Hong Gyum Kim

[6]-Gingerol Suppresses Colon Cancer Growth by Targeting Leukotriene A4 Hydrolase

[6]-Gingerol, a natural component of ginger, exhibits anti-inflammatory and antitumorigenic activities. Despite its potential efficacy in cancer, the mechanism by which [6]-gingerol exerts its chemopreventive effects remains elusive. The leukotriene A(4) hydrolase (LTA(4)H) protein is regarded as a relevant target for cancer therapy. Our in silico prediction using a reverse-docking approach revealed that LTA(4)H might be a potential target of [6]-gingerol. We supported our prediction by showing that [6]-gingerol suppresses anchorage-independent cancer cell growth by inhibiting LTA(4)H activity in HCT116 colorectal cancer cells. We showed that [6]-gingerol effectively suppressed tumor growth in vivo in nude mice, an effect that was mediated by inhibition of LTA(4)H activity. Collectively, these findings indicate a crucial role of LTA(4)H in cancer and also support the anticancer efficacy of [6]-gingerol targeting of LTA(4)H for the prevention of colorectal cancer.

Phosphorylation of Caspase-7 by p21-activated Protein Kinase (PAK) 2 Inhibits Chemotherapeutic Drug-induced Apoptosis of Breast Cancer Cell Lines

p21-activated kinase (PAK) 2, a member of the PAK family of serine/threonine protein kinases, plays an important role in physiological processes such as motility, survival, mitosis, and apoptosis. However, the role of PAK2 in resistance to chemotherapy is unclear. Here we report that PAK2 is highly expressed in human breast cancer cell lines and human breast invasive carcinoma tissue compared with a human non-tumorigenic mammary epithelial cell line and adjacent normal breast tissue, respectively. Interestingly, we found that PAK2 can bind with caspase-7 and phosphorylate caspase-7 at the Ser-30, Thr-173, and Ser-239 sites. Functionally, the phosphorylation of caspase-7 decreases its activity, thereby inhibiting cellular apoptosis. Our data indicate that highly expressed PAK2 mediates chemotherapeutic resistance in human breast invasive ductal carcinoma by negatively regulating caspase-7 activity.

Extracellular Signal-Regulated Kinase 8-Mediated c-Jun Phosphorylation Increases Tumorigenesis of Human Colon Cancer

Extracellular signal-regulated kinase 8 (ERK8), a recently discovered member of the mitogen-activated protein kinase protein family, has been less studied than other family members, leaving its physiologic functions mostly unknown. The biological consequences of overexpression of ERK8 in JB6 Cl41 epidermal skin cells or knockdown of ERK8 in HCT15 colorectal cancer cells was studied. Kinase assays and transient transfection experiments were performed to study the signaling pathway between ERK8 and c-Jun. We found that ERK8 is relatively highly expressed in HCT15 human colorectal cancer cells and plays an important role in the promotion and progression of colorectal cancer. ERK8 promoted neoplastic transformation, and knockdown of ERK8 in HCT15 colorectal cancer cells reduced the tumorigenic properties of these cell lines. Furthermore, a direct interaction between ERK8 and c-Jun was shown. With epidermal growth factor treatment, overexpression of ERK8 in JB6 Cl41 cells caused an increased phosphorylation of c-Jun at Ser(63) and Ser(73), resulting in increased activator protein-1 transactivation. In contrast, knockdown of ERK8 in HCT15 colorectal cancer cells blocked c-Jun phosphorylation. The interaction between ERK8 and c-Jun seems to increase the tumorigenic properties of HCT15 colorectal cancer cells. Thus, ERK8-regulated signaling might serve as a potential therapeutic target in colorectal cancer.

