Overexpression of CD44 Standard Isoform Upregulates HIF-1α Signaling in Hypoxic Breast Cancer Cells

Cluster of differentiation 44 (CD44), a cell surface receptor for hyaluronic acid (HA), is involved in aggressive cancer phenotypes. Herein, we investigated the role of the CD44 standard isoform (CD44s) in hypoxia-inducible factor-1α (HIF-1α) regulation using MCF7 overexpressing CD44s (pCD44s-MCF7). When pCD44s-MCF7 was incubated under hypoxia, levels of HIF-1α, vascular endothelial growth factor, and the HIF-1α response element-derived luciferase activity were significantly increased compared to those in the control MCF7. Incubation of pCD44s-MCF7 cells with HA further increased HIF-1α accumulation, and the silencing of CD44s attenuated HIF-1α elevation, which verifies the role of CD44s in HIF-1α regulation. In addition, the levels of phosphorylated extracellular signal-regulated kinase (ERK) was higher in hypoxic pCD44s-MCF7 cells, and HIF-1α accumulation was diminished by the pharmacological inhibitors of ERK. CD44s-mediated HIF-1α augmentation resulted in two functional outcomes. First, pCD44s-MCF7 cells showed facilitated cell motility under hypoxia via the upregulation of proteins associated with epithelial-mesenchymal transition, such as SNAIL1 and ZEB1. Second, pCD44s-MCF7 cells exhibited higher levels of glycolytic proteins, such as glucose transporter-1, and produced higher levels of lactate under hypoxa. As a consequence of the enhanced glycolytic adaptation to hypoxia, pCD44s-MCF7 cells exhibited a higher rate of cell survival under hypoxia than that of the control MCF7, and glucose deprivation abolished these differential responses of the two cell lines. Taken together, these results suggest that CD44s activates hypoxia-inducible HIF-1α signaling via ERK pathway, and the CD44s-ERK-HIF-1α pathway is involved in facilitated cancer cell viability and motility under hypoxic conditions.

Involvement of NRF2 Signaling in Doxorubicin Resistance of Cancer Stem Cell-Enriched Colonospheres

Cancer stem cells (CSCs) are a subset of tumor cells, which are characterized by resistance against chemotherapy and environmental stress, and are known to cause tumor relapse after therapy. A number of molecular mechanisms underlie the chemoresistance of CSCs, including high expression levels of drug efflux transporters. We investigated the role of the antioxidant transcription factor NF-E2-related factor 2 (NRF2) in chemoresistance development, using a CSC-enriched colonosphere system. HCT116 colonospheres were more resistant to doxorubicin-induced cell death and expressed higher levels of drug efflux transporters such as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) compared to HCT116 monolayers. Notably, levels of NRF2 and expression of its target genes were substantially elevated in colonospheres, and these increases were linked to doxorubicin resistance. When NRF2 expression was silenced in colonospheres, Pgp and BCRP expression was downregulated, and doxorubicin resistance was diminished. Collectively, these results indicate that NRF2 activation contributes to chemoresistance acquisition in CSC-enriched colonospheres through the upregulation of drug efflux transporters.