Prostaglandin E receptor EP4 is a therapeutic target in breast cancer cells with stem-like properties

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 Chemokine Receptor CXCR3 Isoform B Drives Breast Cancer Stem Cells

We are seeking to identify molecular targets that are relevant to breast cancer cells with stem-like properties. There is growing evidence that cancer stem cells (CSCs) are supported by inflammatory mediators expressed in the tumor microenvironment. The chemokine receptor CXCR3 binds the interferon-γ-inducible, ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11 and malignant cells have co-opted this receptor to promote tumor cell migration and invasion. There are 2 major isoforms of CXCR3: CXCR3A and CXCR3B. The latter is generated from alternative splicing and results in a protein with a longer N-terminal domain. CXCR3 isoform A is generally considered to play a major role in tumor metastasis. When the entire tumor cell population is examined, CXCR3 isoform B is usually detected at much lower levels than CXCR3A and for this, and other reasons, was not considered to drive tumor progression. We have shown that CXCR3B is significantly upregulated in the subpopulation of breast CSCs in comparison with the bulk tumor cell population in 3 independent breast cancer cell lines (MDA-MB-231, SUM159, and T47D). Modulation of CXCR3B levels by knock in strategies increases CSC populations identified by aldehyde dehydrogenase activity or CD44+CD24− phenotype as well as tumorsphere-forming capacity. The reverse is seen when CXCR3B is gene-silenced. CXCL11 and CXCL10 directly induce CSC. We also report that novel CXCR3 allosteric modulators BD064 and BD103 prevent the induction of CSCs. BD103 inhibited experimental metastasis. This protective effect is associated with the reversal of CXCR3 ligand-mediated activation of STAT3, ERK1/2, CREB, and NOTCH1 pathways. We propose that CXCR3B, expressed on CSC, should be explored further as a novel therapeutic target

Abstract P2-06-03: Prostaglandin E receptor EP4 is a therapeutic target in breast cancer stem cells

Abstract There is an urgent need to identify mechanisms underlying the survival of cells expressing cancer stem cell or tumor-initiating properties. The cyclooxygenase-2 (COX-2) pathway is highly expressed in many breast tumors and contributes to poor outcomes. The COX-2 product prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on the G-protein-coupled receptor EP4. We compared the expression and function of COX-2 and EP4 in mammosphere-forming and bulk populations derived from a panel of human and murine luminal and basal type tumor cells with different metastatic capacities. Expression of both EP4 and COX-2 were markedly increased in mammosphere-forming cells derived from basal and/or metastatic cells relative to the bulk population, but neither COX-2 or EP4 levels were elevated in mammospheres derived from luminal or non-metastatic cells. Breast cancer stem cells, expressing elevated EP4 are correspondingly more sensitive to inhibition with EP4 antagonists both in vitro and in limiting-dilution assays in vivo. Somewhat to our surprise, cancer stem cells remain relatively sensitive to lysis by Natural Killer cells. We have also shown that EP4 blockade protects NK cells from tumor-induced immune suppression. These studies identify EP4 as a potential therapeutic target in the general tumor cell population and show that EP4 targeting may selectively inhibit cells with tumor-initiating or stem cell properties. EP4 antagonists can directly inhibit cancer stem cells and tumor metastasis and indirectly support NK-mediated mechanisms of tumor control. We are delineating the mechanisms by which EP4 and COX-2 are upregulated in cells with stem-like properties. In addition to EP4 and COX-2, STAT3 is upregulated in stem cells. Inhibition of STAT3 reduces mammosphere-forming capacity. Our studies support a mechanism whereby COX-2/EP4 signaling induces STAT3 to support breast cancer stem cell survival by a feed-forward mechanism. Citation Format: Amy Fulton, Namita Kundu, Tyler Kochel, Xinrong Ma, Jocelyn Reader. Prostaglandin E receptor EP4 is a therapeutic target in breast cancer stem cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-03.</jats:p

Abstract 2257: Multiple drug resistance-associated protein 4 (MRP4) may contribute to breast cancer metastasis by exporting the COX-2 product PGE2

Abstract 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</jats:p

Abstract 524: Cyclooxygenase pathway in ovarian cancer.

