Non-small cell lung cancer (NSCLC) not amenable to surgical resection has a high mortality rate, due to the ineffectiveness and toxicity of chemotherapy. Thus, there remains an urgent need of efficacious drugs that can combat this disease. In this study, we show that targeting the formation of pro-angiogenic epoxyeicosatrienoic acids (EETs) by the cytochrome P450 arachidonic acid epoxygenases (Cyp2c) represents a new and safe mechanism to treat NSCLC growth and progression. In the transgenic murine K-Ras model and human orthotopic models of NSCLC, we found that Cyp2c44 could be downregulated by activating the transcription factor PPARα with the ligands bezafibrate and Wyeth-14,643. Notably, both treatments reduced primary and metastatic NSCLC growth, tumor angiogenesis, endothelial Cyp2c44 expression and circulating EET levels. These beneficial effects were independent of the time of administration, whether before or after the onset of primary NSCLC, and they persisted after drug withdrawal, suggesting the benefits were durable. Our findings suggest that strategies to downregulate Cyp2c expression and/or its enzymatic activity may provide a safer and effective strategy to treat NSCLC. Moreover, as bezafibrate is a well-tolerated clinically approved drug used for managing lipidemia, our findings provide an immediate cue for clinical studies to evaluate the utility of PPARα ligands as safe agents for the treatment of lung cancer in humans.

The prostaglandin and leukotriene families of lipid mediators are formed via two distinct biosynthetic pathways that are initiated by the oxygenation of arachidonic acid by either cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LOX), respectively. The 5-LOX product 5S-hydroxyeicosatetraenoic acid, however, can also serve as an efficient substrate for COX-2, forming a bicyclic diendoperoxide with structural similarities to the arachidonic acid-derived prostaglandin endoperoxide PGH 2 [Schneider C, et al. (2006) J Am Chem Soc 128:720-721]. Here we identify two cyclic hemiketal (HK) eicosanoids, HKD 2 and HKE 2 , as the major nonenzymatic rearrangement products of the diendoperoxide using liquid chromatography-mass spectrometry analyses as well as UV and NMR spectroscopy. HKD 2 and HKE 2 are furoketals formed by spontaneous cyclization of their respective 8,9-dioxo-5S,11R,12S,15S-tetrahydroxy-or 11,12-dioxo-5S,8S,9S,15S-tetrahydroxy-eicosadi-6E,13E-enoic acid precursors, resulting from opening of the 9S,11R-and 8S,12S-peroxide rings of the diendoperoxide. Furthermore, the diendoperoxide is an efficient substrate for the hematopoietic type of prostaglandin D synthase resulting in formation of HKD 2 , equivalent to the enzymatic transformation of PGH 2 to PGD 2 . HKD 2 and HKE 2 were formed in human blood leukocytes activated with bacterial lipopolysaccharide and calcium ionophore A23187, and biosynthesis was blocked by inhibitors of 5-LOX or COX-2. HKD 2 and HKE 2 stimulated migration and tubulogenesis of microvascular endothelial cells, implicating a proangiogenic role of the hemiketals in inflammatory sites that involve expression of 5-LOX and COX-2. Identification of the highly oxygenated hemiketal eicosanoids provides evidence for a previously unrecognized biosynthetic cross-over of the 5-LOX and COX-2 pathways.L eukotrienes and prostaglandins are lipid mediators derived from oxidative modification of arachidonic acid. Both families of eicosanoids exert inflammatory and immunomodulatory functions in disease, as well as homeostatic functions in normal physiological processes (1). Atherosclerosis, asthma, and many types of cancer are prototypical inflammatory diseases that are characterized by concomitant formation of both leukotrienes and prostaglandins (2). The formation of leukotrienes and prostaglandins diverges at the point of the initial oxygenation of the common arachidonic acid substrate by either 5-lipoxygenase (5-LOX) or cyclooxygenase-2 (COX-2), respectively. From there, biosynthesis proceeds along separate and distinct pathways, each utilizing specific enzymes that catalyze complex reactions using highly unstable substrates (3, 4).The possibility of a biosynthetic convergence of the 5-LOX and COX-2 pathways was implicated when the 5-LOX product 5S-hydroxyeicosatetraenoic acid (5S-HETE) was identified as an efficient and specific substrate for oxygenation by recombinant COX-2 (5). The COX-2 oxygenation product of 5S-HETE is a bicyclic diendoperoxide with structural similarities to the prostaglandin (PG) e...

