Chemically Induced Hypoxia Enhances miRNA Functions in Breast Cancer.

Emma Gervin,Mousumi Majumder,Bonita Shin,Sujit Maiti,Reid Opperman,Riley Feser,Mackenzie Cullen

doi:10.3390/cancers12082008

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.

Highlights

Breast cancer is the most common form of cancer, as well as the second leading cancer-related death among Canadian women [1]
We have shown that miR526b and miR655 overexpression in ER-positive breast cancer cell line MCF7 and an ER-negative human epidermal growth factor receptor 2 (HER2)-positive breast cancer cell line SKBR3 promotes epithelial-to-mesenchymal transition (EMT), cell migration, invasion, induction of stem-like cells (SLCs) phenotype, tumor growth, and metastasis in vivo [13,14]
We have previously shown that when we treat poorly metastatic breast cancer tumor cell line MCF7 and primary endothelial cell line human umbilical vein endothelial cells (HUVECs) with cell secretions from miR526b and miR655-overexpressing cells, there is an increase in reactive oxygen species (ROS), SO, and oxidative stress marker thioredoxin reductase 1 (TXNRD1) expression

Summary

Introduction

Breast cancer is the most common form of cancer, as well as the second leading cancer-related death among Canadian women [1]. One of the factors that contribute to the complexity of tumor growth, metastasis, and patient survival in breast cancer is the level of hypoxia (oxygen deficiency) within the tumor microenvironment [2]. Due to their rapid proliferation, cancer cells outgrow the available blood supply. This limits the delivery of oxygen and nutrients to the cells, making the center of the aggressively growing tumor largely hypoxic [2]. Cancerous cells secrete growth factors and stimulants that facilitate tumor-associated angiogenesis in the tumor microenvironment to deliver the required oxygen and nutrients to dividing tumor cells [3]

Results

Discussion

Conclusion