Abstract Objectives: To examine the role of the cyclooxygenase (COX) pathway in ovarian cancer. Cyclooxygenases, specifically COX1 and COX2, are frequently overexpressed in malignancies and lead to the synthesis of prostaglandins. A particularly important prostaglandin, PGE2 is exported from the cell into the extracellular milieu by the multi-drug resistance protein 4 (MRP4) transporter where it acts in a paracrine and autocrine manner by activating a family of G-protein coupled receptors (EP1-4). PGE2 is transported into the cell by the prostaglandin transporter (PGT) and catabolized by 15-prostaglandin dehydrogenase (15-PGDH). It has been previously shown that COX1, rather than COX2, is overexpressed in ovarian cancer. Little is known about the role of other members of this pathway, including EP receptors, PGT, MRP4, and 15-PGDH, in this disease. Methods: HOSE (benign immortalized ovarian epithelial cells) and two ovarian cancer cell lines, SKOV3 and OVCAR3, were grown under standard conditions. Cells were harvested and RNA and protein were isolated. Quantitative RT-PCR was done using the SYBR Green protocol to examine the expression of members of the cyclooxygenase pathway and expressed relative to HOSE cells. Results: In both ovarian cancer cell lines relative to HOSE, expression of COX2, PGT, EP4, and EP1 was decreased. In SKOV3 cells, COX1 and 15-PGDH expression was decreased but MRP4 levels were comparable to levels detected in HOSE cells. In OVCAR3 cells, COX1 and 15-PGDH expression levels were increased and MRP4 was decreased relative to HOSE cells. PGE2 levels in media will be measured to determine the net effect of these changes. Protein levels of the members of the cyclooxygenase pathway will be determined by western blot. Conclusions: It is clear that several members of the cyclooxygenase pathway are expressed differently in malignant versus normal ovarian cells. Studies in progress will determine the functional significance of these differences. Citation Format: Gautam G. Rao, Tyler Kochel, Namita Kundu, Jocelyn Reader, Amy Fulton. Cyclooxygenase pathway in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 524. doi:10.1158/1538-7445.AM2013-524</jats:p

Abstract 3250: Prostaglandin E2 receptor EP1 suppresses breast cancer metastasis

Abstract Cyclooxygenases (COX-1 and COX-2) catalyze the formation of prostaglandins and play a role in the pathogenesis of breast cancer. Prostaglandin E2 (PGE2), the chief COX product in tumors, is the predominant protumorigenic prostanoid and mediates biological effects by binding to each of four EP receptors (EP1-4). Each receptor is coupled to different intracellular signaling pathways; and EP1 is coupled to calcium mobilization and PKC activation. Our published studies indicate that EP1 was detected in the cytoplasm and nucleus of benign ducts and malignant cells in invasive ductal carcinomas, and overall survival for women with tumors negative for nuclear EP1 was significantly worse than for women with any nuclear EP1 expression. Pharmacologic antagonism or reduction of EP1 expression increased metastatic capacity in a murine model of metastatic breast cancer. These data support our hypothesis that EP1 functions as a metastasis suppressor. We now report that murine metastatic mammary tumor cell lines 410.4 and 66.1 have decreased EP1 mRNA expression compared to the non-metastatic cell line 410. Western blot analysis of total and subcellular fractions of EpH4 (normal, immortalized mammary epithelial) and malignant 410, 410.4 and 66.1 cell lines demonstrates that EP1 protein is present in the total and cytoplasmic fractions for all cell lines examined. Nuclear EP1 is detected in EpH4 and non-metastatic 410 cells; however, very little to no nuclear protein is detected in metastatic cell lines 410.4 and 66.1. The absence of nuclear EP1 in metastatic cell lines is consistent with the prognostic data that overall survival for women with tumors negative for nuclear EP1 was significantly worse. Previously, we determined that reduction of EP1 expression leads to increased metastatic capacity; therefore, we investigated the effect of EP1 overexpression on lung colonization in 410.4 and 66.1 cells via tail vein injection in a syngeneic murine model of breast cancer. In 410.4 cells, EP1 overexpression leads to a 57%-97% decrease in metastasis, and in 66.1 cells EP1 overexpression resulted in a 10% - 38% decrease in lung tumor burden compared to vector control mice. The inverse correlation between EP1 expression and metastatic capacity supports our hypothesis that EP1 functions as a metastasis suppressor. We explored potential mechanisms leading to the alteration in the metastatic behavior in response to manipulation of the expression of EP1. We previously published that an EP1 antagonist altered adhesion to laminin in 410.4 cells. Overexpression of EP1 altered the expression of several integrin receptors in 410.4 and 66.1 cell lines including integrins alpha-V, alpha-6 and alpha-3. Also, overexpression of EP1 led to altered adhesion of these cells to several extracellular matrices including fibronectin, collagen, laminin and fibrinogen. Alteration of the adhesive properties of these cells by EP1 could contribute to the metastasis suppressor function of the EP1 receptor. Citation Format: Jocelyn C. Reader, Xinrong Ma, Namita Kundu, Olga Goloubeva, Amy Fulton. Prostaglandin E2 receptor EP1 suppresses breast cancer metastasis. [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 3250. doi:10.1158/1538-7445.AM2015-3250</jats:p

