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