Hsiu-Ni Kung

Glutamine Synthetase Is a Genetic Determinant of Cell Type–Specific Glutamine Independence in Breast Epithelia

Although significant variations in the metabolic profiles exist among different cells, little is understood in terms of genetic regulations of such cell type–specific metabolic phenotypes and nutrient requirements. While many cancer cells depend on exogenous glutamine for survival to justify the therapeutic targeting of glutamine metabolism, the mechanisms of glutamine dependence and likely response and resistance of such glutamine-targeting strategies among cancers are largely unknown. In this study, we have found a systematic variation in the glutamine dependence among breast tumor subtypes associated with mammary differentiation: basal- but not luminal-type breast cells are more glutamine-dependent and may be susceptible to glutamine-targeting therapeutics. Glutamine independence of luminal-type cells is associated mechanistically with lineage-specific expression of glutamine synthetase (GS). Luminal cells can also rescue basal cells in co-culture without glutamine, indicating a potential for glutamine symbiosis within breast ducts. The luminal-specific expression of GS is directly induced by GATA3 and represses glutaminase expression. Such distinct glutamine dependency and metabolic symbiosis is coupled with the acquisition of the GS and glutamine independence during the mammary differentiation program. Understanding the genetic circuitry governing distinct metabolic patterns is relevant to many symbiotic relationships among different cells and organisms. In addition, the ability of GS to predict patterns of glutamine metabolism and dependency among tumors is also crucial in the rational design and application of glutamine and other metabolic pathway targeted therapies.

Acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress.

BackgroundA variety of oncogenic and environmental factors alter tumor metabolism to serve the distinct cellular biosynthetic and bioenergetic needs present during oncogenesis. Extracellular acidosis is a common microenvironmental stress in solid tumors, but little is known about its metabolic influence, particularly when present in the absence of hypoxia. In order to characterize the extent of tumor cell metabolic adaptations to acidosis, we employed stable isotope tracers to examine how acidosis impacts glucose, glutamine, and palmitate metabolism in breast cancer cells exposed to extracellular acidosis.ResultsAcidosis increased both glutaminolysis and fatty acid β-oxidation, which contribute metabolic intermediates to drive the tricarboxylic acid cycle (TCA cycle) and ATP generation. Acidosis also led to a decoupling of glutaminolysis and novel glutathione (GSH) synthesis by repressing GCLC/GCLM expression. We further found that acidosis redirects glucose away from lactate production and towards the oxidative branch of the pentose phosphate pathway (PPP). These changes all serve to increase nicotinamide adenine dinucleotide phosphate (NADPH) production and counter the increase in reactive oxygen species (ROS) present under acidosis. The reduced novel GSH synthesis under acidosis may explain the increased demand for NADPH to recycle existing pools of GSH. Interestingly, acidosis also disconnected novel ribose synthesis from the oxidative PPP, seemingly to reroute PPP metabolites to the TCA cycle. Finally, we found that acidosis activates p53, which contributes to both the enhanced PPP and increased glutaminolysis, at least in part, through the induction of G6PD and GLS2 genes.ConclusionsAcidosis alters the cellular metabolism of several major metabolites, which induces a significant degree of metabolic inflexibility. Cells exposed to acidosis largely rely upon mitochondrial metabolism for energy generation to the extent that metabolic intermediates are redirected away from several other critical metabolic processes, including ribose and glutathione synthesis. These alterations lead to both a decrease in cellular proliferation and increased sensitivity to ROS. Collectively, these data reveal a role for p53 in cellular metabolic reprogramming under acidosis, in order to permit increased bioenergetic capacity and ROS neutralization. Understanding the metabolic adaptations that cancer cells make under acidosis may present opportunities to generate anti-tumor therapeutic agents that are more tumor-specific.

