Sung Gil Chi

Caveolin-1 Increases Aerobic Glycolysis in Colorectal Cancers by Stimulating HMGA1-Mediated GLUT3 Transcription

Caveolin-1 (CAV1) acts as a growth suppressor in various human malignancies, but its expression is elevated in many advanced cancers, suggesting the oncogenic switch of its role during tumor progression. To understand the molecular basis for the growth-promoting function of CAV1, we characterized its expression status, differential roles for tumor growth, and effect on glucose metabolism in colorectal cancers. Abnormal elevation of CAV1 was detected in a substantial fraction of primary tumors and cell lines and tightly correlated with promoter CpG sites hypomethylation. Depletion of elevated CAV1 led to AMPK activation followed by a p53-dependent G1 cell-cycle arrest and autophagy, suggesting that elevated CAV1 may contribute to ATP generation. Furthermore, CAV1 depletion downregulated glucose uptake, lactate accumulation, and intracellular ATP level, supporting that aerobic glycolysis is enhanced by CAV1. Consistently, CAV1 was shown to stimulate GLUT3 transcription via an HMGA1-binding site within the GLUT3 promoter. HMGA1 was found to interact with and activate the GLUT3 promoter and CAV1 increased the HMGA1 activity by enhancing its nuclear localization. Ectopic expression of HMGA1 increased glucose uptake, whereas its knockdown caused AMPK activation. In addition, GLUT3 expression was strongly induced by cotransfection of CAV1 and HMGA1, and its overexpression was observed predominantly in tumors harboring high levels of CAV1 and HMGA1. Together, these data show that elevated CAV1 upregulates glucose uptake and ATP production through HMGA1-mediated GLUT3 transcription, suggesting that CAV1 may render tumor cells growth advantages by enhancing aerobic glycolysis.

Mutational abrogation of the PTEN/MMAC1 gene in gastrointestinal polyps in patients with Cowden disease

BACKGROUND & AIMS To understand the molecular etiology of Cowden disease-associated gastrointestinal polyps, we analyzed the mutational status of PTEN/MMAC1, a recently identified Cowden disease gene located at 10q23, in gastric hamartomas, colonic adenoma, and juvenile polyps of 3 patients with Cowden disease. METHODS Messenger RNA expression, gene deletion, and sequence alteration of PTEN/MMAC1 were evaluated by quantitative polymerease chain reaction (PCR), PCR-single-strand conformation polymorphism, and sequencing analysis. RESULTS Germline missense mutation at codon 289 (AAA to GAA, Lys to Glu) and deletion of the wild-type allele were detected in the polyps of 2 patients with Cowden disease in the same family. Germline allelic deletion and transcriptional silencing of the remaining allele, probably caused by abnormal methylation, were also observed in a gastric hamartoma of 1 patient. CONCLUSIONS The germline mutation and alteration of the remaining allele observed in this study strongly support that PTEN/MMAC1 functions as a tumor suppressor in Cowden disease. This study is the first to show that the mutational abrogation of PTEN/MMAC1 plays a causal role in the genesis of gastrointestinal polyps in Cowden disease, providing molecular genetic evidence that colonic adenoma, juvenile polyp, and gastric hamartoma could be included in the manifestations of Cowden disease.

Caveolin-1 is associated with VCAM-1 dependent adhesion of gastric cancer cells to endothelial cells.

Background/Aims: Cell adhesion molecules play a critical role in the invasion and metastasis of a variety of human tumors. Abnormal expression of VCAM-1 has been demonstrated to correlate with the malignant progression of gastric tumors, but the molecular mechanism underlying the VCAM-1-dependent metastasis has been rarely investigated. To explore the role for tumor cell-expressing adhesion molecules in the carcinoma-endothelium adhesion, we analyzed expression status of adhesion molecules in gastric cancer cells and its association with tumor cell capability of endothelial adhesion. Methods: Endothelial adhesion ability of gastric tumor cells was tested using calcein AM staining assay. Expression of cell surface proteins was determined by Western blot, flow cytometry, and immunofluorescence assays. RNAi-mediated knockdown of gene expression and neutralization with specific antibodies were utilized for functional analysis. Results: One of three cell lines tested was identified to be adhesive to endothelial cells and express VCAM-1. Adherence ability of the cells was dramatically decreased by neutralization of surface VCAM-1. VCAM-1 was co-localized with Caveolin-1 and siRNA-mediated knockdown of Caveolin-1 expression significantly blocked the VCAM-1-dependent cell adhesion. Conclusions: Our data imply important roles for VCAM-1 and Caveolin- 1 in the regulation of metastatic potential of gastric tumor cells.

