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Regulated Antizyme Expression For The Prevention And Treatment Of Breast Cancer
Investigator Initiated Research
Despite advances in early detection as well as improvement in adjuvant hormonal therapy and targeted therapy, over 40,000 American women will die from breast cancer in 2007 and over 178,000 new cases will be diagnosed. Clearly there is a need to better understand the biology of breast cancer and the metabolic pathways that drive the development and progression of the disease in order to develop novel therapeutic agents. Elevated cellular polyamine levels and increased activity of ornithine decarboxylase (ODC, the first enzyme in polyamine biosynthesis) are consistently detected in tumors from many different tissue types, and numerous studies have demonstrated a link between ODC, polyamines and breast cancer. Therefore, we feel that the polyamine pathway represents a promising target to inhibit breast carcinogenesis.
Our long term research goal is to understand the role of the polyamine metabolic pathway in cancer development and progression, and to use this knowledge to develop new and innovative therapies to combat this disease. The objective of this proposal is to demonstrate the critical role of polyamines in breast cancer by utilizing a transgenic mouse model with regulated, mammary-specific antizyme (AZ) expression. AZ is a multifunctional negative regulator of cellular polyamines that inhibits ODC activity, stimulates ODC degradation and suppresses the uptake of exogenous polyamines. We have previously shown that mice with tissue-specific AZ expression are remarkably resistant to tumor development within that tissue and we feel that AZ represents a new paradigm in polyamine-targeted therapy. Our central hypothesis is that AZ expression will prevent mammary carcinogenesis and induce the regression of established mammary tumors. We propose the following aims to test this hypothesis:
Specific Aim 1: Examine the effect of inducible, tissue-specific AZ expression on mammary gland development. We have established a transgenic mouse model with doxycycline-regulated expression of the AZ protein in mammary tissue. The consequences of AZ over-expression to normal mammary gland development will be evaluated in both puberty and pregnancy by qualitative and quantitative analysis of ductal morphogenesis as well as immunohistochemical profiling of mammary gland cell types, cell proliferation and apoptosis.
Specific Aim 2: Determine the ability of AZ to suppress mammary tumor development by crossing MMTV-rtTA/tetO-AZ transgenic mice with MMTV-neu transgenic mice. HER2/ErbB-2/neu is frequently over-expressed in human breast cancer, and targeted expression of neu in the mouse mammary gland leads to spontaneous tumor development. By inducing the expression of AZ within the mammary gland of MMTV-neu mice at various times, we will: a) test the ability of AZ to decrease the growth of established tumors, and b) test the ability of AZ to prevent oncogene-induced tumors.
Specific Aim 3: Develop drugs that will induce the expression of the powerful tumor suppressing protein AZ. Translation of the AZ mRNA normally requires a polyamine-stimulated +1 ribosomal frameshifting event. We will produce a vector to express a transcript with the AZ mRNA fused to enhanced green fluorescent protein (eGFP) mRNA. Therefore, the stimulation of +1 frameshifting will result in increased translation and production of an AZ-eGFP fusion protein. A breast cancer cell-based assay will be utilized to screen a chemical library for small molecules that are capable of inducing +1 ribosomal frameshifting in the AZ mRNA.
This proposal represents an innovative approach targeting a critical tumor-promoting metabolic pathway. The successful completion of these studies in a pre-clinical model relevant to human breast cancer will allow us to demonstrate that AZ blocks mouse mammary tumor development. This critical proof-of-principle will represent a tremendous advance in our knowledge of the role of AZ and polyamines in breast cancer. This will lead to future studies to probe the molecular mechanisms responsible for AZ-induced tumor regression in order to identify novel pathways and targets for breast cancer treatment. Importantly, these studies will also provide a model system to examine minimal residual disease, resistance and relapse mechanisms that may be encountered with future AZ-targeted therapies. Finally, we will identify small molecule lead compounds that could result in novel AZ-inducing therapeutic or preventive agents that will reduce the incidence and mortality of breast cancer in the next decade.
Polyamines are positively charged molecules that bind to many critical components of the cell such as DNA, RNA, proteins and cell membranes. All of the exact functions of polyamines are not known, but extensive studies in many experimental models have clearly demonstrated that polyamines are essential for normal cell growth and survival. An equally large body of evidence shows that the levels of the polyamines and the enzymes involved in their production are greatly elevated in tumor samples compared to adjacent normal tissue. This has stimulated the development of cancer therapies aimed at depleting the amount of polyamines in the cell. Unfortunately, this has been a difficult task and these agents have shown limited ability to treat advanced human cancers, but they have shown great promise in the prevention of tumor development in ongoing clinical trials. We aim to develop a new approach to exploit the obligatory dependence of tumor cell growth and survival on elevated polyamine levels.
Our long term goal is to develop novel therapies for the prevention and treatment of cancer that are based on inhibiting the accumulation of polyamines in the cell. Toward this aim, we will explore the therapeutic potential of the polyamine regulatory protein antizyme (AZ), which blocks both the production and the uptake of polyamines by the cell. Therefore, AZ shuts off both of the two potential sources that cancerous cells can utilize to increase their polyamine content in order to support the growth or maintenance of a tumor. Our hypothesis is that AZ represents a novel and effective targeted therapeutic approach for breast cancer, and we will directly test the ability of AZ to inhibit tumor growth in a mouse model. In this proposal, we will utilize a unique genetically engineered mouse model that we have generated, which gives us the ability to turn the production of AZ protein on or off in the mammary gland of the mouse. We will cross these mice with other genetically engineered mice that are considered a model of human breast cancer because they develop spontaneous tumors that exhibit many of the same genetic alterations and biochemical behaviors as human breast tumors. This will allow us to directly test the ability of AZ to prevent the development of new tumors and to trigger existing tumors to shrink. This will provide a clear demonstration of the critical role of polyamines in breast cancer and the ability of AZ to inhibit the expansion and survival of breast cancer cells. Next, we will develop a screening assay to identify drugs that boost the production of AZ protein in human breast cancer cells. Our innovative approach will identify drugs that stimulate a unique aspect of AZ synthesis that is not shared by any other proteins in the human cell. Therefore, the ultimate goals of these studies are to prove the exciting therapeutic potential of the AZ protein and to discover novel drugs that will increase the production of this inhibitor of tumor growth.
This research has immediate and long-term importance to patients with breast cancer. The immediate impact of our work will be an increased understanding of breast cancer biology and the identification of new targets to attack in breast cancer. This knowledge could lead to the utilization of existing drugs in new combinations for breast cancer treatment. The demonstration that AZ prevents or reverses tumor growth will also stimulate the evaluation of existing drugs that mimic the functions of AZ in clinical trials for breast cancer prevention and therapy. Our drug discovery platform is the first step in the development of new drugs that will directly induce AZ production in breast cancer cells. Our goal is for these molecules to evolve into effective and specific breast cancer prevention and treatment options. This is a more long-term goal, but the exploration of innovative new therapeutic approaches is a critical step that is required to energize our progress in the fight against breast cancer. The discovery of new agents to attack validated targets is essential in order to reduce the toxicity of cancer therapy, to enhance clinical outcomes and to eventually eradicate the disease.