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    Research Grants Awarded

    Therapeutic Strategies To Alter Gene Expression In Breast Cancer

    Grant Mechanism:
    Career Catalyst Research

    Scientific Abstract:
    Therapeutics for breast cancer include hormonal strategies, DNA-damaging agents, cell and DNA replication inhibitors, and receptor tyrosine kinase inhibitors. Our hypothesis, backed by strong preliminary data, elucidates a novel approach to tackle breast cancer by attacking transcription in cancer cells. The transcripts of a number of oncogenes as well as regulators of other key processes such as cell proliferation, angiogenesis, and apoptosis, have relatively short half-lives. Neoplastic cells typically require the activity of one or more of these genes for survival. Thus, treatments that impede RNA synthesis exploit the tumor cells? dependency on these short-lived oncogenes, growth regulators, and survival factors, as they would selectively hinder their expression. Our findings indicate that the ribonucleoside analog, 8-chloro-adenosine (8-Cl-Ado), would be such an agent for the treatment of breast cancer. The rationale comes from our data that metabolites of 8-Cl-Ado are incorporated into RNA and prematurely terminate transcription. In breast cancer cells we have demonstrated that 8-Cl-Ado is metabolized to its active triphosphate form, inhibits transcription, and is cytotoxic in vitro. Additionally, a prodrug form of the analog, 8-Cl-cAMP, has been shown to be cytotoxic to several breast cancer cell lines in vitro and inhibits the growth of human breast cancer xenografts in mice. Moreover, we have found that non-transformed mammary epithelial cells are resistant to 8-Cl-Ado-toxicity and instead possibly undergo a cell cycle arrest. We also established that 8-Cl-Ado suppresses the expression of cyclin E; agents targeting cyclin E would be advantageous for the treatment of breast cancer as high expression levels in these cells have been associated with poor prognosis and therapeutic resistance. Thus, we hypothesize that 8-Cl-Ado would be an effective agent for the treatment of breast cancer via a mechanism involving the depletion of gene products that are essential for the survival of breast cancer cells. Furthermore, these essential gene products may not be required for non-transformed mammary epithelial cells. We propose to further assess the efficacy and mechanism of action of 8-Cl-Ado in the breast cancer cell lines MCF-7 and BT-474 as well as the normal mammary epithelial cell line MCF-10A. SPECIFIC AIMS 1) Examine the metabolism and cell cycle effects of 8-Cl-Ado in non-transformed mammary epithelial cells compared to the breast cancer cell lines. 2) Examine the effects of 8-Cl-Ado on gene expression in breast cancer cells. 3) Investigate the role of the depletion of key genes identified in aim 3 in the 8-Cl-Ado-induced cytotoxicity of breast cancer cells. 4) Assess the activity of 8-Cl-Ado in mouse breast cancer xenografts. EXPERIMENTAL DESIGN Aim 1: Differences in the accumulation and elimination of 8-Cl-ATP and the reciprocal effects on the ATP pool will be measured by HPLC analysis over time during and after treatment. A comparison of uridine incorporation over time after treatment will be used to examine differences in the recovery of RNA synthesis. Cell cycle analysis on 8-Cl-Ado treated cells labeled with BrdU will be used to determine if there are any blocks in the cell cycle progression. Aim 2: The effects of 8-Cl-Ado on gene expression in MCF-10A, MCF-7, T-47D, ZR-75-1, BT-474, and SK-BR-3 breast cancer cell lines will be examined using the Affymetrics HG-U133A 2.0 array. Genes depleted 3 fold or more in all 5 breast cancer cell lines will be identified. These depleted genes will be assessed for their known function and genes that may play a role in the breast cancer cell survival will be considered candidate targets of 8-Cl-Ado cytotoxic activity. Aim 3: To compare pharmacologic versus genetically modified inhibition, the expression of candidate genes, including cyclin E, will be knocked down by siRNA transfection. The effect of their depletion on survival with and without 8-Cl-Ado treatment will be examined by flow cytometery analysis of Annexin V and PI staining and by immunoblot analysis of PARP cleavage. Similarly we will examine the protective effects of their overexpression. Finally, the expression and activity of the 8-Cl-Ado-targeted genes will be examined in the non-transformed MCF-10A cells. Aim 4: MCF-7 or BT-474 will be subcutaneously injected into nude mice. Once the tumors reach ~0.3 cm diameter, 8-Cl-Ado will be given thrice weekly via tail vein injection at a range of doses of 25, 50 and 100 mg/kg/d for 2 weeks and the tumor diameters volumes will be measured. Upon completion of these specific aims we will have determined if 8-Cl-Ado is inhibitory to breast tumor growth in vivo and determined if the cytoprotection in the non-transformed cells is related to difference in the drug metabolism and/or mechanism of action. Furthermore, we will have identified genes altered by the analog treatment and determined if they play a role in 8-Cl-Ado-cytotoxicity. As multiple players of 8-Cl-Ado cytotoxicity are uncovered, it may be possible to examine any cross-talking between these factors to develop a better understanding of the optimal approaches and targets for breast cancer therapeutics. Additionally, our institution has recently received approval from the Food and Drug Administration for a clinical trial of 8-Cl-Ado as an Investigational New Drug. We have obtained GMP-grade 8-Cl-Ado for clinical use, and plan to introduce this agent in the clinic immediately, initially for hematological malignancies and then subsequently in solid tumors. Hence, it will be readily possible to use this agent for patients with breast cancer.

