Prostaglandin E receptor EP4 antagonism inhibits breast cancer metastasis

Cyclooxygenase-2 (COX-2) expression in epithelial tumors is frequently associated with a poor prognosis. In a murine model of metastatic breast cancer, we showed that COX-2 inhibition is associated with decreased metastatic capacity. The COX-2 product, prostaglandin E(2) (PGE(2)), acts through a family of G protein-coupled receptors designated EP1-4 that mediate intracellular signaling by multiple pathways. We characterized EP receptor expression on three murine mammary tumor cell lines and show that all four EP isoforms were detected in each cell. Stimulation of cells with either PGE(2) or the selective EP4/EP2 agonist PGE(1)-OH resulted in increased intracellular cyclic AMP and this response was inhibited with either EP2 or EP4 antagonists. Nothing is known about the function of EP receptors in tumor metastasis. We tested the hypothesis that the prevention of EP receptor signaling would, like inhibition of PGE(2) synthesis, inhibit tumor metastasis. Our results show for the first time that antagonism of the EP4 receptor with either AH23848 or ONO-AE3-208 reduced metastasis as compared with vehicle-treated controls. The therapeutic effect was comparable to that observed with the dual COX-1/COX-2 inhibitor indomethacin. EP3 antagonism had no effect on tumor metastasis. Mammary tumor cells migrated in vitro in response to PGE(2) and this chemotactic response was blocked by EP receptor antagonists. Likewise, the proliferation of tumor cells was also directly inhibited by antagonists of either EP4 or EP1/EP2. These studies support the hypothesis that EP receptor antagonists may be an alternative approach to the use of COX inhibitors to prevent tumor metastasis.     

Frondoside A inhibits breast cancer metastasis and antagonizes prostaglandin E receptors EP4 and EP2

Frondoside A, derived from the sea cucumber Cucumaria frondosa has demonstrable anticancer activity in several models, however, the ability of Frondoside A to affect tumor metastasis has not been reported. Using a syngeneic murine model of metastatic breast cancer, we now show that Frondoside A has potent antimetastatic activity. Frondoside A given i.p. to mice bearing mammary gland-implanted mammary tumors, inhibits spontaneous tumor metastasis to the lungs. The elevated Cyclooxygenase-2 activity in many malignancies promotes tumor growth and metastasis by producing high levels of PGE₂ which acts on the prostaglandin E receptors, chiefly EP4 and EP2. We examined the ability of Frondoside A to modulate the functions of these EP receptors. We now show that Frondoside A antagonizes the prostaglandin E receptors EP2 and EP4. ³H-PGE₂ binding to recombinant EP2 or EP4-expressing cells was inhibited by Frondoside A at low μM concentrations. Likewise, EP4 or EP2-linked activation of intracellular cAMP as well as EP4-mediated ERK1/2 activation were also inhibited by Frondoside A. Consistent with the antimetastatic activity observed in vivo, migration of tumor cells in vitro in response to EP4 or EP2 agonists was also inhibited by Frondoside A. These studies identify a new function for an agent with known antitumor activity, and show that the antimetastatic activity may be due in part to a novel mechanism of action. These studies add to the growing body of evidence that Frondoside A may be a promising new agent with potential to treat cancer and may also represent a potential new modality to antagonize EP4.     

