Rapid acceleration of KRAS-mutant pancreatic carcinogenesis via remodeling of tumor immune microenvironment by PPARδ.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor of pancreatic ductal adenocarcinoma (PDAC), which commonly occurs in the general populations with aging. Although most PanIN lesions (PanINs) harbor oncogenic KRAS mutations that initiate pancreatic tumorigenesis; PanINs rarely progress to PDAC. Critical factors that promote this progression, especially targetable ones, remain poorly defined. We show that peroxisome proliferator-activated receptor-delta (PPARδ), a lipid nuclear receptor, is upregulated in PanINs in humans and mice. Furthermore, PPARδ ligand activation by a high-fat diet or GW501516 (a highly selective, synthetic PPARδ ligand) in mutant KRASG12D (KRASmu) pancreatic epithelial cells strongly accelerates PanIN progression to PDAC. This PPARδ activation induces KRASmu pancreatic epithelial cells to secrete CCL2, which recruits immunosuppressive macrophages and myeloid-derived suppressor cells into pancreas via the CCL2/CCR2 axis to orchestrate an immunosuppressive tumor microenvironment and subsequently drive PanIN progression to PDAC. Our data identify PPARδ signaling as a potential molecular target to prevent PDAC development in subjects harboring PanINs.

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation.

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

The Role of PPAR-δ in Metabolism, Inflammation, and Cancer: Many Characters of a Critical Transcription Factor.

Peroxisome proliferator-activated receptor-delta (PPAR-δ), one of three members of the PPAR group in the nuclear receptor superfamily, is a ligand-activated transcription factor. PPAR-δ regulates important cellular metabolic functions that contribute to maintaining energy balance. PPAR-δ is especially important in regulating fatty acid uptake, transport, and ß-oxidation as well as insulin secretion and sensitivity. These salutary PPAR-δ functions in normal cells are thought to protect against metabolic-syndrome-related diseases, such as obesity, dyslipidemia, insulin resistance/type 2 diabetes, hepatosteatosis, and atherosclerosis. Given the high clinical burden these diseases pose, highly selective synthetic activating ligands of PPAR-δ were developed as potential preventive/therapeutic agents. Some of these compounds showed some efficacy in clinical trials focused on metabolic-syndrome-related conditions. However, the clinical development of PPAR-δ agonists was halted because various lines of evidence demonstrated that cancer cells upregulated PPAR-δ expression/activity as a defense mechanism against nutritional deprivation and energy stresses, improving their survival and promoting cancer progression. This review discusses the complex relationship between PPAR-δ in health and disease and highlights our current knowledge regarding the different roles that PPAR-δ plays in metabolism, inflammation, and cancer.

Oxygenated lipid signaling in tumor-associated macrophages-focus on colon cancer.

Polyunsaturated fatty acids (PUFAs) are enzymatically converted to a variety of bioactive products through insertion of molecular oxygen. PUFA-derived mediators can have either inflammatory or anti-inflammatory/pro-resolving properties, depending upon their specific structures. The relative harm or benefit of these mediators can also be tissue and context dependent. These mediators play important roles in maintaining homeostasis and their dysregulation is involved in pathogenesis of cancers, especially those associated with chronic inflammation. There is a well-established link between colorectal cancer (CRC) and chronic inflammation. The colon harbors a large population of immune cells, which must be tightly regulated in order to maintain the balance between pathogenic and commensal microbes in the gut. Macrophages are key to the process of distinguishing between potentially harmful antigens/microbes and benign or beneficial signals. Macrophages are often associated with tumors (tumor-associated macrophages (TAMs), including CRC. There is some debate as to the prognostic significance of these TAMs in CRC, with some work suggesting a beneficial impact. The purpose of this review is to give an overview of what is currently known regarding PUFA-derived mediator signaling in tumor-associated macrophages in CRC.

15-epi-Lipoxin A4, Resolvin D2, and Resolvin D3 Induce NF-κB Regulators in Bacterial Pneumonia.

