Matrix metalloproteinase-dependent microsomal prostaglandin E synthase-1 expression in macrophages: role of TNF-α and the EP4 prostanoid receptor.

Matrix metalloproteinase (MMP)-9 contributes to the pathogenesis of chronic inflammatory diseases and cancer. Thus, identifying targetable components of signaling pathways that regulate MMP-9 expression may have broad therapeutic implications. Our previous studies revealed a nexus between metalloproteinases and prostanoids whereby MMP-1 and MMP-3, commonly found in inflammatory and neoplastic foci, stimulate macrophage MMP-9 expression via the release of TNF-α and subsequent induction of cyclooxygenase-2 and PGE(2) engagement of EP4 receptor. In the current study, we determined whether MMP-induced cyclooxygenase-2 expression was coupled to the expression of prostaglandin E synthase family members. We found that MMP-1- and MMP-3-dependent release of TNF-α induced rapid and transient expression of early growth response protein 1 in macrophages followed by sustained elevation in microsomal prostaglandin synthase 1 (mPGES-1) expression. Metalloproteinase-induced PGE(2) levels and MMP-9 expression were markedly attenuated in macrophages in which mPGES-1 was silenced, thereby identifying mPGES-1 as a therapeutic target in the regulation of MMP-9 expression. Finally, the induction of mPGES-1 was regulated, in part, through a positive feedback loop dependent on PGE(2) binding to EP4. Thus, in addition to inhibiting macrophage MMP-9 expression, EP4 antagonists emerge as potential therapy to reduce mPGES-1 expression and PGE(2) levels in inflammatory and neoplastic settings.

Matrix metalloproteinase (MMP)-1 and MMP-3 induce macrophage MMP-9: evidence for the role of TNF-alpha and cyclooxygenase-2.

Matrix metalloproteinase (MMP)-9 (gelatinase B) participates in a variety of diverse physiologic and pathologic processes. We recently characterized a cyclooxygenase-2 (COX-2)-->PGE(2)-->EP4 receptor axis that regulates macrophage MMP-9 expression. In the present studies, we determined whether MMPs, commonly found in inflamed and neoplastic tissues, regulate this prostanoid-EP receptor axis leading to enhanced MMP-9 expression. Results demonstrate that exposure of murine peritoneal macrophages and RAW264.7 macrophages to MMP-1 (collagenase-1) or MMP-3 (stromelysin-1) lead to a marked increase in COX-2 expression, PGE(2) secretion, and subsequent induction of MMP-9 expression. Proteinase-induced MMP-9 expression was blocked in macrophages preincubated with the selective COX-2 inhibitor celecoxib or transfected with COX-2 small interfering RNA (siRNA). Likewise, proteinase-induced MMP-9 was blocked in macrophages preincubated with the EP4 antagonist ONO-AE3-208 or transfected with EP4 siRNA. Exposure of macrophages to MMP-1 and MMP-3 triggered the rapid release of TNF-alpha, which was blocked by MMP inhibitors. Furthermore, both COX-2 and MMP-9 expression were inhibited in macrophages preincubated with anti-TNF-alpha IgG or transfected with TNF-alpha siRNA. Thus, proteinase-induced MMP-9 expression by macrophages is dependent on the release of TNF-alpha, induction of COX-2 expression, and PGE(2) engagement of EP4. The ability of MMP-1 and MMP-3 to regulate macrophage secretion of PGE(2) and expression of MMP-9 defines a nexus between MMPs and prostanoids that is likely to play a role in the pathogenesis of chronic inflammatory diseases and cancer. These data also suggest that this nexus is targetable utilizing anti-TNF-alpha therapies and/or selective EP4 antagonists.

Pilot randomized phase II study of celecoxib in oral premalignant lesions.

