Heekyoung Chung

Srg3, a Mouse Homolog of Yeast SWI3, Is Essential for Early Embryogenesis and Involved in Brain Development

ABSTRACT Srg3 (SWI3-related gene product) is a mouse homolog of yeast SWI3,Drosophila melanogaster MOIRA (also named MOR/BAP155), and human BAF155 and is known as a core subunit of SWI/SNF complex. This complex is involved in the chromatin remodeling required for the regulation of transcriptional processes associated with development, cellular differentiation, and proliferation. We generated mice with a null mutation in theSrg3 locus to examine its function in vivo. Homozygous mutants develop in the early implantation stage but undergo rapid degeneration thereafter. An in vitro outgrowth study revealed that mutant blastocysts hatch, adhere, and form a layer of trophoblast giant cells, but the inner cell mass degenerates after prolonged culture. Interestingly, about 20% of heterozygous mutant embryos display defects in brain development with abnormal organization of the brain, a condition known as exencephaly. Histological examination suggests that exencephaly is caused by the failure in neural fold elevation, resulting in severe brain malformation. Our findings demonstrate that Srg3 is essential for early embryogenesis and plays an important role in the brain development of mice.

MTA1 overexpression correlates significantly with tumor grade and angiogenesis in human breast cancers

Metastasis associated antigen 1 (MTA1) is a recently identified candidate metastasis‐associated gene that plays an important role in tumorigenesis and tumor aggressiveness, especially tumor invasiveness and metastasis. We analyzed the relationship between MTA1 expression and variable clinicopathological features and characterized its role in tumor angiogenesis in human breast cancers. Two hundred and sixty‐three breast cancer cases that successfully underwent surgery at Hanyang University Hospital (Seoul, Korea) between January 1989 and December 1997 were enrolled. MTA1 expression was observed by immunohistochemical staining and correlated with intratumoral microvessel density (MVD) and other clinicopathological parameters. MTA1 overexpression correlated significantly with higher tumor grade (grades 1 and 2 vs grade 3, P = 0.009). However, MTA1 expression did not correlate with tumor stage, status of estrogen and progesterone receptors, or axillary lymph node metastasis. Interestingly, MTA1 expression was found to correlate significantly with tumor MVD (P = 0.002). Survival analysis did not show a significant difference between MTA1 overexpression and poorer survival. In conclusion, MTA1 overexpression was found to be closely associated with higher tumor grade and increased tumor angiogenesis. These findings suggest MTA1 as a predictor of aggressive phenotype and a possible target molecule for anti‐angiogenic drugs in breast cancer treatment. (Cancer Sci 2006)

Anticancer effects of wogonin in both estrogen receptor-positive and -negative human breast cancer cell lines in vitro and in nude mice xenografts.

Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. Herein, we investigated the in vitro and in vivo anticancer effects and associated mechanisms of wogonin in human breast cancer. Effects of wogonin were examined in estrogen receptor (ER)‐positive and ‐negative human breast cancer cells in culture for proliferation, cell cycle progression, and apoptosis. The in vivo effect of oral wogonin was examined on tumor xenograft growth in athymic nude mice. The molecular changes associated with the biological effects of wogonin were analyzed by immunoblotting. Cell growth was attenuated by wogonin (50–200 μM), independently of its ER status, in a time‐ and concentration‐dependent manner. Apoptosis was enhanced and accompanied by upregulation of PARP and Caspase 3 cleavages as well as proapoptotic Bax protein. Akt activity was suppressed and reduced phosphorylation of its substrates, GSK‐3β and p27, was observed. Suppression of Cyclin D1 expression suggested the downregulation of the Akt‐mediated canonical Wnt signaling pathway. ER expression was downregulated in ER‐positive cells, while c‐ErbB2 expression and its activity were suppressed in ER‐negative SK‐BR‐3 cells. Wogonin feeding to mice showed inhibition of tumor growth of T47D and MDA‐MB‐231 xenografts by up to 88% without any toxicity after 4 weeks of treatment. As wogonin was effective both in vitro and in vivo, our novel findings open the possibility of wogonin as an effective therapeutic and/or chemopreventive agent against both ER‐positive and ‐negative breast cancers, particularly against the more aggressive and hormonal therapy‐resistant ER‐negative types.

