Breast cancer is the second most common cause of brain metastases, affecting approximately 15% of breast cancer patients. Although the treatment of systemic breast cancer has greatly improved, the incidence of brain metastases appears to be increasing due to the inability of these therapies to enter the brain and eliminate breast cancer cells. Many patients with brain metastases die from resulting neurological complications while survivors often suffer damaging side effects from treatment. Breast cancer metastasis to the brain is an emerging medical problem that needs immediate attention, requiring the development of new and innovative therapeutic approaches.

Our laboratory was the first to describe the overexpression of the proteins Claudin-3 and -4 in primary breast cancers. Claudin-3 and -4 are located on the surface of epithelial cells and participate in the formation of "tight junctions." Important to us from the therapeutic perspective, Claudin-3 and -4 also function as receptors for the potently toxic Clostridium perfringens enterotoxin (CPE). We have shown that CPE can destroy breast cancer cells and breast tumor tissue through binding its receptors, Claudin-3 and -4, in as little as 60 minutes. In addition, the destruction of cells by CPE is highly specific since cells that do not express Claudin-3 or -4 are completely unharmed by the toxin. However, CPE is too toxic to be injected systemically since many normal organs contain epithelial cells that also express these receptors. So how can one take advantage of its cytotoxic effects to cancer cells? In our recent studies, we found that Claudin-3 and -4 proteins were present in all tested cases (5/5) of breast cancer metastases to the brain, but were absent in normal brain tissue. Further, mice that received several injections of CPE into the brain at levels that are extremely toxic to breast cancer cells showed no ill effects. This finding strongly suggests that the blood-brain barrier prevents CPE from entering the general circulation. Thus, the administration of CPE into the brain may provide an innovative method of killing breast cancer metastases without harming the host.

To test this possibility, we will first confirm that Claudin-3 and -4 proteins are present in a large number of cases of breast cancer metastasis to the brain and are absent throughout all brain tissues. Next, we will develop a mouse model of breast cancer metastasis to the brain in which we can directly observe tumor growth and response to treatment in a living animal. This will be accomplished by introducing a gene (firefly luciferase) that emits light into breast cancer cells before implanting them in the brain. The light can then be seen inside of the mouse using a highly sensitive camera. We will use this model to test the ability of CPE to eliminate breast cancer metastasis without harming the brain. To deliver CPE, we will place it into state-of-the-art microscopic, biodegradable polymer beads. These beads are based on the same technology as the Gliadel wafer, a biodegradable polymer disk currently used to deliver drugs in the treatment of brain tumors. These tiny beads will be injected into the brain and will slowly release the CPE as they are degraded over a period of weeks. This technology allows a single injection to be administered and bathes the tumor in CPE, providing maximum cell killing. Further, since Claudin-3 and -4 are present in breast cancer metastases in the brain, but not present in brain tissue, these proteins may also serve as targets for antibody-directed therapy.

In summary, Claudin-3 and -4 will be tested as targets for the treatment of breast cancer metastases to the brain using the novel antitumor agent CPE. We also will develop a mouse model of breast cancer metastasis to the brain that will serve as a powerful tool for the development of new therapies. This study may provide a novel therapeutic agent and a new target for the treatment of breast cancer metastasis to the brain. This work will have immediate translational potential and addresses a very important emerging problem in breast cancer treatment.