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Characterization and Targeting of Cell Surface Expressed Nucleolin in Breast Cancer Angiogenesis
A) Background: Tumor growth requires the formation of new blood vessels accomplished in two ways: by the sprouting of new blood vessels from existing ones and by the recruitment of endothelial progenitor cells from the bone marrow. Tumor blood vessels differ from normal blood vessels by the expression of specific markers that can be used to target drugs to the tumor. Targeting tumor blood vessels has distinct advantages because endothelial cells are accessible to drugs circulating in the blood, and genetically stable non-transformed cells are less likely than tumor cells to develop resistance against drugs.
Our laboratory uses in vivo screening of phage libraries to identify peptides that specifically bind to tumor blood vessels in vivo. One of these peptides, F3, binds to tumor endothelial cells, tumor cells, and a subpopulation of bone marrow cells. F3 is rapidly internalized and transported into the nucleus of these cells (Porkka et al., PNAS, 8, 751, 2002). Dr. Christian has recently shown that F3 binds to cell surface nucleolin in MDA-MB-435 human breast carcinoma cells and endothelial cells. Nucleolin is a major nuclear protein in dividing cells; however, several reports indicate that it is also expressed on the cell surface, where it serves as a receptor for L-selectin and the growth factor midkine. We have confirmed the cell surface expression of nucleolin by FACS analysis, confocal microscopy, and cell surface biotinylation. We have also shown that intravenously injected anti-nucleolin antibodies specifically localize in MDA-MB-435 xenograft vessels and in non-tumoral angiogenic vessels of implanted matrigel plugs. A functional role for cell surface nucleolin in angiogenesis is suggested by our preliminary results showing that anti-nucleolin antibodies inhibit tube formation by endothelial cells in vitro.
B) Objective/Hypothesis: We hypothesize that cell surface nucleolin is important for the angiogenic process, and that it may be necessary for endothelial tube formation or for the recruitment of endothelial progenitor cells (the minor bone marrow cell population that binds F3) to the angiogenic vessels.
C) Specific Aims: 1) To determine whether anti-nucleolin antibodies inhibit tumor growth and angiogenesis in vivo. 2) To study the function of cell surface nucleolin in endothelial cell differentiation and in the recruitment of endothelial progenitor cells to angiogenic sites. 3) To determine the frequency of cell surface nucleolin expression in human breast cancers and their vasculature.
D) Study Design: Anti-nucleolin antibodies prepared against various parts of the nucleolin molecule will be intravenously injected into mice bearing MDA-MB-435 tumors or matrigel plugs. Rabbit IgG will serve as a control. Tumor volume will be monitored, and the tumors and gel plugs will be recovered and their blood vessel density will be determined from sections stained for blood vessel markers. The effect of the antibodies on endothelial cell differentiation will be tested in assays for cell migration, adhesion, and survival. Nucleolin involvement in endothelial progenitor cells will be studied by isolating bone marrow cells from mice that express lacZ under the Tie2 promoter, and by determining their expression of cell surface nucleolin. Nucleolin positive cells will be isolated and tested for their contribution in angiogenesis with and without anti-nucleolin antibody treatment. The expression of cell surface nucleolin in human breast cancers will be studied by immunohistochemistry, using commercially available panels of tumor sections.
E) Potential Outcomes and Benefits: This study will provide information on the expression and functional significance of cell surface nucleolin in tumors and their vasculature. Antibodies reactive with cell surface nucleolin may provide a new tool for tumor treatment that is based either on functional blockade of cell surface nucleolin or specific delivery of drugs into tumors.
Tumor growth is dependent on the formation of new blood vessels, which assures a supply of oxygen and nutrients. The formation of tumor blood vessels, called angiogenesis, is accomplished by at least two different mechanisms. First, cells in already established blood vessels start to divide and second, progenitor cells from the bone marrow attach and integrate into the new blood vessels. Endothelial cells, the cells that form the inner lining of blood vessels, are particularly important in the growth of new blood vessels. Our laboratory works on peptides that distinguish growing blood vessels from normal resting vessels. Such peptides can be used to carry drugs into tumors and thereby increase drug efficacy and reduce side effects.
One of our peptides, F3, is particularly promising. It specifically binds to tumor vessels and other growing vessels, and also binds to the tumor cells. This shared specificity should make possible a two-pronged attack on tumors: targeting both tumor vessels and tumor cells for destruction.
Dr. Christian has identified cell-surface-expressed nucleolin as the receptor for F3 in human breast cancer cells and in the vessels of the tumors that form when these cells are injected into mice. Nucleolin is primarily a nuclear protein, but it can also be present on the cell surface, where it may have a function related to rapid growth of cells. He has also shown that anti-nucleolin antibody specifically concentrates in tumor blood vessels after it is injected into the circulation of mice bearing human breast cancer tumors, and that the antibody blocks the growth of vessels in a cell culture model system, an activity the F3 peptide does not have.
In the proposed project, we will explore the use of the anti-nucleolin antibody in inhibiting the growth of new blood vessels and tumor cells in breast cancer. It is likely that the antibody inhibits some function of cell surface nucleolin, which is important for the growth of tumors and their blood vessels. Part of our study will be directed toward understanding how nucleolin works in this role.
This project will enhance the understanding of the role of blood vessels in the development of breast cancer and may provide a basis for a new cancer treatment.