    Lay Abstract:
    Approximately 240,000 women were diagnosed with breast cancer this year and over 40,000 breast cancer mortalities are expected in 2007 in the U.S. alone. Currently, breast cancer is also the second leading cause of cancer mortalities in women. Given these statistics, there is a need for new breast cancer treatments. Therapeutics for breast cancer include hormonal strategies, DNA-damaging agents, cell and DNA replication inhibitors, and receptor tyrosine kinase inhibitors. Our hypothesis, backed by strong preliminary data, elucidates a novel approach to tackle breast cancer by attacking transcription in cancer cells. The goal of this proposal is to examine the transcription inhibitor, 8-chloro-adenosine (8-Cl-Ado), as a new therapeutic agent for the treatment of breast cancer. 8-Cl-Ado is a ribonucleoside analog that has demonstrated promise in a number of hematological malignancies. Our aim is to extend the application of 8-Cl-Ado into solid tumors, with breast cancer in particular. We hypothesize that 8-Cl-Ado would be an effective agent for the treatment of breast cancer via a mechanism involving the depletion of gene products that are essential for the survival of breast cancer cells. Furthermore, these essential gene products may not be required for non-transformed mammary epithelial cells. In preliminary results, we have shown that 8-Cl-Ado is cytotoxic to cultures of two breast cancer cells lines MCF-7 and BT-474, but not the normal mammary cell line MCF-10A. Our aim is to compare the effects of 8-Cl-Ado and how it is used in MCF-10A cells versus MCF-7 and BT-474 cancer cell lines, to distinguish the drugs effect on malignant and nonmalignant breast cells. This will be accomplished by using a number of techniques to assess the drug?s effects on cellular bioenergy and on biological macromolecules such as nucleic acids and proteins. We will also examine the effects of 8-Cl-Ado on the growth of these cells. We will also assess the activity of 8-Cl-Ado in mice harboring human breast tumors to determine how effective it is in a whole animal system. To evaluate how 8-Cl-Ado-induced cell killing in breast cancer cells over normal cells, one of the techniques that will be employed is the use of gene array analysis to compare the genes affected by 8-Cl-Ado treatment. The result is expected to uncover various genes involved in the survival of the tumors, which when inhibited will lead to tumor cell killing. As more and more of these genes are discovered, it will be possible to examine interactions between these specific genes which will further help us understand the complex nature of cancer. This is expected to then lead to the further development of optimal therapeutic strategies as well as identify additional targets for the treatment of breast cancer. In summary, the goal of the work proposed here is the development of a promising new agent, 8-Cl-Ado, for the treatment of breast cancer. The research will provide a better understanding of how the agent specifically affects malignant breast cells over non-malignant cells. In addition, we will identify specific genes that are involved with breast cancer cell survival, which may aid in the development in future targeted therapies. Ultimately, the proposed research will provide more insight into the factors which promote the growth and survival of breast cancer cells as well as provide a way to increase the survival rate for breast cancer patients. Our research institute has recently received approval from the Food and Drug Administration for a clinical trial of 8-Cl-Ado as an Investigational New Drug. We plan to introduce this agent in the clinic immediately, initially for hematological malignancies and then subsequently in solid tumors. Hence, it will be readily possible to use this agent for patients with breast cancer.