COX-2 dependent regulation of mechanotransduction in human breast cancer cells

The ability of living cells to exert physical forces upon their surrounding is a necessary prerequisite for diverse biological processes, such as local cellular migrations in wound healing to metastatic-invasion of cancer. How forces are coopted in metastasis has remained unclear, however, because the mechanical interplay between cancer cells and the various stromal components has not been experimentally accessible. Current dogma implicates inflammation in these mechanical processes. Using Fourier transform traction microscopy, we measured the force-generating capacity of human breast cancer cells occupying a spectrum of invasiveness as well as basal and inducible COX-2 expression (MCF-7<SUM-149<MDA-MB-231). Compared with non-invasive MCF-7 and moderately-invasive SUM-149, poorly-differentiated MDA-MB-231 cells showed increased cellular dispersion on collagen matrix that was accompanied by emergent distribution of contractile stresses at the interface between the adherent cell and its substrate, defined herein as the traction field. In metastatic MDA-MB-231 cells, the local tractions were precisely tuned to the surrounding matrix rigidity in a physiologic range with the concomitant expression of mechanosensitive integrin β₁. These discrete responses at the single-cell resolution were correlated with PGE₂ secretion and were ablated by shRNA-mediated knockdown of COX-2. Both COX-2-silenced and COX-2-expressing cells expressed EP2 and EP4 receptors, but not EP1 and EP3. Exogenous addition of PGE₂ increased cell tractions and stiffened the underlying cytoskeletal network. To our knowledge this is the first report linking the expression of COX-2 with mechanotransduction of human breast cancer cells, and the regulation of COX-2-PGE₂-EP signaling with physical properties of the tumor microenvironment. Drug treatments aimed at reducing this mechanical interplay may have therapeutic potential in the treatment of breast cancer.     

Cyclooxygenase inhibitors retard murine mammary tumor progression by reducing tumor cell migration, invasiveness and angiogenesis

Tumor-derived prostaglandins (PGs) have been implicated in the progression of murine and human breast cancer. Chronic treatment with a non-selective PG inhibitor indomethacin was shown in this laboratory to retard the development and metastasis of spontaneous mammary tumors in C3H/HeJ female retired breeder mice. The present study examined the role of endogenous prostaglandins in the proliferation/survival, the migratory and invasive behavior and angiogenic ability of a highly metastatic murine mammary tumor cell line, C3L5, originally derived from a C3H/HeJ spontaneous mammary tumor. This cell line was shown to express high levels of cyclooxygenase (COX) -2 mRNA and protein as detected by Northern and Western blotting as well as immunostaining. PGE(2) production by C3L5 cells was primarily owing to COX-2, since this was blocked similarly with non-selective COX inhibitor indomethacin and selective COX-2 inhibitor NS-398, but unaffected with the selective COX-1 inhibitor valeryl salicylate. C3L5 cell proliferation/survival in vitro was not influenced by PGs, since their cellularity remained unaffected in the presence of PGE(2) or NS-398 or PG-receptor (EP1/EP2) antagonist AH6809; a marginal decline was noted only at high doses of indomethacin, which was not abrogated by addition of exogenous PGE(2). Migratory and invasive abilities of C3L5 cells, as quantitated with in vitro transwell migration/invasion assays, were inhibited with indomethacin or NS-398 or AH6809 in a dose-dependent manner; the indomethacin and NS-398-mediated inhibition was partially reversed upon addition of exogenous PGE(2). An in vivo angiogenesis assay that used subcutaneous implants of growth factor-reduced matrigel inclusive of tumor cells showed a significant inhibition of blood vessel formation in these implants in animals treated with indomethacin compared with animals receiving vehicle alone. These studies show that selective and nonselective COX-2 inhibitors retarded tumor progression in this COX-2-expressing murine mammary tumor model by inhibiting tumor cell migration, invasiveness and tumor-induced angiogenesis. The inhibitory effects were not entirely PG dependent; some PG-independent effects were also noted.     

Growth and metastatic behavior of molecularly well-characterized human breast cancer cell lines in mice

