Abstract
The glycolytic response of hypoxic cells is primarily mediated by the hypoxia inducible factor alpha (HIF-1α) but even in the presence of abundant oxygen tumours typically show high rates of glycolysis. Higher levels of HIF-1α in tumours are associated with a poorer prognosis and up-regulation of markers of epithelial mesenchymal transition (EMT) due to HIF-1α actions. We have recently shown that EMT occurs within the CD44high cancer stem cell (CSC) fraction and that epithelial and EMT CSCs are distinguished by high and low ESA expression, respectively. We here show that hypoxia induces a marked shift of the CSC fraction towards EMT leading to altered cell morphology, an increased proportion of CD44high/ESAlow cells, patterns of gene expression typical of EMT, and enhanced sphere-forming ability. The size of EMT fractions returned to control levels in normoxia indicating a reversible process. Surprisingly, however, even under normoxic conditions a fraction of EMT CSCs was present and maintained high levels of HIF-1α, apparently due to actions of cytokines such as TNFα. Functionally, this EMT CSC fraction showed decreased mitochondrial mass and membrane potential, consumed far less oxygen per cell, and produced markedly reduced levels of reactive oxygen species (ROS). These differences in the patterns of oxygen metabolism of sub-fractions of tumour cells provide an explanation for the general therapeutic resistance of CSCs and for the even greater resistance of EMT CSCs. They also identify potential mechanisms for manipulation of CSCs.
Highlights
Tumours are highly glycolytic even in the presence of abundant oxygen, the so-called ‘‘Warburg effect’’ [1,2]
We show that low oxygen levels reversibly increase the size of epithelial to mesenchymal transition (EMT) fractions within HNSCC cell lines and that, compared with epithelial cancer stem cell (CSC) (Epi CSC), EMT CSCs have higher levels of the hypoxic response protein Hypoxia inducible factor 1 alpha (HIF-1a), even under normoxic conditions
To determine the reversibility of EMT upon removal from hypoxia, cells cultured under 0.2% oxygen for 21 days were returned to normoxic conditions and assessed by flow cytometry at 7 day intervals
Summary
Tumours are highly glycolytic even in the presence of abundant oxygen, the so-called ‘‘Warburg effect’’ [1,2]. Tumour invasion and metastasis have become increasingly associated with cancer stem cells (CSCs), a sub-set of cancer cells that is capable of self-renewal, has tumour-initiating ability, and is resistant to therapy [12,13]. Both local tumour invasion and metastasis to distant sites require migratory abilities acquired through epithelial to mesenchymal transition (EMT) of CSCs [14] during which epithelial characteristics are lost and epithelial proteins such as E-cadherin are down-regulated and of mesenchymal proteins such as Vimentin and Twist up-regulated [15]. Induction of EMT in breast cell lines results in cells acquiring the marker phenotype typical of breast CSCs, greater motility, and resistance to therapeutic agents [16,17]
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