Mesangial cells and podocytes express integrins a1b1 and a2b1, which are the two major collagen receptors that regulate multiple cellular functions, including extracellular matrix homeostasis. Integrin a1b1 protects from glomerular injury by negatively regulating collagen production, but the role of integrin a2b1 in renal injury is unclear. Here, we subjected wild-type and integrin a2-null mice to injury with adriamycin or partial renal ablation. In both of these models, integrin a2-null mice developed significantly less proteinuria and glomerulosclerosis. In addition, selective pharmacological inhibition of integrin a2b1 significantly reduced adriamycin-induced proteinuria, glomerular injury, and collagen deposition in wildtype mice. This inhibitor significantly reduced collagen synthesis in wild-type, but not integrin a2-null, mesangial cells in vitro, demonstrating that its effects are integrin a2b1-dependent. Taken together, these results indicate that integrin a2b1 contributes to glomerular injury by positively regulating collagen synthesis and suggest that its inhibition may be a promising strategy to reduce glomerular injury and proteinuria. The most common cause of end stage kidney disease is glomerulosclerosis, which is characterized by excessive collagen deposition in the glomerulus. Although numerous disease processes can initiate glomerular injury, they all result in abnormal glomerular collagen homeostasis with progression. Glomerular collagen turnover is regulated by multiple factors, including growth factors and profibrotic reactive oxygen species (ROSs) as well as cell extracellular matrix interactions mediated by the matrix receptors integrins (reviewed in ref. 1). Of these factors, the mechanisms by which integrins regulate glomerulosclerosis are the most poorly understood.Integrins consist of two noncovalently associated a-and b-subunits that combine to form 24 different heterodimers in mammals (reviewed in ref.2). The integrin extracellular domains contain the ligand binding site and confer ligand specificity, whereas the cytoplasmic domain interacts with the cytoskeleton and regulates cell signaling. Integrins control critical cell functions, including proliferation, survival, migration, and matrix homeostasis (reviewed in refs. 1 and 2). Thus, it is not surprising that integrins regulate tissue responses to injury in whole organisms.The best-studied integrin in the context of glomerular injury is the major collagen IV receptor, integrin a1b1, which is expressed by mesangial cells 3 and podocytes. 4 We have shown that integrin

Highlights d Breast cancer cells treated with CDK4/6 inhibitor secrete chemokines CCL5 and CXCL10 d Chemokine induction is associated with deregulated mTOR, metabolic stress, and ROS d Chemokines induced by CDK4/6 inhibitor facilitate T cell infiltration into tumors d Chemokines induced by CDK4/6 inhibitor augment adoptive T cell therapy

Protein lysine modification by γ-ketoaldehyde isomers derived from arachidonic acid, termed isolevuglandins (IsoLGs), is emerging as a mechanistic link between pathogenic reactive oxygen species and disease progression. However, the questions of whether covalent modification of proteins by IsoLGs are subject to genetic regulation and the identity of IsoLG-modified proteins remain unclear. Herein we show that Nrf2 and Nox2 are key regulators of IsoLG modification in pulmonary tissue and report on the identity of proteins analyzed by LC-MS following immunoaffinity purification of IsoLG-modified proteins. Gene ontology analysis revealed that proteins in numerous cellular pathways are susceptible to IsoLG modification. Although cells tolerate basal levels of modification, exceeding them induces apoptosis. We found prominent modification in a murine model of radiation-induced pulmonary fibrosis and in idiopathic pulmonary fibrosis, two diseases considered to be promoted by gene-regulated oxidant stress. Based on these results we hypothesize that IsoLG modification is a hitherto unrecognized sequelae that contributes to radiation-induced pulmonary injury and IPF.

