribute to Bony Hypertrophy. Arthritis & rheumatology 2014; 66: 1352-1362

145 Kido S, Fujihara M, Nomura K, Sasaki S, Mukai R, Ohnishi R, Kaneko I, Segawa H, Tatsumi S, Izumi H, Kohno K, Miyamoto K. Molecular Mechanisms of Cadmium-Induced Fibroblast growth Factor 23 Upregulation in Osteoblast-Like Cells. Toxicological sciences: an official journal of the Society of Toxicology 2014

146 Ren T, Qing Y, Dai N, Li M, Qian C, Yang Y, Cheng Y, Li Z, Zhang S, Zhong Z, Wang D. Apurinic/apyrimidinic endonuclease 1 induced upregulation of fibroblast growth factor 2 and its receptor 3 induces angiogenesis in human osteosarcoma cells. Cancer science 2014; 105: 186-194

Peer reviewer: ZuoZhang Yang, Department of Orthopedics, Tumor Hospital of Yunnan Province, Tumor Hospital of Yunnan Province, Kunming 650118, Yunnan Province, China.

Refbacks

  • There are currently no refbacks.


Creative Commons License
This work is licensed under a Creative Commons Attribution 3.0 License.