AWARD NUMBER: W81XWH-04-1-0759 TITLE: Development of an Universal Chemo-Sensitizer to Support Breast Cancer Treatment: Based on the Heregulin Sequence

BACKGROUND: Heregulin (HRG) expressing cells are markedly more sensitive to doxorubicin (DOXO). Cells genetically engineered to overexpress HRG are aggressive and have the ability to bypass the normal estrogen requirements for growth. These HRG expressing cells are antiestrogen independent in vitro and in vivo. It has been shown that HRG internalizes to the nucleus, its localization from the nucleus to a diffused cytoplasmic localization modulated by a deletion of the putative nuclear localization signal. Moreover, a deletion of the transmembrane domain and the NLS confers a marked increased sensitivity to DOXO, a commonly used drug for the treatment of hormone-independent breast carcinomas. The increased sensitization achieved by this mutant HRG protein has been attributed to its entrapment in the cells, making it incapable of being secreted to the media and thereby activating the HER-2 signaling pathway. We identified a minimal sequence derived from the HRG protein that is capable of sensitizing breast cancer cells to DOXO without any perceptible adverse effect. Furthermore, we have recently generated small cDNA's sequences derived from the HRG full length sequence and transfected breast cancer cell lines to conclude that these sequences, which do not contain the EGF-like domain of HRG, do not promote cell growth but induce the sensitization of breast cancer cells to most chemotherapeutic drugs used for the treatment of breast cancer including DOXO, Taxol and cisplatin. The GFP-HRG-mutant-protein was localized to the perinuclear region of breast cancer cells after transfection. Further experimentation is critical for the full understanding of the mechanism of response to conventional agents and to evaluate biological compound as chemosensitizer. RATIONALE/PURPOSE: The identification of a region within the HRG sequence capable of sensitizing breast cancer cells to a wide variety of chemotherapeutic agents without inducing growth prompted us to recognize this molecule as a potential “universal chemosensitizer” to be use clinically in combination with many of the chemotherapeutic clinically used today for the treatment of breast cancer. We propose to develop a “biomolecule” derived from these HRG sequences, conjugate it to a protein delivery reagent and test it in breast cancer cells. The purpose of developing such compound is to use it in chemotherapeutic regimens in order to minimize the dosage and systemic toxicity of chemotherapeutic drugs, thus improving their efficacy in breast cancer treatment. We are entering an era in which basic research and clinical medicine are being united by molecularly directed therapeutic decisions. These experiments will allow the effective and use of tools while also suggest the optimum way in which to combine new biological agents with older established drugs. OBJECTIVES: The objective of this concept proposal is to prove the theory of generating a chemosensitizer for breast cancer cells. To develop a biomolecule based on the HRG-mutant-protein as a “universal chemosensitizer” commonly used chemotherapeutic drugs based therapies for the treatment of breast cancer patients, and to determine its ability to sensitize breast cancer cells in vitro and in vivo. METHODS: The putative “universal chemosensitizer” derived the HRG-mutant-protein will be generated in large quantities as recombinant protein using conventional baculovirus technology, already used in our laboratory. The protein will then delivered into cells in culture using the protein delivery system “Chariot” using the BioPorter protein delivery system. This delivery system has a unique lipid formulation that allows direct translocation of proteins into living cells. We will perform anchorage-dependent and -independent growth assays in the presence of the “putative chemosensitizer” and chemotherapeutic drugs, including DOXO, Taxol, cisplatin (CDDP), VP-16, 5FU and others. IC50’s will be determined for the drugs in the presence and absence of increasing concentrations of the “chemosensitizer”. After in vitro assays are concluded, we will extend our studies to the in vivo models generated in our laboratory (1, 2). For the in vivo studies we will take two approaches, initially we will use the breast cancer cells developed to expressed the chemosensitizer’s cDNA (already generated in our laboratory) and treat the mice with chemotherapeutic drugs in a dose and time dependent manner. Tumor growth, regression and sensitization will be some endpoints. BENEFIT: The benefit of this study will be the generation of “universal chemosensitizer” specific for breast cancer. The use of this compound in combination with chemotherapeutic drugs should result in the increased efficacy of a diverse type of chemotherapeutic regimens by lowering their systemic cytotoxicity and preventing, COOH NH2 C Y T O P L A S M IC T R A N S M E M B R A N E