Namita Kundu

Antagonism of CXCR3 Inhibits Lung Metastasis in a Murine Model of Metastatic Breast Cancer

Tumor cells aberrantly express chemokines and/or chemokine receptors, and some may promote tumor growth and metastasis. We examined the expression and function of chemokine receptor CXCR3 in a syngeneic murine model of metastatic breast cancer. By flow cytometry, CXCR3 was detected in all murine mammary tumor cell lines examined. All human breast cancer cell lines examined also expressed CXCR3, as did the immortalized but nontumorigenic MCF-10A cell line. Interaction of CXCR3 ligands, CXCL9, CXCL10, and CXCL11, with CXCR3 on the highly malignant murine mammary tumor cell line 66.1 resulted in intracellular calcium mobilization and chemotaxis in vitro. To test the hypothesis that tumor metastasis is facilitated by CXCR3 expressed by tumor cells, we employed a small molecular weight antagonist of CXCR3, AMG487. 66.1 tumor cells were pretreated with AMG487 prior to i.v. injection into immune-competent female mice. Antagonism of CXCR3 on 66.1 tumor cells inhibited experimental lung metastasis, and this antimetastatic activity was compromised in mice depleted of natural killer cells. Systemic administration of AMG487 also inhibited experimental lung metastasis. In contrast to the antimetastatic effect of AMG487, local growth of 66.1 mammary tumors was not affected by receptor antagonism. These studies indicate that murine mammary tumor cells express CXCR3 which facilitates the development of lung metastases. These studies also indicate for the first time that a small molecular weight antagonist of CXCR3 has the potential to inhibit tumor metastasis.

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.

Increased cyclooxygenase-2 (cox-2) expression and activity in a murine model of metastatic breast cancer.

Elevated prostaglandin E2 (PGE2) production is a common feature of human malignancies. This activity has often been attributed to increased metabolic activity of the cyclooxygenase enzymes, although a direct comparison of these 2 parameters i.e., prostaglandin production and cox protein expression, is rarely performed in the same malignant tissue. Using a murine model of metastatic breast cancer, we show that PGE2 levels are positively correlated with increased tumorigenic and metastatic potential. Because prostaglandin synthesis is a product of 2 isoforms of the cyclooxygenase enzyme, we examined the expression and activity of both isoforms. All tumor cell lines examined, regardless of phenotype, express both cox‐1 and cox‐2 proteins in vitro. In contrast to the uniform cox‐2 expression in vitro, only tumors resulting from the transplantation of metastatic cell lines express cox‐2 in vivo. Cox‐1 is detected in both metastatic and nonmetastatic tumors. Thus, this is the first evidence that, in the tumor milieu, cox‐2 expression can be regulated differently in metastatic vs. nonmetastatic lesions. Examination of PGE2 synthesis in vitro reveals that nearly complete inhibition of prostaglandin synthesis occurs in the presence of either indomethacin, which inhibits both isoforms, or NS398, which is selective for the cox‐2 isoform. Thus, even though cell lines express both isoforms, the majority of the prostaglandin synthesis stems from the activity of the inducible, cox‐2 isoform. Likewise, cell growth is inhibited by both indomethacin and NS398 in a dose‐dependent manner, albeit at higher drug concentrations than required to ablate PGE2 synthesis. Despite the inhibition of prostaglandin synthesis, the cox‐2 enzyme levels (protein and mRNA) were increased by either indomethacin or NS398.

CXCR3 expression is associated with poor survival in breast cancer and promotes metastasis in a murine model

Breast tumor cells express the chemokine receptor CXCR3, which binds the ligands CXCL9, CXCL10, and CXCL11. CXCR3 and other chemokine receptors may mediate tumor metastasis by supporting migration of tumor cells to sites of ligand expression including the lymph nodes, lungs, and bone marrow. We examined the relationship of CXCR3 expression to clinical outcome in 75 women diagnosed with early-stage breast cancer. We detected CXCR3 in malignant epithelium from all tumors. Twelve percent were weakly positive and 64% had moderate levels of CXCR3. Strong CXCR3-positive staining was observed in 24% of tumors. Kaplan-Meier survival curves showed that high CXCR3 expression was associated with poorer overall survival; the unadjusted hazard ratio was 1.56 and it was marginally significant (P = 0.07). When interactions between lymph node status and CXCR3 were considered, the adjusted hazard ratio for CXCR3 was 2.62 (P = 0.02) for women with node-negative disease at diagnosis, whereas the hazard ratio for CXCR3 was not significant for those with node-positive disease. CXCR3 gene silencing inhibited lung colonization and spontaneous lung metastasis from mammary gland–implanted tumors in a murine model. The size or growth rate of the locally growing tumors was not affected. The antimetastatic effect of CXCR3 gene silencing was compromised in mice depleted of Natural Killer cells or with mutations in IFN-γ, suggesting that the role of CXCR3 is not simply to mediate tumor cell trafficking. These studies support the continued examination of CXCR3 as a potential therapeutic target in patients with breast cancer. [Mol Cancer Ther 2009;8(3):490–8]

