Ji Hyeon Park

Design, Synthesis, and Biological Evaluation of Novel Deguelin-Based Heat Shock Protein 90 (HSP90) Inhibitors Targeting Proliferation and Angiogenesis

Deguelin exhibits potent apoptotic and antiangiogenic activities in a variety of transformed cells and cancer cells. Deguelin also exhibits potent tumor suppressive effects in xenograft tumor models for many human cancers. Our initial studies confirmed that deguelin disrupts ATP binding to HSP90 and consequently induces destabilization of its client proteins such as HIF-1α. Interestingly, a fluorescence probe assay revealed that deguelin and its analogues do not compete with ATP binding to the N-terminus of HSP90, unlike most HSP90 inhibitors. To determine the key parts of deguelin that contribute to its potent HSP90 inhibition, as well as its antiproliferative and antiangiogenic activities, we have established a structure-activity relationship (SAR) of deguelin. In the course of these studies, we identified a series of novel and potent HSP90 inhibitors. In particular, analogues 54 and 69, the B- and C-ring-truncated compounds, exhibited excellent antiproliferative activities with IC(50) of 140 and 490 nM in the H1299 cell line, respectively, and antiangiogenic activities in zebrafish embryos in a dose dependent manner (0.25-1.25 μM).

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.

Roles of Arrest-Defective Protein 1225 and Hypoxia-Inducible Factor 1α in Tumor Growth and Metastasis

Background Vascular endothelial growth factor A (VEGFA), a critical mediator of tumor angiogenesis, is a well-characterized target of hypoxia-inducible factor 1 (HIF-1). Murine arrest-defective protein 1A (mARD1A225) acetylates HIF-1α, triggering its degradation, and thus may play a role in decreased expression of VEGFA. Methods We generated ApcMin/+/mARD1A225 transgenic mice and quantified growth of intestinal polyps. Human gastric MKN74 and murine melanoma B16F10 cells overexpressing mARD1A225 were injected into mice, and tumor growth and metastasis were measured. VEGFA expression and microvessel density in tumors were assessed using immunohistochemistry. To evaluate the role of mARD1A225 acetylation of Lys532 in HIF-1α, we injected B16F10-mARD1A225 cell lines stably expressing mutant HIF-1α/K532R into mice and measured metastasis. All statistical tests were two-sided, and P values less than .05 were considered statistically significant. Results ApcMin/+/mARD1A225 transgenic mice (n = 25) had statistically significantly fewer intestinal polyps than ApcMin/+ mice (n = 21) (number of intestinal polyps per mouse: ApcMin/+ mice vs ApcMin/+/mARD1A225 transgenic mice, mean = 83.4 vs 38.0 polyps, difference = 45.4 polyps, 95% confidence interval [CI] = 41.8 to 48.6; P < .001). The growth and metastases of transplanted tumors were also statistically significantly reduced in mice injected with mARD1A225-overexpressing cells than in mice injected with control cells (P < .01). Moreover, overexpression of mARD1A225 decreased VEGFA expression and microvessel density in tumor xenografts (P < .04) and ApcMin/+ intestinal polyps (P = .001). Mutation of lysine 532 of HIF-1α in B16F10-mARD1A225 cells prevented HIF-1α degradation and inhibited the antimetastatic effect of mARD1A225 (P < .001). Conclusion mARD1A225 may be a novel upstream target that blocks VEGFA expression and tumor-related angiogenesis.

AKAP12 Mediates Barrier Functions of Fibrotic Scars during CNS Repair

The repair process after CNS injury shows a well-organized cascade of three distinct stages: inflammation, new tissue formation, and remodeling. In the new tissue formation stage, various cells migrate and form the fibrotic scar surrounding the lesion site. The fibrotic scar is known as an obstacle for axonal regeneration in the remodeling stage. However, the role of the fibrotic scar in the new tissue formation stage remains largely unknown. We found that the number of A-kinase anchoring protein 12 (AKAP12)-positive cells in the fibrotic scar was increased over time, and the cells formed a structure which traps various immune cells. Furthermore, the AKAP12-positive cells strongly express junction proteins which enable the structure to function as a physical barrier. In in vivo validation, AKAP12 knock-out (KO) mice showed leakage from a lesion, resulting from an impaired structure with the loss of the junction complex. Consistently, focal brain injury in the AKAP12 KO mice led to extended inflammation and more severe tissue damage compared to the wild type (WT) mice. Accordingly, our results suggest that AKAP12-positive cells in the fibrotic scar may restrict excessive inflammation, demonstrating certain mechanisms that could underlie the beneficial actions of the fibrotic scar in the new tissue formation stage during the CNS repair process.

Nuclear translocation of hARD1 contributes to proper cell cycle progression.

