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    Research Grants Awarded

    Extracellular Superoxide Dismutase Plus Pi-88 As A Novel Anti-Metastatic Therapy For Breast Cancer

    Grant Mechanism:
    Postdoctoral Fellowships

    Scientific Abstract:
    BACKGROUND AND RATIONALES: Metastasis is the leading cause of cancer-associated morbidity and mortality in patients with breast cancer. There is currently no adequate therapy for preventing and treating breast cancer metastasis. Therefore, development of effective therapies that not only inhibit cancer growth but also suppress metastasis is highly desirable. Antioxidant enzymes superoxide dismutases (SOD), particularly MnSOD, have been shown to suppress the malignant phenotype of various cancer cells including breast cancer. These enzymes modulate the cellular redox status by catalyzing the dismutation of superoxide into hydrogen peroxide (H2O2). There are several points that lead us to propose that extracellular SOD (EcSOD) could provide superior therapeutic benefits than the other antioxidant enzyme gene therapies. First, in a pancreatic cancer tumor xenograft, EcSOD provides the best tumor suppression and animal survival amongst the three SODs. Besides inhibiting tumor growth, EcSOD has also been shown to have both anti-angiogenic and anti-metastatic effects. Most importantly, due to its extracellular localization and its ability to bind to cell surface and be internalized by cells, EcSOD therapy may not simply rely on direct tumor targeting, but could be applied systemically. The secreted EcSOD binds to heparan sulfate (HS) proteoglycan on cell surfaces through its Heparin Binding Domain (HBD). Interestingly, we discovered that the loss of this HBD rendered the protein (EcSOD?HBD) a more potent inhibitor of breast cancer growth and invasion. This implies that an increase in the circulating levels of EcSOD may be therapeutically beneficial for breast cancer treatment. More importantly, combination treatment of EcSOD plus heparin synergistically inhibited breast cancer progression in vitro. Heparin serves as a HS mimetic that inhibits heparanase, a pro-metastatic enzyme found to be overexpressed in many human cancers including breast cancer. Heparanase is one of the key enzymes involved in degrading the heparan sulfate (HS) component of the extracellular matrix, thereby activating various HS-binding factors that contribute to a tumor microenvironment that promotes cancer growth, angiogenesis, and metastasis. Overexpression of heparanase has been shown to correlate with aggressive phenotype and metastasis potential of breast cancers. This indicates that anti-heparanase therapy when used in combination with EcSOD overexpression is a promising strategy in the development of an effective treatment in suppressing breast cancer progression. PI-88 is the only HS mimetic that has entered clinical trials for cancer treatments. It is a potent inhibitor of heparanase and has been shown to inhibit metastasis both in vitro and in vivo. HYPOTHESIS: Therefore, the overall hypothesis of this proposed study is that overexpression of EcSOD in combination with PI-88, will synergistically inhibit breast cancer progression. Our objectives are to understand the roles played by EcSOD and heparanase in breast cancer progression and to determine if EcSOD/EcSOD?HBD will enhance the anti-angiogenic and anti-metastatic activities of PI-88, thus allowing for therapies at lower dosage to reduce PI-88 related toxicity in breast cancer patients. SPECIFIC AIMS AND STUDY DESIGNS: We will test our hypothesis by pursuing three specific aims. Specific Aim (1): Determine if the combination treatment of full-length or HBD-deficient EcSOD plus PI-88 can increase the in vitro tumor suppressive effects on breast cancer cells compared to either treatment alone. Adenovirus mediated vectors will be used to overexpress the EcSODs and PI-88 will be provided by Progen Pharmaceuticals Limited (Australia). In addition, complementary approaches to modulate the cellular oxidative status and inhibit heparanase will be used to further illustrate the feasibility of the therapeutic targets for treating breast cancer. Tempol will be used as the pharmaceutical alternative for EcSOD and siRNA for heparanase will be used as the complimentary genetic approach to inhibit heparanase expression. Outcomes and analyses will include the determination of: growth rate, cell doubling time, clonogenic survival, and invasion potential. Specific Aim (2): Determine the mechanisms involved in the suppression of breast cancer progression by the combination treatment. We will then determine how the treatments inhibit tumor angiogenesis and invasion by analyzing expression levels of pro-angiogenic factors such as HIF-1? and VEGF as well as determining the key players known to be involved in tumor cell invasion and metastasis by degradation of extracellular matrix in breast cancer such as urokinase plasminogen and matrix metalloproteinases. Cellular oxidative status will also be determined. Specific Aim (3): Evaluate if in vivo breast cancer metastasis can be synergistically inhibited by the combination treatment of EcSOD/EcSOD?HBD plus PI-88 in two murine models of breast cancer. A bioluminescent imaging system will be used to monitor the metastasis of the disease in vivo. OUTCOME AND SIGNIFICANCE: We anticipate that the combination treatment with EcSOD, particularly the EcSOD?HBD plus PI-88 will enhance the anti-tumor efficacy of either treatment alone in breast cancer. This study will help to shed light on how oxidative status and heparanase activation influence breast cancer progression. Successful accomplishment of this project may aid in formulating new therapeutic and preventive strategies for treating metastatic breast cancers.

