WNK1–OSR1 Signaling Regulates Angiogenesis-Mediated Metastasis towards Developing a Combinatorial Anti-Cancer Strategy

Abstract

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 (osr1b) is upregulated in endothelial cells in both embryonic and tumor-induced angiogenesis in zebrafish, accompanied by downregulation of protein phosphatase 2A (pp2a) subunit ppp2r1bb. In addition, wnk1a and osr1b are upregulated in two liver cancer transgenic fish models: [tert x p53−/−] and [HBx,src,p53−/−,RPIA], while ppp2r1bb is downregulated in [tert x p53−/−]. 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 [HBx,src,p53−/−,RPIA] 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 tert 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.

Focal adhesion kinase confers lenvatinib resistance in hepatocellular carcinoma via the regulation of lysine‐deficient kinase 1

“…WNK1 was recently reported to play a role in HCC growth and metastasis, 31,32 but the role of WNK1 in lenvatinib resistance remains unknown. To understand the functional significance of WNK1 in lenvatinib resistance, we inhibited WNK1 either with WNK1 inhibitor WNK463 or WNK1 shRNAs.…”

“…WNK1 was recently reported to play a role in HCC growth and metastasis, 31,32 S1). Conversely, overexpression of WNK1 enhanced the resistance to lenvatinib treatment in lenvatinibsensitive Hep3B and Huh7 parental cells (Figure 5E-G, and Supporting Information: Figure 5).…”

Isoform‐independent promotion of contractility and proliferation, and suppression of survival by with no lysine/K kinases in prostate stromal cells

“…28 WNK1-mediated phosphorylation of the native substrate OSR1 was inhibited by WNK463 with an IC 50 of 41 nM in biochemical assays, and of 106 nM if in HEK293 cells following induction of osmotic stress. 28 In subsequent cell culture studies, WNK463 was mostly applied using 1 μM, [49][50][51][52][53][54][55] or sometimes in lower (100-625 nM), 56,57 or higher (4-10 μM) concentrations. 58,59 Off-targets were examined using a concentration of 10 μM in a screening assay with 444 kinases.…”

Ten Years of Research on Fucoidan and Cancer: Focus on Its Antiangiogenic and Antimetastatic Effects

“…Two downstream effectors of WNK1-mediated angiogenesis are the oxidative stress responsive 1 (OSR1) and the protein phosphatase 2A (PPP2R1A) proteins. Up-regulation of WNK1 and OSR1 in endothelial cells favors the process of neo-angiogenesis, and the axis WNK1-ORSR1-PPP2R1A was demonstrated to play a critical role in both endothelial (HUVEC) and hepatoma (HepG2) cells during angiogenesis and cell migration [ 42 ]. The zebrafish model was used in a very recent study to demonstrate how the co-administration of oligo-fucoidan could improve the anti-cancer efficacy of WNK463 (a WNK1 inhibitor) and rafoxanide (an OSR1 inhibitor) in advanced hepatocellular carcinoma [ 42 ] ( Table 1 ).…”

“…Up-regulation of WNK1 and OSR1 in endothelial cells favors the process of neo-angiogenesis, and the axis WNK1-ORSR1-PPP2R1A was demonstrated to play a critical role in both endothelial (HUVEC) and hepatoma (HepG2) cells during angiogenesis and cell migration [ 42 ]. The zebrafish model was used in a very recent study to demonstrate how the co-administration of oligo-fucoidan could improve the anti-cancer efficacy of WNK463 (a WNK1 inhibitor) and rafoxanide (an OSR1 inhibitor) in advanced hepatocellular carcinoma [ 42 ] ( Table 1 ). The main mechanism responsible for the anticancer activity of fucoidan in association with WNK463 and rafoxanide has been ascribed to its ability to counteract the inflammatory response evoked by the aforementioned inhibitors [ 42 ].…”