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    Awarded Grants
    Fibroblast Activation Protein-Alpha Activation of a Bacterial Toxin as Targeted “Anti-Stromal” Therapy for Breast Cancer

    Scientific Abstract:
    PROPOSAL TITLE: Fibroblast Activation Protein-Alpha activation of a bacterial toxin as targeted “anti-stromal” therapy for breast cancer BACKGROUND: Stromal cell production of signaling molecules is involved in neoplastic transformation and can directly regulate growth and survival of established breast cancer cells. Stromal cells also contribute to the production of angiogenic factors that are required for increased neovascularization. Inhibitors of stromal derived growth factors may be effective therapy for breast cancer due to their ability to disrupt cross talk between the stromal compartment and breast cancer cells. A second strategy to disrupt such crosstalk would be to develop therapies that selectively kill stromal cells within breast cancer sites. In order to develop such targeted therapy we will take advantage of the selective expression of fibroblast activation protein alpha (FAP-alpha) by reactive stromal cells present within sites of epithelial cancers. FAP-alpha has been demonstrated to be overexpressed in the stromal compartment of a majority of breast cancers but is not expressed within stroma of normal breast or in stroma of any other adult tissues. FAP-alpha is a membrane bound serine protease with dipeptidyl peptidase, gelatinase and collagenase enzymatic activities. In previous studies, we have defined substrates for the proteolytic activity of prostate cancer proteases using a variety of techniques that include screening of large combinatorial peptide libraries. Peptide substrates for these proteases have been used to produce small molecule prodrugs and bacterial protoxins that are selectively activated by these prostate-tissue specific proteases. For example, we have modified a potent bacterial toxin, proaerolysin, to produce a toxin that is selectively activated by PSA and is selectively cytotoxic to PSA-producing tumors in vitro and in vivo. OBJECTIVE: The objective of this proposal is to determine if selective elimination of breast tumor associated stromal cells would be effective as targeted treatment for breast cancer. SPECIFIC AIMS: The specific aims of the study are: (1) to produce enzymatically active FAP-alpha; (2) to to identify substrates for the proteolytic activity of FAP-alpha; (3) to introduce the FAP-alpha specific peptide substrate sequence into the activation domain of the proaerolysin toxin; (4) to characterize the effect of FAP-alpha activated proaerolysin “anti-stromal” therapy on growth of human breast cancer xenografts. STUDY DESIGN: FAP-alpha is a membrane bound protease. To produce large amounts of active FAP-alpha we will use PCR-based approach to produce a modified gene that encodes for protein that lacks the short cytoplasmic and transmembrane domain and contains a histidine tag for purification. To define specific substrates for the gelatinase and collagenase activity of FAP-alpha , random peptide libraries of ~ 1.5- 2 million peptide sequences will be synthesized using the “one bead-one peptide” splitting and mixing technique. This approach produces peptides bound to polyethylene glycol (PEGA) grafted “beads” in such a way that each individual bead contains many copies of one unique peptide sequence. The peptide will contain a fluorescent molecule at the carboxy-terminus and a quencher at the amino-terminus. Protease cleavage liberates the quencher resulting in a fluorescent bead that is easily removed for peptide sequencing. Peptide substrates will be re-synthesized as fluorescent quenched soluble peptides and FAP-alpha proteolysis will be reconfirmed. The best FAP-alpha sequences will then be introduced into the activation domain of the proaerolysin toxin and FAP-alpha activation of the toxin will be characterized. Antistromal and antitumor effects will be determined by injecting the modified toxin directly into human breast cancer xenografts in vivo and analyzing toxicity to the animal, effects on tumor growth and effects on stromal compartment by immunohistochemical analysis. POTENTIAL OUTCOMES AND BENEFITS of RESEARCH: The studies described in this proposal will define whether therapies that target the stroma of breast cancers rather than the cancer cells themselves can be effective therapy for breast cancer. The bacterial toxin itself could represent a new targeted therapy for breast cancer. In addition the results could provide the rationale for other types of anti-stromal therapies such as small molecule prodrugs and antibody therapies.

    Lay Abstract:
    PROPOSAL TITLE: Fibroblast Activation Protein-Alpha activation of a bacterial toxin as targeted “anti-stromal” therapy for breast cancer A number of different chemotherapy drugs are used to treat breast cancer once it has spread outside of the breast (i.e. metastatic). Most patients usually have a beneficial response at the beginning of therapy. Eventually the therapy has to be stopped because the cancer cells are no longer killed by the therapy. This is because within the cancer, some of the cells are resistant to killing by the drugs being used. It becomes difficult to give more chemotherapy drugs because these drugs often have severe side effects to normal organs. Therefore, new effective therapies are needed for breast cancer that have minimal side effects on normal organs. Breast cancer cells must grow into the supporting tissue of the breast or the supporting tissue of other organs. This supporting tissue is called the stroma. The stroma is made up of cells that produce the structural proteins of the organ. The stroma cells can also produce molecules that can stimulate the growth of breast cancer cells and the growth of new blood vessel cells that are required by the tumor for the supply of nutrients. New therapies that either block the signals from the stromal cells or selectively kill the stromal cells within tumor sites could be effectively slow or preventing the growth of breast cancer cells. Previously it has been shown that stromal cells within sites of breast cancer begin to produce a specific protein called fibroblast activation protein (FAP). FAP is not produced by normal breast cells nor is it produced by any other normal cells within the various organs of the body. FAP is a type of protein called a protease that acts like a “molecular scissors” that can cut other proteins into smaller pieces. In this proposal we plan to define the protein pieces that are specifically recognized by FAP. These protein pieces could be attached to drugs or other types of toxins to produce inactive prodrugs. These prodrugs are activated to killing molecules following release of the protein piece by FAP. Thus, the prodrugs will only become activated within sites of breast cancer where FAP is present in the stroma. These prodrugs would not be activated by other normal tissues including the normal breast tissue and therefore would have minimal side effects. In previous work, we identified a very potent toxin made by a bacteria. This toxin kills by producing large holes in the outer membrane of cells. The toxin is made in an inactive form by the bacteria so that the bacteria themselves are not killed by the toxin. The toxin is activated by a protease that is made by most human cells. In this proposal, we intend to selectively remove the natural activation part of the toxin and introduce the FAP specific activation piece. This will produce an inactive toxin that could be targeted to kill only stromal cells present within the sites of breast cancer and not kill other types of cells. This FAP activated toxin will allow us to determine if selective killing of the stromal cells can slow or eliminate the growth of breast cancers. If the results are positive we could either use the toxin as a new therapy or could develop other types of therapies that are directed selectively to the stromal cells in the cancer rather than the cancer cells themselves.