Epigenetic alteration of PRKCDBP in colorectal cancers and its implication in tumor cell resistance to TNFα-induced apoptosis

Purpose: PRKCDBP is a putative tumor suppressor in which alteration has been observed in several human cancers. We investigated expression and function of PRKCDBP in colorectal cells and tissues to explore its candidacy as a suppressor in colorectal tumorigenesis. Experimental Design: Expression and methylation status of PRKCDBP and its effect on tumor growth were evaluated. Transcriptional regulation by NF-κB signaling was defined by luciferase reporter and chromatin immunoprecipitation assays. Results: PRKCDBP expression was hardly detectable in 29 of 80 (36%) primary tumors and 11 of 19 (58%) cell lines, and its alteration correlated with tumor stage and grade. Promoter hypermethylation was commonly found in cancers. PRKCDBP expression induced the G1 cell-cycle arrest and increased cellular sensitivity to various apoptotic stresses. PRKCDBP was induced by TNFα, and its level correlated with tumor cell sensitivity to TNFα-induced apoptosis. PRKCDBP induction by TNFα was disrupted by blocking NF-κB signaling while it was enhanced by RelA transfection. The PRKCDBP promoter activity was increased in response to TNFα, and this response was abolished by disruption of a κB site in the promoter. PRKCDBP delayed the formation and growth of xenograft tumors and improved tumor response to TNFα-induced apoptosis. Conclusions: PRKCDBP is a proapoptotic tumor suppressor which is commonly altered in colorectal cancer by promoter hypermethylation, and its gene transcription is directly activated by NF-κB in response to TNFα. This suggests that PRKCDBP inactivation may contribute to tumor progression by reducing cellular sensitivity to TNFα and other stresses, particularly under chronic inflammatory microenvironment. Clin Cancer Res; 17(24); 7551–62. ©2011 AACR.

X-Linked Inhibitor of Apoptosis Protein Is an Important Regulator of Vascular Endothelial Growth Factor–Dependent Bovine Aortic Endothelial Cell Survival

Vascular endothelial growth factor (VEGF) is a critical regulator of endothelial cell biology and vascular function. Chronic VEGF treatment has been shown to inhibit tumor necrosis factor–induced apoptosis in endothelial cells. However, the mechanism for this cell survival is unclear. Interestingly, VEGF also enhances the expression of X-linked inhibitor of apoptosis (XIAP), a well-established antiapoptotic factor. XIAP has been shown to suppress apoptosis by blocking caspase activity in cancer cells, but it remains under studied in the endothelium. Therefore, we hypothesized that VEGF affects important endothelial functions, such as apoptosis and cell migration, by regulating XIAP expression and downstream caspase activity. To test this hypothesis, caspase activity, apoptosis, and cell migration were assessed following XIAP overexpression or depletion in bovine aortic endothelial cells. Much like VEGF treatment, ectopic expression of XIAP blocked tumor necrosis factor–induced apoptosis. Surprisingly, the mechanism was caspase-independent. In addition, XIAP-associated cell survival was the result of enhanced nitric oxide (NO) production, and XIAP was partially localized in caveolae. In these lipid rafts, XIAP interacted with a regulator of NO production, caveolin-1, via a binding motif (FtFgtwiY, where the bold letters represent aromatic amino acids) in the baculoviral IAP repeat-3 domain. Endothelial NO synthase binding to caveolin-1 was competitively inhibited by XIAP, suggesting that XIAP acts as a modulator of NO production by releasing endothelial NO synthase from caveolin-1. Further studies showed that endothelial cell migration was also controlled by XIAP-dependent NO. Taken together, these results suggest that XIAP plays a novel role in endothelial cells, interacting with caveolin-1 and acting as a regulator of vascular antiatherogenic function.