Immune-mediated modulation of breast cancer growth and metastasis by the chemokine Mig (CXCL9) in a murine model.

Current immunotherapies are limited by several factors, including the failure to recruit sufficient numbers of immune effector cells to tumors. The chemokine monokine induced by γ-interferon (Mig; CXCL9) attracts activated T cells and natural killer (NK) cells bearing the chemokine receptor CXCR3. We investigated Mig as an immunotherapeutic agent in a syngeneic murine model of metastatic breast cancer. We transfected the highly malignant murine mammary tumor cell line 66.1 to stably express murine Mig cDNA. Immune-competent mice injected with Mig-expressing tumor cells developed smaller local tumors and fewer lung metastases, and they survived longer than mice injected with vector-control tumor cells. Mig-mediated inhibition of local tumor growth was lost in the absence of host T cells. Mig-transduced tumors had increased numbers of CD4+ T cells compared with vector-control tumors, consistent with the T-cell chemoattractant property of Mig, and many tumor-infiltrating host cells expressed CXCR3. NK cells had not been examined previously as a possible effector cell in Mig-based therapies. Our studies now show that NK cells are critical to the mechanism by which Mig limits metastasis. Inhibition of angiogenesis was not implicated as a mechanism of Mig-mediated therapy in this model. These studies support the hypothesis that by manipulating the Mig-CXCR3 gradient, it is possible to direct host immune effector cells to tumors, curtailing both local tumor growth and metastasis. These studies also implicate host NK cells as an additional effector cell critical for Mig-mediated control of metastasis.

Cyclooxygenase inhibitors modulate NK activities that control metastatic disease

Cyclooxygenase (COX) inhibitors have demonstrated efficacy in models of human cancer but the relevant mechanisms have not all been elucidated. Both Cox-dependent as well as Cox-independent mechanisms have been implicated. Using a syngeneic model of metastatic breast cancer, we have investigated the effect of Cox inhibitors on NK functions that are critical to the control of metastatic disease. NK recognition of target cells is governed by a balance of activating and inhibiting receptors that bind ligands including MHC class I. We now show that treatment of tumor cells with the nonselective COX-1/COX-2 inhibitor indomethacin or the selective COX-2 inhibitor celecoxib leads to decreased expression of the MHC class I molecules Ld and Kd . Downregulated class I expression is associated with concomitant increased sensitivity to NK cell-mediated lysis. Both COX inhibitors limit tumor metastasis and this therapeutic effect is dependent on NK but not T cell function. Antimetastatic activity is also lost in the absence of interferon- γ (IFN-γ). Both COX inhibitors also suppress local tumor growth of subcutaneously implanted mammary tumor cells in immune competent Balb/cByJ mice. This therapeutic activity is lost in the absence of either CD4+ or CD8+ T cells, but is not compromised by the loss of NK activity. Thus, the mechanism of tumor inhibition differs in the context of local versus metastatic disease. Taken together, these findings are consistent with a mechanism not previously described, whereby COX inhibitors may relieve MHC-mediated inhibition of NK cytotoxicity leading to recognition and lysis of metastatic tumor cells.

