Wonseok Yang

Regulation of Cell Proliferation and Migration by Keratin19-Induced Nuclear Import of Early Growth Response-1 in Breast Cancer Cells

Purpose: Keratin19 (KRT19) is the smallest known type I intermediate filament and is used as a marker for reverse transcriptase PCR–mediated detection of disseminated tumors. In this study, we investigated the functional analysis of KRT19 in human breast cancer. Experimental Design: Using a short hairpin RNA system, we silenced KRT19 in breast cancer cells. KRT19 silencing was verified by Western blot analysis and immunocytochemistry. We further examined the effect of KRT19 silencing on breast cancer cells by cell proliferation, migration, invasion, colony formation assay, cell-cycle analysis, immunocytochemistry, immunohistochemistry, and mouse xenograft assay. Results: Silencing of KRT19 resulted in increased cell proliferation, migration, invasion, and survival. These effects were mediated by upregulation of Akt signaling as a result of reduced PTEN mRNA expression. Silencing of KRT19 decreased the nuclear import of early growth response-1 (Egr1), a transcriptional factor for PTEN transcription, through reduced association between Egr1 and importin-7. We also confirmed that silencing of KRT19 increased tumor formation in a xenograft model. Conclusions: KRT19 is a potential tumor suppressor that negatively regulates Akt signaling through modulation of Egr1 nuclear localization. Clin Cancer Res; 19(16); 4335–46. ©2013 AACR.

Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression.

Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cells own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

Akt isoform-specific inhibition of MDA-MB-231 cell proliferation

To dissect the isoform-specific roles of Akt in breast cancer cells, constitutively active Akt isoforms were introduced into MDA-MB-231 cells. Both Akt1 and Akt2 efficiently inhibited the growth of MDA-MB-231 cells. Overexpression of Akt1 down-regulated ERK activity inhibiting Ser 259 phosphorylation of c-Raf and subsequent downstream signaling. Akt2 overexpression up-regulated the cell cycle inhibitor p27. Cycloheximide decay assays showed that Akt2 increased the stability and nuclear localization of p27, thus inhibiting the cyclin E/CDK2 complex. These results suggest that the inhibition of cell proliferation by Akt1 and Akt2 is mediated by isoform-specific mechanisms.

HER2 stabilizes survivin while concomitantly down-regulating survivin gene transcription by suppressing Notch cleavage.

In breast cancer, the HER2 (human epidermal growth factor receptor 2) receptor tyrosine kinase is associated with extremely poor prognosis and survival. Notch signalling has a key role in cell-fate decisions, especially in cancer-initiating cells. The Notch intracellular domain produced by Notch cleavage is translocated to the nucleus where it activates transcription of target genes. To determine the combinatory effect of HER2 and Notch signalling in breast cancer, we investigated the effect of HER2 on Notch-induced cellular phenomena. We found the down-regulation of Notch-dependent transcriptional activity by HER2 overexpression. Also, the HER2/ERK (extracellular-signal-regulated kinase) signal pathway down-regulated the activity of γ-secretase. When we examined the protein level of Notch target genes in HER2-overexpressing cells, we observed that the level of survivin, downstream of Notch, increased in HER2 cells. We found that activation of ERK resulted in a decrease in XAF1 [XIAP (X-linked inhibitor of apoptosis)-associated factor 1] which reduced the formation of the XIAP-XAF1 E3 ligase complex to ubiquitinate survivin. In addition, Thr(34) of survivin was shown to be the most important residue in determining survivin stability upon phosphorylation after HER2/Akt/CDK1 (cyclin-dependent kinase 1)-cyclin B1 signalling. The results of the present study show the combinatorial effects of HER2 and Notch during breast oncogenesis.

CD24 regulates cell proliferation and transforming growth factor β-induced epithelial to mesenchymal transition through modulation of integrin β1 stability.

To determine the role of CD24 in breast cancer cells, we knocked down CD24 in MCF-7 human breast cancer cells by retroviral delivery of shRNA. MCF-7 cells with knocked down CD24 (MCF-7 hCD24 shRNA) exhibited decreased cell proliferation and cell adhesion as compared to control MCF-7 mCD24 shRNA cells. Decreased proliferation of MCF-7 hCD24 shRNA cells resulted from the inhibition of cell cycle progression from G1 to S phase. The specific inhibition of MEK/ERK signaling by CD24 ablation might be responsible for the inhibition of cell proliferation. Phosphorylation of Src/FAK and TGF-β1-mediated epithelial to mesenchymal transition was also down-regulated in MCF-7 hCD24 shRNA cells. Reduced Src/FAK activity was caused by a decrease in integrin β1 bound with CD24 and subsequent destabilization of integrin β1. Our results suggest that down-regulation of Raf/MEK/ERK signaling via Src/FAK may be dependent on integrin β1 function and that this mechanism is largely responsible for the CD24 ablation-induced decreases in cell proliferation and epithelial to mesenchymal transition.