Translation initiation on mRNAs bound by nuclear cap-binding protein complex CBP80/20 requires interaction between CBP80/20-dependent translation initiation factor and eukaryotic translation initiation factor 3g.

Background: How the eIF3 complex and ribosomes are recruited during translation on CBP80/20-bound mRNAs remains obscure. Results: CTIF interacts with eIF3g to recruit the eIF3 complex. Conclusion: Translation on CBP80/20-bound mRNAs requires CTIF-eIF3g interaction. Significance: The use of different eIF3 subunits for recruiting eIF3 complex implies that translation on CBP80/20-bound mRNAs differs mechanistically from translation on eIF4E-bound mRNAs. In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.

Exon junction complex enhances translation of spliced mRNAs at multiple steps

Translation of spliced mRNAs is enhanced by exon junction complex (EJC), which is deposited on mRNAs as a result of splicing. Although this phenomenon itself is well known, the underlying molecular mechanism remains poorly understood. Here we show, using siRNAs against Y14 and eIF4AIII and spliced or intronless constructs that contain different types of internal ribosome entry sites (IRESes), that Y14 and eIF4AIII increase translation of spliced mRNAs before and after formation of the 80S ribosome complex, respectively. These results suggest that EJC modulates translation of spliced mRNA at multiple steps.

X-linked inhibitor of apoptosis protein controls α5-integrin-mediated cell adhesion and migration

The association of integrins with caveolin-1 regulates cell adhesion. However, the vascular ramifications of this association remain to be clearly determined. We recently reported that the X chromosome-linked inhibitor of apoptosis protein (XIAP)-caveolin-1 interaction is critical to endothelial cell survival. Thus, we hypothesized that XIAP performs a crucial function in integrin/caveolin-1-mediated endothelial cell survival. In this study, we demonstrated that XIAP is recruited into the alpha(5)-integrin complex via caveolin-1 binding and mediates cell adhesion. We also determined that XIAP is critical to shear stress-stimulated ERK activation in an alpha(5)-integrin-dependent manner but is not important to VEGF-induced ERK activation. This differential activation of ERK is partly attributable to unique localizations of the receptors. Furthermore, we confirmed that XIAP is an essential molecule in the efficient recruitment of focal adhesion kinase (FAK) into the alpha(5)-integrin-associated complex. This alpha(5)-integrin-caveolin-1-XIAP-FAK multicomplex regulates endothelial cell migration via a mechanism that involves shear-dependent ERK activation. Together, our results indicate that XIAP stabilizes the alpha(5)-integrin-associated focal adhesion complex, thereby further regulating endothelial cell adhesion and migration. The findings of this study provide us with greater insight into the molecular mechanisms underlying the control of vascular function by integrins.

Ago2/miRISC-mediated inhibition of CBP80/20-dependent translation and thereby abrogation of nonsense-mediated mRNA decay require the cap-associating activity of Ago2

AGO2 physically interacts with GW182 by anti tag coimmunoprecipitation (View interaction)

Mitochondrial oxidative phosphorylation system is recruited to detergent‐resistant lipid rafts during myogenesis

Since detergent‐resistant lipid rafts play important roles in the signal transduction for myogenesis, their comprehensive proteomic analysis could provide new insights to understand their function in myotubes. Here, the detergent‐resistant lipid rafts were isolated from C2C12 myotubes and analyzed by capillary RPLC/MS/MS. Among the 327 proteins (or protein groups) identified, 28% were categorized to the plasma membrane or raft proteins, 29% to mitochondria, 20% to microsomal proteins, 10% to other proteins, and 13% to unknown proteins. The localization of oxidative phosphorylation (OXPHOS) complexes in the sarcolemma lipid rafts was further confirmed from C2C12 myotubes by cellular fractionation, surface‐biotin labeling, immunofluorescence, and lipid raft fractionation. After adding exogenous cytochrome c, the sarcolemma isolated from myotubes had an ability to consume oxygen in the presence of NADH or succinate. The generation of NADH‐dependent extracellular superoxide was increased by inhibiting or downregulating OXPHOS I, III, and IV in myotubes, indicating that OXPHOS proteins are major sources for extracellular ROS in skeletal muscle. With all these data, we can conclude that OXPHOS proteins are associated with the sarcolemma lipid rafts during C2C12 myogenesis to generate extracellular ROS.