Arrest defective 1 (ARD1) is an acetyltransferase that is highly conserved across organisms, from yeasts to humans. The high homology and widespread expression of ARD1 across multiple species and tissues signify that it serves a fundamental role in cells. Human ARD1 (hARD1) has been suggested to be involved in diverse biological processes, and its role in cell proliferation and cancer development has been recently drawing attention. However, the subcellular localization of ARD1 and its relevance to cellular function remain largely unknown. Here, we have demonstrated that hARD1 is imported to the nuclei of proliferating cells, especially during S phase. Nuclear localization signal (NLS)-deleted hARD1 (hARD1ΔN), which can no longer access the nucleus, resulted in cell morphology changes and cellular growth impairment. Notably, hARD1ΔN-expressing cells showed alterations in the cell cycle and the expression levels of cell cycle regulators compared to hARD1 wild-type cells. Furthermore, these effects were rescued when the nuclear import of hARD1 was restored by exogenous NLS. Our results show that hARD1 nuclear translocation mediated by NLS is required for cell cycle progression, thereby contributing to proper cell proliferation.

Prompt meningeal reconstruction mediated by oxygen-sensitive AKAP12 scaffolding protein after central nervous system injury

The meninges forms a critical epithelial barrier, which protects the central nervous system (CNS), and therefore its prompt reconstruction after CNS injury is essential for reducing neuronal damage. Meningeal cells migrate into the lesion site after undergoing an epithelial-mesenchymal transition (EMT) and repair the impaired meninges. However, the molecular mechanisms of meningeal EMT remain largely undefined. Here we show that TGF-β1 and retinoic acid (RA) released from the meninges, together with oxygen tension, could constitute the mechanism for rapid meningeal reconstruction. AKAP12 is an effector of this mechanism, and its expression in meningeal cells is regulated by integrated upstream signals composed of TGF-β1, RA and oxygen tension. Functionally, AKAP12 modulates meningeal EMT by regulating the TGF-β1-non-Smad-SNAI1 signalling pathway. Collectively, TGF-β1, RA and oxygen tension can modulate the dynamic change in AKAP12 expression, causing prompt meningeal reconstruction after CNS injury by regulating the transition between the epithelial and mesenchymal states of meningeal cells.

The N-terminal ectodomain of Ninjurin1 liberated by MMP9 has chemotactic activity

Ninjurin1 is known as an adhesion molecule promoting leukocyte trafficking under inflammatory conditions. However, the posttranslational modifications of Ninjurin1 are poorly understood. Herein, we defined the proteolytic cleavage of Ninjurin1 and its functions. HEK293T cells overexpressing the C- or N-terminus tagging mouse Ninjurin1 plasmid produced additional cleaved forms of Ninjurin1 in the lysates or conditioned media (CM). Two custom-made anti-Ninjurin1 antibodies, Ab(1-15) or Ab(139-152), specific to the N- or C-terminal regions of Ninjurin1 revealed the presence of its shedding fragments in the mouse liver and kidney lysates. Furthermore, Matrix Metalloproteinase (MMP) 9 was responsible for Ninjurin1 cleavage between Leu(56) and Leu(57). Interestingly, the soluble N-terminal Ninjurin1 fragment has structural similarity with well-known chemokines. Indeed, the CM from HEK293T cells overexpressing the GFP-mNinj1 plasmid was able to attract Raw264.7 cells in trans-well assay. Collectively, we suggest that the N-terminal ectodomain of mouse Ninjurin1, which may act as a chemoattractant, is cleaved by MMP9.

Autoacetylation regulates differentially the roles of ARD1 variants in tumorigenesis

ARD1 is an acetyltransferase with several variants derived from alternative splicing. Among ARD1 variants, mouse ARD1(225) (mARD1(225)), mouse ARD1(235) (mARD1(235)), and human ARD1(235) (hARD1(235)) have been the most extensively characterized and are known to have different biological functions. In the present study, we demonstrated that mARD1(225), mARD1(235), and hARD1(235) have conserved autoacetylation activities, and that they selectively regulate distinct roles of ARD1 variants in tumorigenesis. Using purified recombinants for ARD1 variants, we found that mARD1(225), mARD1(235), and hARD1(235) undergo similar autoacetylation with the target site conserved at the Lys136 residue. Moreover, functional investigations revealed that the role of mARD1(225) autoacetylation is completely distinguishable from that of mARD1(235) and hARD1(235). Under hypoxic conditions, mARD1(225) autoacetylation inhibited tumor angiogenesis by decreasing the stability of hypoxia-inducible factor-1α (HIF-1α). Autoacetylation stimulated the catalytic activity of mARD1(225) to acetylate Lys532 of the oxygen-dependent degradation (ODD) domain of HIF-1α, leading to the proteosomal degradation of HIF-1α. In contrast, autoacetylation of mARD1(235) and hARD1(235) contributed to cellular growth under normoxic conditions by increasing the expression of cyclin D1. Taken together, these data suggest that autoacetylation of ARD1 variants differentially regulates angiogenesis and cell proliferation in an isoform-specific manner.