The cyclooxygenase pathway is strongly implicated in breast cancer progression but the role of this pathway in the biology of breast cancer stem/progenitor cells has not been defined. Recent attention has focused on targeting the cyclooxygenase 2 (COX-2) pathway downstream of the COX-2 enzyme by blocking the activities of individual prostaglandin E (EP) receptors. Prostaglandin E receptor 4 (EP4) is widely expressed in primary invasive ductal carcinomas of the breast and antagonizing this receptor with small molecule inhibitors or shRNA directed to EP4 inhibits metastatic potential in both syngeneic and xenograft models. Breast cancer stem/progenitor cells are defined as a subpopulation of cells that drive tumor growth, metastasis, treatment resistance, and relapse. Mammosphere-forming breast cancer cells of human (MDA-MB-231, SKBR3) or murine (66.1, 410.4) origin of basal-type, Her-2 phenotype and/or with heightened metastatic capacity upregulate expression of both EP4 and COX-2 and are more tumorigenic compared to the bulk population. In contrast, luminal-type or non-metastatic counterparts (MCF7, 410, 67) do not increase COX-2 and EP4 expression in mammosphere culture. Treatment of mammosphere-forming cells with EP4 inhibitors (RQ-15986, AH23848, Frondoside A) or EP4 gene silencing, but not with a COX inhibitor (Indomethacin) reduces both mammosphere-forming capacity and the expression of phenotypic markers (CD44^hi/CD24^low, aldehyde dehydrogenase) of breast cancer stem cells. Finally, an orally delivered EP4 antagonist (RQ-08) reduces the tumor-initiating capacity and markedly inhibits both the size of tumors arising from transplantation of mammosphere-forming cells and phenotypic markers of stem cells in vivo. These studies support the continued investigation of EP4 as a potential therapeutic target and provide new insight regarding the role of EP4 in supporting a breast cancer stem cell/tumor-initiating phenotype.     

The prostaglandin E2 receptor EP2 is required for cyclooxygenase 2-mediated mammary hyperplasia

Expression of cyclooxygenase 2 (COX-2) in breast cancer correlates with poor prognosis, and COX-2 enzyme inhibitors reduce breast cancer incidence in humans. We recently showed that COX-2 overexpression in the mammary gland of transgenic mice induced mammary cancer. Because prostaglandin E2 (PGE2) is the major eicosanoid and because the EP2 subtype of the PGE2 receptor is highly expressed in the mammary tumors, we tested if this G protein-coupled receptor is required for tumorigenesis. We crossed the MMTV-COX-2 transgenic mice with Ep2-/- mice and studied tumor development in bigenic mice. Lack of EP2 receptor strongly suppressed COX-2-induced effects such as precocious development of the mammary gland in virgins and the development of mammary hyperplasia in multiparous female mice. Interestingly, the expression of amphiregulin, a potent mammary epithelial cell growth factor was down regulated in mammary glands of Ep2-/- mice. Total cyclic AMP (cAMP) levels were reduced in Ep2-/- mammary glands suggesting that PGE2 signaling via the EP2 receptor activates the Gs/cAMP/protein kinase A pathway. In mammary tumor cell lines, expression of the EP2 receptor followed by treatment with CAY10399, an EP2-specific agonist, strongly induced amphiregulin mRNA levels in a protein kinase A-dependent manner. These data suggest that PGE2 signaling via the EP2 receptor in mammary epithelial cells regulate mammary gland hyperplasia by the cAMP-dependent induction of amphiregulin. Inhibition of the EP2 pathway in the mammary gland may be a novel approach in the prevention and/or treatment of mammary cancer.     

Mammary Carcinoma Cell Derived Cyclooxygenase 2 Suppresses Tumor Immune Surveillance by Enhancing Intratumoral Immune Checkpoint Activity

Introduction

Systemic inhibition of the inflammatory enzyme cyclooxygenase (COX) 2 decreases the risk of breast cancer and its recurrence. However, the biology of COX-2 in the multicellular tumor microenvironment is poorly defined.

Methods

Mammary tumor onset and multiplicity were examined in ErbB2 transgenic mice that were deficient in mammary epithelial cell COX-2 (COX-2^MECKO) compared to wild type (WT) mice.Tumors were analyzed, by real time PCR, immune-staining and flow cytometry, for proliferation, apoptosis, angiogenesis and immune microenvironment. Lentiviral shRNA delivery was used to knock down (KD) COX-2 in ErbB2-transformed mouse breast cancer cells (COX-2KD), and growth as orthotopic tumors was examined in syngenic recipient mice, with or without depletion of CD8⁺ immune cells.

