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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 (CD44hi/CD24low, 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.


Introduction
Elevated cyclooxygenase 2 (COX-2) expression is common in breast cancer and is associated with a worse prognosis [1,2] but the role of COX-2 pathway members in the behavior of breast cancer stem cells has yet to be defined. The principle COX-2 product in tumors is prostaglandin E 2 (PGE 2 ) which mediates cellular responses by acting on a family of four G protein-coupled receptors (EP1-EP4). Prostaglandin E receptor 4 (EP4) is expressed in a wide range of epithelial malignancies [3][4][5][6][7][8][9][10][11][12] and pharmacologic blockade or genetic silencing of the EP4 receptor inhibits proliferation and migration in vitro and growth and metastasis in vivo [13][14][15][16][17]. These data provide evidence that EP4 and COX-2 are important to the behavior of the general population of tumor cells. Recently, mesenchymal stem cells were shown to create a supportive microenvironment for cancer stem cells by a PGE 2dependent mechanism [18]. We asked if the COX-2 pathway is supportive of breast cancer stem cell survival by examining the expression and function of COX-2 and EP4 in cells with a stem cell phenotype. We now show that both EP4 and COX-2 are highly induced on candidate tumorinitiating/stem cell populations and that EP4 antagonists reduce cancer stem cell properties in vitro and in vivo supporting the hypothesis that EP4 and/or COX-2 may represent novel targets expressed by the most high risk and resistant subpopulations. For some studies, the targeting vector was from OpenBiosystems, Huntsville, AL [21]; for other studies an EP4shRNA was obtained from OriGene, Rockville, MD. MDA-MB-231-luc cells were a generous gift of Dr. Stuart Martin, UMB. Balb/cByJ female mice were purchased from the Jackson Laboratory (Bar Harbor, ME); Balb/c/SCID mice were purchased from Charles Rivers Laboratory (Wilmington, MA). Limiting dilution assays were carried out by injecting the indicated number of cells proximal to the inguinal mammary fat pad. Mice were euthanized on an individual basis when tumors measured 18 mm in largest diameter and lung surface tumor colonies were counted under a dissecting microscope. Tumor volume was calculated by the formula: (a 9 b 2 ) 9 0.5236, where a = longest diameter and b = perpendicular diameter. Lung colonization was evaluated by injecting 1-2 9 10 5 viable tumor cells i.v. into the lateral tail vein of Balb/cByJ female mice. All mice were euthanized on day 18-22 post-transplantation or earlier if moribund. Lungs were examined for tumor colonies.

Live animal imaging
Balb/c/SCID mice were injected i.v. with 1 9 10 5 MDA-MB-231-luc cells treated with 3 lM RQ-15986 or DMSO. On the days indicated, sedated mice were injected with D-Luciferin (Caliper Life Sci., Hopkinton, MA) and wholebody bioluminescence determined by Xenogen system.  Immunohistochemistry A tissue microarray was prepared and described previously [22]. Immunohistochemistry was carried out after antigenretrieval using rabbit anti-human polyclonal EP4 antibody (Lifespan Biosciences, Seattle, WA) followed by secondary antibody (EnVision?dual link System, Dako).
qPCR Quantitative PCR was conducted using SYBR green (Bio-Rad, Hercules, CA) and 400-700 ng cDNA per reaction. Relative expression levels for COX-2 and EP4 were normalized to GAPDH by the DDC t method.

Biostatistics
In vivo data was analyzed by non-parametric Kruskal-Wallis test and pair-wise comparisons were carried out by exact two-sided Wilcoxon test. Tumor incidence data analyzed by generalized linear models approach. Data from in vitro studies analyzed by Student's t test.

