Fiona E. Yull

Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy

The tumor microenvironment (TME) consists of cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that can promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, foster therapeutic resistance, and provide niches for dormant metastases to thrive. An American Association for Cancer Research (AACR) special conference held on November 3-6, 2011, addressed five emerging concepts in our understanding of the TME: its dynamic evolution, how it is educated by tumor cells, pathways of communication between stromal and tumor cells, immunomodulatory roles of the lymphatic system, and contribution of the intestinal microbiota. These discussions raised critical questions on how to include the analysis of the TME in personalized cancer diagnosis and treatment.

The protein kinase IKKepsilon regulates energy balance in obese mice

Obesity is associated with chronic low-grade inflammation that negatively impacts insulin sensitivity. Here, we show that high-fat diet can increase NF-kappaB activation in mice, which leads to a sustained elevation in level of IkappaB kinase epsilon (IKKepsilon) in liver, adipocytes, and adipose tissue macrophages. IKKepsilon knockout mice are protected from high-fat diet-induced obesity, chronic inflammation in liver and fat, hepatic steatosis, and whole-body insulin resistance. These mice show increased energy expenditure and thermogenesis via enhanced expression of the uncoupling protein UCP1. They maintain insulin sensitivity in liver and fat, without activation of the proinflammatory JNK pathway. Gene expression analyses indicate that IKKepsilon knockout reduces expression of inflammatory cytokines, and changes expression of certain regulatory proteins and enzymes involved in glucose and lipid metabolism. Thus, IKKepsilon may represent an attractive therapeutic target for obesity, insulin resistance, diabetes, and other complications associated with these disorders.

Low-Level Laser Therapy Activates NF-kB via Generation of Reactive Oxygen Species in Mouse Embryonic Fibroblasts

BACKGROUND Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation at a cellular level remain unclear. METHODOLOGY/PRINCIPAL FINDINGS In this study, we isolated murine embryonic fibroblasts (MEF) from transgenic NF-kB luciferase reporter mice and studied their response to 810 nm laser radiation. Significant activation of NF-kB was observed at fluences higher than 0.003 J/cm(2) and was confirmed by Western blot analysis. NF-kB was activated earlier (1 hour) by LLLT compared to conventional lipopolysaccharide treatment. We also observed that LLLT induced intracellular reactive oxygen species (ROS) production similar to mitochondrial inhibitors, such as antimycin A, rotenone and paraquat. Furthermore, we observed similar NF-kB activation with these mitochondrial inhibitors. These results, together with inhibition of laser induced NF-kB activation by antioxidants, suggests that ROS play an important role in the laser induced NF-kB signaling pathways. However, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that LLLT also upregulates mitochondrial respiration. CONCLUSION We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive NFkB signaling via generation of ROS. Expression of anti-apoptosis and pro-survival genes responsive to NFkB could explain many clinical effects of LLLT.

Release of Free F2-isoprostanes from Esterified Phospholipids Is Catalyzed by Intracellular and Plasma Platelet-activating Factor Acetylhydrolases

F2-isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F2-isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A2 activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF acetylhydrolase do not release F2-isoprostanes from esterified precursors. The intracellular PAF acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF acetylhydrolases have high affinity for esterified F2-isoprostanes. However, the rate of esterified F2-isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F2-isoprostanes compared with nontransgenic littermates. Our results suggested that PAF acetylhydrolases play key roles in the hydrolysis of F2-isoprostanes esterified on phospholipids in vivo.