Results

Mammary tumor onset was delayed, and multiplicity halved, in COX-2^MECKO mice compared to WT. COX-2^MECKO tumors showed decreased expression of Ki67, a proliferation marker, as well as reduced VEGFA, its receptor VEGFR2, endothelial NOS and the vascular endothelial marker CD31, indicating reduced tumor vascularization. COX-2^MECKO tumors contained more CD4⁺ T helper (T_h) cells and CD8⁺ cytotoxic immune cells (CTL) consistent with increased immune surveillance. The ratio of T_h markers Tbet (T_h1) to GATA3 (T_h2) was higher, and levels of Retnla, a M2 macrophage marker, lower, in COX-2^MECKO tumor infiltrating leukocytes compared to WT, suggesting a prevalence of pro-immune T_h1 over immune suppressive T_h2 lymphocytes, and reduced macrophage polarization to the immune suppressive M2 phenotype. Enhanced immune surveillance in COX-2^MECKO tumors was coincident with increased intratumoral CXCL9, a T cell chemoattractant, and decreased expression of T lymphocyte co-inhibitory receptors CTLA4 and PD-1, as well as PD-L1, the ligand for PD-1. PD-L1 was also decreased in IFNγ-treated COX-2KD mouse mammary cancer cells in vitro and, compared to control cells, growth of COX-2KD cells as orthotopic tumors in immune competent mice was markedly suppressed. However, robust growth of COX-2KD tumor cells was evident when recipients were depleted of CD8⁺ cells.

Conclusions

The data strongly support that, in addition to its angiogenic function, tumor cell COX-2 suppresses intratumoral cytotoxic CD8⁺ immune cell function, possibly through upregulation of immune checkpoints, thereby contributing to tumor immune escape. COX-2 inhibition may be clinically useful to augment breast cancer immunotherapy.     

Selective cyclooxygenase (COX)-1 or COX-2 inhibitors control metastatic disease in a murine model of breast cancer

Using a highly metastatic mammary tumor cell line that expresses both cyclooxygenase (COX) isoforms, we now show that oral administration of either a selective COX-2 inhibitor (celecoxib) or a selective COX-1 inhibitor (SC560) to mice with established tumors results in significant inhibition of tumor growth. Administration of the dual inhibitor, indomethacin, leads to even better growth control. Metastatic capacity is also reduced by treatment of tumor-bearing mice with either COX-1 or COX-2 selective inhibitors. Pretreatment of tumor cells with COX inhibitors also reduces metastatic success, indicating that tumor cells may be a direct target of action by COX inhibitors. Growth of a second cell line, which does not express COX-2 in vivo, is also reduced by celecoxib, implicating both COX-dependent and COX-independent mechanisms.     

Berberine, an isoquinoline alkaloid, inhibits melanoma cancer cell migration by reducing the expressions of cyclooxygenase-2, prostaglandin E₂ and prostaglandin E₂ receptors

Melanoma is the leading cause of death from skin disease due, in large part, to its propensity to metastasize. We have examined the effect of berberine, an isoquinoline alkaloid, on human melanoma cancer cell migration and the molecular mechanisms underlying these effects using melanoma cell lines, A375 and Hs294. Using an in vitro cell migration assay, we show that over expression of cyclooxygenase (COX)-2, its metabolite prostaglandin E? (PGE?) and PGE? receptors promote the migration of cells. We found that treatment of A375 and Hs294 cells with berberine resulted in concentration-dependent inhibition of migration of these cells, which was associated with a reduction in the levels of COX-2, PGE? and PGE? receptors (EP2 and EP4). Treatment of cells with celecoxib, a COX-2 inhibitor, or transient transfection of cells with COX-2 small interfering RNA, also inhibited cell migration. Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of COX-2 or PGE?, enhanced cell migration, whereas berberine inhibited TPA- or PGE?-promoted cell migration. Berberine reduced the basal levels as well as PGE?-stimulated expression levels of EP2 and EP4. Treatment of the cells with the EP4 agonist stimulated cell migration and berberine blocked EP4 agonist-induced cell migration activity. Moreover, berberine inhibited the activation of nuclear factor-kappa B (NF-κB), an upstream regulator of COX-2, in A375 cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, inhibited cell migration. Together, these results indicate for the first time that berberine inhibits melanoma cell migration, an essential step in invasion and metastasis, by inhibition of COX-2, PGE? and PGE? receptors.     

