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    Awarded Grants
    Crosstalk Between Extrinsic and Intrinsic Caspase Activation Pathways in Doxorubicin-Mediated Sensitization of Breast Cancer Cells to TRAIL-Induced Apoptosis

    Scientific Abstract:
    Apoptosis is a form of programmed cell death that is carried out through the concerted action of the caspases, which are a family of cysteine proteases. Caspase activation is classified as occurring via either the extrinsic (death receptor) or intrinsic (mitochondrial) pathway. The death receptor agonist TRAIL holds promise as a cancer therapeutic, as it was shown to cause tumor regression without systemic toxicity in experimental animal models of cancer. However, some breast cancer cells fail to respond to TRAIL. Doxorubicin, a widely-used chemotherapeutic agent, has been reported to sensitize certain TRAIL-resistant breast cancer cells to TRAIL-induced death at clinically relevant concentrations. It is currently unknown what caspase activation events underlie this sensitization. We hypothesize that chemotherapeutic-induced sensitization of breast cancer tumors to TRAIL involves cross-talk between the extrinsic and intrinsic caspase activation pathways. A major hurdle in the understanding of caspase activation events is the lack of specific assays for the individual caspases. Activity based chemical probes hold great promise for the delineation of these caspase activation events. We propose to utilize this technology to complete these specific aims: 1)Develop an affinity labeling protocol to allow for the purification and identification of activated caspases from apoptotic cells; 2)Synthesize caspase-activated fluorescent imaging agents to allow visualization of specific caspase activation events in vivo; 3)Delineate caspase activation events in doxorubicin-induced sensitization of breast cancer cell lines to apoptosis induced by TRAIL. In Aim 1 I will optimize methodologies to purify all activated caspases from apoptotic cells using biotinylated pancaspase inhibitors (both novel and commercially available). Purified caspases will then be identified by immunoaffinity techniques. In Aim 2 probes will be synthesized to emit a fluorescent signal following interaction with a specific caspase. In Aim 3, I will apply these tools to characterize caspase activation in MCF10A and MDA-MB-453 cells in response to TRAIL alone and in combination with doxorubicin. MCF10A is sensitive to TRAIL alone, whereas MDA-MB-453 requires TRAIL in combination with doxorubicin. This research will firmly establish which caspases are involved in doxorubicin-mediated sensitization of these breast cancer cell lines to TRAIL. Probes and techniques developed in this study can be used for the delineation of caspase activation events in other models of breast cancer.

    Lay Abstract:
    Apoptosis is a form of programmed cell death, and ways to specifically trigger apoptosis of breast cancer cells are of great interest in the search for breast cancer therapy. The caspases are a family of proteases, and their activity is essential for the death of the cell during apoptosis. Caspase activation is classified as occurring via either the extrinsic (death receptor) or intrinsic (mitochondrial) pathway. Molecules that trigger caspase activation via the extrinsic apoptotic pathway are promising targets for breast cancer therapy. One such molecule is TRAIL, as it was shown to cause tumor regression without systemic toxicity in experimental animal models of cancer. Unfortunately some breast cancer cells fail to respond to TRAIL. Therefore, ways to sensitize these cells to TRAIL are currently being investigated. Doxorubicin, a widely-used chemotherapeutic agent, has been reported to sensitize certain TRAIL-resistant breast cancer cells to TRAIL-induced death at clinically relevant concentrations. However, it is currently unknown what caspases are activated during this process. Our hypothesis is that chemotherapeutic-induced sensitization of breast cancer tumors to TRAIL involves cross-talk between the extrinsic and intrinsic caspase activation pathways. Our lab is experienced in synthesizing small molecules that function as activity based probes (ABPs) for proteases. These compounds only interact with the active form of the protease. We propose to develop ABPs to determine which caspases are activated during doxorubicin-induced sensitization to TRAIL. In our first aim we will produce ABPs containing a tag for purification. We will use these ABPs to purify and identify all activated caspases from apoptotic cells. In our second aim, we will synthesize ABPs containing a tag that will emit a fluorescent signal following interaction with a specific caspase. These ABPs will allow us to visualize distinct caspase activation events within the cell. In our third aim, we will utilize both sets of tools to understand which caspases are activated, and at what time this activation occurs, in breast cancer cells following treatment with doxorubicin and TRAIL versus treatment with TRAIL alone. This research will firmly establish which caspases are involved in doxorubicin-mediated sensitization of breast cancer cell lines to TRAIL. This type of information is critical for the design of rational treatments for breast cancer to maximize effectiveness, while minimizing harmful side-effects.