Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.

Axl and Mertk Receptors Cooperate to Promote Breast Cancer Progression by Combined Oncogenic Signaling and Evasion of Host Antitumor Immunity.

Despite the promising clinical benefit of targeted and immune checkpoint blocking therapeutics, current strategies have limited success in breast cancer, indicating that additional inhibitory pathways are required to complement existing therapeutics. TAM receptors (Tyro-3, Axl, and Mertk) are often correlated with poor prognosis because of their capacities to sustain an immunosuppressive environment. Here, we ablate Axl on tumor cells using CRISPR/Cas9 gene editing, and by targeting Mertk in the tumor microenvironment (TME), we observed distinct functions of TAM as oncogenic kinases, as well as inhibitory immune receptors. Depletion of Axl suppressed cell intrinsic oncogenic properties, decreased tumor growth, reduced the incidence of lung metastasis and increased overall survival of mice when injected into mammary fat pad of syngeneic mice, and demonstrated synergy when combined with anti-PD-1 therapy. Blockade of Mertk function on macrophages decreased efferocytosis, altered the cytokine milieu, and resulted in suppressed macrophage gene expression patterns. Mertk-knockout mice or treatment with anti-Mertk-neutralizing mAb also altered the cellular immune profile, resulting in a more inflamed tumor environment with enhanced T-cell infiltration into tumors and T-cell-mediated cytotoxicity. The antitumor activity from Mertk inhibition was abrogated by depletion of cytotoxic CD8α T cells by using anti-CD8α mAb or by transplantation of tumor cells into B6.CB17-Prkdc SCID mice. Our data indicate that targeting Axl expressed on tumor cells and Mertk in the TME is predicted to have a combinatorial benefit to enhance current immunotherapies and that Axl and Mertk have distinct functional activities that impair host antitumor response. SIGNIFICANCE: This study demonstrates how TAM receptors act both as oncogenic tyrosine kinases and as receptors that mediate immune evasion in cancer progression.

Pan-TAM Tyrosine Kinase Inhibitor BMS-777607 Enhances Anti-PD-1 mAb Efficacy in a Murine Model of Triple-Negative Breast Cancer.

Tyro3, Axl, and Mertk (TAM) represent a family of homologous tyrosine kinase receptors known for their functional role in phosphatidylserine (PS)-dependent clearance of apoptotic cells and also for their immune modulatory functions in the resolution of inflammation. Previous studies in our laboratory have shown that Gas6/PS-mediated activation of TAM receptors on tumor cells leads to subsequent upregulation of PD-L1, defining a putative PS→TAM receptor→PD-L1 inhibitory signaling axis in the cancer microenvironment that may promote tolerance. In this study, we tested combinations of TAM inhibitors and PD-1 mAbs in a syngeneic orthotopic E0771 murine triple-negative breast cancer model, whereby tumor-bearing mice were treated with pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 alone or in combination. Tyro3, Axl, and Mertk were differentially expressed on multiple cell subtypes in the tumor microenvironment. Although monotherapeutic administration of either pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 mAb therapy showed partial antitumor activity, combined treatment of BMS-777607 with anti-PD-1 significantly decreased tumor growth and incidence of lung metastasis. Moreover, combined treatment with BMS-777607 and anti-PD-1 showed increased infiltration of immune stimulatory T cells versus either monotherapy treatment alone. RNA NanoString profiling showed enhanced infiltration of antitumor effector T cells and a skewed immunogenic immune profile. Proinflammatory cytokines increased with combinational treatment. Together, these studies indicate that pan-TAM inhibitor BMS-777607 cooperates with anti-PD-1 in a syngeneic mouse model for triple-negative breast cancer and highlights the clinical potential for this combined therapy. SIGNIFICANCE: These findings show that pan-inhibition of TAM receptors in combination with anti-PD-1 may have clinical value as cancer therapeutics to promote an inflammatory tumor microenvironment and improve host antitumor immunity.

Reawakening of dormant estrogen-dependent human breast cancer cells by bone marrow stroma secretory senescence.

