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    Research Grants Awarded

    Examining A Novel Link Between Rho Gtpases And The Metabolic Machinery Of Human Breast Cancer Cells

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    A common feature of human breast cancer is excessive signaling through EGF receptor (EGFR) family members and Rho GTPases. Our laboratory has had a long-standing interest in understanding the roles played by Rho GTPases in growth factor-coupled signaling pathways that regulate cell growth and migration. These efforts have led us to discover a previously unappreciated connection between Rho GTPase signaling and the metabolic machinery of the mitochondria. We have uncovered this connection through studies aimed at identifying small molecule inhibitors for activated Rho GTPases, and in particular, by the identification of a small molecule, designated 968. We have found that 968 effectively blocks the ability of cells expressing oncogenic Dbl, an activator (guanine nucleotide exchange factor) of Rho GTPases, as well as cells expressing constitutively active forms of RhoC or Cdc42, to undergo transformation and to exhibit enhanced migration and invasive activity. Likewise, we have found that 968 causes similar effects in human breast cancer cells; for example, it prevents the highly invasive MDA-MB213 cell line from growing in soft agar. Importantly, we have demonstrated that 968 specifically affects fibroblasts transformed by hyper-activated Rho GTPases, as well as human breast cancer cells, whereas it does not inhibit the growth of normal fibroblasts or human mammary epithelial cells. Recently, we identified the mitochondrial enzyme glutaminase, which catalyzes the conversion of glutamine to glutamic acid, as a cellular target for 968. Knock-downs of glutaminase using RNAi effectively eliminated the transforming activity caused by activated Rho GTPases, thus mirroring the results obtained with 968. Interestingly, we also have found that transformed cells show elevated levels of glutaminase activity, compared to their non-transformed counterparts, without any detectable change in the expression of the enzyme. Taken together, these results lead us to propose that Rho GTPases send signals that regulate glutaminase activity in the mitochondria. We further propose that the activation of glutaminase and the ensuing increase in glutamine metabolism result in the enhanced synthesis of growth regulatory proteins that are necessary for the proliferative, survival, and invasive activities that characterize the malignant state. In this application, we proposed to test these ideas through the following lines of investigation: 1) We want to further characterize the role of glutaminase activity in the anchorage-independent growth and invasive activity of human breast cancer cells. Here the objective will be to examine a number of different human breast cancer cell lines and demonstrate that knocking-down glutaminase by RNAi inhibits their ability to exhibit anchorage-independent growth (colony formation in soft agar) and invasiveness. We will verify that similar knock-downs of glutaminase have no effect on the growth of normal mammary epithelial cells. We also want to see whether breast cancer cells that require glutaminase activity for their transforming capability exhibit significantly elevated levels of enzyme activity in their mitochondria. Finally, we want to determine whether the levels of growth regulatory proteins, in particular EGFRs and related family members, are changed when knocking-down glutaminase. 2) We will determine how Rho GTPases mediate the regulation of glutaminase activity in the mitochondria of breast cancer cells. We are especially interested in understanding how RhoC and Cdc42, which signal through distinct target/effector proteins, are able to converge at a common point downstream that enables both of these GTPases to activate glutaminase. One interesting candidate for a common signaling endpoint for RhoC and Cdc42 that we plan to examine is NF-?B, given that it can be activated by these different GTPases and has been implicated in breast cancer development and progression. 3) We will determine how 968 inhibits glutaminase activity. This will involve mechanistic studies aimed at seeing whether 968 blocks the conversion of glutaminase from an inactive dimer to an active tetramer and efforts to determine the 3-dimensional structure for glutaminase bound to the inhibitor. We believe that 968 will provide a template for the design of novel molecules that could have a significant therapeutic benefit for patients with breast cancer. In addition, we expect that the studies outlined in this application will highlight potentially new targets of the metabolic pathway that could be of real value by serving as sites of intervention against this disease.

    Lay Abstract:
    There is now a great deal of appreciation for the roles of receptor tyrosine kinases (e.g. Neu/ErbB2) and their signaling partners in the development of breast cancer. Recently, we have uncovered a novel cellular signaling pathway that culminates in the activation of an enzyme (glutaminase) which influences glutamine metabolism and the metabolic energy of breast cancer cells, and is essential for promoting a number of the key features of the malignant state. This discovery originated from work in our laboratory characterizing a small molecule inhibitor of the signaling activities of a family of proteins (called the Rho proteins) that have been implicated in breast cancer development and in particular, the most aggressive and metastatic forms of the disease. While we originally suspected that the small molecule inhibitor was acting directly on a Rho protein or one of its immediate regulators, we subsequently identified the metabolic enzyme glutaminase as the actual target, which then highlighted a previously unappreciated cellular connection between the Rho proteins and the metabolic machinery of breast cancer cells. What we find to be particularly exciting in terms of therapeutic potential is that it is possible to inhibit the activity of this enzyme without affecting the growth of non-cancerous cells, thereby indicating that glutaminase activity is necessary for the development of the malignant state but is not essential for normal cellular functions. We suspect that the activation of glutaminase contributes to the metabolic events required for the synthesis of proteins that stimulate the growth of breast cancer cells as well as their ability to survive the adverse conditions and stress that characterize the environment of tumor cells, and that promote their invasive and metastatic capability. Thus, we are proposing to further delineate the cellular signals that stimulate glutaminase activity and to determine the types of breast cancer cells where the activation of this enzyme is especially important for the development of the malignant state. We also are very interested in learning more about how the small molecule inhibitor blocks the actions of glutaminase and whether the mechanism of enzyme inhibition sheds light on why the inhibitor is so specific for inhibiting cancer cells without affecting normal cells. It is our hope that these studies will help highlight new types of drugs and therapeutic strategies that can be of benefit against breast cancer, especially those forms of the disease that are most aggressive and may be linked to the aberrant signaling actions of the Rho proteins.