ABSTRACT
NFAT1 is a transcription factor that elicits breast carcinoma cells to become invasive, thus contributing to metastasis. The molecular mechanisms by which NFAT1 operates in this respect are still poorly known. Here, we report that NFAT1 increases lipocalin 2 (LCN2) mRNA and protein expression by binding to specific sites in the LCN2 gene promoter region. We show that the LCN2 protein is required downstream of NFAT1 to increase breast cancer cell invasion. We demonstrate that the NFAT1-LCN2 axis is sufficient to regulate expression of the TNF-like receptor TWEAKR at the RNA level and of its ligand, TWEAK, at the protein level. We show, however, that TWEAKR mediates an anti-invasive effect in breast cancer cells whereas, depending on LCN2 expression, TWEAK has either anti- or pro-invasive capacities. Thus, we identify LCN2 and TWEAKR-TWEAK as crucial downstream effectors of NFAT1 that regulate breast cancer cell motility and invasive capacity.
Subject(s)
Acute-Phase Proteins/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Lipocalins/metabolism , NFATC Transcription Factors/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factors/metabolism , Acute-Phase Proteins/genetics , Animals , Breast Neoplasms/genetics , Cell Line, Tumor , Cytokine TWEAK , Female , Gene Expression Regulation, Neoplastic , Humans , Ligands , Lipocalin-2 , Lipocalins/genetics , Mice , Models, Biological , NIH 3T3 Cells , Neoplasm Invasiveness , Promoter Regions, Genetic/genetics , Protein Binding , Proto-Oncogene Proteins/genetics , Receptors, Tumor Necrosis Factor/genetics , TWEAK Receptor , Tumor Necrosis Factors/genetics , Up-Regulation/geneticsABSTRACT
Glutathione S-transferase P1-1 (GSTP1-1) is a phase II drug metabolism enzyme implicated in carcinogenesis and development of resistance to anti-cancer drugs. It was previously shown that both activating protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) are involved in its regulation. In the present study we examined the inhibitory effect of several chemopreventive agents on the tumor necrosis factor (TNF) alpha- or 12-O-tetradecanoylphorbol 13 acetate (TPA)-induced promoter activity of GSTP1-1, as demonstrated by transient transfection experiments in K562 and U937 leukemia cells. Our results provide evidence for a differential effect of chemopreventive agents such as beta-lapachone, emodin, sanguinarine and capsaicin, which significantly inhibit reporter gene expression as well as TNFalpha- and TPA-induced binding of AP-1 and NF-kappaB, whereas trans-anethole and silymarin do not produce any inhibitory effect. Our results demonstrate the ability of selected chemopreventive agents to decrease GSTP1-1 gene expression mechanisms and could thus contribute to reduce the incidence of glutathione related drug resistance in human leukemia.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , Gene Expression/drug effects , Glutathione Transferase/metabolism , Isoenzymes/metabolism , NF-kappa B/antagonists & inhibitors , Transcription Factor AP-1/antagonists & inhibitors , Chemoprevention , Drug Interactions , Glutathione S-Transferase pi , Glutathione Transferase/genetics , Humans , Isoenzymes/genetics , K562 Cells , NF-kappa B/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factor AP-1/metabolism , Tumor Necrosis Factor-alpha/pharmacology , U937 CellsABSTRACT
Phosphoglycerate mutases catalyze the interconversion of 2- and 3-phosphoglycerate in the glycolytic and gluconeogenic pathways. They exist in two unrelated forms that are either cofactor (2,3-diphosphoglycerate)-dependent or cofactor-independent. The two enzymes have no similarity in amino acid sequence, tertiary structure, or catalytic mechanism. Certain organisms including vertebrates have only the cofactor-dependent form, whereas other organisms can possess the independent form or both. Caenorhabditis elegans has been predicted to have only independent phosphoglycerate mutase. In this study, we have cloned and produced recombinant, independent phosphoglycerate mutases from C. elegans and the human-parasitic nematode Brugia malayi. They are 70% identical to each other and related to known bacterial, fungal, and protozoan enzymes. The nematode enzymes possess the catalytic serine, and other key amino acids proposed for catalysis and recombinant enzymes showed typical phosphoglycerate mutase activities in both the glycolytic and gluconeogenic directions. The gene is essential in C. elegans, because the reduction of its activity by RNA interference led to embryonic lethality, larval lethality, and abnormal body morphology. Promoter reporter analysis indicated widespread expression in larval and adult C. elegans with the highest levels apparent in the nerve ring, intestine, and body wall muscles. The enzyme was found in a diverse group of nematodes representing the major clades, indicating that it is conserved throughout this phylum. Our results demonstrate that nematodes, unlike vertebrates, utilize independent phosphoglycerate mutase in glycolytic and gluconeogenic pathways and that the enzyme is probably essential for all nematodes.