    Lay Abstract:
    Breast cancer is the second most common cause of cancer-related death in women in the US, accounting for 15-17% of all female cancer deaths. At the time of diagnosis, over 60% of breast cancer patients will have disease that has metastasized. While the primary tumor can be removed, there is no adequate therapy for preventing and treating the spread of the tumor to secondary sites. Therefore, development of a therapy that works in broad spectrum of breast cancer and possesses a dual function to inhibit both cancer growth and metastasis will create a significant impact in improving the lives of affected women. It is therefore our objective to address those issues by examining the potential use of an antioxidant enzyme, extracellular superoxide dismutase (EcSOD) via adenovirus-mediated gene therapy in combination with a clinically used anti-metastatic agent, PI-88, in breast cancer therapy. This combination treatment will simultaneously target the cellular oxidative signaling pathways and the heparanase pathways to enhance the anti-tumor effects beyond that of the single treatment alone. Oxidative stress occurs when there is an imbalance between the production of free radicals and the removal of these free radicals by antioxidants. Oxidative stress plays an important role in controlling cellular functions. Small increase in the oxidative stress can stimulate a variety of factors that support greater cell growth, as in the case of cancer development. In contrast, when the oxidative stress is pushed beyond the capacity of cells to repair the oxidative-related damages, cell growth will cease followed by induction of cell death. Most cancer cells show higher oxidative stress than normal cells and the introduction of antioxidants can inhibit tumor cell growth. This implies that increasing antioxidant levels could be an ideal strategy in developing new breast cancer therapies. There are several points that lead us to believe that EcSOD could provide superior therapeutic benefits than other antioxidant gene therapies in treating breast cancer. First, introduction of EcSOD has been shown to dramatically inhibit the growth of pancreas cancer and melanoma cells. Besides inhibiting tumor growth, EcSOD also prevent tumor vascularization and metastasis. Since this EcSOD is an extracellular protein, its application in breast cancer treatment may not rely on direct tumor targeting but its anti-tumor effect could be systemic, meaning that EcSOD gene therapy is more advantageous than standard gene therapy. Besides using the antioxidant enzyme, we will also inhibit the activity of heparanase protein as a combination treatment for breast cancer. Heparanase is not produced by normal cell but is preferentially expressed by cancer cells. It is one of the key enzymes that promote metastasis. The findings that heparanase level is elevated in a wide variety of human cancer cells, including breast cancer has led to an explosion of therapeutic strategies to inhibit its enzymatic activity. PI-88 is the only heparanase inhibitor that has entered clinical trials for cancer treatments. This compound is used as a single therapy or in combination with standard chemotherapy for lung cancer, prostate cancer, liver cancer, and multiple myeloma. It has been shown to inhibit metastasis both in cell culture system and in animal studies. Furthermore, it is less toxic compared to most chemotherapeutic drugs and the ease of its delivery due to the small size makes PI-88 an attractive candidate as an anti-cancer agent. Therefore, we hypothesize that targeting both the cellular oxidative status and heparanase with EcSOD plus PI-88 is a strategically novel and reasoned approach for breast cancer. State-of-the-art molecular biological techniques will be used to examine whether EcSOD overexpression with a virus-mediated gene therapy will enhance the anti-tumor effects of PI-88 in an aggressive and metastatic breast cancer cell line. We will then determine the mechanisms involved in this process to better understand how this treatments inhibits breast cancer progression. EcSOD and PI-88 will also be tested in two breast cancer metastasis models in mice to investigate their additive effect as anti-metastatic agents. A modern small animal imaging system will be used to monitor the growth and metastasis of breast cancer cells as well as to evaluate the effectiveness of the therapy in the animal studies. This study will advance our understanding of how oxidative stress and heparanase pathways influence breast cancer biology and therapy. The fact that EcSOD has not been studied in breast cancer treatment will open up exciting approaches in breast cancer therapy. PI-88 has been awarded Fast Track status by the U.S. Food and Drug Administration (FDA) for the prevention of tumor recurrence following liver resection in liver cancer patients. This designation will speed the process of bringing this potentially clinically very important drug to patients not only with liver cancer but also with other cancers that overexpress heparanase such as breast cancer. Therefore, the combination use of EcSOD with PI-88 is a promising approach to offer alternative treatment for breast cancer patients, especially if the effective dose of PI-88 can be lowered to reduce the PI-88 related side effects. These targeted therapies aiming at attacking the elevated oxidative stress and heparanase pathways involved in the development of breast cancer is advantageous over chemotherapy drugs, since these targeted treatments specifically suppress the progression of cancer cells with minimal effect on healthy cells and could potentially improve survival.