Prostaglandin E2 receptor EP4 as the common target on cancer cells and macrophages to abolish angiogenesis,lymphangiogenesis, metastasis,and stem‐like cell functions

We previously established that COX‐2 overexpression promotes breast cancer progression and metastasis. As long‐term use of COX‐2 inhibitors (COX‐2i) can promote thrombo‐embolic events, we tested an alternative target, prostaglandin E2 receptor EP4 subtype (EP4), downstream of COX‐2. Here we used the highly metastatic syngeneic murine C3L5 breast cancer model to test the role of EP4‐expressing macrophages in vascular endothelial growth factor (VEGF)‐C/D production, angiogenesis, and lymphangiogenesis in situ, the role of EP4 in stem‐like cell (SLC) functions of tumor cells, and therapeutic effects of an EP4 antagonist RQ‐15986 (EP4A). C3L5 cells expressed all EP receptors, produced VEGF‐C/D, and showed high clonogenic tumorsphere forming ability in vitro, functions inhibited with COX‐2i or EP4A. Treating murine macrophage RAW 264.7 cell line with COX‐2i celecoxib and EP4A significantly reduced VEGF‐A/C/D production in vitro, measured with quantitative PCR and Western blots. Orthotopic implants of C3L5 cells in C3H/HeJ mice showed rapid tumor growth, angiogenesis, lymphangiogenesis (CD31/LYVE‐1 and CD31/PROX1 immunostaining), and metastasis to lymph nodes and lungs. Tumors revealed high incidence of EP4‐expressing, VEGF‐C/D producing macrophages identified with dual immunostaining of F4/80 and EP4 or VEGF‐C/D. Celecoxib or EP4A therapy at non‐toxic doses abrogated tumor growth, lymphangiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in treated mice revealed markedly reduced VEGF‐A/C/D and phosphorylated Akt/ERK proteins, VEGF‐C/D positive macrophage infiltration, and proliferative/apoptotic cell ratios. Knocking down COX‐2 or EP4 in C3L5 cells or treating cells in vitro with celecoxib or EP4A and treating tumor‐bearing mice in vivo with the same drug reduced SLC properties of tumor cells including preferential co‐expression of COX‐2 and SLC markers ALDH1A, CD44, OCT‐3/4, β‐catenin, and SOX‐2. Thus, EP4 is an excellent therapeutic target to block stem‐like properties, angiogenesis, and lymphangiogenesis induced by VEGF‐A/C/D secreted by cancer cells and tumor infiltrating macrophages.     

Roles of a prostaglandin E-type receptor, EP3, in upregulation of matrix metalloproteinase-9 and vascular endothelial growth factor during enhancement of tumor metastasis

Cyclooxygenase (COX)-2 is known to correlate with poor cancer prognosis and to contribute to tumor metastasis. However, the precise mechanism of this phenomenon remains unknown. We have previously reported that host stromal prostaglandin E₂ (PGE₂)–prostaglandin E2 receptor (EP)3 signaling appears critical for tumor-associated angiogenesis and tumor growth. Here we tested whether the EP3 receptor has a critical role in tumor metastasis. Lewis lung carcinoma (LLC) cells were intravenously injected into WT mice and mice treated with the COX-2 inhibitor NS-398. The nonselective COX inhibitor aspirin reduced lung metastasis, but the COX-1 inhibitor SC560 did not. The expression of matrix metalloproteinases (MMP)-9 and vascular endothelial growth factor (VEGF)-A was suppressed in NS-398-treated mice compared with PBS-treated mice. Lungs containing LLC colonies were markedly reduced in EP3 receptor knockout (EP3^−/−) mice compared with WT mice. The expression of MMP-9 and VEGF-A was downregulated in metastatic lungs of EP3^−/− mice. An immunohistochemical study revealed that MMP-9-expressing endothelial cells were markedly reduced in EP3^−/− mice compared with WT mice. When HUVEC were treated with agonists for EP1, EP2, EP3, or EP4, only the EP3 agonist enhanced MMP-9 expression. These results suggested that EP3 receptor signaling on endothelial cells is essential for the MMP-9 upregulation that enhances tumor metastasis and angiogenesis. An EP3 receptor antagonist may be useful to protect against tumor metastasis. ( Cancer Sci 2009; 100: 2318–2324)     

