Scientific Report 2007

Molecular and Experimental Medicine

Division of Blood Cell and Vascular Biology

Mechanisms of Breast Cancer Metastasis

B. Felding-Habermann, J.S. Krueger, D. O'Sullivan, W. Hassenpflug, J.S. Forsyth, M. O'Neal, M. Lorger, G. Cattarossi, E.I. Chen, J.R. Yates III, A. Kralli, K.D. Janda, J.F. Kroener*

* Scripps Clinic, La Jolla, California

Patients with breast cancer often respond well to refined surgery and treatments, but development of metastatic disease is still responsible for most deaths in patients who have this neoplasm. Our goal is to develop new therapies that can eliminate the spread of breast cancer, especially to the brain. We therefore seek to understand mechanisms responsible for dissemination of breast cancer and to identify functional targets to prevent and eradicate metastasis of this cancer.

Hyaluronidase Expression and Metastatic Phenotype

Hyaluronic acid, a high molecular weight glycosaminoglycan, is a major component of the extracellular matrix in both normal and tumor tissues. Degradation of stromal hyaluronic acid within a tumor may promote escape of cancer cells from the primary site, and the degradation products strongly promote growth of new blood vessels. We tracked metastatic spreading of human breast cancer cells in immunodeficient mice and compared the gene expression profiles of metastatic lesions from bone, lung, and brain with the profiles of the primary tumors in the mammary fat pad.

With one exception, the gene expression patterns were almost identical. All distant metastases, regardless of the target organ, expressed significantly higher levels of the hyaluronic acid–degrading enzyme hyaluronidase 1 than did their primary tumors. Secretion of the enzyme and biological activity in breast cancer metastases, as well as high serum levels of hyaluronidase 1 in mice with metastases, indicate that hyaluronidase 1 may be predictive of metastatic disease and represent a functional target for the inhibition of metastatic spread.

Changes in Energy Metabolism and Brain Metastasis

Searching for functional characteristics that allow breast cancer cells to spread to the brain, we used multidimensional proteomic analysis to investigate brain metastatic lesions and compare them with the circulating breast cancer cells from which the lesions were derived. Specific changes in protein expression of brain metastases indicated that spread of breast cancer to the brain is supported by a selection or predisposition of cancer cells that can adapt to the unique energy metabolism of the brain. Specific increase in enzymes controlling glycolysis coupled to mitochondrial tricarboxylic acid cycle and oxidative phosphorylation pathways indicated that brain metastatic cells derive energy from glucose oxidation. Importantly, these specialized tumor cells also had enhanced activation of the pentose phosphate pathway and glutathione system to detoxify reactive oxygen species created by the enhanced oxidative metabolism.

Activated Adhesion Receptor Integrin α_vβ₃ in Brain Metastasis

We found that breast cancer cells expressing a specific adhesion receptor, the integrin α_vβ₃, in a high-affinity functional state have an aggressive metastatic phenotype that promotes the spread of breast cancer. Importantly, targeting this activated conformer of the receptor with human antibodies isolated from cancer patients can prevent and inhibit breast cancer metastasis in our mouse model. We found that expression of α_vβ₃, particularly its activated form, is essential for the growth of breast cancer cells in the brain. Direct implantation of tumor cells into the brain of immunodeficient mice and analysis of tumor cell expansion and dissemination by noninvasive bioluminescence imaging and histology indicated that expression of α_vβ₃ and activation of the receptor allow breast cancer cells to survive and proliferate in the growth-restricted microenvironment of the brain.

Thus, we defined functional determinants and molecular markers of human breast cancer metastasis, specifically to the brain. To date, no therapies exist that can effectively combat cerebral breast cancer metastases. Our novel human cell and analytical models (Fig. 1) open the unique opportunity to develop and evaluate new treatment approaches based on molecular mechanisms that we identify as critical for the spread of breast cancer to the brain.

Fig. 1 Development and use of a new human cell model for studies on metastasis of breast cancer to the brain. Top panel, Circulating tumor cells isolated from a breast cancer patient with metastatic disease and established in culture (named BCM2 cells) were injected intravenously into immunodeficient mice. Metastases to the brain and bone were isolated, established in tissue culture, and reinjected into mice. Bottom panel, left, Noninvasive bioluminescence imaging of BCM2 cells labeled with luciferase show the pronounced ability of the cells to colonize the brain of immunodeficient mice and extend down the spine, as often occurs in breast cancer patients with advanced metastatic disease in the brain. Bottom panel, right, Micrographs of metastatic brain lesions from a mouse injected with BCM2 cells (top) and from a breast cancer patient (bottom) show the invasiveness of BCM2 cells and compression of the adjacent, still unaffected, brain tissue. Reprinted from Palmieri, D., Chambers, A.F., Felding-Habermann, B., Huang, S., Steeg, P.S. The biology of metastasis to a sanctuary site. Clin. Cancer Res. 13:1656, 2007.

Publications

Chen, E.I., Hewel, J., Krueger, J.S., Tiraby, C., Weber, M.R., Kralli, A., Becker, K., Yates, J.R. III, Felding-Habermann, B. Adaptation of energy metabolism in breast cancer brain metastases. Cancer Res. 67:1472, 2007.

Kim, Y., Lillo, A.M., Steiniger, S.C., Liu, Y., Ballatore, C., Anichini, A., Mortarini, R., Kaufmann, G.F., Zhou, B., Felding-Habermann, B., Janda, K.D. Targeting heat shock proteins on cancer cells: selection, characterization, and cell-penetrating properties of a peptidic GRP78 ligand. Biochemistry 45:9434, 2006.

Liu, Y., Steiniger, S.C., Kim, Y., Kaufmann, G.F., Felding-Habermann, B., Janda, K.D. Mechanistic studies of a peptidic GRP78 ligand for cancer cell-specific drug delivery. Mol. Pharm. 4:435, 2007.

Palmieri, D., Chambers, A.F., Felding-Habermann, B., Huang, S., Steeg, P.S. The biology of metastasis to a sanctuary site. Clin. Cancer Res. 13:1656, 2007.