The ESCRT Machinery Is Recruited by the Viral BFRF1 Protein to the Nucleus-Associated Membrane for the Maturation of Epstein-Barr Virus

The cellular endosomal sorting complex required for transport (ESCRT) machinery participates in membrane scission and cytoplasmic budding of many RNA viruses. Here, we found that expression of dominant negative ESCRT proteins caused a blockade of Epstein-Barr virus (EBV) release and retention of viral BFRF1 at the nuclear envelope. The ESCRT adaptor protein Alix was redistributed and partially colocalized with BFRF1 at the nuclear rim of virus replicating cells. Following transient transfection, BFRF1 associated with ESCRT proteins, reorganized the nuclear membrane and induced perinuclear vesicle formation. Multiple domains within BFRF1 mediated vesicle formation and Alix recruitment, whereas both Bro and PRR domains of Alix interacted with BFRF1. Inhibition of ESCRT machinery abolished BFRF1-induced vesicle formation, leading to the accumulation of viral DNA and capsid proteins in the nucleus of EBV-replicating cells. Overall, data here suggest that BFRF1 recruits the ESCRT components to modulate nuclear envelope for the nuclear egress of EBV.

Analysis of tumor environmental response and oncogenic pathway activation identifies distinct basal and luminal features in HER2-related breast tumor subtypes

IntroductionBreast cancer heterogeneity occurs as a consequence of the dysregulation of numerous oncogenic pathways as well as many non-genetic factors, including tumor microenvironmental stresses such as hypoxia, lactic acidosis, and glucose deprivation. Although the importance of these non-genetic factors is well recognized, it is not clear how to integrate these factors within the genetic framework of cancer as the next logical step in understanding tumor heterogeneity.MethodsWe report here the development of a series of gene expression signatures to measure the influences of microenvironmental stresses. The pathway activities of hypoxia, lactic acidosis, acidosis and glucose deprivation were investigated in a collection of 1,143 breast tumors, which have been separated into 17 breast tumor subgroups defined by their distinct patterns of oncogenic pathways. A validation dataset comprised of 547 breast tumors was also used to confirm the major findings, and representative breast cancer cell lines were utilized to validate in silico results and mechanistic studies.ResultsThrough the integrative pathway analysis of microenvironmental stresses and oncogenic events in breast tumors, we identified many known and novel correlations between these two sources of tumor heterogeneity. Focusing on differences between two human epidermal growth factor receptor 2 (HER2)-related subgroups, previously identified based on patterns of oncogenic pathway activity, we determined that these subgroups differ with regards to tumor microenvironmental signatures, including hypoxia. We further demonstrate that each of these subgroups have features consistent with basal and luminal breast tumors including patterns of oncogenic signaling pathways, expression of subtype specific genes, and cellular mechanisms that regulate the hypoxia response. Importantly, we also demonstrate that the correlated pattern of hypoxia-related gene expression and basal-associated gene expression are consistent across HER2-related tumors whether we analyze the tumors as a function of our pathway-based classification scheme, using the intrinsic gene list (ERBB2+), or based on HER2 IHC status. Our results demonstrate a cell lineage-specific phenomenon in which basal-like tumors, HER2-related tumors with high hypoxia, as well as normal basal epithelial cells express increased mRNA levels of HIF-1α compared to luminal types and silencing of HIF-1α results in decreased expression of hypoxia-induced genes.ConclusionsThis study demonstrates differences in microenvironmental conditions in HER2-related subgroups defined by distinct oncogenic pathway activities, and provides a mechanistic explanation for differences in the observed hypoxia response between these subgroups. Collectively, these data demonstrate the potential of a pathway-based classification strategy as a framework to integrate genetic and non-genetic factors to investigate the basis of tumor heterogeneity.

Involvement of NO/cGMP signaling in the apoptotic and anti-angiogenic effects of β-lapachone on endothelial cells in vitro