Timosaponin AIII inhibits melanoma cell migration by suppressing COX‐2 and in vivo tumor metastasis

Melanoma is the leading cause of death from skin disease, due in large part to its propensity to metastasize. We examined the effects of timosaponin AIII, a compound isolated from Anemarrhena asphodeloides Bunge, on melanoma cancer cell migration and the molecular mechanisms underlying these effects using B16‐F10 and WM‐115 melanoma cells lines. Overexpression of COX‐2, its metabolite prostaglandin E₂ (PGE₂), and PGE₂ receptors (EP2 and EP4) promoted cell migration in vitro. Exposure to timosaponin AIII resulted in concentration‐dependent inhibition of cell migration, which was associated with reduced levels of COX‐2, PGE₂, and PGE₂ receptors. Transient transfection of COX‐2 siRNA also inhibited cell migration. Exposure to 12‐O‐tetradecanoylphorbal‐13‐acetate enhanced cell migration, whereas timosaponin AIII inhibited 12‐O‐tetradecanoylphorbal‐13‐acetate‐induced cell migration and reduced basal levels of EP2 and EP4. Moreover, timosaponin AIII inhibited activation of nuclear factor‐kappa B (NF‐κB), an upstream regulator of COX‐2 in B16‐F10 cells. Consistent with our in vitro findings, in vivo studies showed that timosaponin AIII treatment significantly reduced the total number of metastatic nodules in the mouse lung and improved histological alterations in B16‐F10‐injected C57BL/6 mice. In addition, C57BL/6 mice treated with timosaponin AIII showed reduced expression of COX‐2 and NF‐κB in the lung. Together, these results indicate that timosaponin AIII has the capacity to inhibit melanoma cell migration, an essential step in the process of metastasis, by inhibiting expression of COX‐2, NF‐κB, PGE_2, and PGE₂ receptors.     

Aluminium chloride promotes tumorigenesis and metastasis in normal murine mammary gland epithelial cells

Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti‐acid drugs, food additives and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. For example, no experimental evidence that aluminium promotes tumorigenesis in cultured mammary epithelial cells exists. We report here that long‐term exposure to concentrations of aluminium—in the form of aluminium chloride (AlCl₃)—in the range of those measured in the human breast, transform normal murine mammary gland (NMuMG) epithelial cells in vitro as revealed by the soft agar assay. Subcutaneous injections into three different mouse strains with decreasing immunodeficiency, namely, NOD SCID gamma (NSG), NOD SCID or nude mice, revealed that untreated NMuMG cells form tumors and metastasize, to a limited extent, in the highly immunodeficient and natural killer (NK) cell deficient NSG strain, but not in the less permissive and NK cell competent NOD SCID or nude strains. In contrast, NMuMG cells transformed in vitro by AlCl₃ form large tumors and metastasize in all three mouse models. These effects correlate with a mutagenic activity of AlCl₃. Our findings demonstrate for the first time that concentrations of aluminium in the range of those measured in the human breast fully transform cultured mammary epithelial cells, thus enabling them to form tumors and metastasize in well‐established mouse cancer models. Our observations provide experimental evidence that aluminium salts could be environmental breast carcinogens.     

