Lipid raft protein flotillin-1 is important for the interaction between SOS1 and H-Ras/K-Ras, leading to Ras activation.

Ras mutations have been frequently observed in human cancer. Although there is a high degree of similarity between Ras isomers, they display preferential coupling in specific cancer types. The binding of Ras to the plasma membrane is essential for its activation and biological functions. The present study elucidated Ras isoform-specific interactions with the membrane and their role in Ras-mediated biological activities. We investigated the role of a lipid raft protein flotillin-1 (Flot-1) in the activations of Ras. We found that Flot-1 was co-localized with H-Ras, but not with N-Ras, in lipid rafts of MDA-MB-231 human breast cells. The amino-terminal hydrophobic domain (1-38) of Flot-1 interacted with the hypervariable region of H-Ras. The epidermal growth factor-stimulated activation of H-Ras required Flot-1 which was not necessary for that of N-Ras in breast cancer cells. Flot-1 interacted with son of sevenless (SOS)-1, which promotes the conversion of Ras-bound GDP to GTP. Notably, Flot-1 was crucial for the interaction between SOS1 and H-Ras/K-Ras in breast and pancreatic cancer cells. Stable knockdown of Flot-1 reduced the in vivo metastasis in a mouse xenograft model with human breast carcinoma cells. A tissue microarray composed of 61 human pancreatic cancer samples showed higher levels of Flot-1 expression in pancreatic tumor tissues compared to normal tissues, and a correlation between K-Ras and Flot-1. Taken together, our findings suggest that Flot-1 may serve as a membrane platform for the interaction of SOS1 with H-Ras/K-Ras in human cancer cells, presenting Flot-1 as a potential target for Ras-driven cancers.

A novel role for flotillin-1 in H-Ras-regulated breast cancer aggressiveness.

Elevated expression and aberrant activation of Ras have been implicated in breast cancer aggressiveness. H-Ras, but not N-Ras, induces breast cell invasion. A crucial link between lipid rafts and H-Ras function has been suggested. This study sought to identify the lipid raft protein(s) responsible for H-Ras-induced tumorigenicity and invasiveness of breast cancer. We conducted a comparative proteomic analysis of lipid raft proteins from invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein flotillin-1 as an important regulator of H-Ras activation and breast cell invasion. Flotillin-1 was required for epidermal growth factor-induced activation of H-Ras, but not that of N-Ras, in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Flotillin-1 knockdown inhibited the invasiveness of MDA-MB-231 and Hs578T TNBC cells in vitro and in vivo. In xenograft mouse tumor models of these TNBC cell lines, we showed that flotillin-1 played a critical role in tumor growth. Using human breast cancer samples, we provided clinical evidence for the metastatic potential of flotillin-1. Membrane staining of flotillin-1 was positively correlated with metastatic spread (p = 0.013) and inversely correlated with patient disease-free survival rates (p = 0.005). Expression of flotillin-1 was associated with H-Ras in breast cancer, especially in TNBC (p < 0.001). Our findings provide insight into the molecular basis of Ras isoform-specific interplay with flotillin-1, leading to tumorigenicity and aggressiveness of breast cancer.

Global gene expression profiling unveils S100A8/A9 as candidate markers in H-ras-mediated human breast epithelial cell invasion.

The goal of the present study is to unveil the gene expression profile specific to the biological processes of human breast epithelial cell invasion and migration using an MCF10A model genetically engineered to constitutively activate the H-ras or N-ras signaling pathway. We previously showed that H-Ras, but not N-Ras, induces MCF10A cell invasion/migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. Thus, these cell lines provide an experimental system to separate the gene expression profile associated with cell invasion apart from cell proliferation/transformation. Analysis of whole human genome microarray revealed that 412 genes were differentially expressed among MCF10A, N-Ras MCF10A, and H-Ras MCF10A cells and hierarchical clustering separated 412 genes into four clusters. We then tested whether S100A8 and S100A9, two of the genes which are most highly up-regulated in an H-Ras-specific manner, play a causative role for H-Ras-mediated MCF10A cell invasion and migration. Importantly, small interfering RNA-mediated knockdown of S100A8/A9 expression significantly reduced H-Ras-induced invasion/migration. Conversely, the induction of S100A8/A9 expression conferred the invasive/migratory phenotype to parental MCF10A cells. Furthermore, we provided evidence of signaling cross-talk between S100A8/A9 and the mitogen-activated protein kinase signaling pathways essential for H-Ras-mediated cell invasion and migration. Taken together, this study revealed S100A8/A9 genes as candidate markers for metastatic potential of breast epithelial cells. Our gene profile data provide useful information which may lead to the identification of additional potential targets for the prognosis and/or therapy of metastatic breast cancer.

