Prostaglandin E receptor EP4 is a therapeutic targ

Authors: Namita Kundu Xinrong Ma Tyler Kochel Olga Goloubeva Paul Staats Keyata Thompson Stuart Martin Jocelyn Reader Yukinori Take Peter Collin Amy Fulton

Publish Date: 2013/11/27

Volume: 143, Issue: 1, Pages: 19-31

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Abstract

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 COX2 pathway downstream of the COX2 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 Mammosphereforming breast cancer cells of human MDAMB231 SKBR3 or murine 661 4104 origin of basaltype Her2 phenotype and/or with heightened metastatic capacity upregulate expression of both EP4 and COX2 and are more tumorigenic compared to the bulk population In contrast luminaltype or nonmetastatic counterparts MCF7 410 67 do not increase COX2 and EP4 expression in mammosphere culture Treatment of mammosphereforming cells with EP4 inhibitors RQ15986 AH23848 Frondoside A or EP4 gene silencing but not with a COX inhibitor Indomethacin reduces both mammosphereforming capacity and the expression of phenotypic markers CD44hi/CD24low aldehyde dehydrogenase of breast cancer stem cells Finally an orally delivered EP4 antagonist RQ08 reduces the tumorinitiating capacity and markedly inhibits both the size of tumors arising from transplantation of mammosphereforming 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/tumorinitiating phenotypeElevated cyclooxygenase 2 COX2 expression is common in breast cancer and is associated with a worse prognosis 1 2 but the role of COX2 pathway members in the behavior of breast cancer stem cells has yet to be defined The principle COX2 product in tumors is prostaglandin E2 PGE2 which mediates cellular responses by acting on a family of four G proteincoupled 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 COX2 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 PGE2dependent mechanism 18 We asked if the COX2 pathway is supportive of breast cancer stem cell survival by examining the expression and function of COX2 and EP4 in cells with a stem cell phenotype We now show that both EP4 and COX2 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 COX2 may represent novel targets expressed by the most high risk and resistant subpopulationsMurine mammary tumor lines 661 and 4104 are highly tumorigenic and metastatic in syngeneic Balb/cByJ mice lines 67 and 410 are poorly tumorigenic and nonmetastatic in the same hosts Human breast cancer cell lines MDAMB231 SKBR3 and MCF7 were obtained from ATCC The EP4 antagonists AH23848 Sigma Chem Co St Louis MO and RQ00015986 hereafter abbreviated RQ15986 and RQ08 gifts of RaQualia Pharma Inc Ref 19 as CJ042794 Frondoside A a gift of Coastside Bio Resources Ref 20 indomethacin Sigma were added to cell cultures to achieve final concentrations as indicated Line 4104 and 661 tumor cells were transfected with a plasmid expressing shRNA targeting the murine EP4 gene or control vector For some studies the targeting vector was from OpenBiosystems Huntsville AL 21 for other studies an EP4shRNA was obtained from OriGene Rockville MD MDAMB231luc 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 × b 2 × 05236 where a = longest diameter and b = perpendicular diameter Lung colonization was evaluated by injecting 1–2 × 105 viable tumor cells iv into the lateral tail vein of Balb/cByJ female mice All mice were euthanized on day 18–22 posttransplantation or earlier if moribund Lungs were examined for tumor colonies

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