Dawn Holt

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]

Prostaglandin E2 EP receptors as therapeutic targets in breast cancer.

Prostaglandins are lipid compounds that mediate many physiological effects. Prostaglandin E2 (PGE2) is the most abundant prostanoid in the human body, and synthesis of PGE2 is driven by cyclooxygenase enzymes including COX-2. Both elevated expression of COX-2 and increased PGE2 levels have been associated with many cancers including breast cancer. PGE2 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.

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.

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 E2 (PGE2) 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-γ. 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.

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 &ggr;. 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 &agr; 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 &ggr; 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.

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.

Abstract 5119: Multiple drug resistance-associated protein 4 (MRP4) may contribute to breast cancer progression by exporting the COX-2 product PGE2.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Cyclooxygenase-2 (COX-2) and its enzymatic product, prostaglandin E2 (PGE2), are elevated in breast cancer and are associated with a poor prognosis and increased metastatic potential. PGE2 initiates various signaling pathways upon binding to each of four cognate EP receptors. We have previously shown that PGE2 signaling through the EP4 receptor increases metastatic potential and supports the survival of breast cancer stem-like cells. Multiple drug resistance-associated protein 4 (MRP4) is responsible for the active export of PGE2 from cells, while the prostaglandin transporter (PGT) imports PGE2 for 15-hydroxyprostaglandin dehydrogenase (15-PGDH)-mediated degradation. The role of neither MRP4 nor PGT has been investigated in breast cancer progression. The purpose of this study is to elucidate the role of MRP4 in PGE2 signaling in breast cancer progression. We hypothesize that elevated expression of MRP4 would cause increased PGE2 export and receptor-mediated signaling and, therefore, enhance metastatic potential, tumor progression, and support breast cancer stem-like cells. We examined MRP4 gene expression data from multiple breast cancer datasets using Oncomine. We found higher expression of ABCC4 in breast cancer versus normal breast. MRP4 was elevated in invasive (IDC) versus localized (DCIS) lesions and elevated in the majority of basal-type breast cancer. To investigate the role of MRP4 further, we examined MRP4 mRNA and protein expression in cell lines representing several molecular subtypes and metastatic capacities. Normal mammary epithelium (MCF10A), luminal (MCF7, T47D), basal (MDA-MB-231, MDA-MB-468, MDA-MB-436, BT549), and Her2-enriched (SKBR3) cell lines were evaluated. As in the primary sample data, MRP4 mRNA and protein expression are elevated in basal and Her2 enriched cell lines (231, 436, BT549, SKBR3) while expression of PGT mRNA and protein is decreased in these cells when compared to cells with lower metastatic potential. This inverse relationship between MRP4 and PGT should lead to higher concentrations of extracellular PGE2 in the tumor microenvironment. We evaluated MRP4 activity by measuring PGE2 export from cells via enzyme immunoassay or resistance to the cytotoxic compound 6-mercaptopurine (6-MP), two substrates of MRP4. Pharmacologic inhibition of MRP4 with MK571 (MRP antagonist) results in decreased efflux of PGE2 and increased sensitivity to 6-MP as expected. Likewise, genetic suppression by RNAi results in lower levels of PGE2 exported from cells and increased sensitivity to 6-MP, confirming altered MRP4 activity. Conversely, ectopic MRP4 overexpression increases PGE2 export. These data support the hypothesis that MRP4 is a critical member of the PGE2 signaling pathway that leads to high extracellular PGE2 and increased PGE2 signaling, implicating MRP4 as a possible therapeutic target in this oncogenic pathway. Citation Format: Tyler J. Kochel, Namita Kundu, Xinrong Ma, Jocelyn Reader, Amy Fulton. Multiple drug resistance-associated protein 4 (MRP4) may contribute to breast cancer metastasis by exporting the COX-2 product PGE2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2257. doi:10.1158/1538-7445.AM2015-2257

Abstract 2845: Prostaglandin E receptor EP4 antagonist RQ00015986 inhibits breast cancer metastasis and protects natural killer cells from PGE-mediated immune suppression

