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What a Shock: No Apoptosis without Heat Shock Protein 90α

Caption: Phospho-HSP90a (red) co-localizes with gamma-H2AX (green) in the apoptotic ring where it plays an essential role in the completion of programmed cell death.
Phospho-HSP90a (red) co-localizes with gamma-H2AX (green) in the apoptotic ring where it plays an essential role in the completion of programmed cell death.

Apoptosis, also known as programmed cell death, consists of a series of reactions designed to systematically chop up a cell and its contents. The process is used to eliminate specific cells during development or to remove old or damaged cells without harming any surrounding cells. Since cancer cells can develop mechanisms to avoid apoptosis, researchers may be able to identify new targets to combat cancer by better understanding the details of the apoptotic process.

One early hallmark of a cell undergoing apoptosis is the apoptotic ring, which is formed by phosphorylated H2AX proteins (γ-H2AX) at the edge of the nucleus. H2AX, a part of the histone complex around which nuclear DNA wraps for packaging into chromosomes, is phosphorylated by the enzyme DNA-PK. Heat shock protein (HSP) 90α, a protein with known ties to cancer, has recently been identified as a potential target of DNA-PK as well as a DNA-PK chaperone. These results suggested to Yves Pommier, M.D., Ph.D, chief of CCR’s Laboratory of Molecular Pharmacology, and colleagues that HSP90α might play a role in forming the apoptotic ring and in promoting apoptosis.

To see whether HSP90α is activated by apoptosis, the researchers treated a number of cell lines with apoptosis-inducing compounds including staurosporine, Fas ligand, and TRAIL. HSP90α phosphorylation increased in response to all three compounds but was highest following TRAIL treatment. Interestingly, phosphorylation of HSP90α occurred at the same rate as γ-H2AX formation after cells were exposed to TRAIL.

Using fluorescently labeled antibodies, the researchers found that, after TRAIL treatment, cells positive for phospho-HSP90α also showed γ-H2AX staining and that both proteins were localized to the nuclear periphery. In addition, cells that were positive for both phosphorylated HSP90α and γ-H2AX had DNA strand breaks, another well-described sign of apoptosis. Since TRAIL-mediated apoptosis requires the activation of caspases, a group of enzymes responsible for cutting up cellular components, the researchers treated the cells with a general caspase inhibitor prior to adding TRAIL. Blocking caspase activity prevented phosphorylation of HSP90α or H2AX, supporting the idea that both proteins are activated via the apoptotic response.

Pommier and colleagues then tested whether DNA-PK was the kinase primarily responsible for HSP90α activation in response to TRAIL. Treatment with a DNA-PK inhibitor blocked TRAIL-mediated phospho-HSP90α formation. Likewise, treating cells lacking DNA-PK protein with TRAIL prevented HSP90α phosphorylation. Activated DNA-PK and phospho-HSP90α also co-localized at the edge of the nucleus.

To determine whether HSP90α activity is essential for apoptosis, the researchers depleted cellular HSP90α using siRNA. Loss of HSP90α led to lower DNA-PK protein levels, reinforcing its role as a DNA-PK chaperone. HSP90α knockdown also inhibited several critical apoptotic processes in response to TRAIL: DNA-PK activation, γ-H2AX formation, DNA fragmentation, and apoptotic body formation. However, caspase 8 activity, which occurs before HSP90α phosphorylation in the apoptotic pathway, was not affected by decreased HSP90α levels.

In contrast to HSP90α siRNA, treating cells with an HSP90 inhibitor actually increased TRAIL-mediated apoptosis. The researchers suggest that this result may not be surprising because the inhibitor can cause DNA-PK and HSP90α to separate, which activates DNA-PK.

Taken together, these studies suggest that activation of HSP90α is an important step in the apoptotic pathway and that phospho-HSP90α may serve as a biomarker for cells undergoing apoptosis. Similarly, HSP90 inhibitors, which are already being tested in clinical trials, may act synergistically with apoptosis-inducing drugs to help eliminate otherwise resistant cancer cells.

Solier S, Kohn KW, Scroggins B, Xu W, Trepel J, Neckers L, Pommier Y. Heat shock protein 90α (HSP90α), a substrate and chaperone of DNA-PK necessary for the apoptotic response. PNAS. Published online July 2, 2012. PubMed Link