Understanding Breast Cancer Cell Metastasis to Bone

Institution: The Burnham Institute for Medical Research
Investigator(s): Sonoko Narisawa, Ph.D. -
Award Cycle: 1998 (Cycle IV) Grant #: 4KB-0106 Award: $436,722
Award Type: New Investigator Awards
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1998)
Breast cancer commonly spreads to bone at a frequency of approximately 70% in patients having distant metastasis. However, the mechanism of bone metastasis is not well understood. One possibility is that the environment within bone marrow, highly rich in growth factors and cytokines, is suitable for the proliferation of breast cancer cells. A more likely explanation is that specific mechanisms of adhesion occur between the bone endothelial (blood vessel lining) cells and breast cancer cells that favors spread to this site. We hypothesize that breast cancer cells released into the circulation adhere to the endothelial cells of bone marrow through a specific receptor interaction, and we will employ a novel molecular technology to study this process.

In our initial experiments we will use a massive collection ('library') of short, random protein sequences that we fix to the surface of virus-like particles called phage. We can then incubate these phage peptide libraries with cultured bone marrow endothelial cells. The small fraction of phage that specifically bind to endothelial cells can be recovered, re-grown in large numbers, and the process repeated to select out a tiny fraction for further study. Thus, the protein sequence on these phage could represent the same recognition site present on breast cancer cells that targets them to bone. Some of these could be already identified genes and proteins, and others should be previously unknown. Next, we will inject the purified phage directly into the circulation of mice- allowing them to briefly circulate, sacrificing the animals, and determining which organ binds the phage most selectively. Naturally, our aim is to find the phage and its protein sequence that targets to bone, thus mimicking the situation for breast cancer. In advanced experiments we can incorporate these bone-targeting phage protein sequences directly into a membrane protein that we place on the surface of cultured mammalian cells. These cells (i.e., surrogate breast cancer cells) will be injected into the circulation of mice to provide a more ‘real world’ test of adhesive function. In final experiments, our studies will involve actual breast cancer cells with different metastatic potential. We would expect that a bone-homing peptide would confer bone localization when placed on the surface of a previously non-metastatic breast cancer cell line.

In addition to identifying novel proteins and mechanisms of breast cancer spread to bone, we will also determine the precise binding site that regulates this process. This puts us much closer to finding a therapeutic inhibitor and working in the direction of blocking this most lethal phase of breast cancer.

Final Report (2001)
Note: The grant was extended 1-yr to complete the aims.

Introduction: Breast cancer exhibits high incidence of bone metastasis. Patients who have developed bone metastasis often suffer from bone fractures, severe pain, hypercalcemia, and paralysis. Despite the fact that bone metastasis diminishes a patient's quality of life dramatically, it is not known why and how breast cancer cells prefer to colonize bone. Furthermore, there is no method to predict the risk of bone metastasis in individual patients. Effective ways to prevent and treat bone metastasis should be developed.

Topic: Metastasis does not occur just randomly, and specific interaction between cancer cells released from the primary organ and the metastasized secondary organ are known in certain types of cancers. In this project, we aim to identify specific interactions that mediate bone metastasis of breast cancer and obtain molecules that travel to bone.

Progress: We have developed a unique cloning method to find adhesion proteins using cultured mammalian bone marrow cells that we established. The method developed in step allowed us to isolated a candidate protein, which is expressed on the surface of bone metastatic breast cancer and prostate cancer cell lines, and induce adhesion to bone marrow cells (target cells), if it is expressed in a experimental control cells. We have modified the method developed in step to a new system using mice. Proteins cloned in this method may not adhere to bone marrow cells; however, this system could be more useful to represent the situation naturally occurring in the human body.

During the course of this funding we had to change many of the technical approaches as proposed initially in 1998. First, the approach of injecting peptide-expressing phage into whole animals experienced many problems. Thus, we established bone stromal cells as an in vitro alternative to using whole mice. And, we used a breast cancer cell line, MDA-MB-231, cDNA library expressed in COS-1 cells as the surrogate vehicle for detecting the bone-homing protein. This led us to propose that a GPI-anchored (i.e., attached to the cell surface through covalent linkage to a glycosyl-phosphatidylinositol) protein, called CD59, was the homing protein of interest. We worked to express CD59 in breast cancer cells to see whether it would allow bone targeting in mice. This approach also was difficult, so we shifted to using Chinese Hamster Ovary (CHO) cells as the CD59 carrier. To date we are still working on this approach for detecting the ability of CD59 to mediate homing to bone.

Impact: If we can identify a protein and/or its partial fragment that specifically travels to bone through the circulation, it could be used in clinical applications, such as in the prevention of bone metastasis, and in drug delivery, or as a diagnostic tool for possible future bone metastasis.

Strategy to clone molecules mediating metastssis of breast cancer to bone
Periodical:American Association for Cancer Research
Index Medicus: AACR
Authors: Narisawa S
Yr: 2001 Vol: 42 Nbr: Abs: 793 Pg: