The Role Of Wnt/β-catenin Pathway In Osteosarcoma Pathogenesis

【Background】Osteosarcoma is the most common primary malignant nonhematological tumor of bone. Osteosaroma has a peak incidence in adolescence. Conventional osteosarcoma frequently affects young adolescents during their longitudinal growth spurt at the metaphysis of long bones, suggesting a correlation between rapid bone turnover and the pathogenesis of osteosarcoma. Prognosis remains less that 20% especially in patients with clinically detectable metastasis at diagnosis or relapsed disease. Limited improvement in survival has been achieved over the past 30 years, regardless of intensifying or modifying chemotherapy.. New target therapy has not been developed yet because of limited progress on the pathogenesis of osteosarocoma.Recently, accumulated evidence show Wnt/β-catenin pathway has emerged as an essential pathway in skeletal development. Wnt acts throughβ-catenin-independent pathways, the so-called non-canonical Wnt pathways (including the Wnt/Ca2+and Wnt/JNK pathways) and through aβ-catenin-dependent or canonical Wnt pathway. The role of the non-canonical Wnt pathway is poorly understood in bone formation, thus this study focus on canonical Wnt pathway.The Wnt pathway is crucial for physiological osteoblast development. Wnt pathway is required for promoting an osteogenic lineage in skeletal stem cells at an early stage. Later on, Wnts stimulate osteoblast proliferation and support osteoblast maturation. Wnt pathway has been shown to be active in osteoblasts in adult mice. The exact role of Wnt coordination osteoblast proliferation and differenation is unclear. Osteosarcoma is characterized by tumor osteoid, which suggest tumor cells retain part of capability as osteoblast but is unable to go terminal differentation. Wnt pathway plays an essential role in bone development, howerer, its role in osteogenic tumor osteosarcoma remains largely unknown.Since mutation of key components APC of Wnt pathway has been found to cause colorectal tumor, increasing evidence suggest that activation of Wnt pathway contributes to a variety of epithelial tumor types development. However, its role in the mesenchymal cells derived tumor osteosarcoma remains unclear. Previous studies have suggested that the Wnt pathway is active in osteosarcoma, based on the detection of Wnt ligands or cytoplasmicβ-catenin staining. However, those approaches are inadequate to demonstrate whether this pathway is active or not given the large family of Wnt pathway, which includes a number of ligands, receptors, co-receptors, and inhibitors. Nuclearβ-catenin expression is the node of Wnt pathway, thus detection nuclearβ-catenin expression is a relatively reliable method.Accumulating evidences suggest that activation of Wnt pathway promotes MSC toward osteoblast development, therefore, manipulating MSC is a promising direction for treatment of osteoporosis and bone regeneration. However, concerns remain that activation of Wnt pathway contributes to tumor development given that numerous studies related active Wnt pathway with tumor development.Thus, in this study, we aim to explore the role of Wnt pathway in osteosarcom by evaluating nuclear P-catenin expression in osteosarcoma biopsies and osteosarcoma cell lines as well as osteoblastomas. Moreover, we assessed the effect of modulating this pathway on cell growth and osteogenic differentiation. Our study will advance the understanding of mechanism of osteosarcoma development and provide a new therapy target for osteosarcoma treatment.【Objectives】1. To determine Wnt pathway status in osteosarcoma.2. To explore the role of Wnt pathway in osteosarcoma cells proliferation and differentiation. [Methods】1. Nuclear P-catenin expression was examined in 52 human osteosarcoma biopsies, 15 osteoblastomas (benign bone tumours) and human osteosarcoma cell lines by immunohistochemistry.2. Wnt pathway activity was measured in osteosarcoma cell lines MG-633. SJSA-11. HOS and U-2-OS by Wnt luciferase reporter assay. Wnt pathway inhibitor DKK-1 expression was examined by qRT-PCR.3. Wnt/β-catenin pathway activity was modulated using a drug GIN (GSK3βinhibitor) and Wnt 3 a and the effect was determined by Wnt luciferase reporter assay, nuclear P-catenin expression and change of Wnt target gene Axin2 mRNA expression.4. The effect of activation Wnt pathway on osteosarcoma cells growth was evaluated by the MTS assay.5. ALP activity and mineralization formation was measured to determine the effect of activation Wnt pathway on osteosarcoma cells differentiation toward osteoblasts.