Oncogenes and Tumor Suppressors COX-2 Elevates Oncogenic miR-526b in Breast Cancer by EP4 Activation

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 inmiR-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 ofmiR-526b in poorly metastatic MCF7and SKBR3 cell lines resulted in increased cellularmigration, invasion, EMTphenotypeandenhanced 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 downregulatedmiR-526b and its functions in vitro. Finally,miR-526b expressionwas 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 aCOX-2 upregulated, oncogenicmiRNApromoting SLCs, the expression of which follows EP4 receptor-mediated signaling, and is a promising biomarker for monitoring and personalizing breast cancer therapy. Mol Cancer Res; 13(6); 1022–33. 2015 AACR.