Laboratory of Osteoblast Biology and Pathology, INSERM U606, Paris, France.
J Bone Miner Res. 2012 Oct;27(10):2118-29. doi: 10.1002/jbmr.1650.
Alterations of Wnt signaling appear to be involved in the pathogenesis of osteosarcoma, presenting mutations of adenomatous polyposis coli (APC) and epigenetic downregulation of Wnt inhibitory factor 1. However, the precise role of Wnt effectors in the bone cancer progression remains unclear. We previously showed that Wnt/β-catenin/T-cell factor (TCF) activation are responsible for the repression of syndecan-2, a key modulator of apoptosis and chemosensitivity in osteosarcoma cells, suggesting a role of Wnt signaling in chemoresistance. In this study, we investigated the functional relationship between syndecan-2, Wnt/β-catenin/TCF signaling and chemosensitivity in these cells. To this goal, we selected resistant osteosarcoma cells from sensitive human cell lines using repeated exposures to doxorubicin. In doxorubicin-responsive but not in doxorubicin-resistant-derived cells syndecan-2 expression was upregulated by doxorubicin treatment. Moreover, syndecan-2 overexpression restored the sensitivity to doxorubicin in resistant-derived cells. We found that syndecan-2 induction by doxorubicin is forkhead box protein O3A (Foxo3a)-dependent. Foxo3a overexpression resulted in increased syndecan-2 expression in sensitive and resistant-derived cells. Doxorubicin modulated Foxo3a binding on syndecan-2 gene promoter and induced Foxo-dependent inhibition of Wnt/TCF activity. Conversely, β-catenin/TCF activation impaired syndecan-2 induction by doxorubicin, indicating that Wnt signaling is competing with the action of the cytotoxic drug. However, β-catenin was also found to be required for Foxo3a activity. Consistently, Dickkopf 1 (DKK1) and secreted frizzled-related protein 1 (sFRP-1) altered doxorubicin action in sensitive cells, whereas inhibition of TCF activity strongly decreased cell viability and increased sensitivity to doxorubicin in sensitive and resistant cells. TCF inhibition also increased the effect of doxorubicin treatment in an orthotopic bone tumor model in mice. Altogether, these data provide evidence that the repression of syndecan-2 by Wnt/β-catenin/TCF signaling contributes to the resistance of osteosarcoma cells to doxorubicin and suggest that TCF inhibition may represent a novel therapeutic strategy in osteosarcoma.
Wnt 信号的改变似乎参与了骨肉瘤的发病机制，表现为腺瘤性结肠息肉病（APC）的突变和 Wnt 抑制因子 1 的表观遗传下调。然而，Wnt 效应物在骨癌进展中的精确作用仍不清楚。我们之前曾表明，Wnt/β-连环蛋白/T 细胞因子（TCF）的激活负责抑制硫酸乙酰肝素蛋白聚糖-2，硫酸乙酰肝素蛋白聚糖-2 是骨肉瘤细胞凋亡和化疗敏感性的关键调节因子，这表明 Wnt 信号在化疗耐药性中起作用。在这项研究中，我们研究了这些细胞中 syndecan-2、Wnt/β-连环蛋白/TCF 信号和化疗敏感性之间的功能关系。为此，我们使用阿霉素反复暴露于敏感的人细胞系中，从敏感的骨肉瘤细胞中选择耐药的骨肉瘤细胞。在用阿霉素处理时，在阿霉素反应性但不在阿霉素耐药衍生细胞中，硫酸乙酰肝素蛋白聚糖-2 的表达上调。此外，硫酸乙酰肝素蛋白聚糖-2 的过表达恢复了耐药衍生细胞对阿霉素的敏感性。我们发现，阿霉素诱导的硫酸乙酰肝素蛋白聚糖-2 诱导依赖于叉头框蛋白 O3A（Foxo3a）。Foxo3a 的过表达导致敏感和耐药衍生细胞中硫酸乙酰肝素蛋白聚糖-2 的表达增加。阿霉素调节 Foxo3a 在硫酸乙酰肝素蛋白聚糖-2 基因启动子上的结合，并诱导 Foxo 依赖性抑制 Wnt/TCF 活性。相反，β-连环蛋白/TCF 的激活损害了阿霉素诱导的硫酸乙酰肝素蛋白聚糖-2 的诱导，表明 Wnt 信号与细胞毒性药物的作用相竞争。然而，还发现β-连环蛋白是 Foxo3a 活性所必需的。一致地，Dickkopf 1（DKK1）和分泌型卷曲相关蛋白 1（sFRP-1）改变了敏感细胞中的阿霉素作用，而 TCF 活性的抑制强烈降低了敏感和耐药细胞的细胞活力并增加了对阿霉素的敏感性。TCF 抑制也增加了阿霉素治疗在小鼠原位骨肿瘤模型中的效果。总之，这些数据提供了证据，表明 Wnt/β-连环蛋白/TCF 信号对硫酸乙酰肝素蛋白聚糖-2 的抑制有助于骨肉瘤细胞对阿霉素的耐药性，并表明 TCF 抑制可能代表骨肉瘤的一种新的治疗策略。
