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    Research Grants Awarded

    Targeting Advanced Breast Cancer Metastasis

    Grant Mechanism:
    Career Catalyst Research

    Scientific Abstract:
    Metastasis, the process whereby tumor cells spread throughout the body, is the leading cause of death in breast cancer patients. However, the determinants of organ-specific metastasis are not well understood. This proposal focuses on addressing one of the priority issues of the Susan G. Komen Foundation: understanding the pathogenesis of breast cancer biology by identifying relevant proteins that are involved in organ specificity of breast cancer metastasis. The hypothesis to be tested is that protein expression profiles in breast cancer cells capable of organ-specific metastasis can be used to distinguish organ specificity among breast cancer cells, and that the differentially regulated proteins contribute to colonization of or survival within the individual target tissues. Metastasis to distant organs is a highly specific process. The origin of the primary tumor seems to determine which distant organs will be colonized. For example, preferred organs for breast cancer metastasis are lymph nodes, bone, lung, brain, and liver. Typically, research on metastasis is performed based on in vivo assays in small animal models, as no single in vitro assay can recapitulate this process. Assays are normally divided into ones for experimental metastasis and ones for spontaneous metastasis. Experimental metastasis is analyzed by injecting tumor cells into the circulatory system and therefore measures only the last steps of the metastastic process, success in colonization of a secondary site. I have employed this assay to obtain brain, lung, and bone metastatic variant cancer cells derived from the original heterogeneous primary breast tumor cell line. Using this experimental metastasis model, I have found changes in the tumorgenecity of the cancer cells that successfully proliferate at sites of metastasis. Specifically, the genes and proteins expressed in those cells differ in part from those expressed by primary tumor cells. Moreover, the changes in expression differ accordingly to the individual metastatic site. These data indicate that the tumor cells are selected for or adapt to the new environments by initiating a unique program of transcriptional and translational regulation. Strikingly, I have found that this reprogramming of genes and protein expression includes factors that enable the cancer cells to respond to growth signals already present in the specific local environment of the secondary tissue. My initial study focused on brain metastases because they are the most feared complication in breast cancer. Nearly 20% of patients with advanced breast cancer are eventually diagnosed with brain lesions, making breast cancer the main source of metastatic brain disease in women. Furthermore, the incidence of brain metastases is rising as patients respond to improved primary cancer therapy and live longer, but no current regimen significantly affects the progression of breast cancer brain metastases. To identify the determinants of breast cancer cell growth in the brain, I examined protein expression profiles for the original cell line and its brain or bone metastatic variants and found a unique protein expression profile for the brain-derived metastatic breast cancer cells. I showed that this protein profile is consistent with a bioenergetic adaptation of the tumor cells and that these metabolic alterations are associated with strongly enhanced tumor cell survival and proliferation in the brain microenvironment. Most excitingly, among the up-regulated proteins in brain metastatic breast cancer cells were extracellular components of the neurotrophin (NT) 3 signaling pathway including the cell surface receptor, TrkC and NT-3. NT-3 a member of the nerve growth factor superfamily, is a secreted protein that promotes survival and proliferation and/or differentiation of neurons through activation of a receptor tyrosine kinase (TrkC). This novel finding provides us with a mechanistic hypothesis about what the nature of the successful adaptation is, a testable hypothesis, and a potential therapeutic opportunity. In this proposal, I intend to explore the specificity and importance of NT-3 signaling in growth of breast cancer metastasis in the brain and to develop function-blocking antibodies as an inhibitor approach. In parallel, I will expand my preliminary results to identify signaling pathways that when disrupted, inhibit metastatic proliferation in the lung and bone. There are three specific aims in this proposal. In aim one, I intend to explore the necessity of neurotrophin-3 growth signaling to adapt primary breast cancer cells to successfully proliferate in the brain. In aim two, I will develop a pharmacological means of disrupting NT-3 signaling to inhibit metastatic growth of breast cancer cells in the brain. In aim three, I intend to identify signaling pathways for breast cancer metastasis at other sites. The findings from the planned study will reveal potential mechanisms that breast cancer cells acquire to colonize and proliferate at specific organ sites. Once identified, differently regulated proteins that facilitate organ-specific metastasis will be invaluable therapeutic targets for developing approaches to prevent metastatic disease. Ultimately, we hope to generate new molecular insights into the organ specificity of breast cancer metastasis and novel therapeutic approaches.

