Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan.
Institute of Biotechnology, National Tsing Hua University, Hsinchu 300044, Taiwan.
Int J Mol Sci. 2022 Oct 11;23(20):12100. doi: 10.3390/ijms232012100.
Lysine-deficient protein kinase-1 (WNK1) is critical for both embryonic angiogenesis and tumor-induced angiogenesis. However, the downstream effectors of WNK1 during these processes remain ambiguous. In this study, we identified that oxidative stress responsive 1b () is upregulated in endothelial cells in both embryonic and tumor-induced angiogenesis in zebrafish, accompanied by downregulation of protein phosphatase 2A (pp2a) subunit . In addition, and are upregulated in two liver cancer transgenic fish models: [] and [], while is downregulated in []. Furthermore, using HUVEC endothelial cells co-cultured with HepG2 hepatoma cells, we confirmed that WNK1 plays a critical role in the induction of hepatoma cell migration in both endothelial cells and hepatoma cells. Moreover, overexpression of OSR1 can rescue the reduced cell migration caused by shWNK1 knockdown in HUVEC cells, indicating OSR1 is downstream of WNK1 in endothelial cells promoting hepatoma cell migration. Overexpression of PPP2R1A can rescue the increased cell migration caused by WNK1 overexpression in HepG2, indicating that PPP2R1A is a downstream effector in hepatoma. The combinatorial treatment with WNK1 inhibitor (WNK463) and OSR1 inhibitor (Rafoxanide) plus oligo-fucoidan via oral gavage to feed [] transgenic fish exhibits much more significant anticancer efficacy than Regorafenib for advanced HCC. Importantly, oligo-fucoidan can reduce the cell senescence marker-IL-1β expression. Furthermore, oligo-fucoidan reduces the increased cell senescence-associated β-galactosidase activity in transgenic fish treated with WNK1-OSR1 inhibitors. Our results reveal the WNK1-OSR1-PPP2R1A axis plays a critical role in both endothelial and hepatoma cells during tumor-induced angiogenesis promoting cancer cell migration. By in vitro and in vivo experiments, we further uncover the molecular mechanisms of WNK1 and its downstream effectors during tumor-induced angiogenesis. Targeting WNK1-OSR1-mediated anti-angiogenesis and anti-cancer activity, the undesired inflammation response caused by inhibiting WNK1-OSR1 can be attenuated by the combination therapy with oligo-fucoidan and may improve the efficacy.
赖氨酸缺陷型蛋白激酶-1（WNK1）对于胚胎血管生成和肿瘤诱导的血管生成都是至关重要的。然而，WNK1 在这些过程中的下游效应物仍然不清楚。在这项研究中，我们发现氧化应激反应蛋白 1b（）在斑马鱼的胚胎血管生成和肿瘤诱导的血管生成中在内皮细胞中上调，同时伴随着蛋白磷酸酶 2A（pp2a）亚基的下调。此外，在两种肝癌转基因鱼模型：[]和[]中，和上调，而在[]中下调。此外，我们使用共培养的 HUVEC 内皮细胞和 HepG2 肝癌细胞，证实了 WNK1 在肝癌细胞迁移中发挥了关键作用，无论是在内皮细胞还是肝癌细胞中。此外，过表达 OSR1 可以挽救 HUVEC 细胞中 shWNK1 敲低引起的细胞迁移减少，表明 OSR1 是内皮细胞中促进肝癌细胞迁移的 WNK1 的下游效应物。过表达 PPP2R1A 可以挽救 WNK1 过表达引起的 HepG2 细胞迁移增加，表明 PPP2R1A 是肝癌细胞的下游效应物。通过口服灌胃联合使用 WNK1 抑制剂（WNK463）和 OSR1 抑制剂（Rafoxanide）加寡聚岩藻聚糖，对[]转基因鱼进行治疗，其抗癌效果明显优于瑞戈非尼治疗晚期 HCC。重要的是，寡聚岩藻聚糖可以降低细胞衰老标志物-IL-1β的表达。此外，寡聚岩藻聚糖降低了 WNK1-OSR1 抑制剂处理的转基因鱼中增加的细胞衰老相关β-半乳糖苷酶活性。我们的结果揭示了 WNK1-OSR1-PPP2R1A 轴在肿瘤诱导的血管生成过程中，在促进肿瘤细胞迁移方面在内皮细胞和肝癌细胞中都起着关键作用。通过体外和体内实验，我们进一步揭示了 WNK1 及其在肿瘤诱导的血管生成过程中的下游效应物的分子机制。通过靶向 WNK1-OSR1 介导的抗血管生成和抗癌活性，通过与寡聚岩藻聚糖联合治疗，可以减轻抑制 WNK1-OSR1 引起的不必要的炎症反应，从而提高疗效。
