Prostaglandin E2 EP receptors as therapeutic targets in breast cancer.

Prostaglandins are lipid compounds that mediate many physiological effects. Prostaglandin E2 (PGE(2)) is the most abundant prostanoid in the human body, and synthesis of PGE(2) is driven by cyclooxygenase enzymes including COX-2. Both elevated expression of COX-2 and increased PGE(2) levels have been associated with many cancers including breast cancer. PGE(2) exerts its effect by binding to the E series of prostaglandin receptors (EP) which are G protein-coupled receptors. Four EP receptor subtypes exist, EP1-4, and each is coupled to different intracellular signaling pathways. As downstream effectors of the COX-2 pathway, EP receptors have been shown to play a role in breast and other malignancies and in cancer metastasis. The role of each EP receptor in malignant behavior is complex and involves the interplay of EP receptor signaling on the tumor cell, on stromal cells, and on host immune effector cells. While preclinical and epidemiological data support the use of nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors (COXibs) for the prevention and treatment of malignancy, toxicities due to COXibs as well as less than promising results from clinical trials have laboratories seeking alternative targets. As knowledge concerning the role of EP receptors in cancer grows, so does the potential for exploiting EP receptors as therapeutic targets for the treatment or prevention of cancer and cancer metastasis.

Antimetastatic activity isolated from Colocasia esculenta (taro).

Breast cancer mortality is primarily due to the occurrence of metastatic disease. We have identified a novel potential therapeutic agent derived from an edible root of the plant Colocasia esculenta, commonly known as taro, which has demonstrable activity in a preclinical model of metastatic breast cancer and that should have minimal toxicity. We have shown for the first time that a water-soluble extract of taro (TE) potently inhibits lung-colonizing ability and spontaneous metastasis from mammary gland-implanted tumors, in a murine model of highly metastatic estrogen receptor, progesterone receptor and Her-2/neu-negative breast cancer. TE modestly inhibits the proliferation of some, but not all, breast and prostate cancer cell lines. Morphological changes including cell rounding were observed. Tumor cell migration was completely blocked by TE. TE treatment also inhibited prostaglandin E2 (PGE2) synthesis and downregulated cyclooxygenase 1 and 2 mRNA expression. We purified the active compound(s) to near homogeneity with antimetastatic activity comparable with stock TE. The active compound with a native size of approximately 25 kDa contains two fragments of nearly equal size. The N-terminal amino acid sequencing of both fragments reveals that the active compound is highly related to three taro proteins: 12-kDa storage protein, tarin and taro lectin. All are similar in terms of amino acid sequence, posttranslational processing and all contain a carbohydrate-binding domain. This is the first report describing compound(s) derived from taro that potently and specifically inhibits tumor metastasis.

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

The COX-2 product prostaglandin E(2) (PGE(2)) contributes to the high metastatic capacity of breast tumors. Our published data indicate that inhibiting either PGE(2) production or PGE(2)-mediated signaling through the PGE(2) receptor EP4 reduces metastasis by a mechanism that requires natural killer (NK) cells. It is known that NK cell function is compromised by PGE(2), but very little is known about the mechanism by which PGE(2) affects NK effector activity. We now report the direct effects of PGE(2) on the NK cell. Endogenous murine splenic NK cells express all four PGE(2) receptors (EP1-4). We examined the role of EP receptors in three NK cell functions: migration, cytotoxicity, and cytokine release. Like PGE(2), the EP4 agonist PGE(1)-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 PGE(2), 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, PGE(1)-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 PGE(2) production or preventing signaling through the EP4 receptor may prevent suppression of NK functions that are critical to the control of breast cancer metastasis.

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-gamma, 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.

Antagonism of the prostaglandin E receptor EP4 inhibits metastasis and enhances NK function.