EP4 is widely expressed in primary human breast cancer and targeting EP4 inhibits metastasis
We examined the expression of EP4 in 44 invasive ductal carcinomas of the breast by immunohistochemistry. EP4 expression was very low or absent in normal ducts (0, 1?, Fig. 1a), malignant epithelium was positive for cytoplasmic EP4 expression. On a scale of 0-3? staining intensity, 21/44 (48 %) specimens had 1? EP4 expression, 13/44 (29 %) were 2? and 10/44 (23 %) were graded as 3? in EP4 staining intensity. Nuclear staining was not observed. EP4 gene silencing or receptor inhibition with small molecule inhibitors block metastasis in a syngeneic murine breast cancer model [13,20,21,23]. In this study, we confirmed, using a second tumor cell line and a different EP4 antagonist (RQ-08), that metastasis is inhibited by EP4 blockade. Line 410.4 tumor cells were implanted into syngeneic Balb/cByJ female mice and oral administration of RQ-08 (30 mg/kg 9 28 days) was initiated on day ?7. When tumors achieved an average diameter of 18 mm, mice were euthanized and metastatic disease was assessed. The growth of primary tumors was modestly inhibited by RQ-08 (not shown) but spontaneous metastasis to the lungs was reduced by 49 % (Fig. 1b, P = 0.04). Metastatic success of human MDA-MB-231-luc cells was also reduced by an EP4 antagonist (Fig. 1c). We studied cell-autonomous effects of EP4 antagonism on the tumor cell alone, by pretreating tumor cells with RQ-15986 (3.0 lM/l) prior to i.v. injection into Balb/SCID mice. At day 1 after i.v. injection of tumor cells, less luciferase signal was detected when EP4 was antagonized. As the surviving tumor cell populations expanded with time, the difference between the two treatment groups became more pronounced. We also created multiple clones of 66.1 expressing EP4shRNA (Fig. 1d). Metastatic potential was reduced by 43, 53, 53, and 84 %, respectively, in comparison to mice injected with vector control cells (Fig. 1e). Thus, EP4 is widely expressed in breast cancer and either genetic or pharmacologic compromise of EP4 activity reduces metastatic potential.
Metastasis and stem cell-associated genes are downregulated in shEP4 cells We employed a qPCR array of known metastasis-associated genes to compare gene expression patterns of 66.1vector versus 66.1shEP4 cells. Table 1 shows genes that were downregulated by at least 1.5-fold in 66.1shEP4 cells compared to 66.1-vector cells that included Csf1, c-met, CXCL12, and CD44. Few genes were upregulated in the context of EP4 silencing, but included the metastasis-suppressor Nme4 (data not shown). The downregulation of Csf1, Timp2, and CD44 in 66.1shEP4 cells was confirmed by qPCR (Fig. 2a). While each of these gene expression changes may be important to the mechanism of metastasis inhibition, we focused our further studies on the reduction in CD44, a phenotypic marker of candidate breast cancer stem/tumor-initiating cells [24,25].
Tumor-initiating cells are identified in a syngeneic model of metastatic breast cancer A true cancer stem cell should have heightened tumorinitiating capacity compared to the general population but a tumor-initiating cell has not been demonstrated in the murine cell lines under study. We cultured 410.4 cells in low-attachment conditions to demonstrate that mammospheres will form. These mammospheres can be dissociated and replated to form secondary and tertiary mammospheres (dns). Limiting dilution assays compared the tumorigenic properties of the general population of 410.4 cells versus mammosphere-forming (MS-1) 410.4 cells. Three of 10 mice injected with 100 of 410.4 bulk cells developed palpable tumors (dashed lines), whereas 8/9 mice injected with MS-1 cells (solid lines) developed progressively growing tumors (Fig. 3c). In an expanded limiting dilution assay using additional cell injection doses ( Table 2) EP4 and COX-2 are upregulated in mammospheres of aggressive breast cancer cells Several laboratories have described the stem cell properties of human breast cancer cell lines [26][27][28] and we confirmed that MDA-MB-231 cells are highly enriched for a stem cell phenotype whereas this population is rare in MCF7 cells, which have a luminal gene signature. Consistent with the literature, more than 95 % of MDA-MB-231 cells are CD44 hi /CD24 low (Fig. 3a). In comparison,  (Fig. 3b), however, these latter cells are highly positive for CD24 expression.  (Fig. 4e), but not in 410 or 67 MS-1 cells (Fig. 4f). Consistent with the mRNA data, increased COX-2 protein was detected in MS-1 cells of MDA-MB-231 and SKBR3 grown in either DME or mammocult media; only a slight increase in COX-2 protein was detected in MCF-7 cells (Fig. 4g). Increased COX-2 in MS-1 cells resulted in more PGE 2 detected in conditioned media of MDA-MB-231 and SKBR3, but not MCF7derived MS-1 cells (Fig. 4h). Thus, in both human and mouse, EP4 and COX-2 are elevated in mammospheres produced by highly tumorigenic, basal-type cell lines. Luminal-type (MCF7) or less tumorigenic, non-metastatic (410, 67) cells do not elevate EP4 or COX-2 expression in MS-1 tumorspheres. were all able to inhibit the size of mammospheres (Fig. 5c-e).
In the presence of AH23848, mammospheres were actually increased in number, but individual spheres were much smaller as indicated by the dramatic reduction in cells/ sphere. AH23848 at 10, 5, 0.5, or 0.05 lM/l reduced sphere cellularity by 78, 69, 55, and 2 %, respectively (Fig. 5c). In contrast, AH23848 did not affect the number of attached cells at any concentration examined (dns). While the number of total spheres was not affected by Frondoside A except at the highest concentration tested (1.0 lM/l), the sphere size was inhibited by 100, 92, 28, and 1 % in a dosedependent manner (Fig. 5d). RQ-15986 (30, 10, 3, and 0.3 lM/l) inhibited sphere-forming cells by 20, 30, 8, or 7 % (Fig. 5e). Taken together, these data indicate that tumor cells with heightened stem cell properties are more sensitive than the general population to EP4 antagonism.

