Role of the EphB4 Receptor Tyrosine Kinase in Breast Cancer

Institution: The Burnham Institute for Medical Research
Investigator(s): Elena Pasquale, Ph.D. -
Award Cycle: 1999 (Cycle V) Grant #: 5JB-0086 Award: $389,758
Award Type: IDEAS II
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (1999)
The formation of breast tumors is a multistep process in which a series of changes (gene mutations) occurring in sequence progressively transform normal cells of the breast into cells that multiply uncontrollably. Additional changes may occur that enable breast cancer cells to detach from the primary tumor and form metastases at distant sites in the body. Some of the genes that become mutated during breast cancer progression have been identified, but many likely remain unknown.

It was recently discovered that a specific protein (receptor tyrosine kinase EphB4) is present at elevated levels in highly malignant human breast tumors. EphB4 is present in all the human breast cancer cell lines that have been examined. In transgenic mouse models of breast cancer, elevated levels of EphB4 were detected in the more aggressive undifferentiated (immature-looking) and metastasizing mammary tumors, but not in the less aggressive non-metastasizing and well differentiated (mature-looking) mammary tumors. Taken together, these data suggest that the EphB4 receptor plays a role in breast cancer, particularly in its more aggressive stages.

EphB4 belongs to the Eph family of receptor tyrosine kinases. Developmental studies have shown that the Eph receptors, by regulating cell adhesion and cell movement in the embryo, are important for the proper organization and integrity of tissues. We have also recently identified a new protein, SHEP1, which may be turned on by Eph receptors, thus promoting breast cancer cell survival. Because tissue disorganization and abnormal cell adhesion, movement, and survival characterize the more advanced stages of cancer, the inappropriate functioning of an Eph receptor in breast tumor cells would be expected to make them more malignant.

We propose to study how changes that either promote or inhibit EphB4 signaling pathways affect the growth, invasiveness, and metastatic potential of human breast cancer cells. We will use nude mice as an animal model to study the properties of tumors derived from genetically engineered breast cancer cells. By taking advantage of a newly developed technology, the cells in which EphB4 signaling pathways have been altered will also be made fluorescent, and therefore will be easy to recognize.

The proposed studies will be the first to explore the function of an Eph receptor in a human breast cancer model. The precise role of the EphB4 receptor in human breast cancer is still a matter of speculation. However, data demonstrating a correlation between EphB4 expression in breast tumors and the degree of malignancy, make a compelling case that the role of EphB4 in breast cancer should be investigated. The fact that the Eph receptors are different from most other receptor tyrosine kinases in that they regulate cell adhesion, migration, and probably cell survival rather than cell division, further supports the idea that EphB4, may have a distinctive role in the more advanced and deadly stages of breast cancer. If this hypothesis is correct, EphB4 could serve as a target gene for therapies to stop breast cancer progression and as a marker of tumors that require more aggressive treatments. Our studies will also provide information on whether SHEP1, a newly identified protein that likely propagates some of the EphB4 signals, could also be a marker for malignancy and a therapeutic target.


Final Report (2001)
The receptor tyrosine kinase EphB4 is present at elevated levels in highly malignant human breast tumors. EphB4 is also present in all the human breast cancer cell lines that have been examined. In transgenic mouse models of breast cancer, elevated levels of EphB4 were detected in the more aggressive and metastasizing mammary tumors. These data suggest that the EphB4 receptor plays a role in breast cancer, particularly in its more aggressive stages. A portion of EphB4 is exposed on the cell surface and binds the exposed portion of a protein called ephrin B2. When this contact occurs, the portion of EphB4 that resides inside the cell transmits signals. In addition, this contact also causes ephrin B2 signaling. Interestingly, ephrin B2 is present in tumor blood vessels and is required for the formation of functional blood vessels in the developing embryo. Therefore, EphB4 in conjunction with ephrin B2 may contribute to breast cancer progression by affecting the properties of both tumor cells and tumor blood vessels.

To examine the role of EphB4 in tumor progression, we engineered human breast tumor cell lines to produce on their surface high levels of only the outer portion of EphB4, which lacks signaling capability. This should increase EphB4 interactions with ephrin B2 and at the same time decrease overall EphB4 signaling. High production of the outer portion of EphB4 on the surface of breast tumor cells increased the growth rate of tumors in a mouse breast cancer model. Analysis of the blood vessels in the tumors suggested that the portion of EphB4 that is exposed on the outer surface of tumor cells binds ephrin B2 in blood vessels, and this stimulates the formation of blood vessels that feed the tumor and promote its growth. Conversely, activation of EphB4 signaling within the tumor cells may inhibit tumor cell growth because cells that were engineered to have increased EphB4 activity stopped proliferating. The results obtained suggest that the outer portion of EphB4 on the surface of tumor cells promotes tumor angiogenesis, and therefore tumor growth. In contrast, high levels of EphB4 signaling may inhibit breast tumor cell proliferation. These data support the idea that the presence of EphB4 protein in breast tumors is a negative prognostic marker, particularly if EphB4 is not highly activated. However, EphB4 expression should be evaluated in conjunction with expression of ephrin B2. The hypothesis emerging from our data is that anti breast cancer therapies that target EphB4 should aim at inhibiting EphB4 expression and/or promoting EphB4 signaling.