A Novel Epithelial-Stromal Model of Metastatic Breast Cancer

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Richard Neve, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11IB-0154 Award: $205,716
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2005)
Metastases, rather than primary tumors, are responsible for most breast cancer related deaths. Yet, we know little about this disease and lack effective animal model systems with which to test effective therapeutics that prevent or treat metastasis. We have identified the receptor tyrosine kinase, EPHA2, as a potential critical regulator of metastasis. In this proposal we will utilize an animal model of the human breast to study how EPHA2 and its ligand, EFNA1, control normal and malignant biology of the breast.

EPHA2 is strongly and specifically expressed in the subset of breast cancers that are predisposed to metastasis. We found EPHA2 and EFNA1 expression is highly compartmentalized in normal breast tissue and EFNA1 expression is lost in invasive cancers. Therefore we will test the hypothesis that EPHA2-EFNA1 interactions in the human breast maintain normal tissue integrity and that loss of EFNA1 is an initiating event in the pathogenesis of metastasis in a subset of breast cancers. Thus, our aims are to provide evidence for a causal role of EPHA2 in breast cancer metastasis. As part of this we aim to use a model that closely reflects the human breast microenvironment. This type of model will provide a more accurate interpretation of gene function in human cancer than existing model systems. We will manipulate gene expression of EPHA2 and EFNA1 in the epithelial and stromal compartments of the breast to identify their impact on morphology and histopathology of the normal breast and metastatic cancer. For these studies, we will use a technique that recapitulates a human mammary gland in the mouse. Combinations of human epithelial and stromal cells from normal or cancerous tissue will be mixed together and implanted under the renal capsule of the mouse. The cells can be manipulated to express specific genes of interest prior to implantation in mice. In this way we can create tissue that can reflects either the normal or the diseased state of a human breast. By studying the morphology, histopathology and metastatic potential of these recombinants we will establish how EPHA2 regulates epithelial morphogenesis and identify whether it plays a causal role in metastasis.

This research has two potential area of impact. First, it will provide a powerful model to identify how epithelial-stromal interactions affect the mechanisms of breast cancer progression, a process still poorly understood for the human disease. Understanding these mechanisms has significant translational potential for therapeutic development. Secondly, it will serve as a “second generation” pre-clinical screen for therapeutic agents. Currently there is a high rate of failure in early phase clinical trials of drugs which showed robust response in mouse xenograft models. A model such as this that more closely resembles the human disease may yield more accurate therapeutic responses.


Final Report (2007)
Metastases, rather than primary tumors, are responsible for most breast cancer related deaths. To understand this disease we need to identify genes which directly regulate breast cell physiology and architecture in the breast. Our laboratory has identified two genes that interact with each and are strongly associated with breast cancer. In this proposal we plan to understand how changes in these genes control normal and malignant biology of the breast.

We hypothesize that in normal breast tissue, the presence of an epithelial cell surface 'sensor' or receptor called ‘EPHA2' interacts with a second protein (EFNA1) in the stromal cells. “The Eph receptor family is the largest family of receptor tyrosine kinases identified to date. The Eph receptors and their membrane-anchored ligands, ephrins, are unique in that they mediate bi-directional signaling. This is concomitant with activation of the Eph receptor tyrosine kinase domain and transduction of the typical forward signal into the receptor-bearing cell. The ligand-receptor interaction also leads to transduction of a reverse signal into the ephrin-bearing cell. The Eph/ephrin signaling mechanism is responsible for diverse and complex biological functions mediated by Eph receptors and ephrin ligands. These include vascular development, tissue-border formation, cell migration, axon guidance, and synaptic plasticity. The role of Eph receptors and ephrins in the processes of development of the cardiovascular system, angiogenesis, and vascular remodeling has been the subject of intense investigation since they were first identified in 1987.”1

We have successfully generated cell lines and molecular tools needed to further study the role of EPHA2 and EFNA1 in normal and cancerous human breast cells. Using these tools we have shown that reduction of EPHA2 protein inhibits invasiveness in cancer cell lines representative of aggressive “triple negative” (i.e., estrogen receptor, progesterone receptor, and Her-2 negative) tumors. Invasiveness can also be reverted when EFNA1 is presented on the surface of an adjacent cell suggesting stromal cells expressing EFNA1 may repress tumor invasive properties. In addition, we have developed a membrane-based screening system that immobilizes EPHNA1 (mimicking stromal cells) to study its effects on cell surface EphA2 using a variety of breast tumor cell lines. These experiments indicate a strong potential for this interaction to inhibit cell spreading and proliferation of tumor cells. Finally, from a therapeutic perspective we have shown that EPHA2 is regulated by EGFR signaling in normal cells, but cancer cells harbor mutations causing EPHA2 expression to be EGFR-independent. The consequences are that these tumor cells become more resistant to EGFR targeted therapeutics, such as gefitinib (Iressa®), erlotinib (Tarceva®), and lapatinib (Tykerb®), that have been approved for clinical use.

This project was designed to prove a causal link between EPHA2 and progression of breast cancer by modeling cell to cell interactions of EPHA2-EFNA1 in the human breast microenvironment. Significant translational potential lies in using this model as a pre-clinical screen for therapeutics targeted to metastasis. Therefore, targeting EPHA2 in these cells may prove to be an effective therapeutic agent to prevent progression of triple negative tumors.

1from J Pathol. 208:453-61 (2006)


Symposium Abstract (2007)
Metastases, rather than primary tumors, are responsible for most breast cancer related deaths. To understand the pathogenesis of this disease we need to identify genes which are directly involved in the process of metastasis. Our laboratory has identified a cell surface sensor, or receptor, (EPHA2) that is strongly associated with metastasis in the breast. We present data on the mechanisms by which this receptor controls normal and malignant biology of the breast.

We sought to evaluate the biologic role of EPHA2 with respect to tumor cell invasion. We have looked at EPHA2 biology using functional assays to asses the invasive potential of the cells. All the EPHA2-positive cells exhibited an aggressive morphology, in agreement with our published measurements of invasion. In contrast, cells lacking EPHA2 formed more differentiated structures, either grape-like strings of cells or disorganized acini-like structures. By applying recombinant EFNA1 (B61, the ligand for EPHA2) or using RNAi to “knock-down” EPHA2 receptor levels in each cell line in 3D we show EPHA2 is required to maintain invasive morphology in most of the cell lines tested. Furthermore, using co-culture assays we show that presentation of EFNA1 by a neighboring cell is sufficient to restrict highly invasive EPHA2-positive tumor cells to islands of growth with distinct borders and more differentiated morphology.

In addition we will present data showing that another cell surface receptor, ERBB3, is found reciprocally expressed with EPHA2 in breast tumors and acts to suppress cell invasion. We have evidence that reciprocal levels of EPHA2 and ERBB3 define distinct biologically and clinically relevant breast tumor subsets reflective of invasiveness and clinical outcome. The clinical implications are that EPHA2 and ERBB3 may have utility as diagnostic markers as well as representing distinct therapeutic targets.