EP4 antagonists inhibit the stem cell phenotype in vitro
To determine if EP4 inhibition would downregulate phenotypic markers of breast cancer stem cells, MDA-MB-231 cells were placed in mammosphere culture and on day 8, RQ-15986 (10 lM/l) was added. On day 10, cultures were analyzed for ALDH expression. In a typical experiment, RQ-15986 treatment reduced the proportion of ALDH ? MDA-MB-231 cells from 30.4 % to 16.7 % (Fig. 6a). RQ-15986, Frondoside A or AH23848 also modestly reduced the percentage of CD44-positive MS-1 cells. In the presence of PBS, 64 % of MDA-MB-231 (MS-1) cells were CD44-positive; Frondoside A (0.25 lM/l) reduced the fraction of CD44-positive cells to 54.3 %. In the presence of DMSO, 53.5 % of cells were CD44-positive; RQ-15986 (10 lM/l) or AH23848 (5 lM/l) reduced the CD44-positive fraction to 45.9 or 46.9 % of cells, respectively. CD44 expression was not changed in attached cells in the presence of EP4 antagonists (dns). CD24 is poorly expressed in MS-1 cells and the degree of expression was not changed by treatment with any EP4 antagonist (dns).

EP4 antagonism inhibits tumor-initiating capacity in vivo
By limiting dilution assays, we assessed the ability of an EP4 antagonist to inhibit tumor-initiating cells in vivo. Balb/cByJ mice were transplanted with 500 or 50 of 410.4 MS-1 cells and, beginning on day ?7, were treated with vehicle or RQ-08 for 28 days and tumor incidence and size were assayed. In two independent experiments, EP4 antagonism was able to significantly inhibit the tumorforming capacity of small numbers of tumor cells as indicated by tumor incidence (Fig. 6b). When 500 cells were injected, tumor incidence was reduced from 90 % to 60 % by RQ-08 (P \ 0.02); RQ-08 inhibited tumor-initiating capacity from 60 % tumor-positive mice to 20 % tumorpositive when 50 cells were injected (P \ 0.058). The calculated stem cell frequency was reduced from 1/126 cells to 1/460 cells by RQ-08 (P \ 0.018). While the average size of tumors is sometimes variable reflecting the different latency in individual mice, it is obvious that RQ-08 has a profound effect on the ability of palpable lesions to expand (Fig. 6c). In animals that developed palpable tumors in spite of RQ-08, the percentage of tumor cells that were CD44 hi /CD24 low was reduced from 84.2 % ± 1.3 (vehicle) to 74.1 ? 1.3 % (P \ 0.006).