Cyclooxygenase-2 (COX-2) is associated with aggressive breast cancers. The COX-2 product prostaglandin E(2) (PGE(2)) acts through four G-protein-coupled receptors designated EP1-4. Malignant and immortalized normal mammary epithelial cell lines express all four EP. The EP4 antagonist AH23848 reduced the ability of tumor cells to colonize the lungs or to spontaneously metastasize from the mammary gland. EP4 gene silencing by shRNA also reduced the ability of mammary tumor cells to metastasize. Metastasis inhibition was lost in mice lacking either functional Natural Killer (NK) cells or interferon-gamma. EP4 antagonism inhibited MHC class I expression resulting in enhanced ability of NK cells to lyse mammary tumor target cells. These studies support the hypothesis that EP4 receptor antagonists reduce metastatic potential by facilitating NK-mediated tumor cell killing and that therapeutic targeting of EP4 may be an alternative approach to the use of COX inhibitors to limit metastatic disease.

A prostaglandin E (PGE) receptor EP4 antagonist protects natural killer cells from PGE2-mediated immunosuppression and inhibits breast cancer metastasis.

Cyclooxygenase-2 is frequently upregulated in epithelial tumors and contributes to poor outcomes in multiple malignancies. The COX-2 product prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on a family of four G protein-coupled receptors (EP1-4). Using a novel small molecule EP4 antagonist (RQ-15986) and a syngeneic murine model of metastatic breast cancer, we determined the effect of EP4 blockade on innate immunity and tumor biology. Natural killer (NK)-cell functions are markedly depressed in mice bearing murine mammary tumor 66.1 or 410.4 cells owing to the actions of PGE2 on NK cell EP4 receptors. The EP4 agonist PGE1-OH inhibits NK functions in vitro, and this negative regulation is blocked by RQ-15986. Likewise, the treatment of tumor-bearing mice with RQ-15986 completely protected NK cells from the immunosuppressive effects of the tumor microenvironment in vivo. RQ-15986 also has direct effects on EP4 expressed by tumor cells, inhibiting the PGE2-mediated activation of adenylate cyclase and blocking PGE2-induced tumor cell migration. The pretreatment of tumor cells with a non-cytotoxic concentration of RQ-15986 inhibited lung colonization, a beneficial effect that was lost in mice depleted of NK cells. The oral administration of RQ-15986 inhibited the growth of tumor cells implanted into mammary glands and their spontaneous metastatic colonization to the lungs, resulting in improved survival. Our findings reveal that EP4 antagonism prevents tumor-mediated NK-cell immunosuppression and demonstrates the anti-metastatic activity of a novel EP4 antagonist. These observations support the investigation of EP4 antagonists in clinical trials.

Modulation of host natural killer cell functions in breast cancer via prostaglandin E2 receptors EP2 and EP4.

Breast malignancies often have high levels of COX-2. 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 (1 of 4 EP expressed on the malignant cell) reduces metastasis by a mechanism that requires natural killer (NK) cells. Tumor-derived PGE2 and exogenous PGE2 are known to have direct inhibitory effects on NK cell functions, but less is known regarding which EP receptors mediate these effects. We now show that several NK functions (lysis, migration, cytokine production) are compromised in tumor-bearing mice and that tumor-produced PGE2 interferes with NK cell functions. PGE2 inhibits the potential of NK cells to migrate, exert cytotoxic effects, and secrete interferon γ. The ability of PGE2 to inhibit NK cells from tumor-bearing mice is by acting on EP2 and EP4 receptors. NK cells from tumor-bearing mice were more sensitive to inhibition by EP4 and EP2 agonists compared with endogenous NK cells from healthy mice. PGE2 was inhibitory to most NK functions of either normal or tumor-bearing mice. In contrast, there was a trend for enhanced tumor necrosis factor α production in response to PGE2 by NK cells from tumor-bearing mice. We also report that a recently described EP4 antagonist, frondoside A, inhibits breast tumor metastasis in an NK-dependent manner and protects interferon γ production by NK cells from PGE2-mediated suppression. Taken together these data show that NK functions are depressed in tumor-bearing hosts relative to normal NK cells and that PGE2 suppresses NK functions by acting on EP2 and EP4 receptors.