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.

The formation of new blood (angiogenesis) and lymphatic (lymphangiogenesis) vessels are major events associated with most epithelial malignancies, including breast cancer. Angiogenesis is essential for cancer cell survival. Lymphangiogenesis is critical in maintaining tumoral interstitial fluid balance and importing tumor-facilitatory immune cells. Both vascular routes also serve as conduits for cancer metastasis. Intratumoral hypoxia promotes both events by stimulating multiple angiogenic/lymphangiogenic growth factors. Studies on tumor-associated lymphangiogenesis and its exploitation for therapy have received less attention from the research community than those on angiogenesis. Inflammation is a key mediator of both processes, hijacked by many cancers by the aberrant expression of the inflammation-associated enzyme cyclo-oxygenase (COX)-2. In this review, we focus on breast cancer and showed that COX-2 is a major promoter of both events, primarily resulting from the activation of prostaglandin (PG) E receptor EP4 on tumor cells, tumor-infiltrating immune cells, and endothelial cells; and the induction of oncogenic microRNAs. The COX-2/EP4 pathway also promotes additional events in breast cancer progression, such as cancer cell migration, invasion, and the stimulation of stem-like cells. Based on a combination of studies using multiple breast cancer models, we show that EP4 antagonists hold a major promise in breast cancer therapy in combination with other modalities including immune check-point inhibitors.

Oxidative stress has a dual role in breast cancer, promoting growth at moderate levels while causing cell death at higher levels, such as during therapeutic interventions that increase reactive oxygen species production. Oncogenic microRNAs miR-526b and miR-655 promote aggressive cancer traits—such as proliferation, migration, invasion, hypoxia response, cancer stem cell properties, and metastasis—via COX-2/EP4/PI3K pathways. These miRNAs and oxidative stress appear to engage in a self-amplifying loop, where miRNA overexpression increases ROS levels, and moderate oxidative stress, in turn, enhances miRNA expression—although the mechanisms are not yet fully understood. This study investigates how overexpressing miR-526b, miR-655, and COX-2 influences breast cancer cell responses to oxidative stress induced by H₂O₂. We examined cell viability, DNA damage, and transcriptomic changes in MCF7, MCF7-miR526b, MCF7-miR655, and MCF7-COX2 cell lines. Overexpression of COX-2 provided the most significant protection against oxidative stress, decreasing apoptosis and promoting cell cycle progression. Cells with miR-526b and miR-655 exhibited distinct yet overlapping stress responses, including decreased expression of DNA damage markers and alterations in p53 signaling. RNA-sequencing and network analyses identified hub genes involved in redox balance, immune, and metabolic pathways, which may have clinical significance (OAS2, TNF, CACNA1C, CALML5). Overall, these findings suggest that miR-526b, miR-655, and COX-2 play novel roles in promoting resistance to oxidative stress through transcriptional reprogramming in breast cancer; the identified markers could serve as potential biomarkers or therapeutic targets.