PURPOSE: Cyclooxygenase-2 (COX-2)-specific inhibition suppresses carcinogenesis in preclinical models and is a promising strategy for preventing oral cancer. In this pilot randomized phase II study, we evaluated the efficacy and safety of the COX-2 inhibitor celecoxib in patients with oral premalignant lesions (OPL). EXPERIMENTAL DESIGN: Patients were randomly assigned to placebo (n=18), celecoxib 100 mg twice daily (n=17), or celecoxib 200 mg twice daily (n=15) for 12 weeks. Six additional patients received celecoxib (400 mg twice daily) in an unblinded extension of the study. Biopsies were obtained at baseline and week 12. All patients entering the study were required to have at least one histologically confirmed early (atypical hyperplasia, atypical hyperkeratosis, or mild dysplasia) or advanced (moderate to severe dysplasia) OPL. RESULTS: Forty-nine patients (46 of 50 randomized and 3 of 6 open label) were evaluable for efficacy analyses. There were no statistically significant differences between the response rates of the randomly assigned arms: placebo, 33.3% (6 of 18); celecoxib 100 mg twice daily, 41.2% (7 of 17); and celecoxib 200 mg twice daily, 20.0% (3 of 15). Two patients responded on celecoxib 400 mg twice daily. Celecoxib was generally well tolerated. Patients with higher baseline COX-2 mRNA levels had an increased risk of disease progression within 3 months. CONCLUSIONS: Celecoxib at 100 or 200 mg twice daily was ineffective in controlling OPLs in this randomized controlled trial. This result and cardiovascular toxicity results of other (large scale) randomized controlled trials of selective COX-2 inhibitors have discouraged the continued investigation of these agents in oral cancer chemoprevention. Better methods for identifying high-risk patients and more active interventions are needed for future oral cancer chemoprevention trials.

Tobacco smoke induces urokinase-type plasminogen activator and cell invasiveness: evidence for an epidermal growth factor receptor dependent mechanism.

Multiple tobacco smoke-related premalignant and malignant lesions develop synchronously or metachronously in various organ sites, including the oral cavity. Both field cancerization and clonal migration seem to contribute to the occurrence of multiple tumors. Although the importance of endogenous factors (e.g., oncogenes) in regulating clonal migration is well established, little is known about the role of exogenous factors. Hence, the main objective of this study was to elucidate the mechanism by which tobacco smoke stimulated the migration of cells through extracellular matrix (ECM). Treatment of MSK-Leuk1 cells with a saline extract of tobacco smoke induced the migration of cells through ECM. Tobacco smoke induced the expression of urokinase-type plasminogen activator (uPA), resulting in plasmin-dependent degradation of ECM and increased cell migration. AG1478, a small-molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, a neutralizing antibody to EGFR, or an antibody to amphiregulin, an EGFR ligand, also blocked tobacco smoke-mediated induction of uPA and cell migration through ECM. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase activity, caused similar inhibitory effects. Taken together, these results suggest that tobacco smoke activated the EGFR-->extracellular signal-regulated kinase 1/2 MAPK pathway, causing induction of uPA. This led, in turn, to increased plasmin-dependent degradation of matrix proteins and enhanced cell migration through ECM. These data strongly suggest that chemicals in tobacco smoke can mimic the effects of oncogenes in regulating uPA-dependent cell invasion through ECM. These findings also strengthen the rationale for determining whether inhibitors of EGFR tyrosine kinase reduce the risk of tobacco smoke-related second primary tumors.

Inhibition of Jun NH2-terminal kinases suppresses the growth of experimental head and neck squamous cell carcinoma.

PURPOSE: This study was carried out to investigate whether c-Jun NH2-terminal kinases (JNK) are potential targets for treating head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: JNK activity was first evaluated in 20 paired samples of human HNSCC. The antitumor activity of SP600125, a reversible nonselective ATP-competitive inhibitor of JNKs, was then investigated both in an HNSCC xenograft model and in vitro using immunohistochemistry, immunoblotting, enzyme immunoassay, flow cytometry, and a Matrigel assay of capillary tube formation. Complementary studies were carried out using small interfering RNA to JNK1/2. RESULTS: JNK activity was increased in human HNSCC compared with normal-appearing epithelium. Treatment of mice bearing HNSCC xenografts with SP600125 resulted in >60% inhibition of tumor growth relative to vehicle-treated animals. Inhibition of tumor growth was associated with significant reductions in both cell proliferation and microvessel density. SP600125 inhibited tumor cell proliferation by causing delays in both the S and G2-M phases of the cell cycle. Inhibition of angiogenesis seemed to reflect effects on both tumor and endothelial cells. The JNK inhibitor suppressed the production of vascular endothelial growth factor and interleukin-8 by tumor cells and also inhibited endothelial cell proliferation and capillary tube formation. Reduced amounts and phosphorylation of epidermal growth factor receptor were found in tumor cells after treatment with SP600125. Small interfering RNA-mediated suppression of JNK1/2 led to reduced tumor cell proliferation and decreased levels of epidermal growth factor receptor, vascular endothelial growth factor, and interleukin-8. CONCLUSIONS: JNK activity is commonly increased in HNSCC. Our preclinical results provide a rationale for evaluating JNK inhibition as an approach to treating HNSCC.

