Targeting Breast Cancer Metastasis

By Jason Socrates Bardi

Associate Professor Brunhilde Felding-Habermann recently sat in her office going over some slides she was preparing about her work on metastatic melanoma and breast cancer cells. She was getting ready to give a talk this week at the 2003 annual retreat of the Department of Molecular and Experimental Medicine (MEM) at The Scripps Research Institute (TSRI).

"I have titled this talk "Targeting Breast Cancer Metastasis," says Felding-Habermann.

She adds that breast cancer is still a pressing public health concern because it has a high propensity to metastasize and when it does, it can be deadly. "Too many patients still die from metastatic breast cancer," she says.

A Dangerous Phenomenon

Metastasis is a dangerous phenomenon in which cancer cells separate from a tumor mass, move through the bloodstream, anchor down in a distant tissue or organ, and begin a new cancer that might compromise the function of that organ. While surgeons can remove cancerous tissue, such procedures are greatly complicated if a tumor establishes new tumors in other organs. Although science and medicine have made tremendous strides in early detection and successful treatment, breast cancer and melanoma still claim tens of thousands of lives a year—usually the end result of metastasis.

The Centers for Disease Control and Prevention (CDC) reports that by the end of 2003, 211,300 U.S. women will be diagnosed with new cases of invasive breast cancer and that an estimated 39,800 women will die of the disease this year.

Similarly, melanoma, the deadliest type of skin cancer, claims many lives. According to the CDC, some 54,000 patients in the United States will be diagnosed with new cases of melanoma in 2003 and 7,700 Americans will die of the disease.

What leads cells to metastasize? What determines where the metastatic cancer cells go? Why do breast cancers typically metastasize and form new tumors in the brain, lungs, liver, and bones specifically? What is it about the vascular cells in these tissues that interacts with the breast cancer cells and allows them to invade, forming a new tumor?

Answering these questions is where basic biomedical scientists like Felding-Habermann and her Scripps colleagues come in.

"Disseminating tumor cells suddenly find themselves having to demonstrate capabilities that they did not need to have in the primary tumor," says Felding-Habermann. "What we are trying to find out is what determines whether or not breast cancer or melanoma cells will metastasize."

Readying the Invasion

Such specific questions are the key to understanding cancer because cancer is a complicated disease. It can be caused by subtle mutations within normal cells. After certain mutations occur, a cancer cell grows resistant to normal programmed cell death, dividing out of control, over and over, and forming a solid tumor. Also common to tumor cells are mutations that lead them to metastasize.

The word "metastasis" comes from the Latin construction meaning to change position, and metastasis depends on the cancerous cells acquiring two separate abilities—increased motility and invasiveness.

Motility, the ability of a cell to get up and go, is an obvious requirement for metastasis. Perhaps not so obvious is the need for cancerous cells to invade tissue, to burrow in and set up shop somewhere else.

These "phenotypic" abilities do not arise out of thin air. Cancerous cells acquire them in part through mutations to their DNA that result in changes of expression of one or more genes. Some mutations turn on or increase the activity of certain key genes, increasing the expression of metalloproteinases for instance; others downregulate them, shutting off production of receptor proteins. Often as these expression levels change, so do the levels of other, related genes.

Changes in gene expression that seem to control metastasis in human breast cancer affect an adhesion receptor called integrin avb3.

Integrins are large protein complexes made up of two different types of polypeptide chains (called the alpha and beta subunits) that come together to form a "heterodimer" that is expressed on the surface of a cell.

They are somewhat top-heavy. A huge portion of the protein is extracellular and sticks out at the surface of the cell, and just a tiny tail of a few dozen amino acids protrudes through the membrane on the inside of the cell.

The large extracellular portions are the domains that bind to molecules on the outside of the cells and mediate the interactions of the cell with surrounding matrix proteins and with other cells.

If tissues were trains and cells were the boxcars, then integrins would be the hooks that hold the boxcars together and keep them on their tracks. This helps to maintain the integrity of tissues in mammals and other multicellular organisms. They are also important in early development for the formation of distinct tissues.

But integrins do more than just hold cells together. They are also crucial mediators of a host of other normal and abnormal biological processes. They are important for inflammation; they are essential for platelet aggregation after vascular injury; and they are involved in cell motility. As such, they are involved in diseases where the normal mechanisms of platelet aggregation go awry—as in heart attacks and strokes—and they are implicated in cancer metastasis.

