Chul Ho Jeong

[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 Sox2 Cooperates in Reprogramming to Pluripotent Stem Cells

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by transduction of reprogramming factors, including Oct4, Sox2, Klf4, and c‐Myc. A coordinated network of these factors was suggested to confer a pluripotency of iPSCs. Together with Oct4, Sox2 plays a major role as a master regulator in ESCs. However, the underlying mechanisms by which Sox2 contributes to self‐renewal or reprogramming processes remain to be determined. Here, we provide new evidence for a phosphorylation‐based regulation of Sox2 activity. Akt directly interacts with Sox2 and promotes its stabilization through phosphorylation at Thr118, which enhances the transcriptional activity of Sox2 in ESCs. Moreover, phosphorylation of Sox2 cooperates in the reprogramming of mouse embryonic fibroblasts by enabling more efficient induction of iPSCs. Overall, our studies provide new insights into the regulatory mechanism of Sox2 in ESCs and also provide a direct link between phosphorylation events and somatic cell reprogramming. STEM CELLS 2010;28:2141–2150

ERK1 and ERK2 regulate embryonic stem cell self-renewal through phosphorylation of Klf4

Understanding and controlling the mechanism by which stem cells balance self-renewal versus differentiation is of great importance for stem cell therapeutics. Klf4 promotes the self-renewal of embryonic stem cells, but the precise mechanism regulating this role of Klf4 is unclear. We found that ERK1 or ERK2 binds the activation domain of Klf4 and directly phosphorylates Klf4 at Ser123. This phosphorylation suppresses Klf4 activity, inducing embryonic stem cell differentiation. Conversely, inhibition of Klf4 phosphorylation enhances Klf4 activity and suppresses embryonic stem cell differentiation. Notably, phosphorylation of Klf4 by ERKs causes recruitment and binding of the F-box proteins βTrCP1 or βTrCP2 (components of an ubiquitin E3 ligase) to the Klf4 N-terminal domain, which results in Klf4 ubiquitination and degradation. Overall, our data provide a molecular basis for the role of ERK1 and ERK2 in regulating Klf4-mediated mouse embryonic stem cell self-renewal.

USP8 Is a Novel Target for Overcoming Gefitinib Resistance in Lung Cancer

Purpose: Common treatment modalities for non–small cell lung cancer (NSCLC) involve the EGF receptor-tyrosine kinase inhibitors (EGFR-TKIs) like gefitinib and erlotinib. However, the vast majority of treated patients acquire resistance to EGFR-TKIs, due, in large part, to secondary mutations in EGFR or amplification of the MET gene. Our purpose was to test ubiquitin-specific peptidase 8 (USP8) as a potential therapeutic target for gefitinib-resistant and -sensitive non–small cell lung cancer (NSCLC). Experimental Design: Testing the effect of knockdown of USP8 and use of a synthetic USP8 inhibitor to selectively kill gefitinib-resistant (or -sensitive) NSCLCs with little effect on normal cells in cell culture and a xenograft mouse model. Results: Knockdown of ubiquitin-specific peptidase 8 (USP8) selectively kills gefitinib-resistant NSCLCs while having little toxicity toward normal cells. Genetic silencing of USP8 led to the downregulation of several receptor tyrosine kinases (RTK) including EGFR, ERBB2, ERBB3, and MET. We also determined that a synthetic USP8 inhibitor markedly decreased the viability of gefitinib-resistant and -sensitive NSCLC cells by decreasing RTK expression while having no effect on normal cells. Moreover, treatment with a USP8 inhibitor led to significant reductions in tumor size in a mouse xenograft model using gefitinib-resistant and -sensitive NSCLC cells. Conclusions: Our results show for the first time that the inhibition of USP8 activity or reduction in USP8 expression can selectively kill NSCLC cells. We propose USP8 as a potential therapeutic target for gefitinib-resistant and -sensitive NSCLC cells. Clin Cancer Res; 19(14); 3894–904. ©2013 AACR.

Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

The N-acetyltransferase arrest defective 1 (ARD1) is an important regulator of cell growth and differentiation that has emerged recently as a critical molecule in cancer progression. However, the regulation of the enzymatic and biological activities of human ARD1 (hARD1) in cancer is presently poorly understood. Here, we report that hARD1 undergoes autoacetylation and that this modification is essential for its functional activation. Using liquid chromatography-tandem mass spectrometry and site-directed mutational analyses, we identified K136 residue as an autoacetylation target site. K136R mutation abolished the ability of hARD1 to promote cancer cell growth in vitro and tumor xenograft growth in vivo. Mechanistic investigations revealed that hARD1 autoacetylation stimulated cyclin D1 expression through activation of the transcription factors beta-catenin and activator protein-1. Our results show that hARD1 autoacetylation is critical for its activation and its ability to stimulate cancer cell proliferation and tumorigenesis.