Tobacco smoke stimulates the transcription of amphiregulin in human oral epithelial cells: evidence of a cyclic AMP-responsive element binding protein-dependent mechanism.

Activation of epidermal growth factor receptor (EGFR)-mediated signaling has been implicated in the pathogenesis of tobacco smoke-induced cancers. Recently, elevated levels of amphiregulin, a ligand of the EGFR, were found in the oral mucosa of smokers. The main objective of this study was to elucidate the mechanism by which tobacco smoke induces amphiregulin. Treatment of a nontumorigenic human oral epithelial cell line (MSK-Leuk1) with a saline extract of tobacco smoke stimulated amphiregulin (AR) transcription resulting in increased amounts of amphiregulin mRNA and protein. Tobacco smoke stimulated the cyclic AMP (cAMP)-->protein kinase A (PKA) pathway leading to increased cAMP-responsive element binding protein-dependent activation of AR transcription. These inductive effects of tobacco smoke were dependent on the aryl hydrocarbon receptor (AhR). In fact, alpha-naphthoflavone, an AhR antagonist, blocked tobacco smoke-mediated induction of binding of cAMP-responsive element binding protein to the AR promoter and thereby suppressed the induction of amphiregulin. Notably, treatment of MSK-Leuk1 cells with tobacco smoke or exogenous amphiregulin stimulated DNA synthesis. An inhibitor of EGFR tyrosine kinase or a neutralizing antibody to amphiregulin abrogated the increase in DNA synthesis mediated by tobacco smoke. Taken together, these findings suggest that tobacco smoke stimulated a signaling pathway comprised of AhR-->cAMP-->PKA resulting in enhanced AR transcription and increased DNA synthesis. The ability of tobacco smoke to induce amphiregulin and thereby enhance DNA synthesis is likely to contribute to the procarcinogenic effects of tobacco smoke.

Levels of cyclooxygenase-2 are increased in the oral mucosa of smokers: evidence for the role of epidermal growth factor receptor and its ligands.

Cyclooxygenase-2 (COX-2) is a promising pharmacologic target for preventing aerodigestive malignancies. In this study, we investigated the effects of tobacco smoke on the expression of COX-2 in oral mucosa. An approximately 4-fold increase in amount of COX-2 mRNA was observed in the oral mucosa of active smokers versus never smokers. Thus, a series of in vitro studies were carried out to elucidate the mechanism by which tobacco smoke induced COX-2. Treatment of a nontumorigenic oral epithelial cell line (MSK-Leuk1) with a saline extract of tobacco smoke (TS) stimulated COX-2 transcription, resulting in increased amounts of COX-2 mRNA, COX-2 protein, and prostaglandin E(2) (PGE(2)) synthesis. Exposure of cells to TS also caused an increase in epidermal growth factor receptor (EGFR) tyrosine kinase activity. Both an inhibitor of EGFR tyrosine kinase activity and a neutralizing anti-EGFR antibody blocked TS-mediated induction of COX-2. To define the mechanism by which TS activated EGFR, the release of amphiregulin and transforming growth factor alpha, two ligands of the EGFR, was measured. Exposure to TS caused a rapid increase in the release of both ligands. TS also markedly induced the expression of mRNAs for amphiregulin and transforming growth factor alpha. Importantly, increased expression of both ligands was also detected in the oral mucosa of active smokers. Taken together, these results suggest that activation of EGFR signaling contributes to the elevated levels of COX-2 found in the oral mucosa of smokers. Moreover, these findings strengthen the rationale for determining whether inhibitors of COX-2 or EGFR tyrosine kinase activity can reduce the risk of tobacco smoke-related malignancies of the aerodigestive tract.

