Glial tumors progress to malignant grades by heightened proliferation and relentless dispersion throughout the central nervous system. Understanding genetic and biochemical processes that foster these behaviors is likely to reveal specific and effective targets for therapeutic intervention. Our current report shows that the fibroblast growth factor-inducible 14 (Fn14), a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Forced Fn14 overexpression stimulates glioma cell migration and invasion, and depletion of Rac1 by small interfering RNA inhibits this cellular response. Activation of Fn14 signaling by the ligand TNF-like weak inducer of apoptosis (TWEAK) stimulates migration and up-regulates expression of Fn14; this TWEAK effect requires Rac1 and nuclear factor-KB (NF-KB) activity. The Fn14 promoter region contains NF-KB binding sites, which mediate positive feedback causing sustained overexpression of Fn14 and enduring glioma cell invasion. Furthermore, Fn14 gene expression levels increase with glioma grade and inversely correlate with patient survival. These results show that the Fn14 cascade operates as a positive feedback mechanism for elevated and sustained Fn14 expression. Such a feedback loop argues for aggressive targeting of the Fn14 axis as a unique and specific driver of glioma malignant behavior.

Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is an extremely rare, aggressive cancer affecting children and young women. We identified germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 69% (9/13) of SCCOHT cases in addition to SMARCA4 protein loss in 82% (14/17) of SCCOHT tumors but in only 0.4% (2/485) of other primary ovarian tumors. These data implicate SMARCA4 in SCCOHT oncogenesis.

The G protein-coupled receptor GPR30 binds 17β-estradiol (E 2 ) yet differs from classic estrogen receptors (ERα and ERβ). GPR30 can mediate E 2 -induced nongenomic signaling, but its role in ERα-positive breast cancer remains unclear. Gene expression microarray data from five cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERα-positive status. We therefore examined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol (DES), ligands that selectively activate GPR30 and ER, respectively, and small interfering RNAs. In expression studies, E 2 and DES, but not G-1, transiently downregulated both ER and GPR30, indicating

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2009.Contributed by V. Craig Jordan, September 14, 2011 (sent for review June 21, 2011) In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17β-estradiol (E 2 ) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E 2 paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E 2 -induced apoptosis by analysis of gene expression across time (2-96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E 2 -induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E 2 in 5C cells compared with both WS8 and 2A cells and hence, were associated with E 2 -induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E 2 inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E 2 -induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E 2 -inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E 2 interacted to superadditively induce apoptosis. Therefore, these data indicate that E 2 induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.aromatase inhibitor | antihormonal resistance | estrogen receptor | gene expression microarrays | selective estrogen receptor modulator E lucidation of the basic structure function relationships of synthetic estrogens based on either stilbene (1) or triphenylethylene (2) was a landmark achievement that continues to have major therapeutic implications to this day. The first successful chemical therapy for the treatment of any cancer was the use of high-dose synthetic estrogen for the treatment of metastatic breast cancer (3). Response rates for patients who were more than a decade beyond menopause were about 30%. Importantly, treatment near menopause was ineffective, and therefore, tumor responsiveness was related to the duration of estrogen deprivation. In 1970, Alexander Haddow commented that "the extraordinary extent of tumor regression observed in...

Cells of Pseudomonas aeruginosa secrete a fluorescent yellow-green siderophore, pyoverdine, when grown under iron-deficient conditions. We describe here the cloning and characterization of a gene, pvdS, which is required for this process. The pvdS gene is required for expression from promoters of at least two pyoverdine synthesis genes and can cause expression from these promoters in Escherichia coli, where they are otherwise inactive. Sequencing of pvdS revealed that it is a member of a subfamily of RNA polymerase sigma factors which direct the synthesis of extracellular products by bacteria. The pvdS gene is expressed only in iron-starved bacteria, and in E. coli cells at least, expression is regulated by the Fur repressor protein. We propose that in iron-rich cells of P. aeruginosa, Fur binds to the pvdS promoter and prevents expression of the gene; under conditions of iron starvation, repression is relieved and PvdS is made, reprogramming the cells for pyoverdine synthesis.Fluorescent pseudomonads are characterized by the production of yellow-green fluorescent pigments. These act as siderophores and are variously termed pyoverdines or pseudobactins. The fluorescence and color are due to the presence of a dihydroxyquinoline group; this is attached to a peptide of 6 to 12 amino acids, and the exact nature of the peptide is species or strain specific (5). A pyoverdine-deficient mutant of the mammalian pathogen Pseudomonas aeruginosa showed greatly reduced virulence in a mouse model of infection (14), so that pyoverdine is a virulence factor for this organism. Synthesis of pyoverdines and pseudobactins is repressed by the presence of excess iron in the growth medium and this is due, at least in part, to reduced transcription of the corresponding biosynthetic genes (18, 31). However, the molecular mechanisms which control expression of these genes are not well understood. Regulation of expression of siderophore synthesis genes is best characterized in Escherichia coli, in which expression of the relevant genes is controlled by the Fur repressor protein. In the presence of iron (Fe 2ϩ ), Fur binds to the promoters of siderophore-related genes and represses transcription. A Furlike protein is also present in P. aeruginosa, and Fur Ϫ mutants of this species are derepressed for pyoverdine synthesis, implicating Fur in the regulation of expression of pyoverdine biosynthetic genes (29). However, there is evidence that transcriptional activators are also required for expression of siderophore genes in pseudomonads (12,19,27,31,33,38,39). A protein from P. aeruginosa which is likely to be an activator of gene expression is required for synthesis of pyochelin, a nonfluorescent siderophore (12). The pfrA gene is required for expression of pseudobactin synthesis genes in P. putida WCS358 (39), and a second gene required for expression of these genes has also been identified (38). A gene required for expression of siderophore genes in P. fluorescens M114 has been described very recently and is likely to encode an altern...

The expression of many virulence factors in Pseudomonas aeruginosa is dependent upon environmental conditions, including iron levels, oxygen, temperature, and osmolarity. The virulence of P. aeruginosa PAO1 is influenced by the iron-and oxygen-regulated gene encoding the alternative sigma factor PvdS, which is regulated through the ferric uptake regulator (Fur). We observed that overexpression of PvdS in strain PAO1 and a ⌬pvdS::Gm mutant resulted in increased pyoverdine production and proteolytic activity compared to when PvdS was not overexpressed. To identify additional PvdS-regulated genes, we compared extracellular protein profiles from PAO1 and the ⌬pvdS::Gm mutant grown under iron-deficient conditions. A protein present in culture supernatants from PAO1 but not in supernatants from ⌬pvdS::Gm was investigated. Amino acid sequence analysis and examination of the genomic database of PAO1 revealed that the N terminus of this 27-kDa protein is identical to that of protease IV of P. aeruginosa strain PA103-29 and is homologous to an endoprotease produced by Lysobacter enzymogenes. In this study, the gene encoding an endoprotease was cloned from PAO1 and designated prpL (PvdS-regulated endoprotease, lysyl class). All (n ‫؍‬ 41) but one of the strains of P. aeruginosa, including clinical and environmental isolates, examined carry prpL. Moreover, PrpL production among these strains was highly variable. Analysis of RNase protection assays identified the transcription initiation site of prpL and confirmed that its transcription is iron dependent. In the ⌬pvdS::Gm mutant, the level of prpL transcription was iron independent and decreased relative to the level in PAO1. Furthermore, transcription of prpL was independent of PtxR, a PvdS-regulated protein. Finally, PrpL cleaves casein, lactoferrin, transferrin, elastin, and decorin and contributes to PAO1's ability to persist in a rat chronic pulmonary infection model.

PAX2 mutations cause renal-coloboma syndrome (RCS), a rare multi-system developmental abnormality involving optic nerve colobomas and renal abnormalities. End-stage renal failure is common in RCS, but the mechanism by which PAX2 mutations lead to renal failure is unknown. PAX2 is a member of a family of developmental genes containing a highly conserved 'paired box' DNA-binding domain, and encodes a transcription factor expressed primarily during fetal development in the central nervous system, eye, ear and urogenital tract. Presently, the role of PAX2 during kidney development is poorly understood. To gain insight into the cause of renal abnormalities in patients with PAX2 mutations, kidney anomalies were analyzed in patients with RCS, including a large Brazilian kindred in whom a new PAX2 mutation was identified. In a total of 29 patients, renal hypoplasia was the most common congenital renal abnormality. To determine the direct effects of PAX2 mutations on kidney development fetal kidneys of mice carrying a Pax2 (1Neu)mutation were examined. At E15, heterozygous mutant kidneys were approximately 60% of the size of wild-type littermates, and the number of nephrons was strikingly reduced. Heterozygous 1Neu mice showed increased apoptotic cell death during fetal kidney development, but the increased apoptosis was not associated with random stochastic inactivation of Pax2 expression in mutant kidneys; Pax2 was shown to be biallelically expressed during kidney development. These findings support the notion that heterozygous mutations of PAX2 are associated with increased apoptosis and reduced branching of the ureteric bud, due to reduced PAX2 dosage during a critical window in kidney development.

Exotoxin A (ETA) is secreted by Pseudomonas aeruginosa under iron-limiting growth conditions. The ETA structural gene, toxA, is regulated at the transcriptional level by the gene products of the regAB operon. The expression of both toxA and regAB is repressed under iron-replete conditions, suggesting a role for the ferric uptake regulator (Fur) in regulation of ETA synthesis; however, the Fur protein does not interact directly with the toxA or the regAB promoters. Evidence is presented that the iron control of ETA synthesis is mediated by a Fur-regulated alternative sigma factor, PvdS, which had initially been identified as a positive activator for the production of the siderophore pyoverdin. In a delta pvdS deletion mutant, ETA was produced at low levels of less than 5% compared to wild type, but still in response to iron starvation, and introduction of a functional pvdS gene on a plasmid fully restored wild-type levels and normal iron regulation of ETA synthesis. Therefore, a functional pvdS locus is essential for ETA production. Neither toxA nor regAB mRNA was detectable in a delta pvdS mutant. Overexpression of pvdS from the tac promoter on a plasmid resulted in a high-level and iron-independent production of ETA in wild-type PAO1, in the delta pvdS strain, but not in a delta regA strain as a host. These findings suggest that PvdS is required for the activation of the regAB promoters. The transcription of regAB and toxA after induction of the P tac-pvdS gene was monitored in cells grown in high-iron medium. While both regAB and toxA were highly expressed during all growth phases under microaerobic conditions, toxA transcripts were detected only during the exponential but not the early stationary phase of growth under aerobic conditions. These results suggest that a second regulatory mechanism besides the Fur-PvdS system is involved in iron regulation of ETA production.