Autophagy and cellular senescence mediated by Sox2 suppress malignancy of cancer cells.

Autophagy is a critical cellular process required for maintaining cellular homeostasis in health and disease states, but the molecular mechanisms and impact of autophagy on cancer is not fully understood. Here, we found that Sox2, a key transcription factor in the regulation of the “stemness” of embryonic stem cells and induced-pluripotent stem cells, strongly induced autophagic phenomena, including intracellular vacuole formation and lysosomal activation in colon cancer cells. The activation occurred through Sox2-mediated ATG10 gene expression and resulted in the inhibition of cell proliferation and anchorage-independent colony growth ex vivo and tumor growth in vivo. Further, we found that Sox2-induced-autophagy enhanced cellular senescence by up-regulating tumor suppressors or senescence factors, including p16INK4a, p21 and phosphorylated p53 (Ser15). Notably, knockdown of ATG10 in Sox2-expressing colon cancer cells restored cancer cell properties. Taken together, our results demonstrated that regulation of autophagy mediated by Sox2 is a mechanism-driven novel strategy to treat human colon cancers.

ERK1 phosphorylates Nanog to regulate protein stability and stem cell self-renewal

Nanog regulates human and mouse embryonic stem (ES) cell self-renewal activity. Activation of ERKs signaling negatively regulates ES cell self-renewal and induces differentiation, but the mechanisms are not understood. We found that ERK1 binds and phosphorylates Nanog. Activation of MEK/ERKs signaling and phosphorylation of Nanog inhibit Nanog transactivation, inducing ES cell differentiation. Conversely, suppression of MEK/ERKs signaling enhances Nanog transactivation to inhibit ES cell differentiation. We observed that phosphorylation of Nanog by ERK1 decreases Nanog stability through ubiquitination-mediated protein degradation. Further, we found that this phosphorylation induces binding of FBXW8 with Nanog to reduce Nanog protein stability. Overall, our results demonstrated that ERKs-mediated Nanog phosphorylation plays an important role in self-renewal of ES cells through FBXW8-mediated Nanog protein stability.

Isoliquiritigenin Induces Apoptosis and Inhibits Xenograft Tumor Growth of Human Lung Cancer Cells by Targeting Both Wild Type and L858R/T790M Mutant EGFR

Background: Non-small-cell lung cancer (NSCLC) exhibits EGFR mutation. Results: Treatment with isoliquiritigenin (ILQ) inhibited growth and induced apoptosis in tyrosine kinase inhibitor-sensitive and -resistant NSCLC cells. ILQ suppressed wild type and mutant (L858R/T790M) EGFR kinase activity and attenuated H1975 lung cancer cell xenograft tumor growth. Conclusion: ILQ directly targets wild type or mutant EGFR. Significance: ILQ could be a potential therapeutic agent against NSCLC. Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR.

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.

AKAP12 regulates vascular integrity in zebrafish.

The integrity of blood vessels controls vascular permeability and extravasation of blood cells, across the endothelium. Thus, the impairment of endothelial integrity leads to hemorrhage, edema, and inflammatory infiltration. However, the molecular mechanism underlying vascular integrity has not been fully understood. Here, we demonstrate an essential role for A-kinase anchoring protein 12 (AKAP12) in the maintenance of endothelial integrity during vascular development. Zebrafish embryos depleted of akap12 (akap12 morphants) exhibited severe hemorrhages. In vivo time-lapse analyses suggested that disorganized interendothelial cell-cell adhesions in akap12 morphants might be the cause of hemorrhage. To clarify the molecular mechanism by which the cell-cell adhesions are impaired, we examined the cell-cell adhesion molecules and their regulators using cultured endothelial cells. The expression of PAK2, an actin cytoskeletal regulator, and AF6, a connector of intercellular adhesion molecules and actin cytoskeleton, was reduced in AKAP12-depleted cells. Depletion of either PAK2 or AF6 phenocopied AKAP12-depleted cells, suggesting the reduction of PAK2 and AF6 results in the loosening of intercellular junctions. Consistent with this, overexpression of PAK2 and AF6 rescued the abnormal hemorrhage in akap12 morphants. We conclude that AKAP12 is essential for integrity of endothelium by maintaining the expression of PAK2 and AF6 during vascular development.