BACKGROUND: Dormant estrogen receptor positive (ER+) breast cancer micrometastases in the bone marrow survive adjuvant chemotherapy and recur stochastically for more than 20 years. We hypothesized that inflammatory cytokines produced by stromal injury can re-awaken dormant breast cancer cells. METHODS: We used an established in vitro dormancy model of Michigan Cancer Foundation-7 (MCF-7) breast cancer cells incubated at clonogenic density on fibronectin-coated plates to determine the effects of inflammatory cytokines on reactivation of dormant ER+ breast cancer cells. We measured induction of a mesenchymal phenotype, motility and the capacity to re-enter dormancy. We induced secretory senescence in murine stromal monolayers by oxidation, hypoxia and estrogen deprivation with hydrogen peroxide (H2O2), carbonyl-cyanide m-chlorophenylhydrazzone (CCCP) and Fulvestrant (ICI 182780), respectively, and determined the effects on growth of co-cultivated breast cancer cells. RESULTS: Exogenous recombinant human (rh) interleukin (IL)-6, IL-8 or transforming growth factor ß1 (TGFß1) induced regrowth of dormant MCF-7 cells on fibronectin-coated plates. Dormant cells had decreased expression of E-cadherin and estrogen receptor α (ERα) and increased expression of N-cadherin and SNAI2 (SLUG). Cytokine or TGFß1 treatment of dormant clones induced formation of growing clones, a mesenchymal appearance, increased motility and an impaired capacity to re-enter dormancy. Stromal injury induced secretion of IL-6, IL-8, upregulated tumor necrosis factor alpha (TNFα), activated TGFß and stimulated the growth of co-cultivated MCF-7 cells. MCF-7 cells induced secretion of IL-6 and IL-8 by stroma in co-culture. CONCLUSIONS: Dormant ER+ breast cancer cells have activated epithelial mesenchymal transition (EMT) gene expression programs and downregulated ERα but maintain a dormant epithelial phenotype. Stromal inflammation reactivates these cells, induces growth and a mesenchymal phenotype. Reactivated, growing cells have an impaired ability to re-enter dormancy. In turn, breast cancer cells co-cultured with stroma induce secretion of IL-6 and IL-8 by the stroma, creating a positive feedback loop.

Crk adaptor protein promotes PD-L1 expression, EMT and immune evasion in a murine model of triple-negative breast cancer.

The tumor infiltration of immune cells in solid cancers can profoundly influence host antitumor responses. In recent years, immunotherapeutic regimens, that target immune checkpoints, demonstrated significant antitumor response by increasing intra-tumoral immune cell populations, including CD8+ effector T cells. However, administration of such immune checkpoint inhibitors is largely inefficacious in inducing immunogenicity and treating breast cancer. Currently, there is a great need to better understand cell autonomous mechanisms of immune evasion in breast cancer to identify upstream therapeutic targets that increase the efficacy of immunotherapy. Here we show that Crk, an SH2 and SH3 domain-containing adaptor protein implicated in focal adhesion signaling, cell migration, and invasion, and frequently up-regulated in human cancers, has an important role in regulating the tumor immune microenvironment. Using a murine 4T1 breast adenocarcinoma model of spontaneous metastasis in immune-competent BALB/C mice, we show that genetic ablation of Crk by CRISPR-Cas9 leads to enhanced anti-tumor immune cell populations, cytotoxic effector and immune surveillance cytokines in primary tumor. Pathologically, this leads to a significant reduction in tumor growth and lung metastasis. Mechanistically, Crk KO suppresses EMT and PD-L1 expression on tumor cells and acts additively with anti-PD1 therapy to suppress tumor growth and metastasis outcomes. Taken together, these data reveal a previously un-described function of Crk adaptor protein expression in tumor cells for cell autonomous regulation of tumor immune microenvironment.

Normalization of TAM post-receptor signaling reveals a cell invasive signature for Axl tyrosine kinase.

BACKGROUND: Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis. METHODS: In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling. RESULTS: Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk. CONCLUSION: These studies demonstrate that TAM show unique post-receptor signatures that impinge on distinct gene expression profiles and tumorigenic outcomes.

Epac1 increases migration of endothelial cells and melanoma cells via FGF2-mediated paracrine signaling.

Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N-sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N-sulfation of HS in melanoma. Therefore, we examined whether Epac1 regulates FGF2-mediated cell-cell communication. Conditioned medium (CM) of melanoma cells with abundant expression of Epac1 increased migration of human umbilical endothelial cells (HUVEC) and melanoma cells with poor expression of Epac1. CM-induced increase in migration was inhibited by antagonizing FGF2, by the removal of HS and by the knockdown of Epac1. In addition, knockdown of Epac1 suppressed the binding of FGF2 to FGF receptor in HUVEC, and in vivo angiogenesis in melanoma. Furthermore, knockdown of Epac1 reduced N-sulfation of HS chains attached to perlecan, a major secreted type of HS proteoglycan that mediates the binding of FGF2 to FGF receptor. These data suggested that Epac1 in melanoma cells regulates melanoma progression via the HS-FGF2-mediated cell-cell communication.

Store-operated Ca2+ entry (SOCE) regulates melanoma proliferation and cell migration.

Store-operated Ca(2+) entry (SOCE) is a major mechanism of Ca(2) (+) import from extracellular to intracellular space, involving detection of Ca(2+) store depletion in endoplasmic reticulum (ER) by stromal interaction molecule (STIM) proteins, which then translocate to plasma membrane and activate Orai Ca(2+) channels there. We found that STIM1 and Orai1 isoforms were abundantly expressed in human melanoma tissues and multiple melanoma/melanocyte cell lines. We confirmed that these cell lines exhibited SOCE, which was inhibited by knockdown of STIM1 or Orai1, or by a pharmacological SOCE inhibitor. Inhibition of SOCE suppressed melanoma cell proliferation and migration/metastasis. Induction of SOCE was associated with activation of extracellular-signal-regulated kinase (ERK), and was inhibited by inhibitors of calmodulin kinase II (CaMKII) or Raf-1, suggesting that SOCE-mediated cellular functions are controlled via the CaMKII/Raf-1/ERK signaling pathway. Our findings indicate that SOCE contributes to melanoma progression, and therefore may be a new potential target for treatment of melanoma, irrespective of whether or not Braf mutation is present.

'Reduced malignancy as a mechanism for longevity in mice with adenylyl cyclase type 5 disruption'.

Disruption of adenylyl cyclase type 5 (AC5) knockout (KO) is a novel model for longevity. Because malignancy is a major cause of death and reduced lifespan in mice, the goal of this investigation was to examine the role of AC5KO in protecting against cancer. There have been numerous discoveries in genetically engineered mice over the past several decades, but few have been translated to the bedside. One major reason is that it is difficult to alter a gene in patients, but rather a pharmacological approach is more appropriate. The current investigation employs a parallel construction to examine the extent to which inhibiting AC5, either in a genetic knockout (KO) or by a specific pharmacological inhibitor protects against cancer. This study is unique, not only because a combined genetic and pharmacological approach is rare, but also there are no prior studies on the extent to which AC5 affects cancer. We found that AC5KO delayed age-related tumor incidence significantly, as well as protecting against mammary tumor development in AC5KO × MMTV-HER-2 neu mice, and B16F10 melanoma tumor growth, which can explain why AC5KO is a model of longevity. In addition, a Food and Drug Administration approved antiviral agent, adenine 9-ß-D-arabinofuranoside (Vidarabine or AraAde), which specifically inhibits AC5, reduces LP07 lung and B16F10 melanoma tumor growth in syngeneic mice. Thus, inhibition of AC5 is a previously unreported mechanism for prevention of cancers associated with aging and that can be targeted by an available pharmacologic inhibitor, with potential consequent extension of lifespan.

Calorie restriction can reverse, as well as prevent, aging cardiomyopathy.

Calorie restriction (CR) is the most widely studied intervention protecting from the adverse effects of aging. Almost all prior studies have examined the effects of CR initiated in young animals. Studies examining the effects of CR on development of aging cardiomyopathy found only partial prevention. The major goal of this study was to determine whether CR initiated after aging cardiomyopathy developed could reverse the cardiomyopathy. Aging cardiomyopathy in 2-year-old mice was characterized by reduced left ventricular (LV) function, cardiac hypertrophy, and increased cardiac apoptosis and fibrosis. When short-term (2 months) CR was initiated after aging cardiomyopathy developed in 20-month-old mice, the decrease in cardiac function, and increases in LV weight, myocardial fibrosis and apoptosis were reversed, such that the aging hearts in these mice were indistinguishable from those of young mice or mice where CR was initiated in young mice. If apoptosis was the mechanism for protecting against aging cardiomyopathy, then total myocyte numbers should have reverted to normal with CR, but did not. However, the alterations in cytoskeletal proteins, which contribute to aging cardiomyopathy, were no longer observed with CR. This is the first study to demonstrate complete prevention of aging cardiomyopathy by CR and, more importantly, that instituting this intervention even later in life can rapidly correct aging cardiomyopathy, which could have important therapeutic implications.