Specialized proresolving mediators (SPMs) decrease NF-κB activity to prevent excessive tissue damage and promote the resolution of acute inflammation. Mechanisms for NF-κB regulation by SPMs remain to be determined. In this study, after LPS challenge, the SPMs 15-epi-lipoxin A4 (15-epi-LXA4), resolvin D1, resolvin D2, resolvin D3, and 17-epi-resolvin D1 were produced in vivo in murine lungs. In LPS-activated human bronchial epithelial cells, select SPMs increased expression of the NF-κB regulators A20 and single Ig IL-1R-related molecule (SIGIRR). Of interest, 15-epi-LXA4 induced A20 and SIGIRR in an lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2) receptor-dependent manner in epithelial cells and in murine pneumonia. This SPM regulated NF-κB-induced cytokines to decrease pathogen-mediated inflammation. In addition to dampening lung inflammation, surprisingly, 15-epi-LXA4 also enhanced pathogen clearance with increased antimicrobial peptide expression. Taken together, to our knowledge these results are the first to identify endogenous agonists for A20 and SIGIRR expression to regulate NF-κB activity and to establish mechanisms for NF-κB regulation by SPMs for pneumonia resolution.

Resolvin D3 and Aspirin-Triggered Resolvin D3 Are Protective for Injured Epithelia.

Acute lung injury is a life-threatening condition caused by disruption of the alveolar-capillary barrier leading to edema, influx of inflammatory leukocytes, and impaired gas exchange. Specialized proresolving mediators biosynthesized from essential fatty acids, such as docosahexaenoic acid, have tissue protective effects in acute inflammation. Herein, we found that the docosahexaenoic acid-derived mediator resolvin D3 (RvD3): 4S,11R,17S-trihydroxydocosa-5Z,7E,9E,13Z,15E,19Z-hexaenoic acid was present in uninjured lungs, and increased significantly 24 to 72 hours after hydrochloric acid-initiated injury. Because of its delayed enzymatic degradation, we used aspirin-triggered (AT)-RvD3: 4S,11R,17R-trihydroxydocosa-5Z,7E,9E,13Z,15E,19Z-hexaenoic acid, a 17R-epimer of RvD3, for in vivo experiments. Histopathological correlates of acid injury (alveolar wall thickening, edema, and leukocyte infiltration) were reduced in mice receiving AT-RvD3 1 hour after injury. AT-RvD3-treated mice had significantly reduced edema, as demonstrated by lower wet/dry weight ratios, increased epithelial sodium channel γ expression, and more lymphatic vessel endothelial hyaluronan receptor 1-positive vascular endothelial growth factor receptor 3-positive lymphatic vessels. Evidence for counterregulation of NF-κB by RvD3 and AT-RvD3 was seen in vitro and by AT-RvD3 in vivo. Increases in lung epithelial cell proliferation and bronchoalveolar lavage fluid levels of keratinocyte growth factor were observed with AT-RvD3, which also promoted cutaneous re-epithelialization. Together, these data demonstrate protective actions of RvD3 and AT-RvD3 for injured mucosa that accelerated restoration of epithelial barrier and function.

Lipoxin Signaling in Murine Lung Host Responses to Cryptococcus neoformans Infection.

Lipoxins (LX) are proresolving mediators that augment host defense against bacterial infection. Here, we investigated roles for LX in lung clearance of the fungal pathogen Cryptococcus neoformans (Cne). After intranasal inoculation of 5,000 CFU Cne, C57BL/6 and C.B-17 mice exhibited strain-dependent differences in Cne clearance, immunologic responses, and lipoxin A4 (LXA4) formation and receptor (ALX/FPR2) expression. Compared with C.B-17 mice, C57BL/6 lungs had increased and persistent Cne infection 14 days after inoculation, increased eosinophils, and distinct profiles of inflammatory cytokines. Relative to C.B-17 mice, bronchoalveolar lavage fluid levels of LXA4 were increased before and after infection in C57BL/6. The kinetics for 15-epi-LXA4 production were similar in both strains. Lung basal expression of the LX biosynthetic enzyme Alox12/15 (12/15-lipoxygenase) was increased in C57BL/6 mice and further increased after Cne infection. In contrast, lung basal expression of the LXA4 receptor Alx/Fpr2 was higher in C.B-17 relative to C57BL/6 mice, and after Cne infection, Alx/Fpr2 expression was significantly increased in only C.B-17 mice. Heat-killed Cne initiated lung cell generation of IFN-γ and IL-17 and was further increased in C.B-17 mice by 15-epi-LXA4. A trend toward reduced Cne clearance and IFN-γ production was observed upon in vivo administration of an ALX/FPR2 antagonist. Together, these findings provide the first evidence that alterations in cellular immunity against Cne are associated with differences in LXA4 production and receptor expression, suggesting an important role for ALX/FPR2 signaling in the regulation of pathogen-mediated inflammation and antifungal lung host defense.