SRG3 Interacts Directly with the Major Components of the SWI/SNF Chromatin Remodeling Complex and Protects Them from Proteasomal Degradation

The mammalian SWI/SNF complex is an evolutionarily conserved ATP-dependent chromatin remodeling complex that consists of nine or more components. SRG3, a murine homologue of yeast SWI3, Drosophila MOIRA, and human BAF155, is a core component of the murine SWI/SNF complex required for the regulation of transcriptional processes associated with development, cellular differentiation, and proliferation. Here we report that SRG3 interacts directly with other components of the mammalian SWI/SNF complex such as SNF5, BRG1, and BAF60a. The SWIRM domain and the SANT domain were required for SRG3-SNF5 and SRG3-BRG1 interactions, respectively. In addition, SRG3 stabilized SNF5, BRG1, and BAF60a by attenuating their proteasomal degradation, suggesting its general role in the stabilization of the SWI/SNF complex. Such a stabilization effect of SRG3 was not only observed in the in vitro cell system, but also in cells isolated from SRG3 transgenic mice or knock-out mice haploinsufficient for the Srg3 gene. Taken together, these results suggest the critical role of SRG3 in the post-transcriptional stabilization of the major components of the SWI/SNF complex.

Induction of Neuronal Vascular Endothelial Growth Factor Expression by cAMP in the Dentate Gyrus of the Hippocampus Is Required for Antidepressant-Like Behaviors

The cAMP cascade and vascular endothelial growth factor (VEGF) are critical modulators of depression. Here we have tested whether the antidepressive effect of the cAMP cascade is mediated by VEGF in the adult hippocampus. We used a conditional genetic system in which the Aplysia octopamine receptor (Ap oa1), a Gs-coupled receptor, is transgenically expressed in the forebrain neurons of mice. Chronic activation of the heterologous Ap oa1 by its natural ligand evoked antidepressant-like behaviors, accompanied by enhanced phosphorylation of cAMP response element-binding protein and transcription of VEGF in hippocampal dentate gyrus (DG) neurons. Selective knockdown of VEGF in these cells during the period of cAMP elevation inhibited the antidepressant-like behaviors. These findings reveal a molecular interaction between the cAMP cascade and VEGF expression, and the pronounced behavioral consequences of this interaction shed light on the mechanism underlying neuronal VEGF functions in antidepression.

BAF60a Interacts with p53 to Recruit the SWI/SNF Complex

To understand the tumor-suppressing mechanism of the SWI/SNF chromatin remodeling complex, we investigated its molecular relationship with p53. Using the pREP4-luc episomal reporter, we first demonstrated that p53 utilizes the chromatin remodeling activity of the SWI/SNF complex to initiate transcription from the chromatin-structured promoter. Among the components of the SWI/SNF complex, we identified BAF60a as a mediator of the interaction with p53 by the yeast two-hybrid assay. p53 directly interacted only with BAF60a, but not with other components of the SWI/SNF complex, such as BRG1, SRG3, SNF5, or BAF57. We found out that multiple residues at the amino acid 108–150 region of BAF60a were involved in the interaction with the tetramerization domain of p53. The N-terminal fragment of BAF60a containing the p53-interacting region as well as small interfering RNA for baf60a inhibited the SWI/SNF complex-mediated transcriptional activity of p53. The uncoupling of p53 with the SWI/SNF complex resulted in the repression of both p53-dependent apoptosis and cell cycle arrest by the regulation of target genes. These results suggest that the SWI/SNF chromatin remodeling complex is involved in the suppression of tumors by the interaction with p53.

Inhibitor of DNA Binding 1 Activates Vascular Endothelial Growth Factor through Enhancing the Stability and Activity of Hypoxia-Inducible Factor-1α

Inhibitor of DNA binding 1 (Id-1) has been implicated in tumor angiogenesis by regulating the expression of vascular endothelial growth factor (VEGF), but its molecular mechanism has not been fully understood. Here, we show the cross talk between Id-1 and hypoxia-inducible factor-1α (HIF-1α), that Id-1 induces VEGF by enhancing the stability and activity of HIF-1α in human endothelial and breast cancer cells. Although both the transcript and proteins levels of VEGF were induced by Id-1, only the protein expression of HIF-1α was induced without transcriptional changes in both human umbilical endothelial cells and MCF7 breast cancer cells. Such induction of the HIF-1α protein did not require de novo protein synthesis but was dependent on the active extracellular response kinase (ERK) pathway. In addition, stability of the HIF-1α protein was enhanced in part by the reduced association of the HIF-1α protein with von Hippel-Lindau protein in the presence of Id-1. Furthermore, Id-1 enhanced nuclear translocation and the transcriptional activity of HIF-1α. Transcriptional activation of HIF-1–dependent promoters was dependent on the active ERK pathway, and the association of HIF-1α protein with cyclic AMP-responsive element binding protein was enhanced by Id-1. Finally, Id-1 induced tube formation in human umbilical endothelial cells, which also required active ERK signaling. In conclusion, we provide the molecular mechanism of the cross talk between HIF-1α and Id-1, which may play a critical role in tumor angiogenesis. (Mol Cancer Res 2007;5(4):321–9)

