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    Research Grants Awarded

    The role of G12 proteins in breast cancer invasion and metastasis

    Study Section:
    Tumor Cell Biology I

    Scientific Abstract:
    Background: The prognosis for patients with localized breast cancer has improved over the last two decades; however, metastatic disease continues to contribute greatly to morbidity and mortality. Thus, understanding the mechanisms that regulate invasion and metastasis could provide crucial information needed to develop successful therapies to prevent metastatic progression of breast cancer. Signaling through the G12 family of G proteins (Ga12 and Ga13) leads to the activation of RhoA/C and inactivation of E-cadherin, both believed to be critical events for promoting invasion and metastatic spread of breast cancer cells. Further, G12 can be activated by receptors that are known to promote breast cancer invasion and metastasis. We have observed that activation of G12 induces a significant increase in human breast cancer cell invasion in vitro , and inhibition of G12 signaling markedly reduces invasion. Moreover, using the mouse breast cancer line 4T1, we have observed that inhibition of G12 signaling results in a dramatic inhibition of metastatic spread in mice. In addition, a preliminary analysis of G12 expression in human breast cancers reveals significantly higher levels of G12 in tumor when compared to benign tissue in the same specimen. This pattern is especially pronounced in inflammatory breast cancers. These data suggest that G12 plays an essential role in breast cancer invasion and metastasis and may be particularly important in inflammatory breast cancers. We propose to determine the role of amplified expression and/or aberrant activation of G12 proteins in breast cancer invasion and metastasis, with particular emphasis on inflammatory breast cancer. Aims: 1) Examine G12 expression using immunohistochemistry in invasive human breast tumors, including inflammatory breast cancers, and evaluate expression in the context of variables such as grade, stage, metastasis, and survival. 2) Investigate the role of G12 in breast cancer cell behavior in vitro , with an emphasis on models of inflammatory breast cancer, and establish the relative contribution of downstream signaling mechanisms . We will use expression of specific inhibitors of G12 signaling and RNA interference to generate tumor cell lines in which the different G12 pathways are selectively activated or inhibited and examine the consequences of this modulation on growth, apoptosis, and invasion. 3) Assess the effects G12 signaling on breast cancer invasion and metastasis in vivo , using mouse models of human breast cancer metastasis that utilize whole-body imaging technology. We will use a mouse mammary fat pad assay and human breast cancer cells engineered to stably express luciferase . Engineered cells will be implanted in the mammary fat pad, and their metastatic spread will be tracked and quantified based on their luciferase signal measured by the Xenogen® system . Benefits: An understanding of the mechanisms involved in breast cancer invasion and metastasis could provide essential information needed for the development of successful therapies for patients with aggressive disease, such as inflammatory breast cancer. Successful completion of these aims could identify G12 proteins as new targets for therapeutic intervention in this deadly disease.

    Lay Abstract:
    Over 200,000 new cases of breast cancer will be diagnosed in the United States this year. Although patient survival for local disease has improved in the past two decades, metastatic disease continues to contribute significantly to the morbidity and mortality of breast cancer. Thus, understanding the mechanisms of just how breast cancer invades and metastasizes could provide critical information needed to develop successful therapies for patients with aggressive breast cancers. Such approaches are desperately needed for subtypes of breast cancers that exhibit a propensity for particularly aggressive behavior, such as inflammatory breast cancer. We have observed that breast cancers express abnormally high levels of signaling molecules known as G12 proteins when compared to benign tissue in the same specimen; this upregulation is particularly evident in inflammatory breast cancers. Although a role for G12 proteins in breast cancer invasion and metastasis has not previously been described, these G12 proteins are activated by numerous factors known to be important in breast cancer progression. Moreover, activation of the G12 proteins leads to the activation of a well known metastasis-promoting molecule termed RhoA and to the inactivation of a molecule that suppresses metastasis, E-cadherin. Based on these findings, we decided to explore the possible involvement of G12 family proteins in the progression of breast cancer. Our preliminary data demonstrate that blocking G12 signaling inhibits breast cancer cell invasion, as well as metastasis in mice bearing aggressive breast tumors. These observations indicate that G12 may play an important role in breast cancer progression. We propose to determine the importance of G12 proteins in breast cancer invasion and metastasis in order to evaluate the effectiveness of targeting these proteins as a therapeutic strategy. We will pursue two complementary approaches to investigate the roles of G12 proteins in breast cancer biology, with particular emphasis on inflammatory breast cancer. First, in aim 1, we will assess G12 expression in human breast cancers and correlate this information with clinical and pathological variables such as stage, grade, metastasis, and survival. Second, in aims 2 and 3, we will expand our analysis using both inflammatory breast cancer cell models and animal models of breast cancer progression to determine which branches of the G12 signaling pathway are important for invasion and metastasis and what factors are activating them. Success of the first aim could provide an immediate clinical benefit by facilitating the diagnosis of potentially aggressive breast cancers, while the studies in aims 2 and 3 might reveal that the G12 proteins play a central - and previously undescribed - role in breast cancer metastasis. Moreover, successful completion of these aims should provide experimental systems useful for discovery of new drugs that block breast cancer invasion and metastasis.