Human Mesenchymal Stem (Stromal) Cells Promote the Resolution of Acute Lung Injury in Part through Lipoxin A4.

Previous studies demonstrated that bone marrow-derived mesenchymal stem (stromal) cells (MSCs) reduce the severity of acute lung injury in animal models and in an ex vivo perfused human lung model. However, the mechanisms by which MSCs reduce lung injury are not well understood. In the present study, we tested the hypothesis that human MSCs promote the resolution of acute lung injury in part through the effects of a specialized proresolving mediator lipoxin A4 (LXA4). Human alveolar epithelial type II cells and MSCs expressed biosynthetic enzymes and receptors for LXA4. Coculture of human MSCs with alveolar epithelial type II cells in the presence of cytomix significantly increased the production of LXA4 by 117%. The adoptive transfer of MSCs after the onset of LPS-induced acute lung injury (ALI) in mice led to improved survival (48 h), and blocking the LXA4 receptor with WRW4, a LXA4 receptor antagonist, significantly reversed the protective effect of MSCs on both survival and the accumulation of pulmonary edema. LXA4 alone improved survival in mice, and it also significantly decreased the production of TNF-α and MIP-2 in bronchoalveolar lavage fluid. In summary, these experiments demonstrated two novel findings: human MSCs promote the resolution of lung injury in mice in part through the proresolving lipid mediator LXA4, and LXA4 itself should be considered as a therapeutic for acute respiratory distress syndrome.

Wood smoke enhances cigarette smoke-induced inflammation by inducing the aryl hydrocarbon receptor repressor in airway epithelial cells.

Our previous studies showed that cigarette smokers who are exposed to wood smoke (WS) are at an increased risk for chronic bronchitis and reduced lung function. The present study was undertaken to determine the mechanisms for WS-induced adverse effects. We studied the effect of WS exposure using four cohorts of mice. C57Bl/6 mice were exposed for 4 or 12 weeks to filtered air, to 10 mg/m(3) WS for 2 h/d, to 250 mg/m(3) cigarette smoke (CS) for 6 h/d, or to CS followed by WS (CW). Inflammation was absent in the filtered air and WS groups, but enhanced by twofold in the bronchoalveolar lavage of the CW compared with CS group as measured by neutrophil numbers and levels of the neutrophil chemoattractant, keratinocyte-derived chemokine. The levels of the anti-inflammatory lipoxin, lipoxin A4, were reduced by threefold along with cyclo-oxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1 in airway epithelial cells and PGE2 levels in the bronchoalveolar lavage of CW compared with CS mice. We replicated, in primary human airway epithelial cells, the changes observed in mice. Immunoprecipitations showed that WS blocked the interaction of aryl hydrocarbon receptor (AHR) with AHR nuclear transporter to reduce expression of COX-2 and mPGES-1 by increasing expression of AHR repressor (AHRR). Collectively, these studies show that exposure to low concentrations of WS enhanced CS-induced inflammation by inducing AHRR expression to suppress AHR, COX-2, and mPGES-1 expression, and levels of PGE2 and lipoxin A4. Therefore, AHRR is a potential therapeutic target for WS-associated exacerbations of CS-induced inflammation.

Maresin 1 biosynthesis during platelet-neutrophil interactions is organ-protective.

Unregulated acute inflammation can lead to collateral tissue injury in vital organs, such as the lung during the acute respiratory distress syndrome. In response to tissue injury, circulating platelet-neutrophil aggregates form to augment neutrophil tissue entry. These early cellular events in acute inflammation are pivotal to timely resolution by mechanisms that remain to be elucidated. Here, we identified a previously undescribed biosynthetic route during human platelet-neutrophil interactions for the proresolving mediator maresin 1 (MaR1; 7R,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid). Docosahexaenoic acid was converted by platelet 12-lipoxygenase to 13S,14S-epoxy-maresin, which was further transformed by neutrophils to MaR1. In a murine model of acute respiratory distress syndrome, lipid mediator metabololipidomics uncovered MaR1 generation in vivo in a temporally regulated manner. Early MaR1 production was dependent on platelet-neutrophil interactions, and intravascular MaR1 was organ-protective, leading to decreased lung neutrophils, edema, tissue hypoxia, and prophlogistic mediators. Together, these findings identify a transcellular route for intravascular maresin 1 biosynthesis via platelet-neutrophil interactions that regulates the extent of lung inflammation.

