Ras MAPK signaling is often deregulated

Ras–MAPK signaling is often deregulated and is constitutively active in many types of cancers including pancreatic, colon, lung, melanoma and breast (Dunn et al., 2005). Activation of PI3K, Rac-1 and JNK was necessary for bFGF-induced fibroblast migration and blocking the activation of PI3K, Rac-1 or JNK has been shown to significantly downregulate the wound healing capacity of these ABT737 (Kanazawa et al., 2010). PI3K–Rac-1–JNK signaling was for collagen I-induced fibroblastic transformation and scattering of NMuMG mammary epithelial cells (Shintani et al., 2006). Further, the cytoplasmic accumulation of JNK activated by constitutively active Rac-1 has been demonstrated in intestinal epithelial cells (Stappenbeck and Gordon, 2001). Taken together, these reports suggest the involvement of Ras–PI3K signaling in regulating the activation of JNK. In our present study, the upstream signal required for the activation and accumulation of cytoplasmic JNK appears to involve Ras–PI3K–Rac-1–Pak-1 pathway as evidenced by the upregulation of these molecules in the cells treated with radiation. Further, cells treated with pUC and pUC+10Gy showed a decrease in the expression of these molecules, indicating that uPAR and cathepsin B regulate the Ras–Pak-1 pathway. Inhibition of Ras–Pak-1 pathway by pUC treatment, alone or in combination with radiation, led to an increase in the expression of MEKK-1. MEKK-1 interacted with p-JNK in the cells treated with pUC and to some extent in the cells treated with 10Gy. In contrast, there was no interaction between these molecules in the cells treated with SV, FLU or FLC. We have previously reported that apoptosis was induced in glioma cells treated with pUC (Malla et al., 2010, 2012b) and to some extent in glioma cells treated with radiation (Malla et al., 2012a). Hence, it is possible that these molecules are interacting only in the cells that are undergoing apoptosis. An increase in the interaction between MEKK-1 and p-JNK was also observed in the cells treated with the Pak-1 inhibitor (IPA3). Further, we observed that the expression of MEKK-1 and p-JNK remained unaltered with the Pak-1 inhibitor. As per the above findings, it can be considered that Pak-1 negatively regulates the binding of MEKK-1 with p-JNK. A similar kind of Pak-1-mediated negative regulation of MEKK-1-dependent JNK pathway was observed in 293 human embryonic kidney cells (Gallagher et al., 2002). Pak-1 constitutively phosphorylates MEKK-1 on serine 67 in resting 293 cells, but its dephosphorylation following exposure to anisomycin allows the binding of JNK to MEKK-1. In conclusion, uPAR and cathepsin B mediate the migration of glioma cells by increasing the localization of cytoplasmic JNK at the focal complexes of the leading edge of glioma cells as depicted in Fig. 8. uPAR and cathepsin B regulate the Ras–Pak-1 pathway by controlling the activation and translocation of JNK. shRNA treatment against uPAR and cathepsin B inhibits the Ras–Pak-1 pathway, thereby inducing the activation and interaction of MEKK-1 with p-JNK. The MEKK-1 and p-JNK complex further translocates into the nucleus, reducing the availability of the cytoplasmic pool of JNK required for the migration of the glioma cells. Taken together, it can be concluded that the cytosolic activity of JNK induces the migration of cells and radiation further enhances this phenomenon, thereby driving these glioma cells towards a more malignant and resistant phenotype. pUC treatment induces nuclear translocation of p-JNK and thus reduces the migration of 5310 and 4910 non-GICs and GICs. Finally, it can be concluded that the regulation of JNK–MAPK through the simultaneous suppression of uPAR and cathepsin B proves to be a potential therapeutic target for inhibiting the migration of glioma cells. The following are the supplementary data related to this article.
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Introduction