Phenotypic drift of metastatic and cell-surface properties of mammary adenocarcinoma cell clones during growth in vitro

We have examined cell clones obtained from a 13762 mammary adenocarcinoma tumor and its spontaneous lung metastasis for phenotypic stability during serial culture passage in vitro. Two clones that varied markedly in their metastatic properties were chosen for further examination. One of these clones (MTC) obtained from the parental transplanted tumor initially failed to metastasize within 23 days post-injection s.c. but gained the ability to form spontaneous pulmonary metastases after several serial passages in vitro. Another clone (MTLn₃) derived from a spontaneous lung metastasis was initially highly metastatic after short-term culture, but lost the potential to form large numbers of spontaneous lung metastases with long-term culture. In contrast to MTA, clone MTLn₃ displayed lymph-node metastasis, and the frequency of lymph-node involvement increased when late-passage cultures of MTLn₃ cells were assayed in vivo. Both clones from late-passage cultures produced larger tumor sizes at the primary (mammary fat pad) injection sites compared to early passage cells. The morphologies of MTC cells changed with serial tissue culture passage, while the morphologies of MTLn₃ cells did not change. The display of fibronectin on MTC cells by immunofluorescence did not change with culture passage; fibronectin was not detected in cultures of clone MTLn₃. Fibronectin was also found on MTC cells by cell surface labelling using lactoperoxidase-catalyzed iodination-sodium dodecylsulfate polyacrylamide gel electrophoresis-autoradio-graphy. lodination of fibronectin on MTC cells did not vary with culture passage, and as in immunofluorescence experiments it was not detected on MTLn₃ cells. There was a decrease in exposure of certain cell surface proteins on MTC cells with culture passage, but we did not detect modifications with this procedure that correlated with culture passage of MTLn₃ cells. We conclude that prolonged culture in vitro can result in modifications of metastatic and cell-surface properties of tumor cell clones.     

Effect of Eicosapentaenoic Acid on E-type Prostaglandin Synthesis and EP4 Receptor Signaling in Human Colorectal Cancer Cells

The ω-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA), in the free fatty acid (FFA) form, has been demonstrated to reduce adenoma number and size in patients with familial adenomatous polyposis. However, the mechanistic basis of the antineoplastic activity of EPA in the colorectum remains unclear. We tested the hypothesis that EPA-FFA negatively modulates synthesis of and signaling by prostaglandin (PG) E₂ in human colorectal cancer (CRC) cells. EPA-FFA induced apoptosis of cyclooxygenase (COX)-2-positive human HCA-7 CRC cells in vitro. EPA-FFA in cell culture medium was incorporated rapidly into phospholipid membranes of HCA-7 human CRC cells and acted as a substrate for COX-2, leading to reduced synthesis of PGE₂ and generation of PGE₃. Alone, PGE₃ bound and activated the PGE₂ EP4 receptor but with reduced affinity and efficacy compared with its “natural” ligand PGE₂. However, in the presence of PGE₂, PGE₃ acted as an antagonist of EP4 receptor-dependent 3′,5′ cyclic adenosine monophosphate induction in naturally EP4 receptor-positive LoVo human CRC cells and of resistance to apoptosis in HT-29-EP4 human CRC cells overexpressing the EP4 receptor. We conclude that EPA-FFA drives a COX-2-dependent “PGE₂-to-PGE₃ switch” in human CRC cells and that PGE₃ acts as a partial agonist at the PGE₂ EP4 receptor.     

Tumor cells are protected from NK-cell-mediated lysis by adhesion to endothelial cells

We have examined the effect of various cell monolayers on the ability of mouse spleen cells to lyse tumor cell targets (natural cytotoxicity reaction). Natural killer (NK) cell activity was reduced by as much as 75% depending on the cell substrate, with the greatest protection afforded by endothelial cells. Cell adhesion to the underlying cell monolayer was directly correlated with the degree of protection from lysis. Since it has previously been shown that tumor cells manifest selective adhesion to endothelial cells from those organs to which they are likely to metastasize, the experiments suggest a means by which natural surveillance mechanisms might be circumvented during the metastatic process.     

