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A novel interaction of Focal Adhesion Kinase and p53 in breast cancer cells
Tumor Cell Biology I
The most frequent alteration detected in breast cancer cells is an inactivation of the tumor suppressor protein p53, that is altered in approximately 40 % of all breast tumors. Focal Adhesion Kinase (FAK) is overexpressed and up-regulated at early stages of breast tumorigenesis. We have recently identified a novel interaction of p53 and FAK proteins. However, the association of FAK and p53 and its role has never been studied in the breast cancer cell biology field. Objective/Hypothesis: We will test a hypothesis that mutations of p53 inactivating its function will correlate with FAK overexpression in breast cancer tumors. We hypothesize that FAK and p53 interact to control cell survival in breast cancer cells. Specific Aims: (1) We will perform a correlation analysis of p53 mutations, p53 expression, and FAK overexpression with the patient prognosis in breast cancer tumors. (2) We will perform mapping of direct interaction of FAK and p53 in breast cancer cells. (3) We will perform co-localization analysis of FAK and p53 interaction in breast tumor samples. (4) We will test the effect of p53 mutations on FAK promoter and binding activities. We will test the effect of FAK and p53 binding on p53 activity and apoptosis. Study Design: First, immunohistochemical staining of population-based series of breast tumor samples will be performed with FAK and p53 antibodies to detect a correlation between p53 and FAK overexpression. Second, we will perform Real-time PCR analyses to detect mRNA level of proteins in breast tumors. We will perform SSCP/sequencing analyses to detect p53 mutations in breast tumor samples and correlate with FAK overexpression and patient prognosis. Third, we will perform phage display and site-directed mutagenesis assays to map a binding site of FAK- p53 protein interaction that will provide a basis for future FAK-p53 targeted therapy. We will perform confocal laser microscopy, FRET analysis and cell fractionation analyses to detect protein co-localization in vivo. We will test effect of p53 mutations on FAK promoter and binding activities. We will test the effect of FAK overexpression on p53 activity and apoptosis. We will test the effect of different p53 mutations on FAK expression by dual-luciferase assay. Potential Outcomes and Benefits of the Research: We have solid evidence that FAK and p53 interaction will play a key role during breast tumorigenesis. The project will be very important for the development of new FAK-p53 targeted breast cancer therapy approaches and breast cancer treatment programs. The project is highly innovative and will provide new data for breast cancer therapy research. The correlation data on p53 mutation/expression and FAK expression with the patient prognosis will provide the basis for novel targeted therapy approaches on breast cancer patients. Thus, all the data will provide a basis for future targeted FAK-p53 therapy against breast cancer tumorigenesis and metastasis.
More than 1,000,000 new cases of breast cancer are diagnosed every year. It is very important to identify different markers of tumors that can be detected at the early stages of tumorigenesis. One of the critical tyrosine kinases that is linked to these processes of tumor invasion and survival is the Focal Adhesion Kinase (FAK). The FAK gene encodes a non-receptor tyrosine kinase that localizes at contact points of cells with extracellular matrix. Focal Adhesion Kinase (FAK) is elevated in a variety of human tumors, including breast cancer tumors. Recently, we have shown that FAK is up-regulated at early stages of breast tumorigenesis and can be detected in pre-malignant tissues. Thus, FAK can be a marker of benign disease that may progress to a cancer. In addition, tumor suppressor gene p53 is found altered in about 40% of breast tumor cases. Thus, to study these proteins is very important for understanding and preventing poor prognosis of breast cancer patients. We have recently found that p53 and FAK proteins can directly and physically associate. However, there are at present no studies on this novel tumor marker functional association in breast cancer cells. Based on our preliminary data, we will perform a correlation analysis between p53 inactivation and FAK overexpression in 600 breast cancer tumors. We hypothesize that tumors with both mutant p53 and overexpressed FAK will have a poorer prognosis. The project will characterize association between FAK and p53 in breast cancer cells. We hypothesize that we will find a precise binding site between p53 and FAK proteins and functionally characterize this interaction. We will test the effect and mechanism of different p53 mutations on FAK expression and binding. We will test the role of FAK and p53 binding on cell death (apoptosis). We hypothesize that FAK overexpression can block transcriptional activity of p53 and can block apoptosis. We will test novel FAK-p53 targeted approaches to cause apoptosis of breast cancer cells. Thus, novel drugs can be used to target these two tumor markers and inhibit breast cancer tumorigenesis and metastasis.