【Results】1. Wnt pathway is inactive in osteosarcoma The expression ofβ-catenin in osteosarcoma was examined by immunohistochemistry. Negative staining of nuclear P-catenin was detected in 90% (47/52) of cases and the remaining five cases showed weak nuclear staining. Most of the cases (47/52) showed membranous and/or cytoplasmic staining. Strong membranous and nuclear P-catenin staining was seen in all 15 osteoblastomas and absence ofβ-catenin staining was found in osteoclasts.Wnt luciferase assay was used to determine the functional activity of Wnt pathway. The corrected Wnt-luciferase activity was around 10 in the positive control colorectal carcinoma cell line SW480; however, it was around 0.002-0.01 (1000-5000 times lower than the positive control) in osteosarcoma cell lines, comparable with the negative control. Consistently, none of the cell lines showed nuclear P-catenin staining.2. Restoration of Wnt signalling by GIN and Wnt3aInactive Wnt pathway was observed in osteosarcoma and active Wnt pathway is required for osteoblast development, thus we decided to investigate whether Wnt pathway can be actived in osteosarcoma cell lines.GIN stimulates Wnt pathway by inhibiting GSK3, thereby preventingβ-catenin degradation in the cytoplasm. Around a 10-to 200 fold increase in Wnt pathway activity occurred in all osteosarcoma cell lines upon GIN (0.2μM) treatment, as determined by the luciferase assay (p< 0.01). Accordingly, strong nuclear p-catenin staining was observed by immunofluorescence in all cell lines after GIN (0.2μM) treatment. Moreover, a 10-to 60 fold increase in Axin2 mRNA expression was found compared with DMSO-treated cells. Wnt3a is a ligand commonly used to activate Wnt signalling upon binding to the receptors. In the positive control C2C12 cell line, BAT-luc activity increased in a dose-dependent manner upon treatment with Wnt3a (50ng/ml,100ng/ml). However, neither a change of BAT-luc activity nor a change of nuclearβ-catenin staining was observed upon rWnt3a treatment in osteosarcoma cell lines.3. DKK1 expression in osteosarcoma cell linesGIN can stimulate Wnt pathway, however, Wnt3a failed, implying the link between Wnt ligands and receptor is impaired.To determine the possible cause of Wnt pathway inactivity, we used qRT-PCR to assess expression of the Wnt inhibitor DKK-1 at the mRNA level. Compared with HeLa cells, relatively high or similar levels of DKK-1 mRNA were found in MG-63, SJSA-1, and HOS cells, although DKK-1 mRNA expression was hardly detectable in U-2-OS cells.4. Restoration of Wnt signalling promotes osteoblast differentiation in SJSA-1 and HOS cells Since Wnt signalling is essential for osteogenic differentiation, we examined whether restoration of Wnt signalling could promote differentiation of osteosarcoma cells. ALP activity (on day 7)and mineralization formation (on day 21) are early and late stage markers of osteoblast differentiation, respectively. We planned to activate Wnt pathway at different time period by adding GIN at different time point, i.e. prior 3 days, prior 3 days till first 3 days, first 3 days and first 7days. Low levels of ALP activity and absence of mineralization were observed in U-2-OS and MG-63, and stimulation of the Wnt pathway did not rescue this phenotype. High levels of ALP activity and mineralization were seen in SJSA-1 and HOS cells. Stimulation of the Wnt pathway by GIN for 3 or 7 days (when differentiation starts) induced a further increase in ALP activity and mineralization. Three day prior treatment with GIN did not induce ALP activity or mineralization.5. Activation of the Wnt pathway inhibits cell proliferation in MG-63 and U-2-OS cellsThe effect of stimulation of the Wnt pathway on cell proliferation was examined. A slight decrease of cell numbers was found for SJSA-1 and HOS cells upon GIN treatment compared with DMSO control group. In contrast, around 50%inhibition of proliferation was observed in MG-63 and U-2-OS upon GIN treatment.【Conclusions】To the best of our knowledge, this is the first time we report that Wnt pathway is inactive in osteosarcoma. Our view is different from previous conclusion that Wnt pathway is active in osteosarcoma, which based on membranous and/or cytoplasm staining ofβ-catenin.We observed negative nuclearβ-catenin expression in most osteosarcoma specimen and osteosarcoma cell lines and strong nuclearβ-catenin staining in osteoblastoma. Moreover, activation of the Wnt pathway inhibits cell proliferation or promotes osteogenic differentiation in osteosarcoma cell lines. Thus, we propose that loss of Wnt/β-catenin pathway activity, which is required for osteoblast differentiation, may contribute to osteosarcoma development.We also explored the reason for inactivation of Wnt pathway. GIN stimulated Wnt pathway, but Wnt3a failed. High expression of DKK-1 was detected in osteosarcoma cells. Our data suggest high expression of Wnt inhibitor may be the cause of inactive Wnt pathway.The oncogenic role of the Wnt/β-catenin pathway in epithelial tumors has been well studied; however our data support a tumor suppressor role of Wnt pathway in osteosarcoma, which originate from mesenchymal cells, implicating the role of Wnt pathway acts in a context dependent manner.