Effects of Endocrine Disruptors on Bombina orientalis P450 Aromatase Activity

To assess the effects of endocrine-disrupting chemicals on the expression and activity of aromatase in the gonads of Bombina orientalis, a common amphibian, we intraperitoneally injected nonylphenol or bisphenol-A and then examined aromatase mRNA levels by RT-PCR as well as aromatase enzymatic activity by tritiated water release assays. To design primers for the RT-PCR, we cloned the B. orientalis aromatase gene using RT-PCR and degenerate primers. The full-length cDNA was obtained by 5′- and 3′-RACE PCR. The complete sequence of the B. orientalis aromatase gene revealed an open reading frame of 1500 bp encoding a deduced protein of 500 amino acids. Semi-quantitative RT-PCR indicated that nonylphenol or bisphenol-A injection did not significantly affect the expression of B. orientalis aromatase mRNA. However, a 48-hr treatment with nonyphenol or bisphenol A reduced aromatase activity to 47% and 32% of the control, respectively. These results suggest that endocrine disrupters can effectively modulate the activity of B. orientalis aromatase without affecting its mRNA levels.

EGFR negates the proliferative effect of oncogenic HER2 in MDA-MB-231 cells

Members of the EGFR family are potent mediators of normal cell growth and development. HER2 possesses an active tyrosine kinase domain, but no direct ligand has been identified. To investigate the differential effect of HER2 in breast cell lines, HER2 was overexpressed in MCF-10A, MCF7 and MDA-MB-231 cells. HER2 overexpression promoted proliferation, survival and migration in MCF-10A and MCF-7 cells. No significant differences were seen in proliferation, survival or migration between MDA-MB-231 vec and HER2 cells. The activity of downstream HER2 proteins increased in MCF-10A HER2 and MCF-7 HER2 cells but not in MDA-MB-231 HER2 cells. Exogenously expressed HER2 failed to associate with EGFR or HER3 in MDA-MB-231 cells, while overexpression of HER2 enhanced HER family dimerization in MCF-10A and MCF-7 cells.

S100A4 negatively regulates β-catenin by inducing the Egr-1-PTEN-Akt-GSK3β degradation pathway.

S100A4, also known as the mts1 gene, has been reported as an invasive and metastatic marker for many types of cancers. S100A4 interacts with various target genes that affect tumor cell metastasis; however, little is known about cellular signaling pathways elicited by S100A4. In the current study, we demonstrate an inhibitory effect of S100A4 on β-catenin signaling in breast cancer cells. By overexpressing S100A4 in MCF-7, MDA-MB-231 and MDA-MB-453 breast cancer cells, we observed the down-regulation of β-catenin expression and β-catenin-dependent TCF/LEF transcriptional activities. The activity of GSK3β, which phosphorylates β-catenin and induces proteasomal degradation of β-catenin, was increased in S100A4-overexpressing cell lines. Blocking Glycogen Synthase Kinase (GSK3β) activity by lithium chloride or Dvl gene overexpression restored β-catenin expression. We also found that increased GSK3β activity was due to decrease in Akt activity resulting from Egr-1-induced phosphatase and tensin homolog (PTEN) expression. S100A4 induced Egr-1 nuclear localization by increasing the association between Egr-1 and importin-7 and this effect was reduced in S100A4 mutants that harbored a defect in nuclear localization signals. Collectively, we verify herein that S100A4 may act as a tumor suppressor in breast cancers by down-regulating the central signaling axis for tumor cell survival.

Flexural wave cloaking via embedded cylinders with systematically varying thicknesses

Simulations of flexural wave cloaking from multiple scattering events that are achieved by embedded cylinders in a thin plate are performed. Minimization of refraction is performed using small surrounding cylinders with varying thickness in radial and angular directions, respectively. The thickness variations render the effective wave speed lower in the radial direction and higher in the angular direction compared to the speed in the surrounding media, which results in the cloaking effect. In order to verify the feasibility of this approach, 15 layers of cylinders are placed around the blocked area. The multiple-scattering method is used to predict wave propagations and to take the interactions between cylinders into account. The effects of the thickness variation on the cloaking performance are analyzed. The results demonstrate that minimal scattering is achieved when the area of interest is surrounded by the thickness-varying cylinders.

Analysis of Aerodynamic Noise Generation from Pantograph Using Panhead Models of Simple-Geometry and Its Reduction

This study presents a result on aero-acoustic characteristics of pantograph panheads. To analyze the fluid flow around the panhead and resulting sound radiation, simple models of panhead were used in the numerical simulations called Lattice-Boltzmann method. The simulation results were verified using the wind tunnel test. The main aerodynamic noise was generated from the vortex shedding which is characterized by the Strouhal number, flow speed and geometry. The reduction in the radiated noise with simultaneously achieving increased lifting force was implemented for the simple rectangular geometry used in this study. Also, it was shown that the radiated sound power was significantly reduced by minimizing vortex shedding using through-holes or streamline shapes.