Abstract 3862: Mechanistic studies of the metastasis suppressor prostaglandin E2 receptor EP1 in breast cancer.

Abstract Prostaglandin E2 (PGE2), the chief cyclooxygenase-2 enzyme (COX-2) product in tumors, is the predominant protumorigenic prostanoid and mediates biological effects by binding to each of four EP receptors (EP1-4). Each receptor is coupled to different intracellular signaling pathways with EP1 coupled to calcium mobilization and PKC. EP4, expressed on malignant breast cells, promotes metastasis, but a role for EP1 in metastasis has not been investigated. Our published studies indicate that EP1 was detected in the cytoplasm and nucleus of benign ducts and malignant cells in invasive ductal carcinomas, and overall survival for women with tumors negative for nuclear EP1 was significantly worse than for women with EP1 expression. Pharmacologic antagonism and reduction of EP1 expression increased metastatic capacity in our murine model of metastatic breast cancer. These data support our hypothesis that EP1 functions as a metastasis suppressor. We now report that murine metastatic mammary tumor cell lines 410.4 and 66.1 have decreased EP1 mRNA expression compared to the non-metastatic cell line 410. We have also identified the presence of a variant EP1 (EP1v) transcript. EP1v has been identified previously in murine mast cell line MC/9 and rat uterus but not in malignant cells. EP1v has a pattern of mRNA expression different than full-length EP1. Compared to the cell line 410, EP1v expression is slightly increased in 410.4 and decreased in 66.1 cell lines. In order to determine the underlying mechanism in which EP1 suppresses metastasis, a metastasis gene array was performed comparing gene expression in EP1-vector, and EP1-silenced 66.1 cell lines and several candidate genes were identified including Fn1, whose protein is altered during epithelial-to-mesenchymal transition. qPCR analysis of Fn1 expression in EP1-silenced and overexpression cell lines revealed an inverse relationship between EP1 and Fn1 expression. A decrease in EP1 expression led to a 4-5 fold increase in Fn1 expression; whereas, increased EP1 expression resulted in a 0.50 fold decrease in Fn1 expression. Like full length EP1, overexpression of EP1v also resulted in a decrease in Fn1 expression. Bioinformatic analysis of the EP1 gene identified several CpG islands. DNA methylation analysis revealed hypermethylation of the CpG island nearest to the promoter in normal, non-metastatic and metastatic murine mammary tumor cell lines. In the quest to identify a clinically relevant strategy to increase expression of this protective receptor, we treated 410.4 and 66.1 cells with demethylating agent 5-azacytidine which resulted in an increase in EP1 expression. Our published studies show that EP1 acts to suppress metastasis and we are currently exploring the contribution of EP1v to this mechanism. These findings suggest that EP1 has the potential to be a new therapeutic target in reducing breast cancer metastasis and increasing overall cancer survival. Citation Format: Jocelyn C. Reader, Xinrong Ma, Namita Kundu, Olga Goloubeva, Amy Fulton. Mechanistic studies of the metastasis suppressor prostaglandin E2 receptor EP1 in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3862. doi:10.1158/1538-7445.AM2013-3862</jats:p