    Lay Abstract:
    For most types of cancer, including breast cancer, the development of on-going metastasis leads to clinically incurable disease. Improvements in surgery and radiotherapy and the development of new chemotherapeutic agents or their use in new combinations have in general only incrementally improved patient survival. Unfortunately, metastasis remains incompletely characterized at the molecular and biochemical levels. Much attention has been focused on what stimulates cancer cells to emigrate from primary tumors, but what governs growth of the cancer cells once they seed distant sites and why there are different preferential sites for different kinds of cancer are largely unknown. Breast cancer preferentially metastasizes to lymph nodes, bone, lung, brain, and liver. Although large numbers of cells that morphologically appear metastatic are shed into the circulation, only a small number of them successfully form organ-specific metastases. Ultimately, the uncontrolled growth of these subpopulations of cancer cells capable of organ-specific metastatic growth causes death. 20% of breast cancer patients with non-localized disease are eventually diagnosed with brain lesions, making breast cancer the main source of metastatic brain disease in women Unfortunately, most metastases, even when advanced, remain undetected until they become symptomatic. The initial presentation generally consists of a seizure episode, and palliative treatment extends survival in such cases typically for only a few weeks to months. To make an impact on clinical mortality of breast cancer, I have focused my research effort to understand how breast cancer cells capable of organ-specific metastasis enter, survive, and grow in targeted organs such as the brain, in hopes of identifying novel markers that can be used for earlier diagnosis and novel therapeutic targets that can be used to disrupt the formation or growth of tissue-specific breast cancer metastases. In my initial studies, I obtained circulating breast cancer cells from a breast cancer patient and introduced them into immunodeficient mice to model the metastatic process. I then isolated the subpopulations of breast cancer cells that succeeded in growing in the lung, bone, and brain preferential metastatic target sites. Strikingly, I found dramatic differences between the original circulating breast cancer cells and the cancer cells that successfully grew at the sites of metastasis. Specifically, the genes and proteins expressed by those cells differed in part from those expressed by the original, primary, tumor cells. Moreover, the changes in protein expression differed accordingly to the individual metastatic site: I found a unique pattern of protein expression in brain metastatic breast cancer cells that distinguished them from the metastases recovered from bone. Based on this unique protein expression pattern, I identified a new mechanism for growth control of breast cancer brain metastases: elevated expression of the growth factor neurotrophin-3 (NT-3), which is known to be important for the brain growth and development, and its signaling receptor, TrkC. Moreover and strikingly, examination of brain metastastic lesions obtained from six breast cancer patients revealed elevated expression of NT-3 in all six samples, validating the significance of this finding as a general feature of brain-specific breast cancer metastases. I plan to pursue three goals in my next period of study, as described in detail in this proposal. In aim one, I will determine the significance of the newly identified NT-3 signaling pathway for brain-specific breast cancer metastasis. In aim two, I will develop function-blocking antibodies capable of disrupting NT-3 signaling as a potential therapeutic agent that could provide early diagnosis and inhibit breast cancer metastasis in the brain. In aim three, I will extend the studies described above to identify proliferative signaling pathways required for the formation of lung and bone metastases that could similarly be targeted by disconnecting the cross-talk between the organ-specific metastatic breast cancer cells and their preferred tissue environment. Results obtained from this proposal will make an impact on the clinical mortality of breast cancer by providing the leads to identify and generate novel diagnostic markers and novel therapeutic targets for patients with advanced metastatic breast cancer.