Integrin ␣1␤1 negatively regulates the generation of profibrotic reactive oxygen species (ROS) by inhibiting epidermal growth factor receptor (EGFR) activation; however, the mechanism by which it does this is unknown. In this study, we show that caveolin-1 (Cav-1), a scaffolding protein that binds integrins and controls growth factor receptor signaling, participates in integrin ␣1␤1-mediated EGFR activation. Integrin ␣1-null mesangial cells (MCs) have reduced Cav-1 levels, and reexpression of the integrin ␣1 subunit increases Cav-1 levels, decreases EGFR activation, and reduces ROS production. Downregulation of Cav-1 in wild-type MCs increases EGFR phosphorylation and ROS synthesis, while overexpression of Cav-1 in the integrin ␣1-null MCs decreases EGFR-mediated ROS production. We further show that integrin ␣1-null MCs have increased levels of activated extracellular signal-regulated kinase (ERK), which leads to reduced activation of peroxisome proliferator-activated receptor ␥ (PPAR␥), a transcription factor that positively regulates Cav-1 expression. Moreover, activation of PPAR␥ or inhibition of ERK increases Cav-1 levels in the integrin ␣1-null MCs. Finally, we show that glomeruli of integrin ␣1-null mice have reduced levels of Cav-1 and activated PPAR␥ but increased levels of phosphorylated EGFR both at baseline and following injury. Thus, integrin ␣1␤1 negatively regulates EGFR activation by positively controlling Cav-1 levels, and the ERK/PPAR␥ axis plays a key role in regulating integrin ␣1␤1-dependent Cav-1 expression and consequent EGFR-mediated ROS production.Integrins are transmembrane receptors for extracellular matrix components and are composed of noncovalently bound ␣ and ␤ subunits. In mammals, 1 of 18 ␣ subunits heterodimerizes with 1 of 8 ␤ subunits to form 24 distinct integrins, each with specific but overlapping functions (4, 29). Integrins regulate cellular processes such as cell adhesion, differentiation (2), cell cycle progression (26), reactive oxygen species (ROS) production (10, 72), and extracellular matrix synthesis and remodeling (23). In addition, integrins interact with and regulate the activity of different growth factor receptors (3), and this cross talk coordinates biological processes by regulating downstream signaling pathways (8,55,58,61). Integrin occupancy causes growth factor receptor autophosphorylation (44), and growth factor receptors and integrins associate following growth factor stimulation or integrin activation (5, 61, 73).Caveolin-1 (Cav-1) is one of the three members of the caveolin family and is a structural protein involved in the formation of caveola-rich membrane domains (64). Caveolae are clearly defined, small, flask-shaped invaginations of the plasma membrane enriched in sphingolipids and cholesterol and are implicated in transcytosis, lipid metabolism, and receptor trafficking (35). Changes in plasma membrane localization and/or expression of Cav-1 can profoundly affect Cav-1-mediated functions. Different factors can regulate Cav-1 expression at both tr...