Common mechanisms for calorie restriction and adenylyl cyclase type 5 knockout models of longevity.

Adenylyl cyclase type 5 knockout mice (AC5 KO) live longer and are stress resistant, similar to calorie restriction (CR). AC5 KO mice eat more, but actually weigh less and accumulate less fat compared with WT mice. CR applied to AC5 KO results in rapid decrease in body weight, metabolic deterioration, and death. These data suggest that despite restricted food intake in CR, but augmented food intake in AC5 KO, the two models affect longevity and metabolism similarly. To determine shared molecular mechanisms, mRNA expression was examined genome-wide for brain, heart, skeletal muscle, and liver. Significantly more genes were regulated commonly rather than oppositely in all the tissues in both models, indicating commonality between AC5 KO and CR. Gene ontology analysis identified many significantly regulated, tissue-specific pathways shared by the two models, including sensory perception in heart and brain, muscle function in skeletal muscle, and lipid metabolism in liver. Moreover, when comparing gene expression changes in the heart under stress, the glutathione regulatory pathway was consistently upregulated in the longevity models but downregulated with stress. In addition, AC5 and CR shared changes in genes and proteins involved in the regulation of longevity and stress resistance, including Sirt1, ApoD, and olfactory receptors in both young- and intermediate-age mice. Thus, the similarly regulated genes and pathways in AC5 KO and CR mice, particularly related to the metabolic phenotype, suggest a unified theory for longevity and stress resistance.

Sexual dimorphism in cardiac triacylglyceride dynamics in mice on long term caloric restriction.

Human studies indicate augmented myocardial lipid metabolism in females, and that sex and obesity interact to predict myocardial fatty acid oxidation and storage. Altered lipid dynamics precede cardiomyopathies, and many studies now address high fat diets. Conversely, caloric restriction (CR), is the most studied model for longevity and stress resistance, including protection against myocardial ischemia. However, no information exists on the effects of long-term caloric restriction (CR) on triacylglyceride (TAG) content and dynamics in the heart. This study explored the effects of CR, sex and age on TAG dynamics in mouse hearts. Male and female SVJ129 mice were fed either normal (ND) or CR diet for 3 or 10 months. In 5-month-old mice, CR similarly decreased cardiac TAG in males (ND: 25.5±4.5 nmol/mg protein; CR: 12.6±2.7, P<0.05) and females (ND: 30.1±4.4; CR: 13.7±1.2) (no significant differences in TAG content were seen between sexes). CR reduced the contribution of exogenous palmitate to oxidative metabolism in males and females, by 15% and 11% respectively, versus ND, without affecting cardiac workload. CR also induced a larger reduction in TAG turnover in male (68%) than female hearts (38%). Interestingly, in 5 month old male mice, CR reproduced the lower TAG turnover rates of middle-aged males (ND 13-month-old male=423±76 nmol/mg protein/min). Thus, long term CR reduces TAG pool dynamics. Despite reduced content, hearts of female mice subjected to CR retained a more dynamic TAG pool than males, while males respond with greater metabolic remodeling of cardiac lipid dynamics.

Gßγ subunits inhibit Epac-induced melanoma cell migration.

BACKGROUND: Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein ßγ subunits (Gßγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gßγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gßγ and Epac in melanoma, which plays a role in regulation of cell migration. METHODS: SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers. RESULTS: The effect of Gßγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gßγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-ß-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gßγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of ß1 and γ2, which is the major combination of Gßγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of ß adrenergic receptor kinase (ßARK-CT), an endogenous inhibitor for Gßγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gßγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of ß1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gßγ with ßARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPßS), a GDP analogue that inactivates Gßγ, restored 8-pMeOPT-induced Ca 2+ elevation even in the presence of mSIRK. These data suggested that Gßγ inhibits Epac-induced Ca 2+ elevation. Subsequently, the mechanism by which Gßγ inhibits Epac-induced Ca 2+ elevation was explored. mSIRK activates Ca 2+ influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration. These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration. CONCLUSION: We found the cross talk of Ca 2+ signaling between Gßγ and Epac, which plays a major role in melanoma cell migration.