Capturing the Arrest of Tumor Cells

One reason why integrins are important for metastasis is that they help the cancer cells attach within the vasculature of new tissues.

When a cancer cell is in the circulation, it may have difficulty locking onto new tissues because of the interference of blood cells, plasma proteins, and the shear force produced by blood flow. Cancer cells need something like a hook to anchor themselves to a particular spot under these dynamic flow conditions, and integrins can be such hooks.

Felding-Habermann uses a video microscopy system with a flow chamber, established by MEM Professor Zaverio M. Ruggeri, to study how tumor cells arrest in flowing blood. The surface in the flow chamber is engineered to mimic the surface of a blood vessel—with a layer of endothelial cells on top of collagen and other matrix components. When she began these experiments, her question was what would happen when she added metastatic breast cancer cells to blood and "perfused" this mixture over components of the vessel wall.

Her videos show the results: the movement of blood across the screen. Colored with a green fluorophor are platelets, those tiny, molecule filled cells in the blood that are necessary for clotting. Also moving across the surface are cancer cells, which are colored with a red fluorophor. When highly aggressive tumor cells are mixed into the blood sample, they get stuck to the surface. Non-metastatic cells just move on with the flow. Figuring out why was the challenge. Felding-Habermann noticed that the platelets, which form tiny clots or thrombi, help the aggressive cancer cells to arrest.

"You see all these thrombi?" she asks, pointing at the green blotches on the screen. "Almost all of them have tumor cells stuck to them or embedded in them. You notice that when I switch to the microscope filter that detects only the red signal from the tumor cells."

The Link Between Integrins and Cancer

Breast cancer cells express a particular integrin, called avb3, that is similar to an integrin expressed on the surface of platelets. Normal breast epithelium does not express this integrin, but cancer cells do.

Integrin avb3 seems to be needed for the cancer cells' arrest.

The cancer cell integrin has the ability to support interactions between the tumor cells and platelets by binding plasma proteins. Once bound, these proteins can form molecular bridges between the tumor cell integrins and platelet integrins. Felding-Habermann noticed that tumor cell integrin avb3 supports this interaction while blood is flowing - but only if the receptor is activated, as on aggressive, metastatic cells.

This way, tumor cells with activated integrins on their surfaces can cross-link when they hit a thrombus of platelets, even if they are tiny, and they can readily attach to the tissue at that location.

"What you get is a cohesion of tumor cells and platelets," says Felding-Habermann.

The tumor cells with active integrins on their surface can also fish out platelets from the bloodstream. Once the tumor cells have platelets on their surface, they become very sticky. This is shown on another one of her videos. Green and red globs pass together over the surface, getting stuck from time to time as they flow. These globs can form microaggregates as another strategy for arresting, she says. The microaggregates can get stuck in narrow capillaries where the tumor cell can then establish a new colony.

To demonstrate that these integrins were necessary for tumor cell arrest, Felding-Habermann and her colleagues selected a cancer cell line without the beta subunit of the integrin. She discovered that those cells cannot arrest on the surface, but if the beta subunit was added back in, the ability of the cancer cells to arrest was restored.

Similarly, Felding-Habermann says that she has compared normal blood to blood without platelets. The platelets in normal blood immediately begin to clump under the microscope, and the cancer cells soon attach to these clumps. However, she found that when one removes the platelets from the bloodstream, the breast cancer cells cannot attach.

To test the clinical relevance of these findings, Felding-Habermann collaborates with physician Alan Saven, director of the Scripps Cancer Center and Head, division of Hematology/Oncology, Scripps Clinic. Saven provides the clinical insight and connects her with clinicians who help her collect blood samples and tissue specimens from patients with metastatic breast cancer. Felding-Habermann has fished out metastatic tumor cells from patient blood and demonstrated that they do indeed interact with platelets by using integrin avb3. Her perfusion experiments proved that all of the isolated metastatic cells carry avb3 in its activated form.


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Associate Professor Brunhilde Felding-Habermann will speak on her work at the Department of Molecular and Experimental Medicine 's annual retreat. Photo by Kevin Fung.