PGE2-mediated upregulation of iNOS in murine breast cancer cells through the activation of EP4 receptors

We report here that endogenous prostaglandin E(2) (PGE(2)) resulting from cyclooxygenase (COX)-2 expression in a highly metastatic murine breast cancer cell line C3L5 upregulates IFN-gamma + LPS-induced nitric oxide (NO) synthase (iNOS) expression and NO production. This action of PGE(2) is mediated through the EP(4) receptor in a cAMP-dependent manner. Both nonselective and selective COX-2 inhibitors suppressed IFN-gamma + LPS-induced NO production, which was largely restored by exogenous PGE(2) or EP(4) receptor agonist PGE(1) alcohol. EP(4) antagonist AH-23848B inhibited NO production with a concomitant downregulation of iNOS mRNA in IFN-gamma + LPS-stimulated cells. cAMP dependence of NO production by cells under inducible conditions was demonstrated by the use of known modulators of intracellular cAMP. Since both COX-2 and iNOS are implicated in breast cancer progression, our findings of EP(4) receptor-mediated upregulation of iNOS in COX-2-expressing breast cancer cells suggest that blocking COX-2 and/or EP(4) may provide a simple therapeutic modality in this tumor model.     

Alterations in sensitivity to nonspecific cell-mediated lysis associated with tumor progression: characterization of activated macrophage- and natural killer cell-resistant tumor variants

Certain "membrane-mutant," lectin-resistant (Lecr) variants derived from the highly metastatic and poorly immunogenic DBA/2 mouse tumor MDAY-D2 previously were found to differ substantially in their ability to grow and to metastasize. In the present study, the parental MDAY-D2 tumor and several wheat germ agglutinin-resistant (WGAr) variants were examined for alterations in sensitivity to activated macrophage (M phi)- and natural killer cell (NK)-mediated lysis. The results indicated that selection in WGA after mutagenic treatment of a metastatic parental tumor cell line (MDAY-D2), which was M phi-sensitive (M phi S) and NK-resistant (NKR), can result in the isolation of a significantly M phi-resistant (M phi R) and NK-sensitive (NKS) tumor variant, MDW4. The in vivo hybridization of the M phi R, NKS, Lecr MDW4 variant with a normal host-derived cell within a primary subcutaneous tumor, previously demonstrated to result in the progressive and selective outgrowth and metastasis of hybrid products, was found to be associated directly with reversion to the M phi S, NKR phenotype of the metastatic parental MDAY-D2 cell line. DMA/2 mice given iv injections of 10(5) M phi R, NKS cells (MDW4 or MDW4-110c1, a cloned line isolated from a subcutaneous primary tumor of an MDW4-injected animal) survived for a significantly prolonged period as compared to animals given injections of either the parental tumor or M phi S, NKR hybrid products isolated from a MDW4 subcutaneous primary tumor (MDW4-110c2) or visceral metastases (MDW4-24a, MDW4-24b, and MDW4-24c). The results clearly indicate an inverse relationship among the tumor variants in their ability to be lysed by either M phi or NK and suggest a central role for NK rather than M phi surveillance in this tumor system.     

Lung‐resident natural killer cells control pulmonary tumor growth in mice

Accumulating evidence indicates the importance of natural killer (NK) cells in controlling tumor growth and metastasis. NK cell subsets display diversities in their function and tissue distribution and Mac‐1^hi CD27^lo NK cells are the predominant population of lung‐resident NK cells. Although the lung is a major organ where primary tumor develops and cancer cells metastasize, there is no clear evidence whether circulating NK cells and/or tissue‐resident NK cells control tumor growth in the lung. In the present study, we examined an antitumor function of lung‐resident NK cells to control pulmonary tumor growth. In an orthotopic lung tumor model, NK cells controlled pulmonary tumor growth, and mature circulating NK cell subsets were increased in tumor‐bearing lungs through a C‐X‐C motif chemokine receptor 3 (CXCR3)‐dependent mechanism. Although such increase in migratory NK cell subsets can be blocked by anti‐CXCR3 treatment, there was no difference in pulmonary tumor growth in anti‐CXCR3‐treated mice compared with control mice. In addition to pulmonary tumor growth, lung‐resident NK cells, but not migratory NK cells, play a dominant role in controlling metastatic growth of cancer cells in lung. These results strongly indicate an importance of lung‐resident NK cells for controlling pulmonary tumor growth.