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    Research Grants Awarded

    Characterizing Mechanisms That Regulate Cdc42 Activation In Breast Cancer

    Grant Mechanism:
    Postdoctoral Fellowships

    Scientific Abstract:
    CHARACTERIZING MECHANISMS THAT REGULATE CDC42 ACTIVATION IN BREAST CANCER Breast Cancer (BC) is the second most prevalent cancer in women and results in the second highest number of deaths, after lung cancer 1. Over 175,000 new cases of breast cancer are expected to be identified in 2007, predicted to result in more than 40,000 deaths 1. Molecular-based therapeutic strategies have resulted in increased BC survival rates 2, but much remains to be understood regarding the molecular mechanisms that lead to breast cancer, and how these mechanisms can be used to develop new therapeutic strategies. Cdc42 is small GTPase of the Rho (Ras homology) family shown to be overexpressed in human breast cancers 3. Overexpression of cdc42 appears to regulate breast cancer development through promoting cell survival pathways and through contributing to actin-mediated cellular motility and invasion. Cdc42 promotes breast cancer cell survival by preventing c-CBL mediated ubiquitylation and degradation of the epidermal growth factor receptor (EGFR) 4,5. Overexpression of cdc42 also leads to increased integrin-based tumor cell motility and invasiveness 6. In addition, engineered point mutations in cdc42 that regulate GTP turnover lead to tumorigenesis in vitro and in vivo 7,8. Importantly, silencing cdc42 expression in MDA-MB-231 and BT20 breast cancer cell lines leads to decreased EGFR protein levels, and decreases cell proliferation and migration 5. This suggests that therapeutics that cause inactivation of cdc42 may be promising for clinical intervention in BC 9. Cdc42 predominately localizes to the Golgi Complex in mammalian cells 10. Recruitment of cdc42 to the Golgi is dependent upon Coat Protein Complex I (COP-I) and its upstream activator, the small GTPase ADP-Ribosylation Factor (ARF) 10,11. COP-I interacts with cdc42 through the ?-COP subunit 12. This interaction is important for cdc42 action in tumorigenesis, since mutations in either ?-COP or cdc42 that block interaction with the COP-I complex inhibit cdc42-mediated cellular transformation 7. These findings suggest that inhibiting the cellular pathways that regulate ARF and COP-I recruitment to the Golgi will inhibit cdc42 activation and can be used as targets to prevent cdc42-mediated tumorigenesis in BC. COP-I is recruited to the Golgi by the activated from of ARF 13. ARFs, like all GTPases, cycle between the inactive GDP-bound and the active GTP-bound forms. ARF activation is regulated by the opposing actions of guanine nucleotide exchange factors (GEFs) that promote GTP exchange and activation, and GTPase activating proteins (GAPs) that promote deactivation 14. The sec7-family GEF GBF1 has previously been implicated in ARF activation and recruitment of COP-I to Golgi membranes 15. The GTPase activating protein ARFGAP1 has been implicated in ARF inactivation and subsequent release of COP-I from Golgi membranes 16. We hypothesize that perturbing mechanisms that recruit COP-I to Golgi membranes will inhibit Golgi recruitment of cdc42, and will suppress cdc42 activation and cdc42-mediated tumorigenesis in breast cancer cells. We will explore this hypothesis through two specific aims: SPECIFIC AIM 1. To define the regulators that mediate cdc42 recruitment to Golgi membranes and to characterize the relationship between Golgi recruitment and activation of cdc42. We propose to use siRNA-mediated genetic strategies and dominant negative approaches to characterize the roles of GBF1 and ARFGAP1 on (1) cdc42 localization, (2) cdc42 activation, and (3) downstream effects of cdc42 activation, including EGFR stabilization and actin remodeling, in breast cancer cells. SPECIFIC AIM 2. To explore inhibition of Golgi recruitment of cdc42 as a potential therapeutic for breast cancer. Using breast cancer cells as a model, we will explore whether ARF-based mechanisms that regulate Golgi recruitment of cdc42 inhibit cdc42-mediated properties associated with cellular transformation and metastasis, including (1) cell survival and apoptosis, (2) anchorage-independent growth, and (3) cellular migration. Completion of this proposal will define a novel set of therapeutic targets for treatment of breast cancer. 1 American_Cancer_Society, 2007. 2 C. A. Hudis, NEJM 357 (1), 39 (2007); C. L. Shapiro and A. Recht, NEJM 344 (26), 1997 (2001). 3 G. Fritz, I. Just, and B. Kaina, International journal of cancer 81 (5), 682 (1999). 4 W. J. Wu, S. Tu, and R. A. Cerione, Cell 114 (6), 715 (2003). 5 D. S. Hirsch, Y. Shen, and W. J. Wu, Cancer research 66 (7), 3523 (2006). 6 P. J. Keely, J. K. Westwick, I. P. Whitehead et al., Nature 390 (6660), 632 (1997). 7 W. J. Wu, J. W. Erickson, R. Lin et al., Nature 405 (6788), 800 (2000). 8 R. Lin, R. A. Cerione, and D. Manor, J Biol Chem 274 (33), 23633 (1999). 9 D. S. Hirsch and W. J. Wu, Expert review of anticancer therapy 7 (2), 147 (2007). 10 J. W. Erickson, C. Zhang, R. A. Kahn et al., J Biol Chem 271 (43), 26850 (1996). 11 R. V. Fucini, J. L. Chen, C. Sharma et al., Mol Biol Cell 13 (2), 621 (2002). 12 J. L. Chen, L. Lacomis, H. Erdjument-Bromage et al., FEBS letters 566 (1-3), 281 (2004). 13 J. G. Donaldson, D. Cassel, R. A. Kahn et al., PNAS 89 (14), 6408 (1992). 14 J. E. Casanova, Traffic (2007); P. A. Randazzo and D. S. Hirsch, Cellular signalling 16 (4), 401 (2004). 15 R. Garcia-Mata, T. Szul, C. Alvarez et al., Mol Biol Cell 14 (6), 2250 (2003). 16 W. Liu, R. Duden, R. D. Phair et al., J Cell Biol 168 (7), 1053 (2005).