Neovascularization is an essential process in tumor development, it is conceivable that anti‐angiogenic treatment may block tumor growth. In angiogenesis, nitric oxide (NO) is an important factor which mediates vascular endothelial cell growth and migration. β‐Lapachone (3,4‐dihydro‐2,2‐dimethyl‐2H‐naphtho‐[1,2‐b]pyran‐5,6‐dione), a natural product extracted from the lapacho tree (Tabebuia avellanedae), has been demonstrated to possess anti‐cancer and anti‐viral effects. Whether β‐lapachone can induce endothelial cell death or has an anti‐angiogenic effect is still an enigma. We investigated the in vitro effect of β‐lapachone on endothelial cells, including human vascular endothelial cell line, EAhy926, and human umbilical vascular endothelial cells (HUVEC). Our results revealed that (1) the intracellular cGMP levels and the mitochondria membrane potential (MMP) decreased, and calpain and caspases were activated, during β‐lapachone‐induced endothelial cell death; (2) co‐treatment with calpain inhibitors (ALLM or ALLN) or the intracellular calcium chelator, BAPTA, but not the general caspase inhibitor, zVAD‐fmk, provided significant protection against apoptosis by preventing the β‐lapachone‐induced MMP decrease and cytoplasmic calcium increase; (3) addition of NO downregulated the β‐lapachone‐induced cGMP depletion and protected the cells from apoptosis by blocking the MMP decrease and the calcium increase; and (4) exogenous NO protects endothelial cells against the cell death induced by β‐lapachone, but not the anti‐angiogenic effect. From all the data above, we demonstrated that NO can attenuate the apoptotic effect of β‐lapachone on human endothelial cells and suggest that β‐lapachone may have potential as an anti‐angiogenic drug. J. Cell. Physiol. 211: 522–532, 2007.

Latent Factor Analysis to Discover Pathway-Associated Putative Segmental Aneuploidies in Human Cancers

Tumor microenvironmental stresses, such as hypoxia and lactic acidosis, play important roles in tumor progression. Although gene signatures reflecting the influence of these stresses are powerful approaches to link expression with phenotypes, they do not fully reflect the complexity of human cancers. Here, we describe the use of latent factor models to further dissect the stress gene signatures in a breast cancer expression dataset. The genes in these latent factors are coordinately expressed in tumors and depict distinct, interacting components of the biological processes. The genes in several latent factors are highly enriched in chromosomal locations. When these factors are analyzed in independent datasets with gene expression and array CGH data, the expression values of these factors are highly correlated with copy number alterations (CNAs) of the corresponding BAC clones in both the cell lines and tumors. Therefore, variation in the expression of these pathway-associated factors is at least partially caused by variation in gene dosage and CNAs among breast cancers. We have also found the expression of two latent factors without any chromosomal enrichment is highly associated with 12q CNA, likely an instance of “trans”-variations in which CNA leads to the variations in gene expression outside of the CNA region. In addition, we have found that factor 26 (1q CNA) is negatively correlated with HIF-1α protein and hypoxia pathways in breast tumors and cell lines. This agrees with, and for the first time links, known good prognosis associated with both a low hypoxia signature and the presence of CNA in this region. Taken together, these results suggest the possibility that tumor segmental aneuploidy makes significant contributions to variation in the lactic acidosis/hypoxia gene signatures in human cancers and demonstrate that latent factor analysis is a powerful means to uncover such a linkage.

In vitro and in vivo wound healing-promoting activities of β-lapachone

Impaired wound healing is a serious problem for diabetic patients. Wound healing is a complex process that requires the cooperation of many cell types, including keratinocytes, fibroblasts, endothelial cells, and macrophages. beta-Lapachone, a natural compound extracted from the bark of the lapacho tree (Tabebuia avellanedae), is well known for its antitumor, antiinflammatory, and antineoplastic effects at different concentrations and conditions, but its effects on wound healing have not been studied. The purpose of the present study was to investigate the effects of beta-lapachone on wound healing and its underlying mechanism. In the present study, we demonstrated that a low dose of beta-lapachone enhanced the proliferation in several cells, facilitated the migration of mouse 3T3 fibroblasts and human endothelial EAhy926 cells through different MAPK signaling pathways, and accelerated scrape-wound healing in vitro. Application of ointment with or without beta-lapachone to a punched wound in normal and diabetic (db/db) mice showed that the healing process was faster in beta-lapachone-treated animals than in those treated with vehicle only. In addition, beta-lapachone induced macrophages to release VEGF and EGF, which are beneficial for growth of many cells. Our results showed that beta-lapachone can increase cell proliferation, including keratinocytes, fibroblasts, and endothelial cells, and migration of fibroblasts and endothelial cells and thus accelerate wound healing. Therefore, we suggest that beta-lapachone may have potential for therapeutic use for wound healing.