Caloric restriction reduces growth of mammary tumors and metastases.

We investigated the effects of caloric restriction (CR) on growth of tumors and metastases in the 4T1 mammary tumor model and found that CR, compared with normal diet, reduced the growth of mammary tumors and metastases and the total number of metastases that originated both spontaneously from the primary tumor and also experimentally from i.v. injection of the tumor cells. CR also decreased proliferation and angiogenesis and increased apoptosis in tumors. CR reduced levels of insulin, leptin, insulin-like growth factor 1, insulin-like growth factor binding protein 3 and increased adiponectin in tumors. We also demonstrated that tumors from CR mice possessed lower levels of transforming growth factor-ß, lower intratumor deposition of collagen IV and reduced invasiveness due to a decrease in tumor secretion of active matrix metalloproteinase 9. Our results suggest that CR-induced metabolic and signaling changes affect the stroma and the tumor cells resulting in a microenvironment that prevents proliferation of breast tumors and their metastases.

Epac1 promotes melanoma metastasis via modification of heparan sulfate.

Our previous report suggested the potential role of the exchange protein directly activated by cyclic AMP (Epac) in melanoma metastasis via heparan sulfate (HS)-mediated cell migration. In order to obtain conclusive evidence that Epac1 plays a critical role in modification of HS and melanoma metastasis, we extensively investigated expression and function of Epac1 in human melanoma samples and cell lines. We have found that, in human melanoma tissue microarray, protein expression of Epac1 was higher in metastatic melanoma than in primary melanoma. In addition, expression of Epac1 positively correlated with that of N-sulfated HS, and N-deacetylase/N-sulfotransferase-1 (NDST-1), an enzyme that increases N-sulfation of HS. Further, an Epac agonist increased, but ablation of Epac1 decreased, expressions of NDST-1, N-sulfated HS, and cell migration in various melanoma cell lines. Finally, C8161 cells with stable knockdown of Epac1 showed a decrease in cell migration, and metastasis in mice. These data suggest that Epac1 plays a critical role in melanoma metastasis presumably because of modification of HS.

Exchange protein directly activated by cyclic AMP increases melanoma cell migration by a Ca2+-dependent mechanism.

Melanoma has a poor prognosis due to its strong metastatic ability. Although Ca(2+) plays a major role in cell migration, little is known about the role of Ca(2+) in melanoma cell migration. We recently found that the exchange protein directly activated by cyclic AMP (Epac) increases melanoma cell migration via a heparan sulfate-related mechanism. In addition to this mechanism, we also found that Epac regulates melanoma cell migration by a Ca(2+)-dependent mechanism. An Epac agonist increased Ca(2+) in several different melanoma cell lines but not in melanocytes. Ablation of Epac1 with short hairpin RNA inhibited the Epac agonist-induced Ca(2+) elevation, suggesting the critical role of Epac1 in Ca(2+) homeostasis in melanoma cells. Epac-induced Ca(2+) elevation was negated by the inhibition of phospholipase C (PLC) and inositol triphosphate (IP(3)) receptor. Furthermore, Epac-induced cell migration was reduced by the inhibition of PLC or IP(3) receptor. These data suggest that Epac activates Ca(2+) release from the endoplasmic reticulum via the PLC/IP(3) receptor pathway, and this Ca(2+) elevation is involved in Epac-induced cell migration. Actin assembly was increased by Epac-induced Ca(2+), suggesting the involvement of actin in Epac-induced cell migration. In human melanoma specimens, mRNA expression of Epac1 was higher in metastatic melanoma than in primary melanoma, suggesting a role for Epac1 in melanoma metastasis. In conclusion, our findings reveal that Epac is a potential target for the suppression of melanoma cell migration, and, thus, the development of metastasis.

Potentially reduced exposure cigarettes accelerate atherosclerosis: evidence for the role of nicotine.

