Regulation on tumor metastasis by Raf kinase inhibitory protein: New insight with reactive oxygen species signaling.

Targeted therapy aiming at the metastatic signal pathway, such as that triggered by receptor tyrosine kinase (RTK), for the prevention of tumor progression is promising. However, RTK-based targeted therapy frequently suffered from drug resistance due to the co-expression of multiple growth factor receptors that may raise compensatory secondary signaling and acquired mutations after treatment. One alternative strategy is to manipulate the common negative regulators of the RTK signaling. Among them, Raf kinase inhibitory protein (RKIP) is highlighted and focused on this review. RKIP can associate with Raf-1, thus suppressing the downstream mitogen-activated protein kinase (MAPK) cascade. RKIP also negatively regulates other metastatic signal molecules including NF-κB, STAT3, and NOTCH1. In general, RKIP achieves this task via associating and blocking the activity of the critical molecules on upstream of the aforementioned pathways. One novel RKIP-related signaling involves reactive oxygen species (ROS). In our recent report, we found that PKCδ-mediated ROS generation may interfere with the association of RKIP with heat shock protein 60 (HSP60)/MAPK complex via oxidation of HSP60 triggered by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. The departure of RKIP may impact the downstream MAPK in two aspects. One is to trigger the Mt→cytosol translocation of HSP60 coupled with MAPKs. The other is to change the conformation of HSP60, favoring more efficient activation of the associated MAPK by upstream kinases in cytosol. It is worthy of investigating whether various RTKs capable of generating ROS can drive metastatic signaling via affecting RKIP in the same manner.

PKCδ mediates mitochondrial ROS generation and oxidation of HSP60 to relieve RKIP inhibition on MAPK pathway for HCC progression.

Both protein kinase C (PKC) and reactive oxygen species (ROS) are well-known signaling messengers cross-talking with each other to activate mitogen-activated protein kinases (MAPKs) for progression of hepatocellular carcinoma (HCC). However, the underlying mechanisms are not well elucidated. Especially, whether mitochondrial ROS (mtROS) is involved and how it triggers MAPK signaling are intriguing. In this study, we found mtROS generation and phosphorylation of MAPKs were mediated by PKCδ in HCCs treated with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Heat shock protein 60 (HSP60), one of the chaperones in mitochondria was the major protein oxidized in TPA-treated HCCs. Moreover, depletion of HSP60 or expression of HSP60 cysteine mutant prevented TPA-induced phosphorylation of MAPKs. To delineate how HSP60 mediated MAPK activation, the role of Raf kinase inhibitor protein (RKIP), a negative regulator of MAPK, was investigated. TPA dissociated RKIP from HSP60 in both mitochondria and cytosol, concurrently with translocation of HSP60 and MAPK from mitochondria to cytosol, which was associated with robust phosphorylation of MAPKs in the cytosol. Moreover, TPA induced opposite phenotypical changes of HCCs, G1 cell cycle arrest, and cell migration, which were prevented by mtROS scavengers and depletion of PKCδ and HSP60. Consistently, TPA increased the migration-related genes, hydrogen peroxide inducible clone5, matrix metalloproteinase-1/3, lamininγ2, and suppressed the cell cycle regulator cyclin E1 (CCNE1) via PKCδ/mtROS/HSP60/MAPK-axis. Finally, c-jun and c-fos were required for TPA-induced expression of the migration-related genes and a novel microRNA, miR-6134, was responsible for TPA-induced suppression of CCNE1. In conclusion, PKCδ cross-talked with mtROS to trigger HSP60 oxidation for release of RKIP to activate MAPK, regulating gene expression for migration, and G1 cell cycle arrest in HCC. Targeted therapy aiming at key players like PKCδ, RKIP, and HSP60 is promising for preventing HCC progression.