Stable knockdown of S100A4 suppresses cell migration and metastasis of osteosarcoma
Stable knockdown of S100A4 suppresses cell migration and metastasis of osteosarcoma
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DOI：
10.1007/s13277-011-0160-y
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发表时间：
2011-06-01
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影响因子：
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通讯作者：
Fukayama, Masashi
中科院分区：
文献类型：
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作者：
Fujiwara, Masahiko;Kashima, Takeshi G.;Fukayama, Masashi
S100A4 是一种 10-12 kDa 钙结合蛋白，在肿瘤进展和转移中发挥功能作用。本研究旨在采用功能丧失方法研究 S100A4 在骨肉瘤 (OS) 转移中的功能。我们之前的表达谱分析表明，S100a4 优先在高度转移的小鼠 OS 细胞系 LM8 中表达。使用新建立的含有绝缘子和转座子的载体引入针对 S100a4 的短发夹核糖核酸 (shRNA)，我们建立了稳定的 LM8 亚克隆，内源性 S100a4 蛋白几乎 100% 沉默。这些转染子在体外显示出细胞迁移的显着抑制，并且在静脉内接种后在体内定植于肺和形成肺转移的能力显着降低，而细胞增殖或细胞与纤连蛋白、层粘连蛋白和I型胶原的附着没有显着变化。 S100a4-shRNA 克隆中，ezrin（一种新兴的 OS 转移相关因子）的表达和磷酸化以及 MMP 的表达保持不变。在61例人OS中，免疫组化分析显示，85.2%样本中的病变细胞表达S100A4蛋白，且免疫反应性主要是细胞质，但也显示偶尔的核定位。软骨母细胞和成骨细胞 OS 亚型比成纤维细胞亚型表达更多的 S100A4。鼠异种移植模型中显示的 S100A4 在 OS 肺转移中的致病作用，以及表达 S100A4 的原代人 OS 的高比例，表明 S100A4 蛋白代表了未来 OS 治疗的重要潜在靶点。
S100A4, a 10-12 kDa calcium-binding protein, plays functional roles in tumor progression and metastasis. The present study aimed to investigate the function of S100A4 in osteosarcoma (OS) metastasis, using a loss-of-function approach. Our previous expression profiling analysis revealed that S100a4 was preferentially expressed in the highly metastatic mouse OS cell line, LM8. Introducing a short hairpin ribonucleic acid (shRNA) targeting S100a4 using a newly established vector containing insulators and transposons, we established stable LM8 subclones with almost 100% silencing of endogenous S100a4 protein. These transfectants showed a significant suppression of cell migration in vitro as well as a marked reduction in their ability to colonize the lung and form pulmonary metastases in vivo following intravenous inoculation, whereas there was no significant change in cell proliferation or cell attachment to fibronectin, laminin, and type I collagen. Expression and phosphorylation of ezrin, an emerging OS metastasis-associated factor, and expression of MMPs, remained the same in S100a4-shRNA clones. In 61 human OS, immunohistochemical analysis showed that lesional cells in 85.2% samples expressed S100A4 protein, and the immunoreactivity was primarily cytoplasmic, but it also showed occasional nuclear localization. Chondroblastic and osteoblastic OS subtypes expressed more S100A4 than fibroblastic subtypes. The causative role of S100A4 in OS lung metastasis shown in the murine xenograft model, together with the high proportion of primary human OS expressing S100A4, suggest that S100A4 protein represents an important potential target for future OS therapy.