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    Research Grants Awarded

    Characterization Of The Molecular And Biological Role That Lip Plays In Metastatic Breast Cancer

    Grant Mechanism:
    Postdoctoral Fellowships

    Scientific Abstract:
    BACKGROUND: Breast cancer remains the second leading cause of cancer related death in women of the Western World; however, in pre-menopausal women, it is considered the leading cause of death. Most of these women do not die from their primary tumor, but rather of metastatic disease in distant organs. Many women with metastatic breast disease have elevated levels of a protein known as C/EBPbeta-LIP in their tumor cells. C/EBPbeta-LIP is a member of the CCAAT/enhancer binding protein (C/EBPbeta) family of transcription factors that are important for mammary gland development and breast cancer. Although there are three C/EBPbeta? protein isoforms (LAP1, LAP2, & LIP), the LIP isoform is believed to play the most critical role in aggressive human breast tumors, and metastatic disease; however, its molecular actions and binding partners are not well defined. OBJECTIVE/HYPOTHESIS: Our preliminary data demonstrate that LIP expression, in the absence of LAP, leads to mammary tumorigenesis in NOD/SCID mice, and deregulated cell-to-cell contact and increased migration/invasion in cultured mammary epithelial cells, yet the mechanism and the downstream effector genes remain to be described. Because loss of cellular contact and migratory/invasive behaviors are hallmarks of metastasis, we hypothesize that LIP not only plays a role in tumorigenesis, but more importantly, may regulate the metastatic behavior of breast cancer cells. This activity may be regulated by either a LIP homodimer or via heterodimerization with other leucine zipper proteins, such as C/EBPdelta. Lastly, we hypothesize that LIP expression contributes to the aggressive nature of some breast cancers by regulating the cell?s resistance to chemotherapeutic agents. To address these questions we propose the following objectives: SPECIFIC AIMS: 1. To characterize the downstream targets by which LIP regulates cellular contact and migratory or invasive behavior in mammary epithelial cells. 2. To identify the functional significance of an interaction between LIP and C/EBPdelta in the regulation of malignant transformation of mammary epithelial cells. 3. To determine the mechanism by which LIP expression regulates the resistance of breast epithelial cells to chemotherapeutic agents. STUDY DESIGN: The invasive potential of our C/EBPbeta-LIP expressing cells observed in culture will be further characterized by transplantation of these cells into NOD/SCID mice, followed by analysis of tumorigenesis and metastatic lesion development using luciferase technology. Gene expression profiles of the LIP expressing cells will be obtained via microarray and analyzed in conjunction with gene expression profiles known to correlate with the anchorage independent, tumorigenic, invasive phenotype observed in breast cancer cells. These findings will be validated with RT-PCR and Western blot analysis, and then correlated with direct LIP targets determined by ChIP, the aggressiveness of the phenotype, and the metastatic in vivo potential of the cells. The functional significance of an interaction between LIP and C/EBPdelta in the regulation of malignant transformation of mammary epithelial cells will be determined using knock down strategies. Additional dimerization partners of LIP will be determined via GST-LIP pull down of LIP interacting proteins from cells with an invasive phenotype followed by mass spectrometry analysis. Lastly, to determine how LIP expression regulates the resistance of breast epithelial cells to chemotherapeutic agents, C/EBPbeta null vector control, or LIP expressing cells will be cultured in doxorubicin/adriamycin (0.1, 1, 10 ug/ml) and monitored for changes in the expression levels of the multidrug transporters (MDR1, BCRP/ABCG2 and TAP1, 2) at days 0, 2, 4, 6 & 12. Correlations between LIP expression, multidrug transporter activity and resistance to therapy will be determined. IMPACT: The research described in this application will have wide ranging impact on the clinical assessment of breast cancer recurrence, which is often observed as distant metastasis, the clinical response to chemotherapeutic agents and the identification of novel targets to improve existing therapeutic strategies. Because elevated LIP expression is associated with aggressive breast cancer, the results obtained from our study may benefit those patients whose cancer is unresponsive to conventional therapies. The results of this study are also likely to improve our understanding of the mechanisms that breast cancer cells use to become metastatic. Additionally, we may learn how LIP expression can decrease the therapeutic response of a tumor by regulating multidrug transporters that normally help cells to remove damaging chemotherapeutic agents. Lastly, our studies are likely to identify new downstream targets and dimerization partners for LIP that may become attractive new therapeutic targets.

    Lay Abstract:
    Breast cancer is the second leading cause of cancer related death in women. Most of these women do not die from their primary breast tumor, but rather, of metastasis or spread to other organs. Many women with metastatic breast disease have elevated levels of a protein known as C/EBPbeta-LIP in their tumor cells. The role of LIP in aggressive, metastatic breast cancer is not clearly understood and our research is focused on understanding how LIP may regulate progression to a more aggressive breast cancer. LIP must bind with other proteins before it can become functional and regulate the behavior of breast cancer cells. We are specifically interested in defining some of these binding partners and identifying the target genes that LIP regulates. Our preliminary data show that expression of LIP can lead to tumorigenesis in mice and migratory/invasive behavior in cultured cells. We hypothesize that LIP not only plays a role in tumorigenesis, but more importantly, may regulate the metastatic behavior of breast cancer cells. We also hypothesize that LIP expression contributes to the aggressive nature of some breast cancers by regulating the cell?s resistance to chemotherapeutic agents. To address these aims, we will characterize the role that LIP plays in the metastatic behavior of breast cancer cells by injecting LIP cells into mice and monitoring metastasis. We will also identify the downstream genes or targets that LIP may be abnormally activating during metastasis. As mentioned above, LIP must bind or interact with another protein in order to have activity. We will determine whether LIP binds with itself or with other proteins, such as C/EBPdelta and how this might affect its regulation of metastasis. Lastly, we will investigate whether LIP expression contributes to the aggressive nature of some breast cancers by regulating the cell?s resistance to chemotherapeutic agents. Some breast cancer cells have membrane transporters that pump anti cancer drugs back out of the cells. If LIP increases the activity of these pumps, then the cells will not die when they come into contact with chemotherapeutic drugs. Thus, LIP may not only increase metastasis, but may also prevent cells from dying during therapy. The research described in this application may help physicians and patients to understand which breast tumors will ultimately metastasize or which tumors will reappear after therapy. Our data may also help to define a subset of patients whose tumors will not respond well to chemotherapy because LIP can regulate the multidrug transporters that help cells to remove damaging chemotherapeutic agents. Lastly, our studies are likely to identify new downstream targets and binding partners for LIP that may become attractive new therapeutic targets.