Celecoxib inhibits prostate cancer growth: evidence of a cyclooxygenase-2-independent mechanism.

PURPOSE: Selective cyclooxygenase-2 (COX-2) inhibitors may suppress carcinogenesis by both COX-2-dependent and COX-2-independent mechanisms. The primary purpose of this study was to evaluate whether celecoxib or rofecoxib, two widely used selective COX-2 inhibitors, possess COX-2-independent antitumor activity. EXPERIMENTAL DESIGN: PC3 and LNCaP human prostate cancer cell lines were used to investigate the growth inhibitory effects of selective COX-2 inhibitors in vitro. To complement these studies, we evaluated the effect of celecoxib on the growth of PC3 xenografts. RESULTS: COX-1 but not COX-2 was detected in PC3 and LNCaP cells. Clinically achievable concentrations (2.5-5.0 micromol/L) of celecoxib inhibited the growth of both cell lines in vitro, whereas rofecoxib had no effect over the same concentration range. Celecoxib inhibited cell growth by inducing a G(1) cell cycle block and reducing DNA synthesis. Treatment with celecoxib also led to dose-dependent inhibition of PC3 xenograft growth without causing a reduction in intratumor prostaglandin E(2). Inhibition of tumor growth occurred at concentrations (2.37-5.70 micromol/L) of celecoxib in plasma that were comparable with the concentrations required to inhibit cell growth in vitro. The highest dose of celecoxib led to a 52% reduction in tumor volume and an approximately 50% decrease in both cell proliferation and microvessel density. Treatment with celecoxib caused a marked decrease in amounts of cyclin D1 both in vitro and in vivo. CONCLUSIONS: Two clinically available selective COX-2 inhibitors possess different COX-2-independent anticancer properties. The anticancer activity of celecoxib may reflect COX-2-independent in addition to COX-2-dependent effects.

Celecoxib, a selective cyclooxygenase 2 inhibitor, protects against human epidermal growth factor receptor 2 (HER-2)/neu-induced breast cancer.

Cyclooxygenase 2 (HER-2) (Cox-2), an inducible form of Cox, is overexpressed in HER-2/neu-positive human breast cancers. The aim of this study was to determine whether celecoxib, a selective Cox-2 inhibitor, protected against HER-2/neu-induced experimental breast cancer. Cox-2 protein was detected in breast carcinomas from mouse mammary tumor virus (MMTV)/neu mice. Treatment with celecoxib (500 ppm) significantly reduced the incidence of mammary tumors in MMTV/neu mice (P = 0.003) and caused about a 50% reduction in mammary prostaglandin E2 (PGE2) levels. Because mammary glands from MMTV/neu mice expressed all four PGE2 receptor subtypes, we speculate that signaling through PGE2 receptors is important for mammary tumorigenesis. These results strengthen the rationale for developing clinical trials to determine whether selective Cox-2 inhibitors possess anticancer properties in humans at risk for breast cancer.

Microsomal prostaglandin E synthase-1 is overexpressed in head and neck squamous cell carcinoma.

Elevated levels of prostaglandin E(2) (PGE(2)) occur in head and neck squamous cell carcinoma (HNSCC) and have been associated with a poor prognosis. Recently, an inducible microsomal prostaglandin E synthase-1 (mPGES) was identified. This enzyme converts the cyclooxygenase product prostaglandin H(2) (PGH(2)) to PGE(2). Given the apparent significance of PGE(2) in carcinogenesis, it is important to elucidate the mechanisms that account for increased amounts of PGE(2) in HNSCC. By immunoblot analysis, mPGES was overexpressed in 11 of 14 (79%) cases of HNSCC compared with adjacent normal tissue. Immunohistochemistry localized mPGES expression to neoplastic epithelial cells. Cell culture was used to determine whether cellular transformation was associated with increased amounts of mPGES. Levels of mPGES protein and mRNA were markedly elevated in HNSCC cell lines (1483 and Ca9-22) versus a nontumorigenic oral epithelial cell line (MSK-Leuk1). Interestingly, treatment of MSK-Leuk1 cells with PGE(2) caused both dose- and time-dependent stimulation of cell growth. Each of the four known receptors for PGE(2) (E-prostanoid receptor subtypes 1-4) was detected in head and neck squamous mucosa. Taken together, these results suggest that overexpression of mPGES contributes to the increased levels of PGE(2) found in HNSCC. Additional studies will be needed to determine whether this enzyme is a bona fide target for anticancer therapy.

