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Novel Signaling Mechanisms of CXCR4-induced Chemotaxis
Background: Recent evidence indicates that metastatic breast adenocarcinoma cells express the G-protein coupled receptor CXCR4 which facilitates directed cell movement (chemotaxis) towards gradients of SDF-1a. SDF-1a is present in surrounding tissues and distinct organs in the body, including bone, lymph nodes, and lungs. Evidence indicates that when cells encounter a chemoattractant gradient they respond by activation and amplification of signaling events on the side facing the gradient. These signals facilitate a pseudopodium extension in the direction of the gradient. Membrane protrusion of a pseudopodium is independent of actual cell body translocation, and is the first sign of morphological polarity. Published data indicates that tyrosine phosphorylation of signaling proteins plays a key role in establishment of cell polarity and movement. We engineered a technology to separate the pseudopodium from the cell body in order to identify the signals in this structure regulated by CXCR4 activation systems.
Objective: To elucidate the mechanisms by which the CXCR4 receptor system regulates cell motility in breast cancer cells and to create tools that predict the metastatic potential of these cells. We hypothesize that phosphotyrosine signals facilitate CXCR4-mediated pseudopodia formation leading to cell chemotaxis and the invasion of distinct organs.
Aim 1: To identify phosphotyrosine signals that may regulate pseudopodial dynamics in response to CXCR4 activation.
Aim 2: To functionally test the proteins identified in Aim1.
Design: We will use our new pseudopodia purification system, state-of-the-art proteomics (e.g., MudPIT, 2-DE), and custom software to identify key signaling proteins in large scale from metastatic tumor cells. Then, we will use siRNA protein knockout technology and in vitro pseudopodia and chemotaxis systems to functionally test these signals. Finally, we will design and implement computer-based tools that correlate protein identification data with functional data in order to predict metastatic and invasive behavior of breast cancer cells.
Relevance: Understanding the molecular mechanisms that cause stationary tumor cells to become migratory is crucial to the treatment of breast cancer, as this is one of the major factors leading to poor patient prognosis and death. Identification of key signals in the CXCR4 receptor system could serve as markers of metastatic potential or may lead to the development of breast cancer-specific pharmacological agents or gene targeting approaches directed at blocking these signals.
The overall objective of this proposal is to understand the biological signals that cause breast cancer cells to migrate to new sites in the body, which is the underlying cause for poor patient prognosis and death. Recent evidence indicates that the CXCR4 receptor on the surface of breast cancer cells serves as an antenna that transmits signals from its ligand, SDF-1a, causing cells to become invasive and migratory. Cell movement in response to a chemical gradient such as SDF-1a is called chemotaxis. The first step of CXCR4-induced chemotaxis occurs when tumor cells sense gradients of SDF-1a released by cells in various tissues, causing an increase in signaling activity on the side of the cells facing the gradient. This signaling leads to changes in the shape of cells - the extension of a membrane protrusion (pseudopodium), which mediates the processes of migration and invasion. New evidence indicates that tissues like lymph nodes, bone, and lung produce SDF-1a, which attracts CXCR4-positive tumor cells to these organs where they proliferate and form a new tumor, but the signals that regulate this process are not known. Therefore, I propose to study these signals using cutting edge laboratory procedures and state-of-the-art protein identification technology.
I hypothesize that proteins with modifications in tyrosine phosphorylation play a key role in regulating breast cancer cell chemotaxis. My laboratory has developed a procedure to isolate the pseudopodium from the cell body in order to directly identify signals that are amplified in the pseudopodium during chemotaxis. We will use proteomics and bioinformatics to identify these proteins and their phosphorylation changes. These methods are designed to rapidly identify proteins in large scale and, in many cases, are automated. Once we have identified these proteins, we will use genetic 'knockout' methods to determine how these proteins mediate pseudopodia formation and breast cancer chemotaxis.
Identifying proteins in the CXCR4 receptor system and elucidating the signaling mechanisms by which they contribute to the spread of breast cancer may serve as biomarkers of cancer progression and metastatic potential. These biomarkers would be of value to the clinician as an indicator of prognosis and in prescribing an appropriate course of treatment. Such information may also lead to the development of breast cancer-specific pharmacological agents or gene targeting approaches directed at blocking these signals.