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

Abstract

BackgroundThymoquinone (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.MethodsNovel 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.ResultsOf 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 pPAKThr212 followed by late increase in pPAKThr423. 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 pPAKThr423 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 pPAKThr423, finally inhibiting MEK-ERK1/2 signaling and disrupting its prosurvival function. pERK1/2 loss was also validated in vivo.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-201) contains supplementary material, which is available to authorized users.

ERK1/2 promoted proliferation and inhibited apoptosis of human cervical cancer cells and regulated the expression of c-Fos and c-Jun proteins

“…MAPK is one of the protein kinases which can be stimulated in many kinds of cancer cells and related to tumorigenesis [10][11][12][13]. Smallmolecule inhibitors targeted MAPK have been wildly used for cancer therapeutics as a biologically viable model.…”

“…It has been reported that ERK1/2 acts as nearly 180 different molecules targets and regulates several diverse cellular functions such as cell growth, cell cycle, proliferation, cell death, cell apoptosis, cell adhesion and transcription [9]. In fact, the ERK1/2 plays the above roles in various cancers, such as breast cancer [10], colorectal cancer [11], prostate cancer [12] and lung cancer [13]. However, not all references support the role of activated ERKs in promoting tumorigenesis, and result consistent with the role in tumor suppression has been reported as well [14].…”

Thymoquinone chemosensitizes colon cancer cells through inhibition of NF-κB

“…Since then, increasing numbers of studies have focused on TQ in cancer therapy. Numerous in vitro and in vivo studies have investigated the antitumor activity of TQ in several types of cancer (6,14,19,(27)(28)(29)33). In tumor protein p53-null myeloblastic leukemia HL-60 cells, TQ induced apoptosis through an intrinsic signaling pathway (34).…”

“…A recent study reported that TQ exerted an antitumor effect through the interruption of pro-survival mitogen-activated protein kinase kinase 7-mitogen-activated protein kinase 1 signaling in colorectal cancer (14). TQ exerted a direct antitumor effect, and also sensitized cancer cells to other therapies (23,(42)(43)(44).…”

“…In the previous decade, the antitumor activity of TQ has been investigated in a number of studies (6)(7)(8). TQ was observed to induce antitumor effects in several types of cancer, including breast (9), lung (10), multiple myeloma (11), pancreatic (12), cervical (13), colon (14) and prostate cancer (15), as well as squamous (16) and hepatocellular carcinoma (17), acute lymphoblastic leukemia (18), glioblastoma (19), osteosarcoma (20), neuroblastoma (21), bladder (22), gastric (23) and ovarian cancer (24). Although the effect of TQ has been investigated in numerous types of cancer, the molecular mechanisms underlying its action remain to be elucidated.…”