Sulindac Compounds Facilitate the Cytotoxicity of β-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

β-lapachone, a major component in an ethanol extract of Tabebuia avellanedae bark, is a promising potential therapeutic drug for various tumors, including lung cancer, the leading cause of cancer-related deaths worldwide. In the first part of this study, we found that apoptotic cell death induced in lung cancer cells by high concentrations of β-lapachone was mediated by increased activation of the pro-apoptotic factor JNK and decreased activation of the cell survival/proliferation factors PI3K, AKT, and ERK. In addition, β-lapachone toxicity was positively correlated with the expression and activity of NAD(P)H quinone oxidoreductase 1 (NQO1) in the tumor cells. In the second part, we found that the FDA-approved non-steroidal anti-inflammatory drug sulindac and its metabolites, sulindac sulfide and sulindac sulfone, increased NQO1 expression and activity in the lung adenocarcinoma cell lines CL1-1 and CL1-5, which have lower NQO1 levels and lower sensitivity to β-lapachone treatment than the A549 cell lines, and that inhibition of NQO1 by either dicoumarol treatment or NQO1 siRNA knockdown inhibited this sulindac-induced increase in β-lapachone cytotoxicity. In conclusion, sulindac and its metabolites synergistically increase the anticancer effects of β-lapachone primarily by increasing NQO1 activity and expression, and these two drugs may provide a novel combination therapy for lung cancers.

Acidic extracellular pH induces p120-catenin-mediated disruption of adherens junctions via the Src kinase-PKCδ pathway.

An acidic microenvironment induces disruption of adherens junctions (AJs) of hepatoma cells. This study investigated the impact of an acidic extracellular pH (pHe) on p120‐catenin (p120‐ctn) serine phosphorylation. pH 6.6 treatment increased intracellular calcium levels, activated protein kinase C (PKC)α and PKCδ, and decreased serine phosphorylation of p120‐ctn. Further knockdown of PKCα and δ by small interference RNA (siRNA) prevented the pH 6.6‐induced downregulation of p120‐ctn at AJ and the serine dephosphorylation of p120‐ctn. Moreover, PP2 pretreatment and siRNA of c‐Src abrogated the pH 6.6‐induced PKCδ activation. Together, the c‐Src‐PKCδ cascade and PKCα regulate the acidic pHe‐induced AJ disruption.

The lipopolysaccharide-induced pro-inflammatory response in RAW264.7 cells is attenuated by an unsaturated fatty acid- bovine serum albumin complex and enhanced by a saturated fatty acid-bovine serum albumin complex

ObjectiveA 1:1 ratio of fatty acid (FA)–albumin complex was chosen to mimic physiological conditions, and the effects of FA–bovine serum albumin (BSA) complexes were tested in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells.MethodsNitric oxide (NO) and various proteins/factors in RAW264.7 cells were quantified as follows: NO by the Griess assay; prostaglandin (PG) E2, interleukin (IL)-6 and tumor necrosis factor (TNF)-α by ELISA; inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 by Western blotting; and NF-κB and CD14/TLR4 by Western blotting or flow cytometry.ResultsBSA- or FA–BSA-treated RAW264.7 cells without LPS stimulation did not show any significant changes in NO or the tested proteins/factors and thus did not have any pro-inflammatory responses. Pre-treatment with unsaturated FA–BSA complexes significantly decreased the production of LPS-induced NO, PGE2, IL-6 and TNF-α, the expression of iNOS, COX-2 and CD14, IκB degradation and NF-κB translocation. On the contrary, pre-treatment with saturated FA–BSA complexes enhanced these LPS-induced pro-inflammatory factors and the subsequent responses.ConclusionsWe concluded that unsaturated FA–BSA complexes, but not saturated FA–BSA complexes, exert an inhibitory effect on the LPS-induced pro-inflammatory response and that this effect may be partially mediated through suppression of the NF-κB signaling pathway. We suggest that an increase of unsaturated FA–BSA complexes may enhance the host’s defense against bacterial infection.