Are EGF-Receptors Activated by IL-8 in Breast Cancer?

Institution: La Jolla Institute for Molecular Medicine
Investigator(s): Ingrid Schraufstatter, M.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7IB-0144 Award: $155,475
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2001)
The most life-threatening aspect of breast cancer lies in the ability of cancer cells to spread from the primary tumor to distant locations in the body, where they form metastases. An early step in the metastatic process is the generation of tumor cells that can migrate through the surrounding tissue and reach the circulatory system. To complete this process they adhere to the endothelial cells that line blood vessels, migrate into the tissue, and grow as new tumors in the afflicted organ. Our interest is in the protein and receptor signaling events that stimulate tumor cell migration. The IL-8-like chemokines, named for their ability to cause cell movement, are a family of such factors, which activate a receptor called CXCR2. This receptor is present on breast cancer cells, but its role in these cells is poorly understood.

Recently we were able to show that IL-8-induced activation of the CXCR2 on endothelial cells (cells that line the vasculature) had an unexpected effect. It caused the activation of a different class of receptors, the epidermal growth factor receptor (EGFR or Her-1). Further, this activation of the EGFR was associated with IL-8-induced cell migration. Our hypothesis is that this same sequence of events might work in breast cancer cells. This is quite logical, because it is already known that the presence of the EGFR is a 'marker' for metastasis and poor clinical prognosis. In this project we propose that IL-8 'hijacks' the machinery of cellular responses used by the EGFR in breast cancer. An additional hypothesis is that we predict that activation of the EGFR response(s) might cause activation of a nuclear factor that tells cells to produce more IL-8. The net result is that IL-8 works both to increase its own production and to cause breast cancer cells to migrate as an integral process in metastasis.

To study the relationship of IL-8, CXCR2, and the EGFR we plan both activity assays and specific inhibitors to dissect key events in the signaling cascade. The sequence we hope to establish is the direct linkage that leads, (1) to activation of the CXCR2, (2) to activation of the EGF-R, and (3) to increased IL-8 production. The biological significance of this pathway will be confirmed in cell migration and invasion assays, which are proven indicators for metastatic potential. Once at this point, we envision future studies using breast cancer animal models to test the effect of inhibitors in a more realistic setting for the disease.

The idea of the vicious cycle that links IL-8 and EGFR activation is novel. This cross-talk between the two distinct receptor systems may ultimately lead to therapies to help women whose breast cancers ware detected too late or whose cancers are particularly aggressive and not well treated by current drugs.

Final Report (2002)
Metastasizing breast cancers still have a poor long-term prognosis, so novel strategies are needed to prevent the dissemination of cancer cells and/or the angiogenic response that is necessary for the blood supply of the growing tumor. In order to develop any new therapies it is necessary to understand basic biochemical pathways used by the cancer cell that may allow metastasis. The CXCR2 (IL-8 receptor type 2) is expressed by microvascular endothelial cells, where it is instrumental for IL-8 mediated tumor angiogenesis. It is also expressed by some breast cancer cells, where it may play a role in cancer dissemination using the migratory machinery that is associated with the activation of all chemokine receptors. In order to develop means to block this migratory response in endothelial cells and in cancer cells that express the CXCR2 it is important to understand the signal transduction cascade involved.

We found in microvascular endothelial cells that activation of the CXCR2 caused transactivation of the epidermal growth factor receptor (EGFR). In turn, inhibition of the EGFR blocked migration of IL-8 stimulated endothelial cells. When analyzing the intermediate steps between CXCR2 and EGFR activation, some steps were identical to those described over the last two years for other G-protein-coupled receptors. Antibody against heparin-binding EGF-like growth factor (HB-EGF) blocked IL-8 mediated cell migration, indicating that HB-EGF was formed from its pro-form to activate the EGF-R, which in turn was necessary for cell migration. Alternatively IL-8 mediated cell migration could be blocked with anti-EGF-R antibody or an inhibitor of the EGF-R. However, while previous reports had shown that membrane metalloproteinases (MMPs) mediated the cleavage of pro-HB-EGF to HB-EGF, inhibitors of membrane metalloproteinases blocked IL-8 mediated cell migration by only 40%. Since pro-HB-EGF had to be activated, and this process had to happen extracellularly - it was blocked by antibodies added to the media. We started a systematic search for protease inhibitors that blocked IL-8-dependent cell migration. While serine protease inhibitors showed no effect, a general inhibitor of cysteine proteases (E-64) blocked cell migration. Among inhibitors of individual cysteine proteases, CA-074 Me, a specific inhibitor of cathepsin B was found to block cell migration. Finally we could show that IL-8 caused release of cathepsin B from endothelial cells grown on collagen. These observations are important, because they connect three factors that have been found individually to be poor prognostic indicators in various cancers: (1) chemokines of the IL-8 family, (2) activation of the EGF-R, and (3) active cathepsin B.

Future directions will be threefold: (1) Further biochemical definition of the pathways just described, (2) in vivo assessment of a combined anti-CXCR2 and anti-EGFR or anti-cathepsin B strategy in a model of mouse breast cancer metastasis, and (3) extend our study to determine whether the CXCR4, which has been shown to be important for metastasis of breast cancer cells uses the same signaling cascade. Since the CXCR4 is widely expressed by breast cancer cells, whereas CXCR2 expression is quite limited, a similar signaling cascade may play a direct role in breast cancer metastasis. Our research suggests an indirect role for CXCR2 primarily through its angiogenic effect.

Since cell migration is so important for cancer cell metastasis as well as for angiogenesis a better understanding of the basic mechanisms involved will open new possibilities for the development of new therapies.

Symposium Abstract (2005)
Inhibition of metastasis is a major goal in breast cancer research, since metastases are the cause of death in breast cancer patients. Essential steps during metastasis are dissociation of the cancer cells from the primary tumor, circulation and survival in the vasculature or lymphatics, chemotaxis to other organs and proliferation at this new site. The chemokine receptor CXCR4 has been found to be functionally expressed on the cell surface of a variety of cancer cells including breast cancer, and the CXCR4 has been shown to be instrumental in metastasis of breast cancer cells in a mouse model.

In this study, we used a xenograft-derived MDA-MB-231 breast cancer cell, which expressed more CXCR4 than the parent cell line and showed an increased response to SDF-1. SDF-1 caused prolonged Akt activation in these cells, a pathway associated with cell survival, an important step during tumor cell dissemination. In addition, in breast cancer patients increased Akt activation has been associated with resistance to tamoxifen and chemotherapy. In our experiments Akt phosphorylation was dependent on coupling to Gi protein and activation of Src kinase. Inhibition of Akt by various means not only prevented Akt activation, but more importantly inhibited SDF-1 mediated migration of these cells in an in vitro chemotaxis assay. Our results highlight the role of Akt in CXCR4 signaling in breast cancer cells. Combined with previous reports showing that Akt is an important downstream signal following EGF-receptor activation, this activation by two bad actors over-expressed in breast cancer cells, makes Akt a promising target for chemotherapy in breast cancer patients.