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Mechanism of BCAR1/p130Cas-Mediated Estrogen-Independent Proliferation of Breast Cancer Cells
Breast cancer affects approximately one in ten women. The disease may be cured when detected early, but when disseminated, current treatments are palliative. In the past decades, endocrine treatments have been widely applied to manage advanced breast cancer. In particular tamoxifen is effective in treatment of estrogen receptor-positive breast cancer, but ultimately fails due to the development of resistance. The mechanistic basis for tamoxifen treatment failure is only partly understood. An in vitro functional screen for genes responsible for estrogen-independent proliferation of breast cancer cells has identified BCAR1, the human homologue of the rat p130Cas gene. Quantitative analysis of 2593 primary breast tumors using a BCAR1-ELISA has shown that high expression of the BCAR1 protein is a marker of poor prognosis and predicts failure of response of recurrent disease to tamoxifen treatment. BCAR1/p130Cas and HEF1 are members of a family of adaptor proteins and share a similar domain structure. Our recent experiments demonstrate that, despite the similarity, HEF1 does not support estrogen-independent cell proliferation. In contrast, BCAR1/HEF1 chimerical proteins retaining the central domain of BCAR1 are fully functional.
OBJECTIVE / HYPOTHESIS.
Our main objective is to establish how over-expression of BCAR1 mediates estrogen-independent proliferation of breast cancer cells and to understand the role of BCAR1 in breast cancer progression. We hypothesize that BCAR1 recruits specific signaling molecules and thus activates a signaling cascade leading to estrogen-independent cell proliferation.
1. Identification of protein interaction sites in BCAR1 critical for proliferation control in breast cancer cells.
2. Identification of the interaction partners essential for proliferation control.
3. Inferring the putative signaling pathway responsible for estrogen-independent proliferation of breast cancer cells from the identified partners.
4. Functional testing of key regulators of the putative signaling pathway for estrogen-independent cell proliferation.
5. Establishment of the role of identified BCAR1 interaction partners and key regulators of proliferation signaling in breast cancer progression.
To locate the protein binding motifs essential for estrogen-independent cell proliferation within the central domain of BCAR1, we will expand our series of BCAR1/HEF1 chimera. In addition, specific protein binding motifs will be mutated to establish their role in cell proliferation. To identify the interaction partners we will apply immune-precipitation on cell lines expressing various chimerical and mutated BCAR1 variants. Western blotting and mass spectroscopy methods will be used to identify the binding partners critical for estrogen-independent cell proliferation. Based on the identity of the required interaction partners, we will be able to propose a signaling cascade operational in our cells. This signaling pathway will be validated in our cell models by targeted interference with the putative key regulators using small molecule inhibitors, RNAi and/or transfection of dominant variants of these molecules. To assess the role of the identified BCAR1 interaction partners and key regulators of the signaling cascade leading to estrogen independence in breast cancer progression, these molecules will be measured and analyzed in a large series of primary breast tumors with complete documented clinical follow up.
POTENTIAL OUTCOME AND BENEFITS OF THE RESEARCH.
The results of these studies should provide detailed insight in the mechanism of endocrine resistance and early recurrence of breast tumors with high levels of BCAR1. This information may be exploited for improved diagnosis, therapy selection for individual patients, and allow for the development of novel therapies based on specific targets.
Breast cancer affects about one in ten women and is a major cause of death. Cure is only achieved when the tumor has not yet metastasized (i.e. spread to the surrounding tissues or across the body). In metastasized breast disease, both chemotherapy and endocrine therapy can be effective for relatively short periods. Ultimately, the disease becomes resistant to treatment and life threatening. Tamoxifen has been used for over three decades as an endocrine treatment of metastasized breast cancer and achieves responses in about half of the patients. Tamoxifen interferes with the normal growth stimulative effects of the female sex steroid hormone estrogen. In addition, tamoxifen is well tolerated and produces modest side effects in comparison to chemotherapy. However, the positive effect of tamoxifen treatment lasts for a limited time and ultimately the disease becomes resistant to the treatment. The reasons for development of tamoxifen resistance are not yet understood. We have initiated research to unravel the mechanism of tamoxifen resistance in order to facilitate the development of novel (combination) therapies, which can control the disease for much longer periods.
Our fundamental research has led to the identification of the BCAR1 gene, which causes tamoxifen resistance in a breast cancer cell model. Furthermore, we have shown that about one-fifth of the patients possess high levels of BCAR1 protein in the primary tumor. These patients have an increased risk of early recurrence of the disease and are unlikely to benefit from tamoxifen treatment. These results stress the importance of BCAR1 in breast cancer progression.
The BCAR1 protein has been shown to play an important role in many cellular processes essential for tumor cell spreading and survival. To accomplish this, BCAR1 binds to different proteins exhibiting various functions, depending on the specific cell type and the particular biological process. The goal of our proposed studies is to resolve the mechanism of BCAR1 action in growth of our breast cancer cell model and in breast cancer progression in patients. We plan to identify the proteins, which bind to BCAR1 and are essential for breast cancer cell growth and tamoxifen resistance. We will also determine the role of these proteins in clinical breast tumor specimens to confirm their importance in malignant progression of human breast disease.
The results of this study will provide new insights in breast cancer progression and response to endocrine treatments. Furthermore, the generated tools may prove to be helpful for appropriate therapy selection of individual patients and may identify targets for novel treatment strategies of metastasized breast cancer.