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The cyclooxygenase-2 (COX-2) pathway is upregulated in breast and other malignancies. The COX-2 product prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on a family of G-protein-coupled receptors (EP1-EP4). In breast and colon malignancies, EP4 is a principle mediator of pro-tumorigenic and pro-metastatic activities. Our previous studies demonstrated that targeting EP4 with shRNA or by pharmacologic antagonists of EP4 leads to reduced metastasis in a preclinical model of metastatic breast cancer. These studies and others establish EP4 as a potential therapeutic target, however, there is a paucity of effective EP4 antagonists available for preclinical and clinical investigation. Using a novel orally available EP4 selective antagonist, RQ00015986, we have evaluated efficacy in a syngeneic murine model of metastatic breast cancer. Murine and human breast cancer cells express EP4 and receptor occupation with PGE2 (binds all EP) or with PGE1-OH (EP4 agonist) results in adenyl cyclase activation and elevated intracellular cAMP. RQ00015986, in uM/L concentrations, was able to prevent EP4-mediated adenyl cyclase activation in murine mammary tumor cell lines 66.1 and 410.4. Breast tumor cells migrate in response to either PGE2 or PGE1-OH and RQ00015986 inhibited this response. Pretreatment of 66.1 cells with RQ00015986 prior to i.v. injection into syngeneic mice resulted in 58-69% inhibition in numbers of lung tumor colonies. Natural Killer (NK) cells are critical to the control of metastatic dissemination. The ability of RQ00015986 to inhibit lung tumor colonization was completely lost in hosts depleted of mature NK cells. We have reported previously that NK cell functions in tumor-bearing mice are severely depressed through the actions of tumor-derived PGE2 acting on EP4 receptors expressed on the NK cell. The capacities of NK cells to migrate, to produce cytokines and to lyse mammary tumor target cells are each inhibited through PGE2. In the presence of RQ00015986, the capacity of cultured NK cells to migrate and to produce Interferon-α was restored to normal levels. More interestingly, treatment of tumor-bearing mice with RQ00015986 in vivo also restored NK cell functions. This report provides evidence that a novel EP4 antagonist may have therapeutic potential in cancer and may act by antagonizing EP4 prometastatic functions on the malignant cell as well as protecting NK effector cells from PGE2-mediated immune suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2845. doi:1538-7445.AM2012-2845

Abstract 3437: A natural plant product to inhibit breast cancer metastasis

In the United States, breast cancer death is the second leading cause of cancer deaths in women, primarily due to the occurrence of metastatic disease. We have identified a potential novel therapeutic agent derived from an edible root of the plant Colocasia esculenta, commonly known as taro, which has significant antimetastatic 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 (95-99%) as well as spontaneous metastasis from mammary gland-implanted tumors (85%), in a murine model of highly metastatic ER, PR and Her-2/neu negative breast cancer. TE modestly inhibits the proliferation of some, but not all, breast and prostate cancer cell lines and also causes morphologic changes including cell rounding. TE inhibits prostaglandin E2 (PGE2) levels and because PGE2 is a potent immune modulator, we examined the role of immunity in the therapeutic response to TE. We first examined the effect of TE on Natural Killer (NK) cell activities. TE modestly increases NK-mediated tumor cell lysis in vitro. We determined the effect of NK depletion on TE efficacy in vivo. The ability of TE to inhibit metastasis was modestly compromised in NK-depleted mice indicating that NK cells play a minor role in TE-mediated antimetastatic activity. To examine the role of T and B lymphocytes, TE efficacy was examined in Balb/c SCID mice. Antimetastatic activity of TE was completely lost in SCID mice lacking mature T and B cells. Thus, T and /or B cell functions contribute to the mechanism by which TE inhibits metastasis. In published studies we have identified the active component of TE as one of three closely related taro proteins, tarin, taro lectin and 12-kDa storage protein. Current studies are determining how these candidate proteins affect immune functions to inhibit breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3437. doi:1538-7445.AM2012-3437

Abstract 5308: Targeting the Cox2 pathway prostaglandin EP receptors to regulate NK cell functions

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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 indicates 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. In order to better understand the mechanism by which inhibiting PGE2 production or signaling reduces metastatic disease, we examined the role of PGE2 in regulating protective host effector 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. NK effector function is regulated by complex interactions of recognition receptors expressed on NK cells and cognate ligands expressed on target cells. Both stimulatory and inhibitory signals are delivered through these receptor-ligand interactions. We have previously shown that both Cox inhibitors and antagonists of the EP4 receptor modulate the expression of NK cell stimulatory (H60) and inhibitory (MHC class I) ligands on the tumor target cell in favor of NK recognition and killing. We now report the direct effects of PGE2 on the NK cell. Murine splenic NK cells express all four PGE2 receptors (EP1-4). We examined the role of EP receptors in three NK cell functions; cytokine release, cytotoxicity, and migration. Activating all four NK-EP receptors with PGE2 leads to inhibited IFNγ secretion, induced TNFα production, less migration of NK cells and reduced ability to lyse tumor target cells. Using agonists and antagonists of individual EP receptors, we showed that only certain EP receptors contribute to loss of function. Next, we showed that all NK-EP receptor expression is downregulated during progressive tumor growth, which produced an environment that induced a less mature NK phenotype. EP2 and EP4 are linked to protein kinase A and elevations in intracellular cAMP. NK cells from tumor bearing mice respond poorly to stimulation with 10µM PGE2 and intracellular cAMP levels were increased by only 1.45-1.78 fold. In contrast, cAMP was induced by 6.08-10.58-fold in NK cells from normal mice. Thus, the tumor milieu results in changes in both NK-EP expression levels and EP receptor functions. NK mediated cytotoxicity, cytokine release, and migration were also compromised in tumor bearing mice. These results taken together support a mechanism whereby inhibiting PGE2 production or preventing signaling through some, but not all EP receptors may prevent suppression of NK functions that are critical to control breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5308.