MicroRNAs (miRs) are small regulatory molecules emerging as potential biomarkers in cancer. Previously, it was shown that COX-2 expression promotes breast cancer progression via multiple mechanisms, including induction of stem-like cells (SLC), owing to activation of the prostaglandin E2 receptor EP4 (PTGER4). COX-2 overexpression also upregulated microRNA526b (miR-526b), in association with aggressive phenotype. Here, the functional roles of miR-526b in breast cancer and the mechanistic role of EP4 signaling in miR-526b upregulation were examined. A positive correlation was noted between miR-526b and COX-2 mRNA expression in COX-2 disparate breast cancer cell lines. Stable overexpression of miR-526b in poorly metastatic MCF7 and SKBR3 cell lines resulted in increased cellular migration, invasion, EMT phenotype and enhanced tumorsphere formation in vitro, and lung colony formation in vivo in immunodeficient mice. Conversely, knockdown of miR-526b in aggressive MCF7-COX-2 and SKBR3-COX-2 cells reduced oncogenic functions and reversed the EMT phenotype, in vitro. Furthermore, it was determined that miR-526b expression is dependent on EP4 receptor activity and downstream PI3K-AKT and cyclic AMP (cAMP) signaling pathways. PI3K-AKT inhibitors blocked EP4 agonist-mediated miR526b upregulation and tumorsphere formation in MCF7 and SKBR3 cells. NF-kB inhibitor abrogates EP agonist-stimulated miRNA expression in MCF7 and T47D cells, indicating that the NF-kB pathway is also involved in miR-526b regulation. In addition, inhibition of COX-2, EP4, PI3K, and PKA in COX-2-overexpressing cells downregulated miR-526b and its functions in vitro. Finally, miR-526b expression was significantly higher in cancerous than in noncancerous breast tissues and associated with reduced patient survival. In conclusion, miR-526b promotes breast cancer progression, SLC-phenotype through EP4-mediated signaling, and correlates with breast cancer patient survival.Implications: This study presents novel findings that miRNA 526b is a COX-2 upregulated, oncogenic miRNA promoting SLCs, the expression of which follows EP4 receptor-mediated signaling, and is a promising biomarker for monitoring and personalizing breast cancer therapy.

Background: A tumor cell subset known as stem like cells (SLC) defy conventional therapies, requiring identification of SLC-specific therapeutic targets. We established that elevated COX-2 expression by breast cancer cells promotes tumor progression via inactivation of host immune cells, enhancement of cancer cell migration and invasiveness, and promotion of tumor-associated angiogenesis and lymphangiogenesis primarily by activation of the PGE-2 receptor EP4 on tumor and host cells. Cardiovascular side effects of long term use of COX-2 inhibitors argued for an alternative target, potentially EP4. Objectives/approaches: We asked whether COX-2 or EP4 additionally stimulates SLC in breast cancer. COX-2 and HER-2, often co-expressed in human breast cancer, are both major determinants of cancer progression. To define the roles of COX-2 in HER2 +ve and -ve cells, COX-2 cDNA was stably transfected into COX-2-low, HER-2-ve, ER+ve, non-metastatic MCF-7 and COX-2-ve, ER-ve, HER-2-high SKBR-3 human breast cancer cell lines to derive MCF-7-COX-2 and SKBR-3-COX-2 cells which were tested for changes in phenotype and functions in vitro and in vivo. Results: Both COX-2 over-expressing lines showed (1) EMT (low E-Cadherin and high Vimentin, Twist and N-Cadherin), (2) higher proliferation/ migration/ invasion, (3) upregulation of EP4 and angiogenic/ lymphangiogenic factors VEGF-A/C/D, (4) markedly increased SLC contents indicated by clonogenic spheroid formation for successive generations in vitro, increased ALDH activity and co-expression of COX-2 and multiple SLC markers in spheroids. (5) COX-2 expression markedly increased during spheroid formation in COX-2 disparate cell lines, indicating a selection of COX-2 expressing cells. (6) EP4 activation of COX-2 low cell lines with PGE2 or EP4 agonists markedly increased spheroid-forming efficiency. (7) Functional changes listed above (2-5) were blocked with COX-2 inhibitors or an EP4 antagonist (EP4A), indicating dependence on COX-2 and EP4 activity. (8) MCF-7-COX-2 cells showed dramatically increased lung colony forming ability and growth at 4-6 wks in NOD/SCID/GUSB null mice (identified by GUSB staining), which were blocked with EP4A treatment or EP4 knockdown; (9) MCF-7-COX-2 (as low as 5000) cells showed increased orthotopic tumorigenicity in NOD/SCID/IL-2Rγ deficient mice on transplantation for successive generations. (10) Finally, expression of COX-2 and EP4 were positively correlated with the SLC marker ALDH in human breast cancer tissues, and associated with poor patient survival. Conclusion/ Significance: EP4 represents a novel therapeutic target to eradicate SLCs in breast cancer. EP4 antagonists may spare cardiovascular side effects of COX-2 inhibitors, primarily attributed to cardio protective effects of PGI2. (Supported by the CBCF, Ontario Chapter and OICR grants to PKL, TBCRU and CIHR-STP fellowships to MM, CBCF fellowship to EL and a gift of EP4A, RQ-15986 by RaQualia Pharma). Citation Format: Mousumi Majumder, Xiping Xin, Ling Liu, Gillian Bell, Erin Landman, Mauricio Rodriguez- Torres, Lynne-Marie Postovit, David Hess, Peeyush K. Lala. Stem like cells in human breast cancer: EP4 as a therapeutic target. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3905. doi:10.1158/1538-7445.AM2014-3905

<p>Supplementary Figure 2. Representative images for migrant MCF7 and SKBR3 miR526b over expressed cells</p>

<p>Supplementary Figure Legends. Description of supplementary Figures 1-4</p>

<p>Supplementary Figure 1. Validation of Transfection/COX-2 mRNA in breast cancer cell lines/ miR-526b expression levels in a panel of breast cancer cell lines</p>