Endoplasmic reticulum stress enhances fibrotic remodeling in the lungs

Evidence of endoplasmic reticulum (ER) stress has been found in lungs of patients with familial and sporadic idiopathic pulmonary fibrosis. We tested whether ER stress causes or exacerbates lung fibrosis by (i) conditional expression of a mutant form of surfactant protein C (L188Q SFTPC) found in familial interstitial pneumonia and (ii) intratracheal treatment with the protein misfolding agent tunicamycin. We developed transgenic mice expressing L188Q SFTPC exclusively in type II alveolar epithelium by using the Tet-On system. Expression of L188Q SFTPC induced ER stress, as determined by increased expression of heavy-chain Ig binding protein (BiP) and splicing of X-box binding protein 1 (XBP1) mRNA, but no lung fibrosis was identified in the absence of a second profibrotic stimulus. After intratracheal bleomycin, L188Q SFTPC-expressing mice developed exaggerated lung fibrosis and reduced static lung compliance compared with controls. Bleomycin-treated L188Q SFTPC mice also demonstrated increased apoptosis of alveolar epithelial cells and greater numbers of fibroblasts in the lungs. With a complementary model, intratracheal tunicamycin treatment failed to induce lung remodeling yet resulted in augmentation of bleomycin-induced fibrosis. These data support the concept that ER stress produces a dysfunctional epithelial cell phenotype that facilitates fibrotic remodeling. ER stress pathways may serve as important therapeutic targets in idiopathic pulmonary fibrosis.

Duration and Intensity of NF-κB Activity Determine the Severity of Endotoxin-Induced Acute Lung Injury

Activation of innate immunity in the lungs can lead to a self-limited inflammatory response or progress to severe lung injury. We investigated whether specific parameters of NF-κB pathway activation determine the outcome of acute lung inflammation using a novel line of transgenic reporter mice. Following a single i.p. injection of Escherichia coli LPS, transient NF-κB activation was identified in a variety of lung cell types, and neutrophilic inflammation resolved without substantial tissue injury. However, administration of LPS over 24 h by osmotic pump (LPS pump) implanted into the peritoneum resulted in sustained, widespread NF-κB activation and neutrophilic inflammation that culminated in lung injury at 48 h. To determine whether intervention in the NF-κB pathway could prevent progression to lung injury in the LPS pump model, we administered a specific IκB kinase inhibitor (BMS-345541) to down-regulate NF-κB activation following the onset of inflammation. Treatment with BMS-345541 beginning at 20 h after osmotic pump implantation reduced lung NF-κB activation, concentration of KC and MIP-2 in lung lavage, neutrophil influx, and lung edema measured at 48 h. Therefore, sustained NF-κB activation correlates with severity of lung injury, and interdiction in the NF-κB pathway is beneficial even after the onset of lung inflammation.

Inhibition of NF-kappaB activity results in disruption of the apical ectodermal ridge and aberrant limb morphogenesis.

In Drosophila, the Dorsal protein establishes the embryonic dorso–ventral axis during development. Here we show that the vertebrate homologue of Dorsal, nuclear factor-kappa B (NF-κB), is vital for the formation of the proximo–distal organizer of the developing limb bud, the apical ectodermal ridge (AER). Transcription of the NF-κB proto-oncogene c-rel is regulated, in part, during morphogenesis of the limb bud by AER-derived signals such as fibroblast growth factors. Interruption of NF-κB activity using viral-mediated delivery of an inhibitor results in a highly dysmorphic AER, reduction in overall limb size, loss of distal elements and reversal in the direction of limb outgrowth. Furthermore, inhibition of NF-κB activity in limb mesenchyme leads to a reduction in expression of Sonic hedgehog and Twist but derepresses expression of the bone morphogenetic protein-4 gene. These results are the first evidence that vertebrate NF-κB proteins act to transmit growth factor signals between the ectoderm and the underlying mesenchyme during embryonic limb formation.

