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    Research Grants Awarded

    A Src Driven Biomarker And Mediator Of Breast Cancer Metastasis

    Grant Mechanism:
    Postdoctoral Fellowships

    Scientific Abstract:
    Src kinases are activated in breast cancers, but the mechanisms by which they promote tumorigenesis remains unknown. Considerable experimental evidence suggests that src kinases mediate the metastatic phenotype. However the molecular mechanisms that mediate tumor metastasis and the biomarkers of this phenotype have not yet been defined. Our laboratory has cloned a novel substrate of src kinases named Trask that may be the functionally revealing mediator of breast cancer metastasis. Trask was purified and cloned in our lab as the most highly tyrosine phosphorylated protein in mitotic MDA-468 breast cancer cells. Trask is a transmembrane glycoprotein widely expressed in epithelial tissues and is abundantly expressed in human breast ductal epithelium and epithelial cell lines. However the phosphorylation of Trask is highly restricted in normal cells. Epithelial cells undergo phosphorylation of Trask during mitosis. This phosphorylation temporally correlates with the rounded state of mitotic cells and persists into early G1 and is lost only when G1 cells have re-spread. Consistent with a role in detached cells or in regulation of cell shape, Trask phosphorylation is also markedly induced by forced rounding and detachment of trypsinized interphase cells, and phosphorylation persists until cells have been allowed to re-spread and re-attach. Trask knockdown markedly impairs cell migration and lamellipodial extension, cell proliferation, and interferes with cell rounding and detachment in breast epithelial cells. These experimental data have suggested that Trask plays a role in the regulation of epithelial cell adhesion, cytoskeletal remodeling, or possibly membrane remodeling. In contrast to normal breast ductal epithelium or breast epithelial cell lines, Trask is aberrantly and constitutively phosphorylated in many breast cancers and in many breast cancer cell lines. Trask phosphorylation correlates with detachment and growth in suspension such that cancer cells growing in the rounded non-adherent state have highly phosphorylated Trask. Furthermore, the forced overexpression and constitutive phosphorylation of Trask in adherent breast cancer cells induces growth in suspension. The naturally and fully suspended Du4475 breast cancer cells have constitutively phosphorylated Trask throughout the cell cycle and these cells undergo apoptotic cell death when treated with src inhibitors. We believe that the src-induced phosphorylation of Trask regulates membrane or cytoskeleton changes that are functionally important in epithelial cells while detached from substratum or from their architectural framework. This is important during mitosis, during cell migration and wound healing, during epithelial-mesenchymal transition, and particularly important in metastasis. Breast cancer cells may even require Trask phosphorylation for survival during metastatic migration. In our first aim, we will study the requirement for Trask in breast cancer metastasis using mouse genetic models. We have successfully generated a floxed allele of Trask in mouse ES cells and are currently breeding chimeric mouse. After germline transmission and appropriate backcrossings, we propose to introduce this allele into and eliminate Trask function in MMTV-PyMT mice which develop a highly metastatic form of breast cancer driven by src overactivity. The experimental hypothesis is that the development of tumors or tumor metastases will be significantly reduced or absent if Trask function is lost. In our second aim, we will use in vitro models of human breast cancers to determine whether the src-induced phosphorylation of Trask is a biomarker of src inhibitor sensitivity. The sensitivity of a panel of breast cancer cell lines to src inhibitors will be determined in vitro and in vivo models. For the in vitro models, the sensitivity of breast cancer cells will be studied in adherent and in suspended growth and compared with the already established level of Trask phosphorylation. This is important since Trask may be a marker of sensitivity only while cancer cells are detached. For in vivo studies, we have established a model of breast cancer metastasis using mammary fat pad implantation and resection of breast cancer cells with subsequent detection of lymph node metastases using bioluminescent in vivo imaging. The metastatic propensity of breast cancer cells with highly phosphorylated Trask will be compared to those will unphosphorylated Trask. In addition, the ability of src inhibitor therapy to prevent the development of metastases will be studied. The experimental hypothesis is that the constitutive phosphorylation of Trask is a marker of breast cancer sensitivity to src inhibitors, or that breast cancers are transiently sensitive to src inhibitors during periods of Trask phosphorylation such as when they are detached, migrating, or in circulation. Mortality from breast cancer is directly related to tumor metastasis. Any advance in the field of metastasis has the potential to impact this most lethal consequence of breast cancer. Our work can potentially identify subsets of breast cancer patients in whom metastases can be prevented by src inhibitor therapy. Further down the road, our work can lead to additional targets, such as Trask itself, or other proteins involved in this pathway, which can serve as targets of newer anti-metastatic drugs.

