Thymoquinone-induced conformational changes of PAK1 interrupt prosurvival MEK-ERK signaling in colorectal cancer.

Background: Thymoquinone (TQ) was shown to reduce tumor growth in several cancer models both in vitro and in vivo. So far only a few targets of TQ, including protein kinases have been identified. Considering that kinases are promising candidates for targeted anticancer therapy, we studied the complex kinase network regulated by TQ.
Methods: Novel kinase targets influenced by TQ were revealed by in silico analysis of peptide array data obtained from TQ-treated HCT116wt cells. Western blotting and kinase activity assays were used to determine changes in kinase expression patterns in colorectal cancer cells (HCT116wt, DLD-1, HT29). To study the viability/apoptotic effects of combining the PAK1 inhibitor IPA-3 and TQ, crystal violet assay and AnnexinV/PI staining were employed. Interactions between PAK1 and ERK1/2 were investigated by co-immunoprecipitation and modeled by docking studies. Transfection with different PAK1 mutants unraveled the role of TQ-induced changes in PAK1 phosphorylation and TQ's effects on PAK1 scaffold function.
Results: Of the 104 proteins identified, 50 were upregulated ≥ 2 fold by TQ and included molecules in the AKT-MEK-ERK1/2 pathway. Oncogenic PAK1 emerged as an interesting TQ target. Time-dependent changes in two PAK1 phosphorylation sites generated a specific kinase profile with early increase in pPAK(Thr212) followed by late increase in pPAK(Thr423). TQ induced an increase of pERK1/2 and triggered the early formation of an ERK1/2-PAK1 complex. Modeling confirmed that TQ binds in the vicinity of Thr212 accompanied by conformational changes in ERK2-PAK1 binding. Transfecting the cells with the non-phosphorylatable mutant T212A revealed an increase of pPAK(Thr423) and enhanced apoptosis. Likewise, an increase in apoptosis was observed in cells transfected with both the kinase-dead K299R mutant and PAK1 siRNA. Using structural modeling we suggest that TQ interferes also with the kinase domain consequently disturbing its interaction with pPAK(Thr423), finally inhibiting MEK-ERK1/2 signaling and disrupting its prosurvival function. pERK1/2 loss was also validated in vivo.
Conclusions: Our study shows for the first time that the small molecule TQ directly binds to PAK1 changing its conformation and scaffold function. Because TQ affects the central RAF/MEK/ERK1/2 pathway, the combination of TQ with targeted therapies is worth considering for future anticancer treatments.