Discussion
EP4 expressed on the malignant cell contributes to metastatic behavior in a cell-autonomous manner as demonstrated by reduced metastatic potential of tumor cells in which EP4 expression has been silenced or antagonized by small molecule inhibitors. Tumor-derived PGE 2 also acts to suppress the anti-metastatic activities of NK cells by acting on EP4 expressed on the NK cell [23,29,30]. Other EP4-positive immune effector cells are also inhibited by PGE 2 [31][32][33]. Thus, the function of multiple EP4 receptorpositive cells are affected directly by EP4 antagonism.
EP4 promotes multiple cancers [4][5][6][7][8][9][10][11][12][13][14][15][16][17] but little is known about the role of the COX-2 pathway in cancer stem cells. Mesenchymal stem cells are stimulated by IL-1 to produce PGE 2 that creates a supportive microenvironment for cancer stem cells [18]. Stromal fibroblasts support the induction of MCF7-derived stem cells; in that model, PGE 2 is not sufficient to directly act on the progenitor stem cells [34]; rather an indirect mechanism is proposed in which PGE 2 acts indirectly through the cancer-associated fibroblast to induce a stem cell phenotype. The relevant EP receptor was not identified in that study.
Because mammary tumor cells have elevated endogenous COX-2 activity, we investigated the role of PGE 2 and EP4 in an autocrine mechanism that would support breast cancer stem cells. We now show that a sub-population enriched in tumor-initiating cells upregulates both EP4 and COX-2. Upregulation of these genes was only observed in metastatic and/or basal-type human (MDA-MB-231, SKBR3) and murine (410.4, 66.1) cell lines but not in nonmetastatic or luminal-type cells suggesting that tumor-initiating cells may contribute to the aggressive phenotype and may be relatively more sensitive than the non-stem cell population to direct inhibition by either EP4 antagonists or COX inhibitors.
The upregulation of EP4 expression in mammospheres is functionally linked with increased tumor-initiating cell capacity in vivo of mammosphere-forming cells. MS-1 cells were more tumorigenic than the bulk population with an increase in stem cell frequency. Importantly, tumorigenic potential of MS-1 cells was significantly reduced by an EP4 antagonist in vivo corresponding to a loss of CD44 hi /CD24 low cells. In the relatively few RQ-treated  Interestingly, the COX-1/COX-2 inhibitor indomethacin did not affect sphere size, indicating that it is not PGE 2 synthesis per se, but more likely to be PGE 2 -mediated cell signaling that supports sphere formation. The fraction of CD44 hi or ALDH1 ? cells, both phenotypic markers of tumor-initiating cells, were reduced by each of three EP4 antagonists. We did not observe appreciable upregulation of COX-2 in MCF7 tumorspheres consistent with the luminal properties of this cell line. Singh et al. detected a rare COX-2 highly positive cell in MS-1 cultures of MCF7; in these cells, the stem cell factor Oct4 was often co-expressed [35]. Those authors propose that the COX-2 hi , Oct4? is a breast cancer stem cell, consistent with the current report. Taken together, these studies support the hypothesis that EP4-mediated activation can, in an autocrine or paracrine manner drive stem cell survival. Our studies show, for the first time, that a clinically relevant EP4 antagonist can inhibit breast cancer stem cells. Blocking EP4 inhibits breast cancer metastasis and tumorigenic capacity in vivo and stem cell properties including mammosphere-forming ability and phenotypic markers of breast cancer stem cells in vitro. EP4 antagonism may prove to be safer and more effective than global COX-2 inhibition. The demonstrated efficacy of EP4 antagonists in preclinical models of breast and other malignancies supports the continued evaluation of EP4 as a new therapeutic target in advanced malignancy.