Chromatin remodeling, development and disease

Development is a stepwise process in which multi-potent progenitor cells undergo lineage commitment, differentiation, proliferation and maturation to produce mature cells with restricted developmental potentials. This process is directed by spatiotemporally distinct gene expression programs that allow cells to stringently orchestrate intricate transcriptional activation or silencing events. In eukaryotes, chromatin structure contributes to developmental progression as a blueprint for coordinated gene expression by actively participating in the regulation of gene expression. Changes in higher order chromatin structure or covalent modification of its components are considered to be critical events in dictating lineage-specific gene expression during development. Mammalian cells utilize multi-subunit nuclear complexes to alter chromatin structure. Histone-modifying complex catalyzes covalent modifications of histone tails including acetylation, methylation, phosphorylation and ubiquitination. ATP-dependent chromatin remodeling complex, which disrupts histone-DNA contacts and induces nucleosome mobilization, requires energy from ATP hydrolysis for its catalytic activity. Here, we discuss the diverse functions of ATP-dependent chromatin remodeling complexes during mammalian development. In particular, the roles of these complexes during embryonic and hematopoietic development are reviewed in depth. In addition, pathological conditions such as tumor development that are induced by mutation of several key subunits of the chromatin remodeling complex are discussed, together with possible mechanisms that underlie tumor suppression by the complex.

Notch1 confers a resistance to glucocorticoid-induced apoptosis on developing thymocytes by down-regulating SRG3 expression

We previously have reported that SRG3 is required for glucocorticoid (GC)-induced apoptosis in the S49.1 thymoma cell line. Activation of Notch1 was shown to induce GC resistance in thymocytes. However, the specific downstream target of Notch1 that confers GC resistance on thymocytes is currently unknown. We found that the expression level of SRG3 was critical in determining GC sensitivity in developing thymocytes. The expression of SRG3 also was down-regulated by the activated form of Notch1 (NotchIC). The promoter activity of the SRG3 gene also was down-regulated by NotchIC. Expression of transgenic SRG3 resulted in the restoration of GC sensitivity in thymocytes expressing transgenic Notch1. These results suggest that SRG3 is the downstream target of Notch1 in regulating GC sensitivity of thymocytes.

The antitumor effect of LJ-529, a novel agonist to A3 adenosine receptor, in both estrogen receptor–positive and estrogen receptor–negative human breast cancers

Agonists to A3 adenosine receptor (A3AR) have been reported to inhibit cell growth and/or induce apoptosis in various tumors. We tested the effect of a novel A3AR agonist generically known as LJ-529 in breast cancer cells. Anchorage-dependent cell growth and in vivo tumor growth were attenuated by LJ-529, independently of its estrogen receptor (ER) α status. In addition, apoptosis was induced as evidenced by the activation of caspase-3 and c–poly(ADP)ribose polymerase. Furthermore, the Wnt signaling pathway was down-regulated and p27kip was induced by LJ-529. In ER-positive cells, the expression of ER was down-regulated by LJ-529, which might have additionally contributed to attenuated cell proliferation. In ER-negative, c-ErbB2-overexpressing SK-BR-3 cells, the expression of c-ErbB2 and its downstream extracellular signal-regulated kinase pathway were down-regulated by LJ-529. However, such effect of LJ-529 acted independently of its receptor because no A3AR was detected by reverse transcription-PCR in all four cell lines tested. In conclusion, our novel findings open the possibility of LJ-529 as an effective therapeutic agent against both ER-positive and ER-negative breast cancers, particularly against the more aggressive ER-negative, c-ErbB2-overexpressing types. [Mol Cancer Ther 2006;5(3):685–92]