Bonita Shin

In aggressively growing tumors, hypoxia induces HIF-1α expression promoting angiogenesis. Previously, we have shown that overexpression of oncogenic microRNAs (miRNAs, miRs) miR526b/miR655 in poorly metastatic breast cancer cell lines promotes aggressive cancer phenotypes in vitro and in vivo. Additionally, miR526b/miR655 expression is significantly higher in human breast tumors, and high miR526b/miR655 expression is associated with poor prognosis. However, the roles of miR526b/miR655 in hypoxia are unknown. To test the relationship between miR526b/miR655 and hypoxia, we used various in vitro, in silico, and in situ assays. In normoxia, miRNA-high aggressive breast cancer cell lines show higher HIF-1α expression than miRNA-low poorly metastatic breast cancer cell lines. To test direct involvement of miR526b/miR655 in hypoxia, we analyzed miRNA-high cell lines (MCF7-miR526b, MCF7-miR655, MCF7-COX2, and SKBR3-miR526b) compared to controls (MCF7 and SKBR3). CoCl₂-induced hypoxia in breast cancer further promotes HIF-1α mRNA and protein expression while reducing VHL expression (a negative HIF-1α regulator), especially in miRNA-high cell lines. Hypoxia enhances oxidative stress, epithelial to mesenchymal transition, cell migration, and vascular mimicry more prominently in MCF7-miR526b/MCF7-miR655 cell lines compared to MCF7 cells. Hypoxia promotes inflammatory and angiogenesis marker (COX-2, EP4, NFκB1, VEGFA) expression in all miRNA-high cells. Hypoxia upregulates miR526b/miR655 expression in MCF7 cells, thus observed enhancement of hypoxia-induced functions in MCF7 could be attributed to miR526b/miR655 upregulation. In silico bioinformatics analysis shows miR526b/miR655 regulate PTEN (a negative regulator of HIF-1α) and NFκB1 (positive regulator of COX-2 and EP4) expression by downregulation of transcription factors NR2C2, SALL4, and ZNF207. Hypoxia-enhanced functions in miRNA-high cells are inhibited by COX-2 inhibitor (Celecoxib), EP4 antagonist (ONO-AE3-208), and irreversible PI3K/Akt inhibitor (Wortmannin). This establishes that hypoxia enhances miRNA functions following the COX-2/EP4/PI3K/Akt pathways and this pathway can serve as a therapeutic target to abrogate hypoxia and miRNA induced functions in breast cancer. In situ, HIF-1α expression is significantly higher in human breast tumors (n = 96) compared to non-cancerous control tissues (n = 20) and is positively correlated with miR526b/miR655 expression. In stratified tumor samples, HIF-1α expression was significantly higher in ER-positive, PR-positive, and HER2-negative breast tumors. Data extracted from the TCGA database also show a strong correlation between HIF-1α and miRNA-cluster expression in breast tumors. This study, for the first time, establishes the dynamic roles of miR526b/miR655 in hypoxia.

In eukaryotes, overproduction of reactive oxygen species (ROS) causes oxidative stress, which contributes to chronic inflammation and cancer. MicroRNAs (miRNAs) are small, endogenously produced RNAs that play a major role in cancer progression. We established that overexpression of miR526b/miR655 promotes aggressive breast cancer phenotypes. Here, we investigated the roles of miR526b/miR655 in oxidative stress in breast cancer using in vitro and in silico assays. miRNA-overexpression in MCF7 cells directly enhances ROS and superoxide (SO) production, detected with fluorescence assays. We found that cell-free conditioned media contain extracellular miR526b/miR655 and treatment with these miRNA-conditioned media causes overproduction of ROS/SO in MCF7 and primary cells (HUVECs). Thioredoxin Reductase 1 (TXNRD1) is an oxidoreductase that maintains ROS/SO concentration. Overexpression of TXNRD1 is associated with breast cancer progression. We observed that miR526b/miR655 overexpression upregulates TXNRD1 expression in MCF7 cells, and treatment with miRNA-conditioned media upregulates TXNRD1 in both MCF7 and HUVECs. Bioinformatic analysis identifies two negative regulators of TXNRD1, TCF21 and PBRM1, as direct targets of miR526b/miR655. We validated that TCF21 and PBRM1 were significantly downregulated with miRNA upregulation, establishing a link between miR526b/miR655 and TXNRD1. Finally, treatments with oxidative stress inducers such as H₂O₂ or miRNA-conditioned media showed an upregulation of miR526b/miR655 expression in MCF7 cells, indicating that oxidative stress also induces miRNA overexpression. This study establishes the dynamic functions of miR526b/miR655 in oxidative stress induction in breast cancer.