Osteopontin (OPN) plays a pivotal role in cancer cell invasion and metastasis. Although OPN has a large number of phosphorylation sites, the functional significance of OPN phosphorylation in cancer cell motility remains unclear. In this study, we attempted to investigate whether phosphorylated OPN secreted from cancer cells affect cancer cell migration. Quantitative PCR and Western blot analyses revealed that MDA-MB435S, A549, and H460 cells highly expressed OPN, whereas the OPN expression levels in H358, MIAPaca-2, and Panc-1 cells were quite low or were not detected. Compared with the cancer cell lines with a low OPN expression, the high OPN-expressing cancer cell lines displayed a higher cell migration, and the cell migration was suppressed by the anti-OPN antibody. This was confirmed by the OPN overexpression in H358 cancer cells with a low endogenous OPN. Phos-tag ELISA showed that phosphorylated OPN was abundant in the cell culture media of A549 and H460 cells, but not in those of MDA-MB435S cells. Moreover, the A549 and H460 cell culture media, as well as the MDA-MB435S cell culture media with a kinase treatment increased cancer cell motility, both of which were abrogated by phosphatase treatment or anti-OPN antibodies. These results suggest that phosphorylated OPN secreted from cancer cells regulates cancer cell motility.

中文翻译：

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癌细胞分泌的磷酸化骨桥蛋白诱导癌细胞运动

骨桥蛋白（OPN）在癌细胞侵袭和转移中起关键作用。尽管 OPN 具有大量的磷酸化位点，但 OPN 磷酸化在癌细胞运动中的功能意义仍不清楚。在这项研究中，我们试图研究癌细胞分泌的磷酸化 OPN 是否影响癌细胞迁移。定量 PCR 和蛋白质印迹分析显示 MDA-MB435S、A549 和 H460 细胞高度表达 OPN，而 H358、MIAPaca-2 和 Panc-1 细胞中的 OPN 表达水平相当低或未检测到。与OPN低表达的癌细胞系相比，OPN高表达的癌细胞系表现出更高的细胞迁移，并且细胞迁移被抗OPN抗体抑制。具有低内源性 OPN 的 H358 癌细胞中的 OPN 过表达证实了这一点。Phos-tag ELISA 显示磷酸化的 OPN 在 A549 和 H460 细胞的细胞培养基中含量丰富，而在 MDA-MB435S 细胞中则不存在。此外，A549 和 H460 细胞培养基以及经过激酶处理的 MDA-MB435S 细胞培养基增加了癌细胞的运动性，这两者都被磷酸酶处理或抗 OPN 抗体消除。这些结果表明癌细胞分泌的磷酸化 OPN 调节癌细胞运动。以及激酶处理的 MDA-MB435S 细胞培养基增加了癌细胞的运动性，这两者都被磷酸酶处理或抗 OPN 抗体消除。这些结果表明癌细胞分泌的磷酸化 OPN 调节癌细胞运动。以及激酶处理的 MDA-MB435S 细胞培养基增加了癌细胞的运动性，这两者都被磷酸酶处理或抗 OPN 抗体消除。这些结果表明癌细胞分泌的磷酸化 OPN 调节癌细胞运动。