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    A Novel Antiangiogenic and Antimetastatic Agent for Breast Cancer

    Scientific Abstract:
    A NOVEL ANTIANGIOGENIC AND ANTIMETASTATIC AGENT FOR BREAST CANCER BACKGROUND: The attachment of metastatic breast cancer cells is highly dependent on the successful induction by the transformed cell of normal cell-matrix interactions, so that newly established colonies can integrate with physiological systems. These interactions involve surface interactions between tumor cell and surrounding tissues; a class of cell surface proteins known as integrins are particularly important in these interactions. A potent inhibitor of integrin binding, the disintegrin contortrostatin (CN), is the topic of this proposal. Disintegrins are small, RGD-containing, soluble peptides from snake venoms that bind with high affinity to integrins on the surface of tumor and endothelial cells. CN uses its unique dimeric structure and high affinity integrin binding to inhibit breast cancer growth and angiogenesis. We have recently developed a liposomal-delivery system for CN (LCN) that shields CN from immune recognition and dramatically increases its circulatory half-life. We can now target intravenous delivery of CN to tumor cells in a breast cancer animal model and produce impressive inhibition of tumor growth, metastasis, and angiogenesis. OBJECTIVE/HYPOTHESIS: CN targets a greater range of pathogenic events than other antiangiogenic agents, but it is similar to other antiangiogenic agents in not targeting the basic metabolic machinery of the tumor cells. Therefore, it has low cytotoxicity in its therapeutic effect. Antiangiogenic agents have been tested in breast cancer, and Avastin, a human monoclonal antibody against vascular endothelial growth factor (VEGF), resulted in shrinking some tumors, but did not prolong lifespan. However, the validity of antiangiogenic therapy was demonstrated by the positive results of the clinical trial of Avastin in colon cancer patients. We hypothesize that CN is unique in targeting both angiogenesis and metastatic cell migration through binding to multiple integrins and, therefore, has greater potential than many antiangiogenic agents in clinical development. One of the central lessons from efforts to develop antiangiogenic agents is the advantage of combining these agents with a conventional cytotoxic agent. Even more effective has been the combination of metronomic therapy (a cytotoxic agent given chronically at low dose) with an antiangiogenic agent. One of the consequences of metronomic therapy is the inhibition of angiogenesis. We hypothesize that when CN is given in combination with metronomic therapy (using a cytotoxic drug) there will be a synergistic inhibition of tumor progression as compared to either agent alone. SPECIFIC AIMS: Aim #1: Examine activity of CN ± chemotherapeutic agents on breast cancer and endothelial cell proliferation, apoptosis and cell cycle regulation in vitro. Aim #2: Examine therapeutic efficacy of liposomal CN alone, or in combination with metronomic delivery of a chemotherapeutic agent in an orthotopic, xenograft model of metastatic human breast cancer in nude mice. STUDY DESIGN: In view of the expectation that antiangiogenic agents will act most efficiently when combined with chemotherapy or other antiangiogenic agents, we will examine the effect of combination treatment on breast cancer cell lines, as well as on early passage HUVEC cells. We will determine if the combination of CN with cyclophosphamide (CTX) or paclitaxel, exhibits synergism in inhibition of tumor or endothelial cell proliferation, or induction of apoptosis, or alters cell cycle proteins in these cells. Based on the recent report of the effective combination of antiangiogenic agents with low-dose chemotherapeutic drugs, we will examine potential synergism in vivo by combining LCN with low-dose CTX. We will combine intravenous LCN with orally administered CTX in the MDA-MB-435 breast cancer model in nude mice and examine possible synergism of inhibitory action on tumor growth, apoptosis, and angiogenesis. POTENTIAL OUTCOMES AND BENEFITS OF THE RESEARCH: The antitumor activity of CN is based on its high affinity interaction with integrins a5b1, avb3 and avb5 on both cancer and vascular endothelial cells. This diverse mechanism of action provides CN with a distinct advantage over many antiangiogenic agents that only block a single angiogenic pathway. Results of these studies on potential synergistic combinations with cytotoxic chemotherapy will have a direct impact on clinical translatability of this most promising form of therapy for breast cancer patients.

    Lay Abstract:
    A NOVEL ANTIANGIOGENIC AND ANTIMETASTATIC AGENT FOR BREAST CANCER Breast cancer is the second leading cause of cancer death in women in the United States with an estimated 40,000 deaths in 2002. In advanced (metastatic) breast cancer different treatment combinations often fail to control the cancer. Inhibition of new blood vessel growth into tumors (antiangiogenic therapy) is currently a promising approach for treatment of breast cancer. Contortrostatin (CN) is an antiangiogenic protein isolated from southern copperhead snake venom. CN blocks the ability of both breast cancer cells and newly growing blood vessel cells to migrate or invade. Thus, it has an inhibitory effect both on the ability of the tumor to spread and on blood vessels to grow into the tumor. In earlier studies we found that daily intratumor injection of CN effectively blocked human breast cancer growth, cancer-induced angiogenesis, and cancer spread in a mouse model of human breast cancer. There were, however, two important limitations to the clinical translation of CN: first, we needed to develop a targeted intravenous (IV) system to deliver CN to breast cancer, and second, we needed to produce a synthetic version of CN. The first of these problems has been overcome by the development of an encapsulated form of CN that can be delivered IV. Encapsulation of CN involves generating a lipid (or fat-like) coating that under the appropriate conditions forms a sphere around CN. These lipid spheres are called liposomes and they are stable in blood. Blood vessels involved in tumor angiogenesis are disorganized ('leaky') compared to normal vessels in the body, thereby allowing passive accumulation of liposomal CN (LCN) in the tumor; CN is then released from liposomes in the tumor where it inhibits cancer growth and dissemination. To address the second problem, a synthetic version of CN, we have developed a bacterial system to produce synthetic, or recombinant, CN (rCN). This technology will eliminate the continued need for snake venom, a limiting factor in terms of clinical development. Therapeutic effectiveness of the LCN delivery system has been proven in the mouse model of human breast cancer. Liposomal delivery has also allowed us to overcome two associated problems with IV delivery of CN. First, since long-term, repetitive IV administration of LCN will be required in breast cancer patients, it is possible that the human immune system will react against the snake venom protein, neutralizing its activity after prolonged exposure. Our investigations show that the immune response of mice to LCN is essentially eliminated, compared to native CN. Second, studies in mice show that the length of time CN remains in the circulation is very short, but is dramatically increased for LCN. This will enable more effective delivery of LCN to primary or metastatic tumors in breast cancer patients. A number of investigators have demonstrated the antiangiogenic activity of chemotherapeutic agents, particularly when administered with more frequent dosing. It has been suggested that for successful antiangiogenic therapy, combinations of antiangiogenic agents that target different angiogenic pathways be considered. Recently, investigators have shown the merit of continuous low-dose administration of chemotherapeutic agents (metronomic dosing) in combination with antiangiogenic agents. It was also found that continuous low-dose therapy with chemotherapeutic agents may have a highly selective effect on newly growing blood vessel cells. Thus, continuous or frequent administration of low doses of certain chemotherapeutic drugs may be an effective mechanism of antiangiogenic therapy, particularly when combined with specific antiangiogenic agents. In view of these findings, we hypothesize that CN in combination with other antiangiogenic agents targeting different pathways, or in combination with low-dose (metronomic) chemotherapy, will provide the most productive therapeutic approach for clinical application. We will, therefore, examine in test tube experiments and in a human breast cancer animal model combination therapy with agents that should complement the action of CN. These results will further the clinical translational potential of this most promising therapy for breast cancer.