    Lay Abstract:
    Breast cancer is the second most prevalent cancer in women and results in the second highest number of deaths, after lung cancer. Over 175,000 new cases of breast cancer are expected to be identified in 2007, predicted to result in more than 40,000 deaths. Early breast cancer therapeutics blocked tumor growth by non-specifically targeting all rapidly dividing cells, leading to debilitating side effects, including gastrointestinal problems and hair loss. More recent therapeutics target molecules that have been specifically identified to promote breast cancer growth and development. This strategy specifically targets breast cancer cells, sparing non-cancerous cells and causing significantly fewer side effects. Therefore, identification of new therapeutic targets specific to breast cancer is a promising strategy to develop therapeutics that are highly effective, but with fewer side effects. Cdc42 is a protein that has been shown to be present in very high levels in breast cancers. This protein directly regulates breast cancer growth by contributing to the pathways that allow breast cancer cells to multiply rapidly. In addition, cdc42 also regulates the ability of breast cancer cells to migrate to distant areas of the body to seed new tumors far from the original site. Therefore, inactivation of this protein could be key to blocking the ability of breast cancer cells both to multiply and to seed new tumors (metastases). Importantly, it has been shown that elimination of cdc42 in breast cancer models inhibits these processes, suggesting that cdc42 is an important therapeutic target. Cdc42 must be recruited to the correct location in the cell in order to play its role in breast cancer. Previous studies have shown that blocking recruitment of cdc42 to the Golgi Complex, an intracellular organelle, inhibits the role of cdc42 in breast cancer. Targeting cdc42 to the Golgi requires a group of proteins collectively known as COP-I. The mechanisms that target COP-I to the Golgi are known. Therefore, we hypothesize that if we can block recruitment of COP-I to the Golgi, we will block recruitment of cdc42 as well. This should lead to breast cancer cell death and a block in the ability of breast cancer cells to migrate to distant locations. We propose to inhibit COP-I recruitment by molecular approaches, and to determine whether these strategies block cdc42-activated pathways and inhibit breast cancer cell growth and migration. By defining these mechanisms, we will identify new targets for breast cancer therapies. Because the cdc42 pathway is selectively hyperactivated in breast cancer cells, these therapies should selectively target breast cancer cells, sparing other cells in the body. This will lead to increased effectiveness of treatment and decreased side effects for the patient. We hope that our strategy will lead to increased survival for victims of breast cancer and decreased discomfort due to treatment.