Roles of calcium-binding proteins, S100A8 and S100A9, in invasive phenotype of human gastric cancer cells.

Gastric cancer is one of the most common malignancies and is a frequent cause of cancer-related death in Korea. Cure rate of gastric cancer is quite low because of local invasion and metastasis. S100 proteins are calcium-binding proteins which exert various calcium-mediated cellular functions including cell growth, differentiation, migration and signal transduction. S100A8 and S100A9 are overexpressed in many human tumors and have been shown to be implicated in tumor development or progression. In the present study, we investigated the role of S100A8 and S100A9 in invasive phenotype of a human gastric cancer cell line, SNU484. Expression of S100A8 and S100A9 were detected in SNU484 cells. When the expression of these proteins was suppressed by small-interfering RNA (siRNA) targeting S100A8 or S100A9, the invasive and migratory phenotypes of SNU484 cells were significantly inhibited. The siRNAs for S100A8 and S100A9 inhibited matrix metalloproteinase (MMP)-2 expression in SNU484 cells as evidenced by gelatin zymogram assay, immunoblot analysis and reverse transcription (RT)-PCR. These results demonstrate that S100A8 and S100A9 are required for transcriptional activation of MMP-2 gene in SNU484 cells. Taken together, this study revealed a functional contribution of S100A8 and S100A9 proteins to processes required for malignant progression including invasion, migration and proteinase expression in SNU484 human gastric cancer cells.

Diversity of denitrifying bacteria isolated from Daejeon Sewage Treatment Plant.

The diversity of the denitrifying bacterial populations in Daejeon Sewage Treatment Plant was examined using a culture-dependent approach. Of the three hundred and seventy six bacterial colonies selected randomly from agar plates, thirty-nine strains that showed denitrifying activity were selected and subjected to further analysis. According to the morphological and biochemical properties, the thirty nine isolates were divided into seven groups. This grouping was supported by an unweighted pair group method, using an arithmetic mean (UPGMA) analysis with fatty acid profiles. Restriction pattern analysis of 16S rDNA with four endonucleases (AluI, BstUI, MspI and RsaI) again revealed seven distinct groups, consistent with those defined from the morphological and biochemical properties and fatty acid profiles. Through the phylogenetic analysis using the 16S rDNA partial sequences, the main denitrifying microbial populations were found to be members of the phylum, Proteobacteria; in particular, classes Gamma proteobacteria (Aeromonas, Klebsiella and Enterobacter) and Beta proteobacteria (Acidovorax, Burkholderia and Comamonas), with Firmicutes, represented by Bacillus, also comprised a major group.

Denbinobin, a phenanthrene from dendrobium nobile, inhibits invasion and induces apoptosis in SNU-484 human gastric cancer cells.

Dendrobium nobile is widely used as an analgesic, an antipyretic, and a tonic to nourish the stomach in traditional medicine. Mounting evidence suggests an antitumor activity of denbinobin, a major phenanthrene isolated from stems of Dendrobium nobile. The present study aimed to investigate the inhibitory effect of denbinobin on the invasive ability of human cancer cells. The cytotoxicity of denbonobin was examined in several human cancer cell lines including SK-Hep-1 hepato-carcinoma cells, SNU-484 gastric cancer cells, and HeLa cervix cancer cells. Because SNU-484 cells showed the lowest IC50 value, we examined the effect of denbinobin on the invasive ability of SNU-484 cells. The present study revealed, for the first time, that denbinobin inhibits the invasive phenotype of SNU-484 human gastric cancer cells in a dose-dependent manner. Expressions of matrix metalloproteinase (MMP)-2 and MMP-9 were significantly decreased by denbinobin, suggesting that MMP-2/-9 may be responsible for the anti-invasive activity of denbinobin. We also provide evidence that denbinobin induces apoptosis through down-regulation of Bcl-2 and an up-regulation of Bax. Taken together, this study demonstrates that denbinobin inhibits invasion and induces apoptosis in highly invasive SNU-484 human gastric cancer cells. Given that gastric cancer has been estimated to be one of the most common causes of cancer-related death among Asians and the major cause of death from gastric cancer is the metastatic spread of the disease, our findings may provide useful information regarding the application of denbinobin as a chemopreventive agent that could prevent or alleviate metastatic gastric cancer.

