Identification of a stem-like cell population by exposing metastatic breast cancer cell lines to repetitive cycles of hypoxia and reoxygenation

Abstract Palbociclib, a CDK4/6 inhibitor, has been granted accelerated approval by US FDA for hormone receptor-positive HER2-negative metastatic breast cancer. To determine potential biomarkers of palbociclib sensitivity to assist in patient selection and clinical development, we investigated the effects of palbociclib in a panel of molecularly characterized breast cancer cell lines. We quantified palbociclib sensitivity and c-myc expression in 11 breast cancer cell lines, 124 breast cancer samples, and The Cancer Genome Atlas database. We found non-TNBC subtypes were more sensitive to palbociclib than TNBC. Activation of c-myc led to differential palbociclib sensitivities, and further inhibition of c-myc enhanced palbociclib sensitivity. Moreover, we identified for the first time a c-myc/miR-29b-3p/CDK6 axis in breast cancer that could be responsible for c-myc-induced palbociclib insensitivity, in which c-myc activation resulted in downregulation of miR-29b-3p, further activated CDK6 and inhibited cell-cycle arrest at G1 phase. Moreover, downregulated (inactived) c-myc-induced oncogenic addiction could increase palbociclib efficacy, using both Xenograft model and patient-derived tumor xenograft (PDTX) model. Our finding extends the concept of combined blockade of the CDK4/6 and c-myc signaling pathways to increase palbociclib sensitivity, making c-myc a promising biomarker for palbociclib sensitivity in breast cancer.

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Chemically Induced Hypoxia Enhances miRNA Functions in Breast Cancer

Cancers ◽

10.3390/cancers12082008 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2008 ◽

Cited By ~ 1

Author(s):

Emma Gervin ◽

Bonita Shin ◽

Reid Opperman ◽

Mackenzie Cullen ◽

Riley Feser ◽

...

Keyword(s):

Breast Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Metastatic Breast ◽

Breast Tumors ◽

Cancer Cell Lines ◽

Breast Cancer Cell Lines ◽

Mcf7 Cells ◽

Cox 2

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.

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