Lysyl Oxidase Inhibits Ras-Mediated Transformation by Preventing Activation of NF-κB

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Abstract
Lysyl oxidase (LO), which catalyzes the oxidation of lysine residues, was previously shown to have anti-oncogenic activity on ras-transformed cells. Since oncogenic Ras mediates transformation, in part, through the activation of the transcription factor nuclear factor-κB (NF-κB), we tested here the effects of LO on NF-κB activity. Expression of LO in ras-transformed NIH 3T3 cells led to decreased NF-κB binding and activity, as well as the expression of the NF-κB target gene c-myc. Importantly, ectopic expression of LO led to a dramatic decrease in colony formation by ras-transformed NIH 3T3 cells, a finding comparable to the expression of the IκBα dominant-negative mutant, which could be rescued by p65/p50 NF-κB subunit expression. LO was unable to directly inhibit the activity of ectopically expressed p65 and c-Rel NF-κB subunits, suggesting that LO affected an upstream signaling pathway(s) induced by Ras. Consistent with this hypothesis, LO expression decreased both the rate of IκBα turnover and the activities of IKKα and IKKβ. Moreover, the ectopic expression of a constitutively active version of either kinase reversed the negative effects of LO. Ras can induce NF-κB via both the phosphatidylinositol 3-kinase (PI3K)/Akt and Raf/MEK pathways. LO potently downregulated the PI3K and Akt kinases, while partially inhibiting MEK kinase activity. Expression of a constitutively activated, myristylated Akt or PDK1 was able to counteract the effect of LO on NF-κB, whereas constitutively activated Raf was only partially effective. Importantly, LO blocked membrane localization of Akt and PDK1 in Ras-transformed cells. Overall, these results strongly argue that the anti-oncogenic effects of LO on ras-mediated transformation are due to its ability to inhibit signaling pathways that lead to activation of NF-κB.