Genetic reduction of insulin-like growth factor-1 mimics the anticancer effects of calorie restriction on cyclooxygenase-2-driven pancreatic neoplasia.

Risk of pancreatic cancer, the fourth deadliest cancer in the United States, is increased by obesity. Calorie restriction (CR) prevents obesity, suppresses carcinogenesis in many models, and reduces serum levels of IGF-1. In the present study, we examined the impact of CR on a model of inflammation-associated pancreatitis and pancreatic dysplasia, with a focus on the mechanistic contribution of systemic IGF-1. Administration of a 30% CR diet for 14 weeks decreased serum IGF-1 levels and hindered pancreatic ductal lesion formation and dysplastic severity, relative to a higher calorie control diet, in transgenic mice overexpressing COX-2 [bovine keratin-5 promoter (BK5.COX-2)]. These findings in CR mice correlated with reductions in Ki-67-positive cells, vascular luminal size, VEGF expression, and phosphorylation and total expression of downstream mediators of the IGF-1 pathway. Cell lines derived from BK5.COX-2 ductal lesions (JC101 cells) formed pancreatic tumors in wild-type FVB mice that were significantly reduced in size by a 14-week CR regimen, relative to the control diet. To further understand the impact of circulating levels of IGF-1 on tumor growth in this model, we orthotopically injected JC101 cells into liver-specific IGF-1-deficient (LID) mice. The approximate 65% reduction of serum IGF-1 levels in LID mice resulted in significantly decreased burden of JC101 tumors, despite modestly elevated levels of circulating insulin and leptin. These data show that CR prevents development of dysplasia and growth of pancreatic cancer through alterations in IGF-1, suggesting that modulation of this pathway with dietary and/or pharmacologic interventions is a promising pancreatic cancer prevention strategy.

P53 genotype as a determinant of ER expression and tamoxifen response in the MMTV-Wnt-1 model of mammary carcinogenesis.

Clinical studies show that estrogen receptor-α (ER) expressing tumors tend to have better prognosis, respond to antiestrogen therapy and have wild-type p53. Conversely, tumors with inactivating mutations in p53 tend to have worse outcomes and to be ER-negative and unresponsive to antihormone treatment. Previous studies from our laboratory have shown that p53 regulates ER expression transcriptionally, by binding the ER promoter and forming a complex with CARM1, CBP, c-Jun, RNA polymerase II and Sp1. In this study, the MMTV-Wnt-1 transgenic mouse model was used to demonstrate that p53 regulation of ER expression and function is not solely an in vitro phenomenon, but it is also operational in mammary tumorigenesis in vivo. The expression of ER and the ability to respond to tamoxifen were determined in mammary tumors arising in p53 wild type (WT) or p53 heterozygous (HT) animals carrying the Wnt-1 transgene. In p53 WT mice, development of ER-positive tumors was delayed by tamoxifen treatment, while tumors arising in p53 HT mice had significantly reduced levels of ER and were not affected by tamoxifen. P53 null tumors were also found in the p53 HT mice and these tumors were ER-negative. ER expression was upregulated in mouse mammary tumor cell lines following transfection with WT p53 or treatment with doxorubicin. These data demonstrate that p53 regulates ER expression in vivo, and affects response to tamoxifen. Results also provide an explanation for the concordant relationship between these prognostic proteins in human breast tumors.

Overexpression of cyclooxygenase-2 (COX-2) in the mouse urinary bladder induces the expression of immune- and cell proliferation-related genes.

