Association of EP2 receptor and SLC19A3 in regulating breast cancer metastasis

Abstract

Breast cancer is the most common cancer in women worldwide. Triple-negative breast cancer patients have higher metastatic rate than patients with other breast cancer subtypes. Distant metastasis is one of the causes leading to the high mortality rates. Cyclooxygenase-2 (COX2) is associated with breast cancer metastasis and the downstream prostaglandin E2 (PGE2) exerted its effect through EP receptors (EP1-EP4). However, the exact molecular events of EP receptors in breast cancer metastasis remain undefined. Expressions of EP receptors were determined during cancer development in NOD-SCID mice inoculated with MB-231 and MB-231-EP2 clone. EP2 overexpressing stable clone was constructed to investigate the proliferation and invasion potentials in vivo and in vitro. Drug transporter array was used to identify EP2 receptor-associated drug transported genes in breast cancer metastasis. Localization of EP2 receptor in primary tissues and xenografts were examined by immunostaining. Stable EP2-expression cells formed larger tumors than parental cells in mice model and was highly expressed in both primary and metastatic tissues. Silencing of EP2 receptor by siRNA and antagonist (AH 6809) significantly decreased cell proliferation and invasion, concomitant with reduced MMP-2 and MMP-9 expressions. Results from array data showed that expression of SLC19A3 was markedly increased in EP2 siRNA transfected cells. Ectopic expression of SLC19A3 retarded cell proliferation, invasion and MMPs expressions. Notably, SLC19A3 had a lower expression in primary tissues and was negatively correlated with EP2 receptor expression. Our novel finding revealed that EP2 receptor regulated metastasis through downregulation of SLC19A3. Thus, targeting EP2-SLC19A3 signaling is a potential therapeutic therapy for treating metastatic breast cancer.

Keywords: EP2 receptor; MMP-2; MMP-9; SLC19A3; breast cancer metastasis.

Figure 1

Effect of EP2 receptors inhibition on cell proliferation and invasion in MB-231 cells. A. Expression levels of EP1, EP2, EP3 and EP4 receptors were measured and quantified using RT-PCR after 1 week, 2 weeks, and 3 weeks post-injection of MB-231 cells. EP2 receptors were highest among the four receptors. B. Cell proliferation was determined by MTT assay. All experiments were performed in triplicate. C. Cell invasion potential was measured using Matrigel Invasion Chamber. D. Expression of MMP-2, MMP-9, and Cyclin D1 were quantified by real-time RT-PCR. **P<0.01 and ***P<0.001 are considered as statistically significance.

Figure 2

Involvement of EP receptors in breast cancer metastasis. A. Mice were injected with MB-231 and MB-231-EP2 clone in NOD-SCID mice. Tumor volume was measured every 7 days, and metastatic tumors were monitored using in vivo imaging system. B. Immunostaining of EP receptors were performed in primary tumor and metastatic tumor tissues. ***P<0.001 is considered as statistically significance.

Figure 3

Drug transporter gene expression profiling of control-siRNA and EP2-siRNA treated MB-231 cells.

Figure 5

A. Effect of EP2 siRNA or antagonist in EMT-related genes. B. Expression of SLC19A3 in human primary tissues. C. A negative correlation between EP2 receptor and SLC19A3 expression in paired primary tissues (n = 12). *P<0.05 is considered statistically significance.

Figure 4

Effect of SLC19A3 on cell proliferation and invasion in MB-231 cells. A. Cell proliferation was measured by MTT assay. B. Cell invasion potential was measured by MTT. C. Expressions of MMP-2 and MMP-9 were measured by real-time RT-PCR. *P<0.05 and **P<0.01 are considered statistically significance.

Figure 6

A schematic diagram showing the proposed mechanism of breast cancer metastasis induced by EP2 receptor. EP2 receptor regulates SLC19A3 expression, thereby activates MMPs and leads to the development of metastasis.