Sublethal oxidative stress inhibits tumor cell adhesion and enhances experimental metastasis of murine mammary carcinoma

We have postulated that murine mammary tumor progression is fueled, in part, by tumor-associated macrophages that deliver sub-lethal oxidative stress to tumor cells. In the present study, we determined whether oxidative stress would affect murine mammary tumor cell attachment to laminin and fibronectin, critical functions in the metastatic process. Sublethal oxidative stress generated by exposure of cells to hydrogen peroxide (H2O2, 1–1000 μM/L) inhibited tumor cell attachment to immobilized laminin or fibronectin. This oxidant effect was blocked in the presence of catalase which removes H2O2. The inhibitory effect on attachment was rapid, with significant inhibition occurring at 5 min; total inhibition was achieved at 60 min with 1 mM H2O2. The oxidative stress effect was partially reversible at 20 h post-treatment and occurred at concentrations of H2O2 that do not adversely affect cell viability or growth. Pretreatment of tumor cells with H2O2 or hypoxanthanine and xanthine oxidase (to generate superoxide radical and H2O2) prior to intravenous injection, enhanced experimental lung tumor colony formation. The enhancement of experimental metastatic potential with enzyme-generated oxidative stress was completely reversed by catalase; the H2O2-mediated enhancement was only partially reversed with catalase. Thus, treatments that inhibit tumor cell attachment to extracellular matrix proteins in vitro enhance experimental metastasis in vivo.

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.

Prostaglandin E 2 (PGE 2 ) suppresses natural killer cell function primarily through the PGE 2 receptor EP4

The COX-2 product prostaglandin E2 (PGE2) contributes to the high metastatic capacity of breast tumors. Our published data indicate that inhibiting either PGE2 production or PGE2-mediated signaling through the PGE2 receptor EP4 reduces metastasis by a mechanism that requires natural killer (NK) cells. It is known that NK cell function is compromised by PGE2, but very little is known about the mechanism by which PGE2 affects NK effector activity. We now report the direct effects of PGE2 on the NK cell. Endogenous murine splenic NK cells express all four PGE2 receptors (EP1-4). We examined the role of EP receptors in three NK cell functions: migration, cytotoxicity, and cytokine release. Like PGE2, the EP4 agonist PGE1-OH blocked NK cell migration to FBS and to four chemokines (ITAC, MIP-1α, SDF-1α, and CCL21). The EP2 agonist, Butaprost, inhibited migration to specific chemokines but not in response to FBS. In contrast to the inhibitory actions of PGE2, the EP1/EP3 agonist Sulprostone increased migration. Unlike the opposing effects of EP4 vs. EP1/EP3 on migration, agonists of each EP receptor were uniformly inhibiting to NK-mediated cytotoxicity. The EP4 agonist, PGE1-OH, inhibited IFNγ production from NK cells. Agonists for EP1, EP2, and EP3 were not as effective at inhibiting IFNγ. Agonists of EP1, EP2, and EP4 all inhibited TNFα; EP4 agonists were the most potent. Thus, the EP4 receptor consistently contributed to loss of function. These results, taken together, support a mechanism whereby inhibiting PGE2 production or preventing signaling through the EP4 receptor may prevent suppression of NK functions that are critical to the control of breast cancer metastasis.

Cyclooxygenase inhibitors block cell growth, increase ceramide and inhibit cell cycle

We have shown previously in a model of metastatic breast cancer that murine mammary tumor cells express both cyclooxygenase-1 (Cox-1) and Cox-2 isoforms. Growth and metastasis of these tumors in syngeneic hosts are inhibited by either selective Cox-1 (SC560) or selective Cox-2 (celecoxib) inhibitors. To gain insight into the relevant mechanisms involved in the therapeutic response, we determined the effect of Cox inhibitors on tumor cell behavior in vitro. We now report that either selective Cox-1 or Cox-2 drugs inhibited cell replication, but only at concentrations that are no longer selective for either isoform. Growth delay by either nonselective or selective inhibitors was associated with changes in cell morphology including cell rounding; these changes were reversed upon removal of drug. Unlike many other cell types examined, treatment of these mammary tumor cells with Cox inhibitors was not associated with detectable apoptosis. Growth inhibition, induced by either selective or nonselective Cox inhibitors, was accompanied by increased intracellular levels of the sphingolipid ceramide by 1.7–2.6-fold in comparison to vehicle-treated cells. Ceramide changes are associated with cell cycle arrest and we observed that all the Cox inhibitors examined increased significantly the number of cells in G0/G1 and reduced the S phase fraction. Likewise, addition of a cell-permeable form of ceramide (C6-ceramide) could mimic the effect of Cox inhibitors on both cell cycle and cell growth inhibition. Thus, mammary tumor cells are growth restricted by Cox inhibitors. These effects are associated with changes in ceramide levels and a block in cell cycle progression.