By Jamshed Arslan Pharm.D.
Triple-negative breast cancer (TNBC) is difficult to treat because it does not express the receptors (estrogen, progesterone, and HER2) against which effective therapies are available. TNBC defeats the body's regulation on unchecked growth, but its winning strategy remained unclear until recently. A research team, led by Dr. William Kaelin at the Dana-Farber Cancer Institute in Boston, recently discovered that TNBC cells release glutamate to disable a cancer-stunting enzyme, EglN1. This enzyme targets the transcription factor hypoxia-inducible factor (HIF-1α) for degradation under normal oxygen conditions. HIF-1α enables cells to adapt to low oxygen conditions by promoting genes involved in glycolysis, angiogenesis, and energy conservation. The team found that TNBC cells stabilize HIF-1α for survival by releasing glutamate.
The initial clue of this novel survival mechanism came when immunohistochemical analysis revealed an increase in HIF-1α in breast cancer tissue microarrays. Immunoblots and real-time PCR showed that enhanced HIF-1α protein levels correlated with increased expression of HIF-1α-responsive genes. Interestingly, accumulation of HIF-1α was not preceded by an increase in HIF-1α mRNAs, indicating that a post-translational mechanism regulates HIF-1α protein expression. However, levels of the enzyme (EglN1) responsible for the post-translational modification, prolyl-hydroxylation, which degrades HIF-1α, were not reduced in TNBC cells, suggesting the presence of some factor that stabilizes HIF-1α.
Immunohistochemistry: ER alpha/NR3A1 Antibody (CL1196) [NBP2-34478] - Staining of human breast cancer shows moderate to strong nuclear positivity in a subset of tumor cells.
Some of the crucial properties of the unknown factor included a size of less than 3 kDa, and an insensitivity to boiling, RNases, DNase, and proteinase K treatments. Among all the potential candidates revealed by the NMR spectroscopy analysis of HPLC fractions with the highest HIF-inducing activity, only L-glutamate fit the bill. Other potential molecules did not meet the criteria: in contrast to L-glutamate, the addition of D-glutamate or L-glutamine to fresh media did not induce HIF-1α in TNBC cells. Pharmacological inhibition and RNA interference experiments further showed that release of L-glutamate from TNBC cells was mediated by the xCT cystine-glutamate antiporter. As the name suggests, this antiporter is responsible for both the uptake of cystine (oxidized dimer of amino acid cysteine) and the extracellular release of glutamate.
Researchers found that abundance of extracellular glutamate inactivates the antiporter, interrupting the uptake of cystine and decreasing the intracellular cysteine. In vitro assays showed that EglN1 activity correlates with the levels of cysteine. Thus, reduced intracellular cysteine inactivates EglN1, resulting in downregulation of HIF-1α-degradation. All in all, when EglN1 activity is low, there is nothing much cells can do to stop HIF-1α from promoting tumor growth.
Kaelin's team is the first one to indicate that TNBC's victory over cellular anti-cancerous mechanisms is dependent, at least partly, on a single molecule: glutamate. They can also pride themselves in being the first to discover that EglN1 senses cysteine. They found that under oxidizing conditions and when intracellular cysteine is low, EglN1 undergoes inactivation through oxidation of its own cysteine residues. Their research also provides at least two viable pharmacotherapy strategies against TNBCs: antagonism of extracellular L-glutamate and inhibition of L-glutamate release from cancer cells. Blocking the self-serving glutamate from TNBCs can hopefully provide an effective strategy against this refractory disease.
Explore Hypoxia Signaling Targets
Jamshed Arslan, Pharm D.
University of Alabama at Birmingham, School of Medicine
Dr. Arslan studies cell signaling in mitochondrial defects in C. elegans and transgenic mice.
References
Briggs, Kimberly J., et al. "Paracrine Induction of HIF by Glutamate in Breast Cancer: EglN1 Senses Cysteine." Cell, vol. 166, no. 1, 2016, pp. 126–139. doi: 10.1016/j.cell.2016.05.042.
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