Role of NEK6 in Tumor Promoter-induced Transformation in JB6 C141 Mouse Skin Epidermal Cells

NEK6 (NIMA-related kinase 6) is a homologue of the Aspergillus nidulans protein NIMA (never in mitosis, gene A). We demonstrate that overexpression of NEK6 induces anchorage-independent transformation of JB6 Cl41 mouse epidermal cells. Tissue arrays and Western immunoblot analysis show that NEK6 is overexpressed in malignant tissues and several cancer cell lines. Our data also show that NEK6 interacts with STAT3, an oncogenic transcription factor, and phosphorylates STAT3 on Ser727, which is important for transcriptional activation. Additional studies using NEK6 mutants suggested that the phosphorylation on both Ser206 and Thr210 of NEK6 is critical for STAT3 phosphorylation and anchorage-independent transformation of mouse epidermal cells. Notably, knockdown of NEK6 decreased colony formation and STAT3 Ser727 phosphorylation. Based on our findings, the most likely mechanism that can account for this biological effect involves the activation of STAT3 through the phosphorylation on Ser727. Because of the critical role that STAT3 plays in mediating oncogenesis, the stimulatory effects of NEK6 on STAT3 and cell transformation suggest that this family of serine/threonine kinases might represent a novel chemotherapeutic target.

Polycomb (PcG) Proteins, BMI1 and SUZ12, Regulate Arsenic-induced Cell Transformation

Background: Chronic and low-dose arsenic exposure causes cancer in humans through an as yet unknown mechanism. Results: PcG proteins, Bmi1 and Suz12, are required for arsenic-induced cell transformation through their inhibition of tumor suppressors. Conclusion: PcG proteins play a critical role in cell transformation caused by low-dose arsenic exposure. Significance: PcG protein function is enhanced by arsenic and is required for arsenic-induced carcinogenesis. Inorganic arsenic is a well-documented human carcinogen associated with cancers of the skin, lung, liver, and bladder. However, the underlying mechanisms explaining the tumorigenic role of arsenic are not well understood. The present study explored a potential mechanism of cell transformation induced by arsenic exposure. Exposure to a low dose (0.5 μm) of arsenic trioxide (As2O3) caused transformation of BALB/c 3T3 cells. In addition, in a xenograft mouse model, tumor growth of the arsenic-induced transformed cells was dramatically increased. In arsenic-induced transformed cells, polycomb group (PcG) proteins, including BMI1 and SUZ12, were activated resulting in enhanced histone H3K27 tri-methylation levels. On the other hand, tumor suppressor p16INK4a and p19ARF mRNA and protein expression were dramatically suppressed. Introduction of small hairpin (sh) RNA-BMI1 or -SUZ12 into BALB/c 3T3 cells resulted in suppression of arsenic-induced transformation. Histone H3K27 tri-methylation returned to normal in BMI1- or SUZ12-knockdown BALB/c 3T3 cells compared with BMI1- or SUZ12-wildtype cells after arsenic exposure. As a consequence, the expression of p16INK4a and p19ARF was recovered in arsenic-treated BMI1- or SUZ12-knockdown cells. Thus, arsenic-induced cell transformation was blocked by inhibition of PcG function. Taken together, these results strongly suggest that the polycomb proteins, BMI1 and SUZ12 are required for cell transformation induced by organic arsenic exposure.

Phosphorylation of Histone H2B Serine 32 Is Linked to Cell Transformation

Various types of post-translational modifications of the histone tails have been revealed, but a few modifications have been found within the histone core sequences. Histone core post-translational modifications have the potential to modulate nucleosome structure and DNA accessibility. Here, we studied the histone H2B core domain and found that phosphorylation of H2B serine 32 occurs in normal cycling and mitogen-stimulated cells. Notably, this phosphorylation is elevated in skin cancer cell lines and tissues compared with normal counterparts. The JB6 Cl41 mouse skin epidermal cell line is a well established model for tumor promoter-induced cell transformation and was used to study the function of H2B during EGF-induced carcinogenesis. Remarkably, cells overexpressing a nonphosphorylatable H2BS32A mutant exhibited suppressed growth and EGF-induced cell transformation, possibly because of decreased activation of activator protein-1, compared with control cells overexpressing wild type H2B. We identified ribosomal S6 kinase 2 (RSK2) as the kinase responsible for H2BS32 phosphorylation. Serum-starved JB6 cells contain very little endogenous H2BS32 phosphorylation, and EGF treatment induced this phosphorylation. The phosphorylation was attenuated in RSK2 knock-out MEFs and RSK2 knockdown JB6 cells. Taken together, our results demonstrate a novel role for H2B phosphorylation in cell transformation and show that H2BS32 phosphorylation is critical for controlling activator protein-1 activity, which is a major driver in cell transformation.

Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling

Phosphatase and tensin homolog (PTEN) loss or mutation consistently activates the phosphatidylinositol 3‐kinase (PI3‐K)/Akt signaling pathway, which contributes to the progression and invasiveness of prostate cancer. Furthermore, the PTEN/PI3‐K/Akt and Ras/MAPK pathways cooperate to promote the epithelial–mesenchymal transition (EMT) and metastasis initiated from prostate stem/progenitor cells. For these reasons, the PTEN/PI3‐K/Akt pathway is considered as an attractive target for both chemoprevention and chemotherapy. Herein we report that eupafolin, a natural compound found in common sage, inhibited proliferation of prostate cancer cells. Protein content analysis indicated that phosphorylation of Akt and its downstream kinases was inhibited by eupafolin treatment. Pull‐down assay and in vitro kinase assay results indicated that eupafolin could bind with PI3‐K and attenuate its kinase activity. Eupafolin also exhibited tumor suppressive effects in vivo in an athymic nude mouse model. Overall, these results suggested that eupafolin exerts antitumor effects by targeting PI3‐K.

The Crystal structure of the active form of the C-Terminal kinase domain of mitogen- and stress-activated protein kinase 1

Mitogen- and stress-activated protein kinase 1 (MSK1) is a growth-factor-stimulated serine/threonine kinase that is involved in gene transcription regulation and proinflammatory cytokine stimulation. MSK1 is a dual kinase possessing two nonidentical protein kinase domains in one polypeptide. We present the active conformation of the crystal structures of its C-terminal kinase domain in apo form and in complex with a nonhydrolyzable ATP analogue at 2.0 A and 2.5 A resolutions, respectively. Structural analysis revealed substantial differences in the contacts formed by the C-terminal helix, which is responsible for the inactivity of other autoinhibited kinases. In the C-terminal kinase domain of MSK1, the C-terminal alphaL-helix is located in the surface groove, but forms no hydrogen bonds with the substrate-binding loop or nearby helices, and does not interfere with the proteins autophosphorylation activity. Mutational analysis confirmed that the alphaL-helix is inherently nonautoinhibitory. Overexpression of the single C-terminal kinase domain in JB6 cells resulted in tumor-promoter-induced neoplastic transformation in a manner similar to that induced by the full-length MSK1 protein. The overall results suggest that the C-terminal kinase domain of MSK1 is regulated by a novel alphaL-helix-independent mechanism, suggesting that a diverse mechanism of autoinhibition and activation might be adopted by members of a closely related protein kinase family.

Computational and Biochemical Discovery of RSK2 as a Novel Target for Epigallocatechin Gallate (EGCG)

The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2), as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals. The computer prediction was confirmed by an in vitro kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains in vitro and ex vivo. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting phosphorylation of histone H3 at Ser10. Overall, these results indicate that RSK2 is a novel molecular target of EGCG.

Histone XH2AX Is Required for Xenopus Anterior Neural Development CRITICAL ROLE OF THREONINE 16 PHOSPHORYLATION

A role for histone H2AX, one of the variants of the nucleosome core histone H2A, has been demonstrated in DNA repair, tumor suppression, apoptosis, and cell cycle checkpoint function. However, the physiological function and post-translational modification of histone H2AX during vertebrate development have not been elucidated. Here, we provide evidence showing that Xenopus histone H2AX (XH2AX) has a role in the anterior neural plate for eye field formation during Xenopus embryogenesis. A loss-of-function study clearly demonstrated a critical role of XH2AX in anterior neural specification. Through a differentiation assay with Xenopus animal cap embryonic stem cells, we confirmed that XH2AX is required for the activin-induced anterior neural specification of the ectoderm. Furthermore, we found that Chk1 is an essential kinase to phosphorylate histone XH2AX at Thr16, which is involved in the biological function of this histone. Taken together, our findings reveal that XH2AX has a specific role in anterior neural formation of Xenopus, which is mediated through phosphorylation of XH2AX at Thr16 by Chk1.