C-Met-Akt pathway-mediated enhancement of inhibitory c-Raf phosphorylation is involved in vitamin K1 and sorafenib synergy on HCC growth inhibition

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Sorafenib is an FDA-approved agent for treatment of human hepatocellular carcinoma (HCC), but tumor shrinkage is minor. We therefore developed a strategy to combine K vitamins with sorafenib to treat HCC, and found that this combination enhanced sorafenib-induced HCC cell growth inhibition. To explore the mechanisms involved, we examined the role of Raf kinase, since both vitamins K and sorafenib were reported to inhibit tumor cell growth via Raf signaling pathway. We found that whereas lower concentration of vitamin K1 (25 μM) or sorafenib (2.5 μM) alone slightly induced c-Raf phosphorylation at both Ser-43 and Ser-259, combination vitamin K1 plus sorafenib resulted in strong c-Raf phosphorylation at these two serine residues. A Raf kinase activity assay confirmed that combination vitamin K1 plus sorafenib had a synergistic inhibitory effect on it. Since c-Raf phosphorylation at Ser-43 and Ser-259 can be regulated by either PKA or Akt kinase, we examined the effects of both vitamin K1 and sorafenib on their phosphorylation. Although vitamin K1 or sorafenib alone induced PKA phosphorylation, no enhanced phosphorylation effects on PKA were found using this combination. However, vitamin K1 enhanced sorafenib-induced c-Met phosphorylation at Tyr-1349, a DEP-1 protein phosphatase acting site, and consequently induced phosphorylation of PI3K-Akt. Both PI3K inhibitor Ly294002 as well as dominate negative Akt plasmid transfection antagonized vitamin K1 plus sorafenib actions on c-Raf phosphorylation and cell growth inhibition, suggesting that c-Met-PI3K-Akt signaling pathway mediated inhibitory c-Raf phosphorylation may play a central role in vitamin K1 plus sorafenib synergy in inhibiting HCC cell growth.