Estrogen Protects against Obesity-Induced Mammary Gland Inflammation in Mice.

Obesity is a risk factor for the development of hormone receptor (HR)-positive breast cancer in postmenopausal women. Obesity causes subclinical inflammation in white adipose tissue (WAT), characterized by macrophages surrounding dead or dying adipocytes forming crown-like structures (CLS). Estrogen synthesis is catalyzed by aromatase. Previously, we demonstrated CLS and elevated levels of proinflammatory mediators and aromatase in the mammary glands of obese mice and breast tissue of obese women. Here, we tested the hypothesis that supplemental estrogen could prevent or reverse WAT inflammation (WATi) and related molecular changes in the mammary gland. C57BL/6J mice were ovariectomized (OVX) to simulate the postmenopausal state. Supplementation with 17ß-estradiol (E2) protected against high fat diet (HFD)-induced weight gain and mammary glands WATi. Expression of proinflammatory mediators (Cox-2, TNFα, IL1ß) and aromatase were also reduced in the mammary glands of mice that received supplemental E2. Next, to determine whether E2 supplementation can reverse WATi, obese OVX mice were treated with E2 or placebo and then continued on HFD. E2 supplementation induced weight loss, reversed mammary gland inflammation, and downregulated expression of proinflammatory mediators and aromatase. Finally, we determined whether the protective effects of E2 were mediated by estrogen receptor-α (ERα). Knocking out ERα in ovary intact mice fed a HFD led to weight gain, WATi and elevated levels of proinflammatory mediators and aromatase mimicking the effects of OVX. Taken together, our findings indicate that estrogen via ERα protects against weight gain, WATi and associated increases in proinflammatory mediators and aromatase in the mammary gland.

Obesity-dependent changes in interstitial ECM mechanics promote breast tumorigenesis.

Obesity and extracellular matrix (ECM) density are considered independent risk and prognostic factors for breast cancer. Whether they are functionally linked is uncertain. We investigated the hypothesis that obesity enhances local myofibroblast content in mammary adipose tissue and that these stromal changes increase malignant potential by enhancing interstitial ECM stiffness. Indeed, mammary fat of both diet- and genetically induced mouse models of obesity were enriched for myofibroblasts and stiffness-promoting ECM components. These differences were related to varied adipose stromal cell (ASC) characteristics because ASCs isolated from obese mice contained more myofibroblasts and deposited denser and stiffer ECMs relative to ASCs from lean control mice. Accordingly, decellularized matrices from obese ASCs stimulated mechanosignaling and thereby the malignant potential of breast cancer cells. Finally, the clinical relevance and translational potential of our findings were supported by analysis of patient specimens and the observation that caloric restriction in a mouse model reduces myofibroblast content in mammary fat. Collectively, these findings suggest that obesity-induced interstitial fibrosis promotes breast tumorigenesis by altering mammary ECM mechanics with important potential implications for anticancer therapies.

Dietary polyphenols suppress elevated levels of proinflammatory mediators and aromatase in the mammary gland of obese mice.