The mechanisms whereby cyclooxygenase-2 (COX-2) overexpression may contribute to bladder carcinogenesis remain unknown. We recently developed a transgenic mouse model overexpressing COX-2 under the control of a bovine keratin 5 (BK5) promoter causing a high incidence of transitional cell hyperplasia (TCH) in the bladder with a proportion of lesions progressing to invasive carcinoma. Microarray gene analysis was employed to determine the effects of COX-2 overexpression on gene expression profiles in the urinary bladder. Statistical analysis revealed that 70 genes were upregulated and 60 were downregulated by twofold or more in bladders from transgenic compared to wild-type mice. Expression Analysis Systematic Explorer (EASE) analysis revealed that genes associated with Immune/Stress Response and Cell Cycle/Proliferation biological processes were overexpressed in the transgenic mice. Relevant downregulated genes included three transforming growth factor (TGF)-beta related genes, Tgfb2, Tgfb3, and Tgfbi. The growth factor epiregulin was the most highly induced gene among those validated by qRT-PCR in TCH of BK5.COX-2 mouse bladders in parallel with increased staining for Ki67. Prostaglandin E(2) (PGE(2)) directly induced the expression of epiregulin mRNA in bladders from wild-type FVB mice ex vivo. We further determined that recombinant epiregulin increased both cell proliferation and Erk phosphorylation in UMUC-3 bladder cancer cells. These results indicate that the response of the mouse urinary bladder to elevated COX-2 expression includes enhanced inflammatory response and induction of cell proliferation. The growth factor epiregulin may play a role in bladder carcinogenesis and may serve as a novel target for the prevention and treatment of bladder cancer.

Paracrine overexpression of insulin-like growth factor-1 enhances mammary tumorigenesis in vivo.

Insulin-like growth factor-1 (IGF-1) stimulates proliferation, regulates tissue development, protects against apoptosis, and promotes the malignant phenotype in the breast and other organs. Some epidemiological studies have linked high circulating levels of IGF-1 with an increased risk of breast cancer. To study the role of IGF-1 in mammary tumorigenesis in vivo, we used transgenic mice in which overexpression of IGF-1 is under the control of the bovine keratin 5 (BK5) promoter and is directed to either the myoepithelial or basal cells in a variety of organs, including the mammary gland. This model closely recapitulates the paracrine exposure of breast epithelium to stromal IGF-1 seen in women. Histologically, mammary glands from transgenic mice were hyperplastic and highly vascularized. Mammary glands from prepubertal transgenic mice had significantly increased ductal proliferation compared with wild-type tissues, although this difference was not maintained after puberty. Transgenic mice also had increased susceptibility to mammary carcinogenesis, and 74% of the BK5.IGF-1 mice treated with 7,12-dimethylbenz[a]anthracene (20 microg/day) developed mammary tumors compared with 29% of the wild-type mice. Interestingly, 31% of the vehicle-treated BK5.IGF-1 animals, but none of the wild-type animals, spontaneously developed mammary cancer. The mammary tumors were moderately differentiated adenocarcinomas that expressed functional, nuclear estrogen receptor at both the protein and mRNA levels. These data support the hypothesis that tissue overexpression of IGF-1 stimulates mammary tumorigenesis.

The resistance to the tumor suppressive effects of COX inhibitors and COX-2 gene disruption in TRAMP mice is associated with the loss of COX expression in prostate tissue.

Over-expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) has been demonstrated to play a significant role in the tumorigenesis of colon, lung, breast, bladder and skin. However, inconsistent and controversial reports on the expression and activity of COX-2 in prostate cancer raised the question of whether COX-2 plays a pivotal role in prostate carcinogenesis. To address this question, we examined the effects of COX-2 inhibition on prostate tumorigenesis in the transgenic adenocarcinoma mouse prostate (TRAMP) model. Three-week-old TRAMP mice were fed control, celecoxib- or indomethacin-supplemented diets for 27 weeks. A TRAMP/COX-2 knockout mouse model was also generated to determine the effects of the loss of the COX-2 gene on prostate tumorigenesis in TRAMP mice. These studies demonstrated that neither non-steroidal anti-inflammatory drugs (NSAIDs) nor genetic disruption of COX-2 was inhibitory in terms of tumor and metastases incidence, lobe weight or types of pathological lesions. A careful analysis of wild-type and TRAMP tissues was undertaken for the expression of cyclooxygenase-1 (COX-1) and COX-2 using immunoblotting, quantitative real time polymerase chain reaction (qRT-PCR) and immunohistochemistry approaches in TRAMP dorsal prostate tissue from 10- and 16-week-old, as well as tumor from 30-week-old mice. We found that the expression of COX-1 and COX-2 dramatically decreased during TRAMP carcinogenesis. Using the probasin promoter, a COX-2 over-expressing mouse model was also generated but failed to show any pathology in any of the prostate lobes. Collectively, our results suggest that COX-2 may not play a tumorigenic role during prostate carcinogenesis in the TRAMP model.