Identification of H-Ras-specific motif for the activation of invasive signaling program in human breast epithelial cells.

Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR), consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras-specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform-specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.

ErbB2-enhanced invasiveness of H-Ras MCF10A breast cells requires MMP-13 and uPA upregulation via p38 MAPK signaling.

Overexpression of ErbB2 has been frequently found in mammary carcinoma. We have previously shown that the aberrant activation of H-Ras induces human breast cell invasion and migration. The present study was aimed at investigating the effect of ErbB2 overexpression on H-Ras-induced breast cell invasion and to elucidate the underlying mechanisms. Herein, we show that overexpression of ErbB2 promotes invasive and migratory abilities of H-Ras-activated MCF10A human breast epithelial cells through upregulation of matrix metalloproteinase (MMP)-13 and urokinase-type plasminogen activator (uPA). We also demonstrate that the p38 MAPK is an important signaling molecule in the ErbB2-induced upregulation of MMP-13 and uPA and invasion/migration of H-Ras MCF10A cells overexpressing ErbB2. The present study elucidating the molecular mechanism underlying ErbB2-induced promotion of H-Ras MCF10A cell invasion may provide invaluable information for understanding breast cancer progression and establishing therapeutic interventions for breast cancer.

The p38 MAPK inhibitors for the treatment of inflammatory diseases and cancer.

BACKGROUND: The p38 mitogen-activated protein kinase (MAPK) is activated by various pro-inflammatory and stressful stimuli. Mounting evidence suggests that the p38 MAPK signaling cascade is involved in various biological responses other than inflammation such as cell proliferation, differentiation, apoptosis and invasion, suggesting that the p38 MAPK can serve as a potential therapeutic target for the treatment of not only inflammatory diseases but also cancer. METHODS: The unique characteristics of p38 MAPK are summarized with regard to activation and function of p38 MAPK signaling cascades. We then discuss the involvement of p38 MAPK in diseases and the implications of the possible therapeutic use of p38 MAPK inhibitors. The p38 MAPK inhibitors that have been used in the in vitro/in vivo systems as well as in the clinical trials are summarized. RESULTS/CONCLUSION: The p38 MAPK plays an important role in key cellular processes related to inflammation and cancer. Understanding the signal transduction mechanisms and gene regulation by p38 MAPK provides useful information in the development of p38 MAPK inhibitors with therapeutic benefits with reduced side effects. In this review, we summarize and present the list of p38 MAPK inhibitors in in vitro/in vivo studies as well as in clinical trials.

Overexpression of ErbB2 induces invasion of MCF10A human breast epithelial cells via MMP-9.

Metastasis is the principal cause of death from breast cancer. ErbB2 (HER-2/neu) has been identified as an important regulator of metastatic potential of breast cancer. The present study investigated the molecular mechanism underlying the role of ErbB2 in malignant phenotypic conversion of MCF10A human breast epithelial cells which originally have 'normal' cell character. Here we report that ErbB2 induces invasion and migration of MCF10A cells though up-regulation of matrix metalloproteinase (MMP)-9. We also observed a marked reduction of an epithelial cell marker, E-cadherin, and an induction of vimentin in ErbB2-MCF10A cells, suggesting that epithelial-mesenchymal transition may play a role in the ErbB2-induced invasion and migration of MCF10A cells. Overexpression of ErbB2 significantly activated p38 MAPK and Akt, while Raf-1/MEK/ERK pathway was not activated by ErbB2. Using pharmacological inhibitors, we further show that p38 MAPK and Akt signaling pathways are crucial for the ErbB2-induced MMP-9 up-regulation, invasion and migration of MCF10A cells. Given that ErbB2 is one of the most important oncogenes in human breast cancer and thus is an attractive therapeutic target, our findings may provide a molecular basis for the promoting role of ErbB2 in breast cancer progression.