In postmenopausal women, obesity is a risk factor for the development of hormone receptor-positive breast cancer driven by estrogen. After menopause, aromatization of androgen precursors in adipose tissue is a major synthetic source of estrogen. Recently, in mouse models and women, we identified an obesity-inflammation-aromatase axis. This obesity-induced inflammation is characterized by crown-like structures (CLS) consisting of dead adipocytes encircled by macrophages in breast white adipose tissue. CLS occur in association with NF-κB activation, elevated levels of proinflammatory mediators, and increased aromatase expression. Saturated fatty acids released from adipocytes have been linked to obesity-related white adipose tissue inflammation. Here we found that stearic acid, a prototypic saturated fatty acid, stimulated Akt-dependent activation of NF-κB resulting in increased levels of proinflammatory mediators [TNF-α, interleukin (IL)-1ß, COX-2] in macrophages leading, in turn, to the induction of aromatase. Several polyphenols (resveratrol, curcumin, epigallocatechin gallate) blocked these inductive effects of stearic acid. Zyflamend, a widely used polyherbal preparation that contains numerous polyphenols, possessed similar suppressive effects. In a mouse model of obesity, treatment with Zyflamend suppressed levels of phospho-Akt, NF-κB binding activity, proinflammatory mediators, and aromatase in the mammary gland. Collectively, these results suggest that targeting the activation of NF-κB is a promising approach for reducing levels of proinflammatory mediators and aromatase in inflamed mouse mammary tissue. Further investigation in obese women is warranted.

Caloric restriction reverses obesity-induced mammary gland inflammation in mice.

Obesity is a risk factor for the development of hormone receptor-positive breast cancer in postmenopausal women. Estrogen synthesis is catalyzed by aromatase. Recently, we identified an obesity→inflammation→aromatase axis in mouse models and women. In mouse models of obesity, inflammatory foci characterized by crown-like structures (CLS) consisting of dead adipocytes encircled by macrophages were found in the mammary gland. CLS of the breast were found in most overweight and obese women. CLS were associated with adipocyte hypertrophy, activation of NF-κB, elevated levels of proinflammatory mediators and aromatase, and increased expression of the progesterone receptor (PR). Collectively, these findings provide a plausible explanation for the link between obesity, chronic inflammation, and postmenopausal breast cancer. Here, we investigated whether caloric restriction (CR) reversed the inflammatory state and related molecular changes in the mammary gland of obese mice. Obese ovariectomized C57BL/6J mice were subjected to 30% CR for 7 or 14 weeks. Findings in CR mice were compared with the results in mice fed a high-fat diet ad libitum or with control mice fed a low-fat diet. CR was associated with more than a 75% decrease in mammary CLS/cm(2). Reduced histologic inflammation following CR was associated with decreased adipocyte diameter and monocyte chemoattractant protein-1 (MCP-1) levels, reduced NF-κB binding activity, and normalization of levels of proinflammatory mediators, aromatase, and PR. In summary, obesity-related inflammation of the mammary gland and elevated aromatase and PR levels were reversed with CR. Our results provide a rationale for determining whether weight loss can reverse breast inflammation associated with obesity in women.

Increased levels of COX-2 and prostaglandin E2 contribute to elevated aromatase expression in inflamed breast tissue of obese women.

UNLABELLED: Obesity is a risk factor for hormone receptor-positive breast cancer in postmenopausal women. Estrogen synthesis is catalyzed by aromatase, which is encoded by CYP19. We previously showed that aromatase expression and activity are increased in the breast tissue of overweight and obese women in the presence of characteristic inflammatory foci [crown-like structures of the breast (CLS-B)]. In preclinical studies, proinflammatory prostaglandin E(2) (PGE(2)) is a determinant of aromatase expression. We provide evidence that cyclooxygenase (COX)-2-derived PGE(2) stimulates the cyclic AMP (cAMP) → PKA signal transduction pathway that activates CYP19 transcription, resulting in increased aromatase expression and elevated progesterone receptor levels in breast tissues from overweight and obese women. We further demonstrate that a measure of in-breast inflammation (CLS-B index) is a better correlate of these biologic end points than body mass index. The obesity → inflammation → aromatase axis is likely to contribute to the increased risk of hormone receptor-positive breast cancer and the worse prognosis of obese patients with breast cancer. SIGNIFICANCE: We show that obesity-associated inflammatory foci in the human breast are associated with elevated COX-2 levels and activation of the PGE2 → cAMP → PKA signal transduction pathway resulting in increased aromatase expression. These findings help to explain the link among obesity, low-grade chronic inflammation, and breast cancer with important clinical implications.