Airway Epithelium Controls Lung Inflammation and Injury through the NF-κB Pathway

Although airway epithelial cells provide important barrier and host defense functions, a crucial role for these cells in development of acute lung inflammation and injury has not been elucidated. We investigated whether NF-κB pathway signaling in airway epithelium could decisively impact inflammatory phenotypes in the lungs by using a tetracycline-inducible system to achieve selective NF-κB activation or inhibition in vivo. In transgenic mice that express a constitutively active form of IκB kinase 2 under control of the epithelial-specific CC10 promoter, treatment with doxycycline induced NF-κB activation with consequent production of a variety of proinflammatory cytokines, high-protein pulmonary edema, and neutrophilic lung inflammation. Continued treatment with doxycycline caused progressive lung injury and hypoxemia with a high mortality rate. In contrast, inducible expression of a dominant inhibitor of NF-κB in airway epithelium prevented lung inflammation and injury resulting from expression of constitutively active form of IκB kinase 2 or Escherichia coli LPS delivered directly to the airways or systemically via an osmotic pump implanted in the peritoneal cavity. Our findings indicate that the NF-κB pathway in airway epithelial cells is critical for generation of lung inflammation and injury in response to local and systemic stimuli; therefore, targeting inflammatory pathways in airway epithelium could prove to be an effective therapeutic strategy for inflammatory lung diseases.

Dehydration activates an NF-κB–driven, COX2-dependent survival mechanism in renal medullary interstitial cells

Renal prostaglandin (PG) synthesis is mediated by cyclooxygenase-1 and -2 (COX1 and COX2). After dehydration, the maintenance of normal renal function becomes particularly dependent upon PG synthesis. The present studies were designed to examine the potential link between medullary COX1 and COX2 expression in hypertonic stress. In response to water deprivation, COX2, but not COX1, mRNA levels increase significantly in the renal medulla, specifically in renal medullary interstitial cells (RMICs). Water deprivation also increases renal NF-kappaB-driven reporter expression in transgenic mice. NF-kappaB activity and COX2 expression could be induced in cultured RMICs with hypertonic sodium chloride and mannitol, but not urea. RMIC COX2 expression was also induced by driving NF-kappaB activation with a constitutively active IkappaB kinase alpha (IKKalpha). Conversely, introduction of a dominant-negative IkappaB mutant reduced COX2 expression after hypertonicity or IKKalpha induction. RMICs failed to survive hypertonicity when COX2 was downregulated using a COX2-selective antisense or blocked with the selective nonsteroidal anti-inflammatory drug (NSAID) SC58236, reagents that did not affect cell survival in isotonic media. In rabbits treated with SC58236, water deprivation induced apoptosis of medullary interstitial cells in the renal papilla. These results demonstrate that water deprivation and hypertonicity activate NF-kappaB. The consequent increase in COX2 expression favors RMIC survival in hypertonic conditions. Inhibition of RMIC COX2 could contribute to NSAID-induced papillary injury.

Epithelial NF-κB activation promotes urethane-induced lung carcinogenesis

Chronic inflammation is linked to carcinogenesis in several organ systems. In the lungs, NF-κB, a central effector of inflammatory responses, is frequently activated in non-small-cell lung cancer, but its role in tumor promotion has not been studied. Several lines of evidence indicate that ethyl carbamate (urethane)-induced lung tumor formation, a prototypical mouse model of multistage lung carcinogenesis, is potentiated by inflammation. We found that mouse strains susceptible to lung tumor formation (FVB, BALB/c) exhibited early NF-κB activation and inflammation in the lungs after urethane treatment. However, a resistant strain (C57B6) failed to activate NF-κB or induce lung inflammation. In FVB mice, we identified urethane-induced NF-κB activation in airway epithelium, as well as type II alveolar epithelial cells and macrophages. Using an inducible transgenic mouse model (FVB strain) to express a dominant inhibitor of NF-κB specifically in airway epithelial cells, we found that urethane-induced lung inflammation was blocked and tumor formation was reduced by >50%. Selective NF-κB inhibition resulted in increased apoptosis of airway epithelial cells at 2 weeks after urethane treatment in association with a marked reduction of Bcl-2 expression. These studies indicate that NF-κB signaling in airway epithelium is integral to tumorigenesis in the urethane model and identify the NF-κB pathway as a potential target for chemoprevention of lung cancer.