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Imaging the Efficacy of Heat Shock Protein 90 Inhibitors in Human Breast Cancers
Background: The heat shock protein 90 (Hsp90) chaperone system is highly upregulated in human breast cancer cells and the interactions between Hsp90 and its co-chaperone p23 is crucial for efficient protein folding. Hsp90 is a novel chemotherapy target since some of its client proteins are over-expressed, and/or crucial for breast cancer progression and resistance to chemotherapy. Different small molecule Hsp90 inhibitors have been developed to inhibit Hsp90/p23 interactions, and lead to misfolding and subsequent degradation of Hsp90 client proteins and simultaneous inhibition of multiple signal transduction pathways pertaining to breast cancer. However, the nature of Hsp90/p23 interactions and the efficacies of the Hsp90 inhibitors in disrupting these interactions in the native breast tumor microenvironment have not been well characterized. We have developed and validated a split Renilla luciferase (RL) protein-fragment-assisted complementation (SRL-PFAC) technology for non-invasive monitoring of protein-protein interactions by bioluminescence imaging, both in cell culture and in living mice. We will now study Hsp90/p23 interactions using SRL-PFAC. Hypothesis: SRL-PFAC technology can be used to monitor Hsp90/p23 interactions and evaluate the efficacies of different classes of small molecule Hsp90 inhibitors in disrupting these interactions in breast cancer cells in culture and in small living subjects. Specific Aims : Aim1 : We will test the hypothesis that SRL-PFAC can be used to monitor the Hsp90/p23 interaction in breast cancer cells in their native tumor environment. Aim 2 : We will test the hypothesis that Hsp90/p23 split reporters developed in specific aim 1 can be used to evaluate the efficacy of geldanamycin-based Hsp90 inhibitors in disrupting Hsp90/p23 interaction. Aim 3 : We will test the hypothesis that Hsp90/p23 split reporters developed in specific aims 1 and 2 can be used to evaluate the efficacy of other classes of Hsp90 inhibitors that are currently undergoing pre-clinical evaluation. Study Design: SRL-PFAC reporter constructs expressing Hsp90 and p23 will be stably transfected into breast cancer cells and the efficacy of different classes of Hsp90 inhibitors in disruption of Hsp90/p23 interaction will be determined by bioluminescence imaging in cell culture and murine studies. The specificity of interactions will be determined by non-interacting protein partners and mutants controls and confirmed by co-immunoprecipitation western blotting. Potential outcome and benefits : The ability to monitor Hsp90/p23 interactions in intact breast cancer cells in their native tumor microenvironment is crucial for validating mechanisms of existing Hsp90 inhibitors as well as pre-clinical testing of novel Hsp90 inhibitors. This is because: (1) The effects of drug on target can be directly studied, (2) in vivo pharmacokinetics and delivery can be directly optimized, (3) repeated monitoring of efficacy can be accomplished with molecular imaging and (4) individual variations can be studied much better because each mouse serves as its own control. This will ultimately lead to significant improvement in the efficacy in new generation of drugs for cancer treatment including those that target the Hsp90/p23 system as well as other protein-protein interactions.
Breast cancer is the most common cancer diagnosed in women and is the leading cause of cancer death in women ages 20-59. In breast cancer, the heat shock protein 90 (Hsp90) is expressed at a higher level compared to that of normal breast cells. In order to function properly, each protein inside the cells needs to be folded into the right shape. Interactions between Hsp90 and another protein called p23 are essential for controlling the folding of proteins that are important for growth, spread to other parts of the body and in the failure of chemotherapy treatment in breast cancer. Different drugs (Hsp90 inhibitors) have been developed to target the Hsp90 machinery, and two of these drugs are now being tested in human breast cancer clinical trials by the National Cancer Institute. These Hsp90 inhibitors prevent Hsp90 from working with p23 in protein folding. As a result, other proteins that the breast cancer cells need are destroyed and they stop growing. However, the details of protein folding by Hsp90 with p23, and the way by which Hsp90 inhibitors kill breast cancer cells are not completely understood. Traditionally, the methods for studying how chemotherapy affects protein folding in breast cancer cells requires euthanizing animals during different stages of treatment and collecting their tumors prior to analyses. This process can be very time consuming and may not accurately reflect what really happens in the breast tumor environment. To overcome these limitations, our laboratory has developed novel technologies to study how proteins work with each other in intact tumor cells within living animals. Using special cameras, we have been able to ‘see’ how chemotherapy affects protein interactions in cancer cells in the same animal at different time points. In this study, we are proposing to investigate how Hsp90 interacts with p23, and how the Hsp90 inhibitors affect this interaction in the natural breast tumor environment. We want to validate the Hsp90 inhibitors that are already in clinical trials and test new ones that are being developed. We will also compare our methods with other current techniques and perform control studies. Ultimately, we hope to develop new Hsp90 inhibitors that will significantly improve breast cancer therapy and quality of life in breast cancer patients.