Chemically Induced Hypoxia Enhances miRNA Functions in Breast Cancer

Abstract

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.

EP4 as a Therapeutic Target to Impede Breast Cancer-associated Angiogenesis and Lymphangiogenesis

“…In addition, potential tumor markers for aggressive forms of cancer may help early diagnosis and therapeutic monitoring. As discussed in this review, COX-2 induced miRNAs miR655 and miR526b enhances tumor associated angiogenesis and lymphangiogenesis [35,57] and can serve as biomarkers for breast cancer. In conclusion, the evidence highlighted in this review provides a rationale for the pursuit of EP4 antagonists in combination therapies to treat TNBC and other cancers that display high levels of angiogenesis and lymphangiogenesis..…”

“…Angiogenesis is an essential physiological process; however, TAA is one of the main hallmarks of cancer progression [55]. In growing tumors, hypoxia is a major mechanism for upregulation of VEGFs, leading to angiogenesis [56,57]. Cancer can lead to abnormal development of blood vessels to promote metastasis.…”

“…PTEN is a tumor suppressor gene that inhibits the action of the HIF1-α [122,123], which, when expressed, can lead to the expression of angiogenic factors, such as VEGF [124]. In both miR526b and miR655 expressing MCF-7 cell lines, expression of PTEN is reduced [35] and both miRNA regulates the expression of HIF1-α in breast cancer [57]. In a more recent publication, the expression of HIF1-α was compared among different breast cancer cell lines that expressed miR526b or miR655 [57].…”

“…In both miR526b and miR655 expressing MCF-7 cell lines, expression of PTEN is reduced [35] and both miRNA regulates the expression of HIF1-α in breast cancer [57]. In a more recent publication, the expression of HIF1-α was compared among different breast cancer cell lines that expressed miR526b or miR655 [57]. Higher production of this angiogenic transcription factor was observed in cells that expressed these miRNAs when they were put under hypoxic conditions induced by cobalt chloride.…”

Hypoxia-Induced Non-Coding RNAs Controlling Cell Viability in Cancer

“…Recently, in silico bioinformatics analysis showed that hypoxia-upregulates miR526b/miR655 stabilize HIF proteins targeting the transcription factors NR2C2, SALL4, and ZNF207 that, in turn, regulate PTEN (a negative regulator of HIF-1α) and NFκB1 (positive regulator of COX-2 and EP4) [ 128 ].…”