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Synergism Between c-Src and EGFR Intracellular Trafficking in Breast Cancer
c-Src and the Src family of tyrosine kinases (SFKs) have been extensively studied in terms of their function, structure, and role in cell growth, signaling, and cancer. Dual overexpression of c-Src and EGFR (EGF Receptor) occurs in several breast cancer cell lines; this correlates to a Src-dependent increase in activation of and/or phosphorylation of EGFR downstream effectors. Despite extensive studies of the functional interaction between c-Src and EGFR, and the role of c-Src in modulating EGFR down-regulation, little is known about interactions in the trafficking pathways for c-Src and EGFR.
Evidence exists that c-Src associates with the plasma membrane and endosomal membranes in fibroblasts. Since endosomes recycle to and from the plasma membrane, it is logical to ask whether c-Src internalizes from the plasma membrane via the endocytic pathway. Moreover, given the synergism between c-Src and EGFR, and the finding that internalized EGFR can signal from endosomes, we hypothesize that c-Src and EGFR traffic together through the endocytic pathway and that growth factor signaling modulates c-Src trafficking and function. The research outlined herein addresses these questions using both biochemical and cell biological techniques.
First, we will determine whether c-Src endocytoses from the plasma membrane using an ELISA-based internalization assay developed in the Resh laboratory. Using this assay, we will delineate the sequence requirements for c-Src internalization, and whether internalization is sensitive to mitogenic stimuli and endocytic inhibitors. Second, we will identify the endocytic compartments with which c-Src associates by comparing co-localization of c-Src-GFP to known markers of the endocytic pathway. We will determine how c-Src traffics within the cell, and whether c-Src and EGFR co-localize and remain associated along the endocytic pathway using time-lapse confocal imaging (FRAP and FRET). Ultimately, we will test our findings in a physiologically relevant system by examining c-Src trafficking in breast cancer cell lines, which highly overexpress both c-Src and EGFR. Reagents that inhibit c-Src internalization will be developed, and the effects on EGFR signaling in breast cancer cells will be determined.
Given that c-Src and EGFR are abundantly overexpressed in breast cancer, it is critical to understand how c-Src moves around within the cell and how EGF/EGFR signaling influences this process. A deeper comprehension of this process is the first step towards designing novel therapeutic strategies for metastatic breast cancer.
Breast cancer is the second leading cause of cancer-related deaths in women in the United States. The incidence of breast cancer has risen from 1 in 20 women in 1960 to 1 in 8 today. Breast cancer occurs when cells in breast tissue start growing abnormally at a much higher rate than healthy breast cells. These unhealthy cells can invade surrounding tissue in a process which is known as metastasis. Early stage or non-metastatic breast cancer can very often be cured; however, there is no curative therapy for metastatic breast cancer and prognosis is poor.
Many proteins tend to be abnormally expressed in breast cancer, and two such proteins are the proto-oncogene c-Src and the human epidermal growth factor receptor, EGFR. The former is found associated with cellular membranes, the lipid envelope that separates a cell’s inside from outside as well as compartments within the cell itself. The latter is exposed on the cell’s surface and binds epidermal growth factor (EGF), its cognate ligand, which transmits signals to within the cell that promote cell growth and survival. EGFR and c-Src have been shown to interact in advanced stages of breast cancer and can contribute to increased aggressiveness in breast tumours.
Much of the research to date has largely focussed on understanding the role of c-Src on EGFR’s capacity to signal and move or “traffic” within the cell. However, there is a paucity of research in studying the converse, namely the movement of c-Src within the cell, and how EGFR signalling cascades might be influencing this process. We propose to study this problem in the following way.
Many proteins are found in specific locations or “addresses” within the cell, which are determined by “zip codes” that are intrinsically encoded within them. We will first determine if c-Src possesses a “zip code” of its own, and where exactly c-Src resides within the cell. We will then test if c-Src movement is affected by EGFR signalling, specifically if c-Src simply “tags along” with EGFR when it traffics within the cell. Additionally, we will determine if blocking or altering c-Src movement, by changing its “zip code” for instance, affects EGFR signalling. Ultimately, we will test the hypotheses derived from our cell model system to a physiologically relevant setting using breast cancer cell lines that contain high levels of both c-Src and EGFR.
We believe that deepening our understanding of c-Src movement within the cell and its synergism with EGFR will provide critical insight into the molecular and cellular mechanisms of disease progression in breast cancer.