The development of radiation-induced pulmonary fibrosis represents a critical clinical issue limiting delivery of therapeutic doses of radiation to non-small cell lung cancer. Identification of the cell types whose injury initiates a fibrotic response and the underlying biological factors that govern that response are needed for developing strategies that prevent or mitigate fibrosis. C57BL/6 mice (wild type, Nrf2 null, Nrf2flox/flox, and Nrf2Δ/Δ; SPC-Cre) were administered a thoracic dose of 12 Gy and allowed to recover for 250 days. Whole slide digital and confocal microscopy imaging of H&E, Masson’s trichrome and immunostaining were used to assess tissue remodeling, collagen deposition and cell renewal/mobilization during the regenerative process. Histological assessment of irradiated, fibrotic wild type lung revealed significant loss of alveolar type 2 cells 250 days after irradiation. Type 2 cell loss and the corresponding development of fibrosis were enhanced in the Nrf2 null mouse. Yet, conditional deletion of Nrf2 in alveolar type 2 cells in irradiated lung did not impair type 2 cell survival nor yield an increased fibrotic phenotype. Instead, radiation-induced ΔNp63 stem/progenitor cell mobilization was inhibited in the Nrf2 null mouse while the propensity for radiation-induced myofibroblasts derived from alveolar type 2 cells was magnified. In summary, these results indicate that Nrf2 is an important regulator of irradiated lung’s capacity to maintain alveolar type 2 cells, whose injury can initiate a fibrotic phenotype. Loss of Nrf2 inhibits ΔNp63 stem/progenitor mobilization, a key event for reconstitution of injured lung, while promoting a myofibroblast phenotype that is central for fibrosis.

Integrin ␣1␤1 is a collagen receptor that down-regulates collagen and reactive oxygen species (ROS) production, and mice lacking this receptor show increased ROS levels and exacerbated glomerular sclerosis following injury. Caveolin-1 (Cav-1) is a multifunctional protein that is tyrosine-phosphorylated in response to injury and has been implicated in ROSmediated injury. Cav-1 interacts with integrins, and integrin ␣1␤1 binds/activates T cell protein-tyrosine phosphatase (TCPTP), which is homologous to the tyrosine phosphatase PTP1B known to dephosphorylate Cav-1. In this study, we analyzed whether phosphorylated Cav-1 (pCav-1) is a substrate of TCPTP and if integrin ␣1␤1 is essential for promoting TCPTP-mediated Cav-1 dephosphorylation. We found that Cav-1 phosphorylation is significantly higher in cells lacking integrin ␣1␤1 at base line and following oxidative stress. Overexpression of TCPTP leads to reduced pCav-1 levels only in cells expressing integrin ␣1␤1. Using solid phase binding assays, we demonstrated that 1) purified Cav-1 directly interacts with TCPTP and the integrin ␣1 subunit, 2) pCav-1 is a substrate of TCPTP, and 3) TCPTP-mediated Cav-1 dephosphorylation is highly increased by the addition of purified integrin ␣1␤1 or an integrin ␣1 cytoplasmic peptide to which TCPTP has been shown to bind. Thus, our results demonstrate that pCav-1 is a new substrate of TCPTP and that integrin ␣1␤1 acts as a negative regulator of Cav-1 phosphorylation by activating TCPTP. This could explain the protective function of integrin ␣1␤1 in oxidative stress-mediated damage and why integrin ␣1-null mice are more susceptible to fibrosis following injury.Caveolin-1 (Cav-1) is a key component of caveolae, plasma membrane invaginations enriched in sphingolipids and cholesterol, which function in lipid metabolism, transcytosis, and receptor trafficking (1). Cav-1 interacts with and regulates the localization and function of various transmembrane proteins (1, 2). For instance, Cav-1 negatively regulates TGF-␤ signaling (3) as well as EGF receptor expression and activation (4). In addition, Cav-1 participates in integrin-mediated signaling, as demonstrated by the finding that Cav-1-mediated integrin ␤1 endocytosis is critical for regulation of fibronectin turnover (5).Cav-1 is phosphorylated at tyrosine 14 by the Src family kinases in response to various stimuli, including growth factor-mediated signaling, mechanical stretch, and hyperosmolarity (6 -10). Cav-1 is also phosphorylated in response to oxidative stress, since hydrogen peroxide induces Cav-1 phosphorylation in endothelial cells (11-13), fibroblasts (7,10,14) and renal cells (15). Finally, Cav-1 phosphorylation increases in vivo in brain injury (16) and in the spinal cord following experimental autoimmune encephalomyelitis (17). However, whether increased pCav-1 contributes to or protects from injury is controversial at present. In this context, increased expression of pCav-1 is associated with cell survival after oxidative stress (15); however, in contrast to these...