Department of Pediatrics, Kyoto University Graduate School of Medicine, Shougoin, Sakyo-ku, Japan.
Clin Exp Metastasis. 2010 Dec;27(8):619-30. doi: 10.1007/s10585-010-9352-x. Epub 2010 Sep 26.
The outcomes of osteosarcoma patients still remain poor because of intractable pulmonary metastasis. We previously established a highly metastatic osteosarcoma cell line, LM8 from Dunn mouse osteosarcoma by in vivo selection. We herein aimed to clarify the characteristic biological features related with high metastatic potential and new target molecules to suppress pulmonary metastasis of osteosarcoma, using this syngeneic spontaneous metastatic model. LM8 cells acquired fibroblastic morphology with striking filopodia on the cell surface. Immunostaining showed faint stress fiber formation and peripherally localized integrin β1, and biochemical analyses showed the activated Cdc42 and autophosphorylation of focal adhesion kinase (FAK) in LM8 cells when compared to Dunn cells. LM8 cells had activated motility in single cell migration mode. LM8 migration was increased by a Rho-associated kinase (ROCK) inhibitor, Y-27632, while decreased by Cdc42 silencing using RNA interference system. We found that a clinically approved camptothecin analog, irinotecan suppressed the migration, Cdc42 activity, and autophosphorylation of FAK, and attenuated integrin β1 distribution selectively in LM8 cells. Daily oral administration of irinotecan significantly reduced the rate and size of pulmonary metastasis in syngeneic C3H mice. The fibroblastic morphology and activated cell migration with the dependency on Cdc42 but not Rho-ROCK signaling pathway argued that LM8 moved in mesenchymal mode of cell migration. This activated mesenchymal migration was a key component of the pulmonary metastasis of LM8 cells. The inhibition of mesenchymal migration by irinotecan, in addition to its cytotoxic effects, might be effective in preventing pulmonary metastasis of osteosarcoma.
骨肉瘤患者的预后仍然很差，因为其存在难治性肺转移。我们之前通过体内选择，从 Dunn 鼠骨肉瘤中建立了一个高转移性骨肉瘤细胞系 LM8。在此，我们旨在使用这种同基因自发转移模型，阐明与高转移潜能相关的特征性生物学特征和抑制骨肉瘤肺转移的新靶分子。LM8 细胞获得成纤维细胞形态，细胞表面有明显的丝状伪足。免疫染色显示应力纤维形成微弱，整合素 β1 位于细胞周围，生化分析显示与 Dunn 细胞相比，LM8 细胞中的 Cdc42 被激活，黏着斑激酶（FAK）发生自身磷酸化。LM8 细胞在单细胞迁移模式下具有激活的迁移能力。Rho 相关激酶（ROCK）抑制剂 Y-27632 可增加 LM8 迁移，而使用 RNA 干扰系统沉默 Cdc42 可降低迁移。我们发现一种临床批准的喜树碱类似物伊立替康可抑制迁移、Cdc42 活性和 FAK 的自身磷酸化，并选择性地降低 LM8 细胞中整合素 β1 的分布。伊立替康的每日口服给药可显著降低同基因 C3H 小鼠肺转移的速度和大小。成纤维细胞形态和激活的细胞迁移依赖于 Cdc42，但不依赖于 Rho-ROCK 信号通路，表明 LM8 以间质细胞迁移的方式迁移。这种激活的间质迁移是 LM8 细胞发生肺转移的关键组成部分。伊立替康抑制间质迁移（除了其细胞毒性作用外）可能对预防骨肉瘤肺转移有效。