The tobacco industry markets potentially reduced exposure products (PREPs) as less harmful or addictive alternatives to conventional cigarettes. This study compared the effects of mainstream smoke from Quest, Eclipse, and 2R4F reference cigarettes on the development of atherosclerosis in apolipoprotein E-deficient (apoE -/-) mice. Mice were exposed to smoke from four cigarette types for 12 weeks beginning at age of 12 weeks, and in a separate study for 8 weeks, beginning at age of 8 weeks. In both studies, mice exposed to smoke from high-nicotine, high-tar Quest 1, and 2R4F cigarettes developed greater areas of lipid-rich aortic lesions than did non-smoking controls. Exposure to smoke from the lower-nicotine products, Eclipse, and Quest 3, was associated with smaller lesion areas, but animals exposed to smoke from all of the tested types of cigarette had larger lesions than did control animals not exposed to smoke. Urinary levels of isoprostane F2 alpha VI, increased proportionally to cigarette nicotine yield, whereas induction of pulmonary cytochrome P4501A1 was proportional to tar yield. Lesion area was associated with both nicotine and tar yields, although in multiple regression analysis only nicotine was a significant predictor of lesion area. Smoke exposure did not alter systolic blood pressure (SBP), heart rate (HR), blood cholesterol, or leukocyte count. Taken together, these observations suggest that smoking may accelerate atherosclerosis by increasing oxidative stress mediated at least in part via the actions of nicotine.

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.

Role of protein kinase C-dependent signaling pathways in the antiangiogenic properties of nafoxidine.

We analyzed the effect of nafoxidine on the earlier biological processes of angiogenesis and explored the role of different signaling pathways involved in the in vitro response of endothelial cells (HUVEC). Nafoxidine significantly inhibited adhesion, spreading, migration and invasion of HUVEC at concentrations ranging from 1 to 2.5 microM. Endothelial cord formation on Matrigel was inhibited by nafoxidine and cotreatment with phorbol-12-myristate-13-acetate (PMA) clearly prevented the antiangiogenic effect of the antiestrogen. On the contrary, cotreatment with the PKC inhibitor bisindolylmaleimide potentiated inhibition of cord formation. PMA also inhibited the nafoxidine-induced secretion of metalloproteinase-2 and tissue inhibitor of metalloproteinases-1 in HUVEC monolayers. Cotreatment with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the cAMP analog N6,2'-o-dibutyryladenosine 3',5'-cyclic monophosphate prevented the inhibition of endothelial cord formation induced by nafoxidine. Our work presents evidence about the signaling pathways involved in the antiangiogenic effect of nafoxidine, suggesting that PKC-dependent signaling pathways are essential in angiogenesis during endothelial cord formation.

Bryostatin-1 stimulates the transcription of cyclooxygenase-2: evidence for an activator protein-1-dependent mechanism.

Bryostatin-1 (bryostatin) is a macrocyclic lactone derived from Bugula neritina, a marine bryozoan. On the basis of the strength of in vitro and animal studies, bryostatin is being investigated as a possible treatment for a variety of human malignancies. Severe myalgias are a common dose-limiting side effect. Because cyclooxygenase-2 (COX-2)-derived prostaglandins can cause pain, we investigated whether bryostatin induced COX-2. Bryostatin (1-10 nM) induced COX-2 mRNA, COX-2 protein, and prostaglandin biosynthesis. These effects were observed in macrophages as well as in a series of human cancer cell lines. Transient transfections localized the stimulatory effects of bryostatin to the cyclic AMP response element of the COX-2 promoter. Electrophoretic mobility shift assays and supershift experiments revealed a marked increase in the binding of activator protein-1 (AP-1)(c-Jun/c-Fos) to the cyclic AMP response element of the COX-2 promoter. Pharmacological and transient transfection studies indicated that bryostatin stimulated COX-2 transcription via the protein kinase C-->mitogen-activated protein kinase-->AP-1 pathway. All-trans-retinoic acid, a prototypic AP-1 antagonist, blocked bryostatin-mediated induction of COX-2. Taken together, these results suggest that bryostatin-mediated induction of COX-2 can help to explain the myalgias that are commonly associated with treatment. Moreover, it will be worthwhile to evaluate whether the addition of a selective COX-2 inhibitor can increase the antitumor activity of bryostatin.