    Lay Abstract:
    Mortality from breast cancer is invariably related to the development of tumor metastasis. Once developed, the progression of breast cancer metastases is virtually unstoppable and current therapies only minimally delay its natural course. Significant advances in the treatment of metastatic breast cancer have been awaiting a better understanding of the molecular mechanisms that underlay tumor metastasis so that rationally designed therapies could be developed to target this lethal aspect of breast cancer. Considerable evidence suggests that the src family of tyrosine kinases, which are overactive in many breast cancers, are important in driving tumor metastasis. But little is known about the mechanisms by which src kinases promote metastases, and there are currently no biomarkers that can identify in which patients they may be playing an important role. Our lab has been studying how src kinases promote tumor metastases and we believe we have discovered an important new substrate of src kinases that may finally shed light on the molecular mechanisms of breast cancer metastasis and a biomarker that may identify breast cancers that depend on this pathway for dissemination. This new target of src is called Trask. Our preliminary evidence suggests that Trask functions in regulating the shape and movement of cells. This is a function that can be particularly important in tumor metastasis. We have studied many samples of normal breast tissues and breast cancers and breast cancer cell lines and have found that src kinases seem to abnormally phosphorylate Trask only in some breast cancers, but not in normal breast cells. When we eliminate Trask expression in breast epithelial cells, their movements and migration are markedly inhibited. When we overexpress Trask in breast cancer cells, they detach and separate from the other cells, a process that is similar to tumor metastasis. These data have led us to believe that Trask may be a very important target of src, and one which is important in development of tumor metastasis. A breast cancer cell line with highly phosphorylated Trask undergoes apoptotic cell death when treated with src inhibitors. These data have led us to believe that Trask is an important target of src kinases in breast cancer cells and phosphorylated Trask may be the biomarker that identifies breast cancer cells that are being driven to metastasize by src kinases. The central hypothesis is that Trask play an important role in metastasis of breast cancer cells. Thus, it is expected that inhibition or elimination of Trask could result in abolishment or significant reduction in metastases. We will use a mouse model to determine whether Trask is required for breast cancer metastasis. Our lab is developing a mouse model where the Trask gene can be knocked out. We will then knock out the Trask gene in an existing mouse model that develops metastatic breast cancer. If our hypothesis is correct, knocking out the Trask gene will prevent the development of metastases. We are also going to determine whether Trask is a suitable biomarker to be used in clinical trials of src inhibitors. We will determine whether breast cancer cells with highly phosphorylated Trask are especially sensitive to src inhibitors. We will also determine whether Trask phosphorylation identifies specific periods of time, such as during detachment and migration, that breast cancer cells may be sensitized to src inhibitors. This study explores the mechanisms for cancer dissemination. It takes into accounts novel approaches and ideas that have not been pursued before. If hypothesis that Trask is required for metastasis is correct it will be a breakthrough in our understanding of the metastatic process. The aim of this study is to provide genetic and pharmacological evidence that phosphorylation of Trask is instrumental in cancer dissemination. This will not only establish mechanistic evidence, but will provide a desperately needed biomarker for testing src inhibitors in clinical studies. Furthermore, since Trask is expressed on the cell surface, it can serve as the target for newer generations of drugs that seek to suppress the process of metastasis.